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Archives of Clinical Microbiology

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Editor in Chief Dr. Ahmed Sherif Attia

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Department of Microbiology and Immunology Faculty of Pharmacy, Cairo University

ACMicrob is a hybrid journal with rapid publication of articles in all fields and areas of microbiology and infectious diseases.

Published by IMedPub publishing house. ISSN: 1989-8436

2010

Content
Issue
1:1 1:2 1:3 1:4 1:5 2:1 2:2

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Article
Launch Editorial Archives of Clinical Microbiology: A Versatile Journal in the Field. Clinical virology: An oxymoron or a useful tool? The Gastrointestinal Tract: A Friend or Foe to Listeria monocytogenes? Comparative Study of the Effect of Bile on the Listeria monocytogenes Virulent Strain EGD-e and Avirulent Strain HCC23. Human cytomegalovirus infection in pregnant women and neonates: A new risk factor for cardiovascular disease? Archives of Clinical Microbiology: New Vision Interleukin (IL)-1a, IL-7 and IL-13 Profiles among Nigerians with Trypanosoma brucei gambiense Infection Phytochemical Screening And Hepatoprotective Properties Of The Aqueous Root Bark Extract Of Sarcocephalus latifolius (Smith) Bruce (African Peach) Hospital-Acquired Bloodstream Infections in Cancer Patients between 2005 and 2007 in a Turkish University Hospital.Segregation Analysis Varicella Pneumonia in an Immunocompromised Patient: a case report Strongyloides Colitis presented with acute abdomen The role of Herbal Medicine use in HIV/AIDS treatment Multi-system organ failure following administration of yellow fever vaccine: a case report Genetic diversity of Aeromonas spp. isolates from animal demonstrated by Random amplified Polymorphic DNA analysis. Extended spectrum -lactamase in clinical isolates of Escherichia coli in a tertiary care centre. Investigations of Recurrent outbreaks of unknown fever, establish rural dengue activity in West Midnapore, a costal district in West Bengal, India. Helicobacter pylori infection: efficacy of probiotics and role of genome wide association studies Genetic Heterogeneity of Prevotella Strains Involved in Dentoalveolar Abscess Antimicrobial and antioxidant efficacy of various solvent extracts of seeds and fruits rind of Caesalpinia pulcherrima Swartz.

2:3

2:4 3:1 3:2 3:3 3:4 3:5 4:1 4:2 4:3 4:4 4:5

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Launch Editorial Archives of Clinical Microbiology: A Versatile Journal in the Field.


Aamir Shahzad
Max F. Perutz Laboratories, University of Vienna, Vienna Bio Center, Vienna, Austria. aamir.shahzad@univie.ac.at

This editorial is written to give introduction of Archives of Clinical Microbiology (ACM). Archives of Clinical Microbiology (ACM) is a new peer reviewed, international journal in the field of Clinical microbiology and infectious diseases which covera all aspects of these fields. Archives of Clinical Microbiology (ACM) aims to provide latest updates on modern research in the field to scientific community around the world. Archives of Clinical Microbiology (ACM) journal have world famous scientists in journals editorial board.1 As, being international journal, Archives of Clinical Microbiology (ACM) editorial board is represented by famous scientists from all continents of the world. Archives of Clinical Microbiology (ACM) covers all aspects of basic and clinical microbiology subjects relevant to infectious diseases including current research on diagnosis, management, treatment, preventive measures, vaccination, and methodology. Clinical microbiology relevant immunology, pathophysiology, genetics, epidemiological, anthropology and genomics studies are also welcome. There are many journals relevant to clinical microbiology field but all these journals are limited in their scope and are also confined to only lab science research or only cover clinical research. Archives of Clinical Microbiology (ACM) is aimed to combine both lab and clinical research related to microbiology and infectious diseases. Archives of Clinical Microbiology (ACM) is an open access journal with rapid publication of articles.2 Articles are subject to standard peer review process which will be fast process as compared to other journals in the field. Articles will be immediately published after their acceptance. The published articles will be freely available to the readers throughout the world without any need to pay any charges to get access to full articles. It means that published articles will have more readers as compared to printed journals articles which charge a subscription fees. Archives of Clinical Microbiology (ACM) charges very low article processing charges (APCs) as compared to many open access journals.3 Further more, to encourage submissions from lower income nations, Archives of Clinical Microbiology (ACM) is offering special discounts in article processing charges (APCs). Archives of Clinical Microbiology (ACM) publish original research papers, case reports, short communications, personal views, comprehensive and mini review articles and also short letter to editor articles. I invite you to submit your precious research work in Archives of Clinical Microbiology (ACM).

References and Links:


1. Archives of Clinical Microbiology (ACM) Editorial Board (website link) [link to http://imedpub.com/ojs/index.php/acmicrob/about/editorialTeam] 2. Why publish with iMedPub? (website link) [link to http://imedpubjournals.ning.com/notes/index/show?noteKey=Why_publish_with_iMedPub%3F] 3. iMedPub Article processing charges (APCs) policy (website link) [link to http://imedpubjournals.ning.com/notes/Article-processing_charges]

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Archives of Clinical Microbiology (ACMicrob) is a new peer reviewed, international journal, with world famous scientists on the editorial board. ACMicrob is an open access journal with rapid publication of articles in all fields and areas of microbiology and infectious diseases. ACMicrob covers all aspects of basic and clinical microbiology relevant to infectious diseases including current research on diagnosis, management, treatment, preventive measures, vaccination, and methodology. Clinical microbiology relevant immunology, pathophysiology, genetics, epidemiological, and genomics studies are also welcome.

Submit your manuscript here:

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Clinical virology: An oxymoron or a useful tool?


Charles H. Calisher.
Professor, Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences Colorado State University 3195 Rampart Rd., Delivery Code 1690, Foothills Campus Fort Collins, CO 80523-1690 TEL: (970) 491-2987 FAX: (970) 491-8707 e-mail: calisher@cybersafe.net

If a patient sees a physician because the patient has this, that or the other signs and symptoms and is told he or she has a virus infection, they might begin to wonder whether they have walked or been carried into a shoe repair shop by mistake. Such impromptu diagnoses, even if correct, are neither useful nor inexpensive and one would be better off staying home in bed and getting some rest. The physician should order some sort of lab test that would help focus on what is ailing the patient or, at the very least, what is not. Nonetheless, it is unlikely that he or she could do anything for the patient anyway. A bit of tincture of time is called for in most virus infections. So, excluding the virus infections that can be treated (influenza viruses, herpesviruses, human immunodeficiency virus, respiratory syncytial virus, Lassa virus and a few others), there is not much the physician can do anyway, no matter the viral disease. For the most part, it is a race between the virus and the immune responses, so one is on ones own. When recovered, at least one knows one will hopefully not have to deal again with that particular virus. On the other hand, for the good of the population, if not for the good of the individual patient, it is always useful to know what is circulating in a population.. Knowing what agent is or might be circulating in the community and recognizing the pattern of its circulation (the classic what?, who?, when?, where?, and how?) often times can easily lead to prevention of other cases. Without knowing what we are dealing with, we are at the mercy of the disease. Therefore, the epidemiologic characteristics of a disease are second in importance only to identifying the disease and one cannot make a proper primary diagnosis without some laboratory backup. Theres the rub! Diagnosis. We virologists like to talk about geographic distributions, nucleotide sequences, electron microscopic characters, seasonality, and all sorts of other diagnostic techniques that are mostly (not completely) ineffective and untimely with respect to diagnosis of the patient, except as they relate to the rest of the community epidemiology. What is needed, obviously, are rapid, accurate and precise techniques. A rapid diagnosis may be, but is not always helpful. An accurate diagnosis may be easy, but is not always dependable. A precise diagnosis may be easy, but it may not be possible with certain commercial or in-house kits, techniques, and other procedures. Rapidity, accuracy and precision are key diagnostic elements, with experience and overview absolutely necessary resources. The results of a test which indicates a 10% probability of either a rhinovirus or tick-borne encephalitis virus infection is less than useful and results in wasted precious time and effort (and money). Because the need for rapid and precise techniques is great and a profit might be made by developing them, many civilian and military investigators, universities, and commercial interests have pitched headlong into this area of study and product development. It is a great time to be alive and interested in viral diagnosis. The twin, or at least related, fields of diagnosis and treatment clearly are moving towards prevention of diseases and interruption or retardation of viral replication and spread. Nonetheless, as do all scientific advances, they move slowly, meticulously, sometimes misleadingly, and sometimes erroneously towards the goals; five steps forward and one-step backwards, but forward in the end. Basic knowledge of immunology, pathophysiology, and treatment is helpful in managing the patients illness, but such treatment can be similar to finding a black box in a large and unlighted room, unless the
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clinician has some idea of the cause of the illness. Taking a good guess can be very useful if the guess is correct, but taking a bad guess can be disastrous for both the patient and for the community. When Nipah virus infections are diagnosed (clinically or using inaccurate laboratory assays) as Japanese encephalitis or measles, when West Nile virus infections are diagnosed as St. Louis encephalitis virus infections, or any time when infection with virus X is diagnosed as infection with virus Y, we should all be embarrassed and perhaps a great deal less arrogant than we are. As much attention should be paid to a sample at 4 PM on a Friday as at 11 AM on a Tuesday; the patient and the clinician are waiting. Reports should be required to be made available as soon as possible so that the people who need to make larger decisions can have time to make them. Information, sometimes withheld for political reasons, should be disseminated as quickly and as honestly as possible. Diagnosis is, or should be, a team effort, beginning with the very basics: accuracy, rapidity and precision. The bottom line in all this is the word clinical: (A) the patient, (B) the patient, and (C) the patient. If the information we obtain is not bedside-relevant and patient-oriented, the information we obtain is useful, but only as a foundation for other studies or for cocktail party chatter. The aim of this new journal is to publish timely articles regarding all aspects of basic and clinical microbiology relevant to infectious diseases, including current research on diagnosis, management, treatment, preventive measures, vaccination, and methodology. Clinical microbiology-relevant immunology, pathophysiology, genetics, epidemiological, and genomics studies also will be published. I wish the publisher and editors good luck in receiving enough first class manuscripts to meet their goals. If they are hard-nosed about what is acceptable to the journal and what is not, they will not need luck.

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Archives of Clinical Microbiology (ACMicrob) is a new peer reviewed, international journal, with world famous scientists on the editorial board. ACMicrob is an open access journal with rapid publication of articles in all fields and areas of microbiology and infectious diseases. ACMicrob covers all aspects of basic and clinical microbiology relevant to infectious diseases including current research on diagnosis, management, treatment, preventive measures, vaccination, and methodology. Clinical microbiology relevant immunology, pathophysiology, genetics, epidemiological, and genomics studies are also welcome.

Submit your manuscript here:

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2010 Vol.1 No. 1:3

The Gastrointestinal Tract: A Friend or Foe to Listeria monocytogenes?


Janet R. Donaldson
Department of Biological Sciences, Mississippi State University, Box GY, Mississippi State, MS 39762 Correspondence: email: Donaldson@biology.msstate.edu, tel: (662) 325-9547, fax: (662) 325-7582

Listeria monocytogenes is a gram-positive bacillus and is the causative agent of listeriosis. Approximately 2500 cases of listeriosis and 500 deaths are reported each year, making it the deadliest food borne pathogen [1]. The CDC has reported that the incidence of L. monocytogenes has decreased by 36% between 1996 and 2006. However, outbreaks still occur, making it a cause for concern among the medical and food industries. During its infection cycle, L. monocytogenes is subjected to a variety of elements that are in place by the host to combat infections. The bacterium enters the body through ingestion of contaminated food (most likely) and must then resist the dangerous environments encountered throughout the gastrointestinal tract. Some of these stressors are acidic environment of the stomach, intracellular states within intestinal epithelial cells and macrophages, reduced oxygen content, and bile. Bile is composed of a multitude of components, of which bile salts have been shown to provide protection against pathogenic bacteria [2]. It has been proposed that resistance to high concentrations of bile salts is critical to the establishment of listeriosis [3]. Studies have found that L. monocytogenes replicates extracellularly within the lumen of the gallbladder, where bile salt concentrations can be as high as 15% or more [4, 5]. It is surprising that this facultative intracellular pathogen chooses to grow extracellularly in this potentially harmful environment. It has been proposed that L. monocytogenes grows extracellularly in the gallbladder in order to be more efficient at spreading throughout the gastrointestinal tract. Recently, it was found that bile actually enhanced the ability of L. monocytogenes to form biofilms [6]. The study was conducted using 0.3% bile salts, which is a concentration similar to that found in the small intestine. It was proposed that this might allow for enhanced colonization by Listeria in the intestines. Therefore it is possible that the cells are forming biofilms in the intestines, but the gallbladder provides a location for the cells to replicate before establishing an invasive infection. Findings from our lab suggest that pathogenic strains may be more resistant to high concentrations of bile salts than avirulent strains [7]. We found that extensive surface deformities were present on an avirulent strain and were not observed in a virulent strain following exposure to high concentrations of bile salts (20%). The cell width of the virulent strain increased, while the width of the avirulent strain actually decreased [7]. We proposed that bile salts alter the cell membrane of both virulent and avirulent strains, but the effect is much more severe in the avirulent strain. The compromised membrane then allows for the influx of bile salts, which then target the DNA and induce ds breaks, leading to cell death.

Even though these findings have contributed significantly to our understanding of how bile resistance influences the establishment of invasive infections, much work is still needed in this area in order to determine mechanisms involved in bile resistance in enterics. Many bile resistance genes have been found in L. monocytogenes, but it is unlikely that these genes are the only means for bile resistance since they are also found in bile sensitive strains [7]. Therefore, studies are needed that analyze the global expression patterns presented by enterics in these stressful environments. Recently, it was found that exposure to the acidic environment of the stomach preconditions L. monocytogenes to be more invasive [8]. They found that L. monocytogenes treated with acid or salt prior to infections were better equipped to grow within Caco-2 epithelial cells. Another study also found that exposure of L. monocytogenes to acid selects for acidtolerant bacteria, which in turn were more virulent than wild-type strains [9]. These (and other) studies suggest that the sequential exposure of L. monocytogenes to the stressors encountered throughout the gastrointestinal tract may actually increase resistance to downstream stressors, 3 thus making the bacterium more virulent. Research is needed to determine whether exposure to bile salts also precondition bacteria for intracellular infections or even biofilm formation and whether this preconditioning might be dependent upon the pathogenic potential of the bacterium. In conclusion, many studies suggest that bile tolerance is a major contributor to the infection cycle of L. monocytogenes and other enterics as well. The gastrointestinal tract exposes L. monocytogenes to varying concentrations of bile salts, which could possibly result in bacteria better adapted to survive within the gastrointestinal tract. Deciphering the mechanisms utilized to tolerate these environments will help develop methods to circumvent enteric diseases.

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References
1. Farber, J.M. and P.I. Peterkin, Listeria monocytogenes, a food-borne pathogen. Microbiol Rev, 1991. 55(3): p. 476-511. 2. Merritt, M.E. and J.R. Donaldson, Effect of Bile Salts on DNA and Membrane Integrity of Enteric Bacteria. J Med Microbiol, 2009. 3. Gunn, J.S., Mechanisms of bacterial resistance and response to bile. Microbes Infect, 2000. 2(8): p. 907-13. 4. Hardy, J., J.J. Margolis, and C.H. Contag, Induced biliary excretion of Listeria monocytogenes. Infect Immun, 2006. 74(3): p. 1819-27. 5. Hardy, J., et al., Extracellular replication of Listeria monocytogenes in the murine gall bladder. Science, 2004. 303(5659): p. 851-3. 6. Begley, M., C. Kerr, and C. Hill, Exposure to bile influences biofilm formation by Listeria monocytogenes. Gut Pathog, 2009. 1(1): p. 11. 7. Merritt, M.E., A.M. Lawrence, and J.R. Donaldson, Comparative Study of the Effect of Bile on the Listeria monocytogenes Virulent Strain EGD-e and Avirulent Strain HCC23 Arch Clinical Micro, 2009. accepted. 8. Olesen, I., F.K. Vogensen, and L. Jespersen, Gene transcription and virulence potential of Listeria monocytogenes strains after exposure to acidic and NaCl stress. Foodborne Pathog Dis, 2009. 6(6): p. 669-80. 9. ODriscoll, B., C.G. Gahan, and C. Hill, Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence. Appl Environ Microbiol, 1996. 62(5): p. 1693-8.

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Archives of Clinical Microbiology (ACMicrob) is a new peer reviewed, international journal, with world famous scientists on the editorial board. ACMicrob is an open access journal with rapid publication of articles in all fields and areas of microbiology and infectious diseases. ACMicrob covers all aspects of basic and clinical microbiology relevant to infectious diseases including current research on diagnosis, management, treatment, preventive measures, vaccination, and methodology. Clinical microbiology relevant immunology, pathophysiology, genetics, epidemiological, and genomics studies are also welcome.

Submit your manuscript here:

http://www.acmicrob.com

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Comparative Study of the Effect of Bile on the Listeria monocytogenes Virulent Strain EGD-e and Avirulent Strain HCC23.
Megan E. Merritt1, Amanda M. Lawrence2, and Janet R. Donaldson1*
Virology Division. Molecular Diagnostic Laboratories, LLC 2439 Kuser Road, Hamilton, NJ 08690-3303. Financial support: This work was supported by MDL. 1Department of Biological Sciences, Mississippi State University, Mississippi State, MS and 2 Electron Microscopy Center, Mississippi State University, Mississippi State, MS *Correspondence: email: donaldson@biology.msstate.edu; Tel: (662) 325-9547. Fax: (662) 325-7582.

Abstract Listeria monocytogenes is an enteric pathogen that can replicate within bile. Tolerance to bile has been suggested to be related to the pathogenic potential of the bacterium. We analyzed whether bile exerts different types of damage to the cell membrane of the virulent strain EGD-e and the avirulent strain HCC23. The growth and morphology of the serovar 1/2a strain EGD-e and the serovar 4a strain HCC23 were analyzed under both aerobic and anaerobic conditions following exposure to bile salts. Our data indicate that exposure to bile salts significantly impairs the growth of HCC23 as compared to EGD-e. Additionally, scanning electron microscopy and transmission electron microscopy analyses indicate that bile salts alter the cell envelope of EGDe and HCC23 differently. Our results indicate that differences exist in the ability of the virulent strain EGD-e and avirulent strain HCC23 to tolerate bile, suggesting that bile resistance may be directly related to pathogenicity.

Introduction
Listeria monocytogenes is a gram-positive bacterial pathogen and is the causative agent of the food-borne illness listeriosis. L. monocytogenes is responsible for nearly 28% of reported food-related deaths each year in the United States [1]. This bacterium is able to proliferate in a wide range of environments, making it a dangerous source of food contamination [2-5]. The implementation of a zero-tolerance policy for the presence of L. monocytogenes in ready-to-eat (RTE) food products in 1989 led to a drastic reduction in the incidence of L. monocytogenes contaminated RTE-products. However, there are still many reported cases of L. monocytogenes contamination and listeriosis annually [6], indicating that it is essential to understand the pathogenesis associated with this microbe. Resistance to bile is considered a major virulence determinant for enterics [7]. The establishment of listeriosis is dependent upon the ability of L. monocytogenes to survive the acidic and bile environments encountered in the gastrointestinal system and to invade and replicate within the epithelial cells lining the intestinal tract [8]. Several mechanisms have been identified that allow for the resistance of L. monocytogenes to bile, including the bilE operon that excludes bile from the cytoplasm and bsh, which encodes a bile salt hydrolase involved in converting bile to a less toxic form [9-11]. In addition, the virulence regulator prfA regulates expression of these bile tolerance genes [9-11], indicating the necessity for bile resistance in the pathogenesis of L. monocytogenes. Bile is mainly composed of bile salts, cholesterol, and phospholipids. The detergent activity of bile is primarily attributed to the bile salt component [7]. L. monocytogenes is highly resistant to bile salts [9, 12]. However, it has not been analyzed whether this capability is directly related to pathogenicity. To determine if bile resistance could be related to the virulence capability of L. monocytogenes, we examined the effect that bile salts have on the survival and maintenance of the membrane integrity of the virulent strain EGD-e and the naturally isolated avirulent strain HCC23 under aerobic and anaerobic conditions. EGD-e is a serovar 1/2a strain that was initially described in 1926 following an
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outbreak of illness among laboratory animals [13]. HCC23 is a serovar 4a strain that was initially isolated from the brain of a healthy channel catfish and was subsequently found to be avirulent through mouse infection studies [14]. We found that bile salts affected the membranes of both EGD-e and HCC23, but this effect was much more severe in the avirulent strain. These results suggest that the pathogenic potential of L. monocytogenes could be directly related to bile tolerance, but bile tolerance should not be used as a sole indicator for virulence capability. We present these data and suggest a model for how bile may act as a bacteriostatic agent against L. monocytogenes.

Methods
Bacterial strains and anaerobic growth conditions. The L. monocytogenes strains EGD-e and HCC23 were grown in brain heart infusion (BHI) media at 37C. All experimental methods were performed with cultures grown either aerobically or anaerobically. Anaerobic conditions were achieved by placing Wheaton serum bottles containing 1 ml of bile-infused BHI media in a vinyl anaerobic chamber for 48 hr (Type B, Coy Laboratory Products, Inc.), after which bottles were capped with aluminum seals. Syringes were used to inoculate cultures and remove samples. Anaerobic conditions for BHI plates were achieved by incubating the plates in a BBL Gas Pak System. Anaerotest strips were used to verify anaerobic conditions. Growth analysis in the presence of bile salts. Fresh overnight cultures of EGD-e or HCC23 were diluted 1:100 in 2 ml of BHI medium containing 0%, 10%, or 20% oxgall, sodium glycodeoxycholate (GDCA), or sodium taurodeoxycholate hydrate (TDCA) and were grown at 37C in a shaking incubator under either aerobic or anaerobic conditions. Bile salts were purchased from Sigma Aldrich. For each time point, 2 l of culture were used for OD600 measurements with a Nanodrop ND-1000. Pathlengths for the Nanodrop readings were adjusted in accordance with the manufacturer. Three independent experiments were averaged for each

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bile salt under both anaerobic and aerobic conditions. Additionally, cultures grown in 0%, 10%, or 20% oxgall, GDCA, or TDCA for 8 hr at 37C were plated onto BHI agar and incubated overnight at 37C. Analyses of growth on agar plates were performed for three, independent experiments under both aerobic and anaerobic conditions. Cultures treated with bile under anaerobic conditions were plated and incubated under anaerobic conditions. Scanning electron microscopy. Fresh overnight cultures of EGD-e or HCC23 were diluted 1:100 in 2 ml of BHI media infused with either 0% or 20% oxgall. After a 6 hr shaking incubation at 37C, cells were centrifuged at 8,000 x g for 3 min at room temperature (Eppendorf Centrifuge 5415R). The resulting bacterial cell pellets were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M cacoldylate buffer, washed in 0.1 M cacoldylate, post-fixed in 1% (v/v) osmium tetraoxide in 0.1 M cacoldylate buffer, rewashed in distilled water, dehydrated in an ethanol series, and dried in an hexamethyldisilazane (HMDS) series. Samples were sputter coated with goldpalladium (Polaron SEM coating system) prior to observation with a field emission scanning electron microscope (JEOL JSM-6500F). Samples were prepared from three independent experiments of EGD-e or HCC23 treated with either 0% or 20% oxgall under both aerobic and anaerobic conditions. The length and width of 20 individual cells from each independent experiment were collected for analysis using the JEOL-PC-SEM 6500 software provided with the microscope. A mean average was calculated for cell length and cell width for data collected from the control cells (0% oxgall) and treated cells (20% oxgall). The mean average from the control cells was compared to the mean average from the bile treated EGD-e or HCC23 cells using a student t-test. A p-value < 0.05 indicated that the parameter measured (cell length or cell width) had significantly changed following treatment with oxgall. Transmission electron microscopy. Sample preparations for TEM observations were performed as indicated above for the SEM with the following exceptions. After the dehydration step, the cells were treated in a stepwise resin/ethanol series and embedded into resin at 68 70C overnight. The cells were then sectioned using an ultramicrotome (Reichert-Jung Ultracut E) and viewed under a transmission electron microscope (JEOL JEM-100CXII). The width of the cell wall, cell membrane, and cell envelope for 20 individual cells from each independent experiment was collected for analysis. Microscopic analyses were performed on three independent experiments for cells grown under aerobic or anaerobic conditions with either 0% or 20% oxgall. A mean average was calculated for the cell wall, cell membrane, and cell envelope thickness for control cells (0% oxgall) and bile-treated cells (20% oxgall). The mean average from the control was compared to the mean average from the treated EGD-e or HCC23 using a student t-test. A p5 value < 0.05 indicated that the parameter measured (cell membrane, wall, or envelop thickness) had significantly changed following treatment with oxgall.

shown for TDCA and GDCA). The growth of HCC23 in the presence of bile salts was much more impaired than that observed for EGD-e. Increasing the concentration of bile salts exaggerated the growth deficiency exhibited by HCC23 (Fig. 1A). However, the avirulent strain did appear to maintain minimal cell viability through spectrophometric analysis. To determine if HCC23 was still viable following exposure to bile salts, cells were plated after 8 hr of incubation in the presence of oxgall, TDCA, or GDCA. Our results indicated that approximately 5% of the HCC23 cells remained viable in the presence of 20% oxgall as compared to untreated controls (3.8 x 108 treated with 20% oxgall as compared to 6.7 x 109 untreated viable at 8 hr). The growth of EGD-e in the presence of bile salts was also impaired as compared to the control EGD-e cells (Fig. 1A). However, in contrast to HCC23 bile-exposed cells, growth of EGD-e increased to the level of control cells following an extended lag period (Fig. 1A). Under normal growth conditions, EGD-e exhibited a 1 hr lag phase. In the presence of 10% oxgall, TDCA, or GDCA this phase was extended to 2 hr. In the presence of 20% bile salts, this growth period was extended to 6 hr (Fig. 1A). The effect of the bile salt on EGD-e and HCC23 was similar regardless of whether oxgall or conjugated GDCA or TDCA was used as the treatment (data not shown). To determine if similar growth patterns were observed in environmental conditions that potentially mimic those found within the human digestive system, we analyzed the ability of EGD-e and HCC23 to grow in the presence of bile salts under anaerobic conditions (Fig. 1B). In general, the growth of both strains was less prolific under anaerobic conditions. Both strains exhibited a decrease in growth with an increase in the concentration of bile salt. EGD-e had a 2hr lag phase in BHI media under anaerobic growth. This was slightly longer that that observed for aerobic conditions. Treatment of EGD-e with 10% bile salts increased the lag phase to 4 hr. However, exposure to 20% bile salts had a greater inhibitory affect on the growth of EGD-e under anaerobic conditions (Fig. 1B). These results were further analyzed by plating cells following an 8-hr incubation in the presence of 20% bile salts on BHI agar under anaerobic conditions. Approximately 17% of EGD-e treated with 20% oxgall survived as compared to untreated controls cells (3.71 x 109 treated with 20% oxgall as compared to 2.24 x 1010 untreated viable at 8 hr).
A EGD HCC23

Results and Discussion


Analysis of EGD-e and HCC23 growth in the presence of bile salts. L. monocytogenes must be able to resist the damaging effects of bile salts in order to establish infections. With the recent finding that L. monocytogenes is able to replicate extracellularly within the gallbladder [15, 16] where bile salt concentrations can be 15% or higher, it has been suggested that resistance to high concentrations of bile salts is essential for the pathogenesis of this pathogen [7]. Additionally, it was recently found that bile aids in the formation of biofilms of L. monocytogenes [17], suggesting that this feature may allow for the survival of L. monocytogenes in the high bile environment of the gallbladder. To determine if the pathogenic potential of L. monocytogenes could potentially be related to bile tolerance, we examined the ability of the avirulent strain HCC23 and the virulent strain EGD-e to grow in the presence of 0%, 10%, or 20% of oxgall, GDCA, or TDCA under both aerobic and anaerobic conditions. Under aerobic conditions growth was affected for both EGD-e and HCC23 in the presence of oxgall, TDCA, and GDCA (Fig. 1A, data not
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B

Time (hrs)

Time (hrs)

EGD

HCC23

Time (hrs)

Time (hrs)

FIGURE 1. Growth of EGD-e and HCC23 in the presence of oxgall. EGD-e (!) and HCC23 (!) were grown under either aerobic conditions (A) or anaerobic conditions (B) in 0% (blackfilled), 10% (hashed), or 20% (white-filled) oxgall. Graphs represent the average of three independent experiments.

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HCC23 did not have a longer lag phase under anaerobic conditions as was observed with EGD-e. However, cell growth was never able to reach the same concentration as that observed under aerobic conditions. Treatment with 10% and 20% bile salts under anaerobic conditions produced similar growth patterns as observed under aerobic conditions for HCC23 (Fig. 1). These results suggest that EGD-e may be better equipped to adapt to this stressful environment, repair DNA and membrane damage that might have been introduced by the bile salts, and resume replication to some extent. Bile salts induce morphological changes in both EGD-e and HCC23. Utilizing scanning electron microscopy we were able to visually observe the different effects that bile salts have on the cell surface of these strains of L. monocytogenes. Since similar growth patterns were observed for oxgall, TDCA, and GDCA treated cells, only oxgall was used for morphological analyses. Additionally, since differences were seen in the ability of EGD-e to grow in the presence of 20% oxgall under different environmental conditions, morphological differences were analyzed in bile treated cells grown under aerobic and anaerobic conditions. Changes in cell length and width were examined for three independent experiments for control and bile-treated EGD-e and HCC23 grown under aerobic and anaerobic conditions. In aerobic conditions nearly 78% of HCC23 treated with 20% oxgall exhibited visible surface deformities, indicating a loss of rigidity to the surface (Fig. 2A). These distortions were not present in the untreated HCC23, confirming that these alterations to the membrane were due to the bile salts. EGD-e showed less deterioration to the cell wall when exposed to oxgall; only 22% of the bile treated EGD-e cells exhibited damage to the cell surface under aerobic conditions (Fig. 2A). Additionally, these changes were not observed under control conditions. The length and width of control and bile-treated cells were measured to determine if oxgall altered the shape of L. monocytogenes under aerobic conditions. Oxgall did not significantly alter the cell length of either EGD-e or HCC23 (Table 1). However, the cell width of both strains was significantly altered in the presence of bile salts under aerobic conditions.
TABLE 1. Average cell length and width of HCC23 and EGD-e cells in the presence of 0% or 20% oxgall under aerobic and anaerobic conditions. Average ( m) Strain, % oxgall HCC23, 0% HCC23, 20% EGD-e, 0% EGD-e, 20% Cell Length aerobic 1.329 1.34 1.347 1.376 anaerobic 1.518 1.406* 1.45 1.376 Cell Width aerobic 0.519 0.375 0.376 0.512* anaerobic 0.503 0.429* 0.529 0.508
FIGURE 2. Scanning electron micrographs of EGDe and HCC23 grown under aerobic (A) or anaerobic (B) conditions. Cells were examined following a 6 hr treatment with either 0% or 20% oxgall. Scale bars represent 1m.

*Indicates signi cant changes (p<0.05) in bile treated cells compared to untreated cells.

The cell width of HCC23 decreased in the presence of 20% oxgall. Interestingly, the cell width of EGD-e significantly increased in the presence of oxgall (0.376 m control v. 0.512 m bile treated). This correlated with SEM micrographs that showed indentations along the surface of the cell wall in HCC23, while the rigidity and structure of the EGD-e cell membrane remained intact with little visible change (Fig. 2). To determine if similar deformities occurred under anaerobic conditions, EGD-e and HCC23 treated with 20% oxgall under anaerobic conditions were observed by SEM. Similar to the observations under aerobic condition, bile salts induced significant amounts of damage to the surface of HCC23 (Fig. 2B). Under anaerobic conditions 73% of HCC23 cells exhibited cell surface deformities, while only 27% of EGD-e exhibited minimal damage. In anaerobic conditions growth in the presence of oxgall only significantly changed the cell morphology for HCC23 (Table 1). HCC23 exhibited a significant decrease in the cell length (1.518m control v. 1.406m bile treated) and cell width (0.503 m control v. 0.429 m bile treated (Table 1). It is possible that
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HCC23 becomes shorter and thinner as cytoplasmic material is lost through the compromised membrane. This has been demonstrated in Lactobacilli and Bifidobacteria, where it has been shown that bile salts dissipate the transmembrane electron potential [18]. This disrupts the membrane integrity and allows the leakage of protons, potassium ions, and other cellular components out of the cell [18]. The fact that EGD-e expanded in cell length could indicate a mechanism utilized to keep the membrane intact. Bile salts alter the cell envelope of EGD-e and HCC23. To further investigate the effect that bile salts have on the cell envelope of EGD-e and HCC23, cells exposed to 20% oxgall under aerobic and anaerobic conditions were examined using a transmission electron microscope. Cells were analyzed for alterations in the thickness of the cell membrane, cell wall (peptidoglycan layer), and cell envelope (cell membrane and peptidoglycan layer). Comparing the thickness of the cell wall, membrane, and envelope between the control cells and biletreated cells of both strains revealed that bile affected the cell envelope of both strains under aerobic and anaerobic conditions (Fig. 3). Oxgall
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treated HCC23 and EGD-e grown under aerobic conditions showed a significant decrease in the average cell wall and cell envelope thickness when compared to their respective control (Table 2). EGD-e also had a significant decrease in the thickness of the cell membrane. Bile salts induced significant changes to the cell wall, membrane, and envelope of both strains under anaerobic conditions (Fig. 3B, Table 2). The average thickness of the HCC23 cell wall, membrane, and envelope significantly decreased when grown in the presence of bile under anaerobic conditions. While the cell wall and cell envelope of EGD-e decreased, the cell membrane thickness increased when grown in the presence of oxgall (3.5 nm control v. 5.74 nm bile-treated). This decrease in the thickness of the layers of the membrane was due, whether directly or indirectly, to the detergent effect of bile salts.
Table 2. Average thickness of cell wall, membrane, and envelope of HCC23 and EGD-e cells in the presence of 0% and 20% oxgall under aerobic and anaerobic conditions.

Strain, % oxgall HCC23, 0% HCC23, 20% EGD-e, 0% EGD-e, 20%

aerobic anaerobic

Cell Wall

Cell Membrane
aerobic anaerobic

aerobic anaerobic

Cell Envelope

FIGURE 3. Transmission electron micrographs of EGD-e and HCC23 grown under either aerobic (A) or anaerobic (B) conditions. Cells were examined following a 6 hr treatment with either 0% or 20% oxgall. Scale bars represent 0.5m.

20.31 25.57 15.57*13.15* 17.79 25.87 15.91*20.17*

5.02 5.58 8.54 5.21*

3.72 3.32* 3.5 5.74*

25.02 29.07 21.13*16.25* 27.32 29.47 20.91*26.06*

Even though the thickness of the cell envelope of EGD-e was significantly affected, the overall shape of the cell was not altered. This suggests that EGD-e has a mechanism for excluding the bile salts from the cell. Recently, a bile exclusion system (bilE) in L. monocytogenes was characterized for its ability to prohibit bile salts from entering the cell [11]. bilE expression was also shown to be regulated by the main virulence regulator prfA, which indicates that the exclusion of bile is related to virulence. Yet, even in this previous study, radiolabelled bile salts were still able to accumulate within the cell whether bilE was present or mutated, thus indicating that other mechanisms allow for the export of the bile once it has penetrated the membrane. Interestingly, HCC23 (NC_011660) also contains the bilE operon and shows 91% sequence similarity to the bilE gene in EGD-e (NC_003210). It also shows 97% similarity to the bsh gene of EGD-e (lmo2067) and 99% similarity to the proposed bile salt dehydrolase, btlB gene (lmo0754). HCC23 lacks pva (lmo0446), which is the only other gene identified for bile resistance in L. monocytogenes. This suggests that there may be other genes in EGD-e that are yet to be characterized for their involvement with tolerance and adaptation to bile. This area needs to be further explored in order to determine the factors that contribute to bile resistance. Visible differences were also observed in the nucleoid and the cytoplasm of EGD-e and HCC23 treated with 20% oxgall (Fig. 3). Under aerobic conditions, 28% of HCC23 bile-treated cells appeared to have fragmented DNA (Fig. 3A). This affect was exaggerated under anaerobic conditions, where nearly 54% of the HCC23 bile-treated cells exhibited damage. Under both aerobic and anaerobic conditions, only 9% of EGD-e exhibited this same nucleoid damage. Cells were also seen to have areas where the cell membrane was dissociated from the cytoplasm. This was observed in 8% of HCC23 and only 2% of EGD-e under both aerobic and anaerobic growth conditions (Fig. 3). The areas where the membrane seemed to be dissociated from the cytoplasm correlated with the invaginations observed through the SEM. This could be due to the compromised membrane due to displacement of the phospholipids by the bile salts. However, it cannot be excluded that the morphological changes observed at the membrane could be due to autolytic enzymes activated through programmed cell death and are therefore not directly caused by the bile salts [19]. HCC23 accumulates bile salts within the cytoplasm. TEM micrograph examinations also indicated that HCC23 had patterns
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of intracellular darkening following 6 hr of exposure to oxgall. To determine if this cytoplasmic defect could be attributed to an influx of bile salts, we examined HCC23 following a 3-hr and 6-hr exposure to 0% or 20% oxgall in anaerobic conditions. TEM micrographs indicated that a visible darkening occurred within the cytoplasm of these cells. After 3 hr of bile exposure, approximately 13% of the treated HCC23 cells exhibited areas of partial darkening in the cytoplasm (Fig. 4). By 6 hr 20% of HCC23 exhibited this phenotype. HCC23 cells that were not exposed to oxgall did not have any areas of intracellular darkening within the cytoplasm (Fig. 4). Additionally, no areas of cytoplasmic darkening were observed within EGD-e in the presence or absence of bile salts (Fig. 3). The increase in intracellular darkening in the cytoplasm of HCC23 could indicate intracellular accumulation of bile salts. Because our SEM data indicated that the membrane of HCC23 was being significantly altered in the presence of bile salts it is no surprise that the accumulation of bile salts was specific to this strain. Bile salts that are able to breach the cell wall and accumulate within the cytoplasm could then exert DNA damage and arrest replication. This idea is supported by the fact that more HCC23 cells had fragmented

FIGURE 4. Transmission electron micrographs of HCC23 with areas of cytoplasmic darkening. Cells were grown under anaerobic conditions and analyzed at 3 hr and 6 hr posttreatment with either 0% or 20% oxgall. Scale bars represent 0.5m.

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nucleoids compared to EGD-e. If DNA damage occurred the cell would become non-functional, which would explain the decrease in growth, especially in HCC23, in increasing concentrations of bile salts. However, it is also possible that the darkening is an artifact from processing the samples for TEM analysis. Regardless, this would still indicate that the HCC23 strain had a compromised membrane, as this intracellular darkening was not observed in EGD-e. From these data and literature supporting the detergent properties of bile salts on lipids [20] , we propose a model for the potential effect of bile on the viability of L. monocytogenes (Figure 5). Bile salts act on the phospholipids and fatty acids of the membrane to disrupt the proton and potassium ion pumps, thus altering the transmembrane electron gradient and allowing for the influx of bile salts into the cell and efflux of cellular components out of the cell. Bile salts move into the cytoplasm and are targeted to the nucleoid. The bile salts then induce DNA damage, most likely through reactive oxygen species. If the damage is too profound for repair, the cell will cease to replicate. DNA damage induced by bile salts is well described in gramnegative bacteria [21-23] but remains in its infancy in gram-positive bacteria, though a recent study did find the induction of the nucleotide excision repair gene uvrA in L. monocytogenes cells in presence of bile salts [24]. The effect that bile has on the DNA of L. monocytogenes needs to be further analyzed.
FIGURE 5. Potential model for growth arrest in L. monocytogenes avirulent strains in presence of bile. Bile salts damage the structure of the cell membrane, resulting in the loss of the electron chemical gradient, influx of bile salts into the cell, and the efflux of intracellular components. Bile salts accumulate within the cytoplasm and target the DNA. Extensive membrane damage will lead to extensive DNA damage, which will result in replication and growth arrest.

Acknowledgements This work was supported by research funds provided to JRD by the Department of Biological Sciences and the Research Initiation Program at Mississippi State University. We would also like to thank Chris Holly and Cassandre Man-Bourdon for help with the EM analyses.

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Referencias
1. Mead, P.S., Slutsker,L., Dietz, V., McCaig, L.F.,Bresee,J.S., Shapiro, C., Griffin,P.M. and Tauxe, R.V., Food-Related Illness and Death in the United States. Emerging Infectious Diseases, 1999. 5(5): p. 607-625. 2. Cataldo, G., et al., Acid adaptation and survival of Listeria monocytogenes in Italianstyle soft cheeses. J Appl Microbiol, 2007. 103(1): p. 185-93. 3. Farber, J.M. and P.I. Peterkin, Listeria monocytogenes, a food-borne pathogen. Microbiol Rev, 1991. 55(3): p. 476-511. 4. Gahan, C.G., B. ODriscoll, and C. Hill, Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Appl Environ Microbiol, 1996. 62(9): p. 3128-32. 5. Graves, L.M. and B. Swaminathan, PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis. Int J Food Microbiol, 2001. 65(1-2): p. 55-62. 6. Crum, N.F., Update on Listeria monocytogenes infection. Curr Gastroenterol Rep, 2002. 4(4): p. 287-96. 7. Gunn, J.S., Mechanisms of bacterial resistance and response to bile. Microbes Infect, 2000. 2(8): p. 907-13. 8. Gahan, C.G. and C. Hill, Gastrointestinal phase of Listeria monocytogenes infection. J Appl Microbiol, 2005. 98(6): p. 1345-53. 9. Begley, M., et al., Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes. Infect Immun, 2005. 73(2): p. 894-904. 10. Dussurget, O., et al., Listeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis. Mol Microbiol, 2002. 45(4): p. 1095-106. 11. Sleator, R.D., et al., A PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes. Mol Microbiol, 2005. 55(4): p. 1183-95. 12. Begley, M., C.G. Gahan, and C. Hill, Bile stress response in Listeria monocytogenes LO28: adaptation, cross-protection, and identification of genetic loci involved in bile resistance. Appl Environ Microbiol, 2002. 68(12): p. 6005-12.

13. Murray, E.G.D., R.A. Webb, and M.B.R. Swann, A disease of rabbit characterised by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus: Bacterium monocytogenes (n. sp.). J Pathol. Bacteriol., 1926. 29: p. 407-439. 14. Erdenlig, S., A.J. Ainsworth, and F.W. Austin, Pathogenicity and production of virulence factors by Listeria monocytogenes isolates from channel catfish. J Food Prot, 2000. 63(5): p. 613-9. 15. Hardy, J., et al., Extracellular Replication of Listeria monocytogenes in the Murine Gall Bladder. Science, 2004. 303(5659): p. 851-853. 16. Hardy, J., J.J. Margolis, and C.H. Contag, Induced Biliary Excretion of Listeria monocytogenes. Infect. Immun., 2006. 74(3): p. 1819-1827. 17. Begley, M., C. Kerr, and C. Hill, Exposure to bile influences biofilm formation by Listeria monocytogenes. Gut Pathog, 2009. 1(1): p. 11. 18. Kurdi, P., et al., Mechanism of Growth Inhibition by Free Bile Acids in Lactobacilli and Bifidobacteria. J. Bacteriol., 2006. 188(5): p. 1979-1986. 19. Lewis, K., Programmed death in bacteria. Microbiol Mol Biol Rev, 2000. 64(3): p. 503-14. 20. Garidel, P., et al., Membranolytic activity of bile salts: influence of biological membrane properties and composition. Molecules, 2007. 12(10): p. 2292-326. 21. Bernstein, C., et al., Bile salt activation of stress response promoters in Escherichia coli. Curr Microbiol, 1999. 39(2): p. 68-72. 22. Kandell, R.L. and C. Bernstein, Bile salt/acid induction of DNA damage in bacterial and mammalian cells: implications for colon cancer. Nutr Cancer, 1991. 16(3-4): p. 227-38. 23. Prieto, A.I., F. Ramos-Morales, and J. Casadesus, Bile-induced DNA damage in Salmonella enterica. Genetics, 2004. 168(4): p. 1787-94. 24. Kim, S.H., et al., Role of uvrA in the growth and survival of Listeria monocytogenes under UV radiation and acid and bile stress. J Food Prot, 2006. 69(12): p. 3031-6.

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Human cytomegalovirus infection in pregnant women and neonates: A new risk factor for cardiovascular disease?
John A. Blaho,*+
Virology Division. Molecular Diagnostic Laboratories, LLC 2439 Kuser Road, Hamilton, NJ 08690-3303. Financial support: This work was supported by MDL. *Correspondence: John A. Blaho Virology Division MDL Company +Present address: Simons Center for Systems Biology Institute for Advanced Study Princeton, NJ 08540 Email: blaho@ias.edu

Abstract Human cytomegalovirus (HCMV) is a member of the herpes virus family which may cause significant morbidity in immunocompromised patients, including neonates. No vaccine exists for HCMV but appropriate hygienic precautions can limit the spread of the virus to mothers and, subsequently, neonates. Neonatal HCMV has been associated with congenital birth defects while HCMV infection of healthy adults may cause a mild mononucleosis. Recent evidence suggests that HCMV infection may be associated with vascular cell proliferation and increased arterial blood pressure. This commentary questions whether neonatal HCMV infection is a new risk factor for heart disease.

Introduction
Human cytomegalovirus (HCMV) is a member of the Herpesvirideae family of large enveloped DNA viruses. There are eight herpesviruses that are known to infect and cause morbidity and mortality in humans. The definitive characteristic of the herpesvirus family is the ability of these viruses to cause both acute lytic infections, as well as long term persistent latent infections. Thus, once an individual has been infected by HCMV, they remain infected with the virus for the remainder of their life. Throughout the course of an infected individuals life, the virus may reactivate to generate productively replicating virus particles, which may be spread to other, noninfected individuals. Since many HCMV infections are asymptomatic or silent, infected individuals may not know that they pose a risk for transmission to others. It is for this reason that asymptomatic HCMV infections in pregnant mothers pose a significant risk to fetuses and neonates. The consequence of such infections on the coronary heart health of children is the focus of this Commentary. HCMV and vascular cell proliferation. One of the initial correlates of HCMV with vascular disease was the finding of increased HCMV-specific antibodies in atherosclerosis patients who required surgery compared to those who did not [1]. Subsequently, HCMV DNA was detected in atherosclerotic coronary arteries [2]. Perhaps most importantly, anti-HCMV ganciclovir therapy was shown to lower atherosclerosis following heart transplants [3]. It should be noted that these early associations were not universally accepted [4, 5]. Since that time, it has been realized that most clinical HCMV isolates replicate in cultured cells derived from the vasculature, including epithelial, endothelial, smooth muscle, and monocyte-derived macrophage cells [6, 7]. A recently developed rat heart transplant model provided data consistent with HCMV infection stimulating the expression of cellular angiogenesis genes, as well as increasing production of cytokines and growth factors [8]. It now appears that HCMV infection induces the differentiation and migration of monocytes [9], which are likely the major cell type harboring reactivation of latent HCMV. These, and presumably other, modifications to the vasculature as a consequence of HCMV likely impact the pathophysiology of vascular disease.

HCMV infection and high blood pressure. The direct mechanism through which viruses might cause pulmonary hypertension remains unknown. It is conceivable that the anti-viral inflammatory response against HCMV plays an important role in the process. In fact, inflammation-dependent hypertension has been validated in a mouse animal model system [10]. A recent analysis utilizing murine CMV (MCMV) in mice demonstrated that viral infection led to increased arterial blood pressure [11]. Coincident with MCMV replication was increased expression of proinflammatory cytokines. The significance of these studies is their implication that management of CMV infection may limit development of atherosclerosis and hypertension in humans. Cardiovascular disease risk in children. Since 1988, the incidence of hypertension among persons twenty years of age and older has been steadily increasing. According the 2003-2004 National Health and Nutrition Examination Survey, obesity plays a major role. Approximately seventeen percent of individuals aged between two and nineteen years of age are overweight. In light of this growing childhood obesity epidemic, it may be time now to consider the implications of congenital HCMV in the development of cardiovascular disease. Neonatal arterial thrombosis due to severe congenital HCMV infection has been reported [12]. Thus, HCMV exposure during gestation may turn out to be an important additional cardiovascular risk factor. Mechanisms of transmission. HCMV infections are found in all socioeconomic groups throughout all geographic locations in the world. It has been estimated by the United States Centers for Disease Control (CDC) that between 50 and 80% of the adult population in the United States is seropositive for HCMV [13]. While seroprevalence increases with age, the majority of these infections occur prior to puberty. In most cases, transmission of HCMV occur person to person by close physical interaction, which involves contact with secretion and excretion of bodily fluids including saliva, blood, urine, tears, semen, vaginal fluid, and breast milk. Another source of HCMV transmission is receipt of solid organ or hematopoietic stem cell transplantation from an infected donor. HCMV is a member of the TORCHES group of pathogens that can cross the placenta so it also spreads by vertical transmission from mother to child.

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HCMV infection during pregnancy. Most HCMV infections, including late-gestational in utero and neonatal, are subclinical. However, HCMV is the most common virus transmitted from infected pregnant mother to child. Approximately one-third of women who have a primary HCMV infection during pregnancy pass the virus on to the neonate [13]. Thus, approximately one in 150 children are born with congenital HCMV infections. Congenital HCMV infection is defined by detection of the virus in the newborns urine, blood, or saliva within three weeks of birth. Children with congenital HCMV present with small body size, jaundice, petechiae, hypotonia, and hepatosplenomegaly. They may also be prone to lethargy and seizures. Women who are planning to become pregnant may receive an HCMV blood test, which detects the presence of immunoglobulin molecules directed against the virus. If the test is positive for HCMV, it is unlikely that the baby will be at risk for congenital HCMV. Seronegative mothers must take hygienic precautions (listed above). HCMV and breastfeeding. HCMV may be transmitted through breast milk. The newborn population most at risk includes extremely immature (preterm) neonates. The rate of HCMV reactivation in the mother coincides with the seroprevalance of HCMV in the mother so maternal serology is important. Accordingly, the current recommendation of the American Academy of Pediatrics is for breastfeeding of both term and preterm infants [14]. Consequences of congenital HCMV in neonates. In the United States, between 1 and 4% of seronegative mothers get a primary HCMV infection during their pregnancy [13]. If this infection results in HCMV transmission to the unborn child in utero during the first trimester, it may lead to birth defects. The most common consequence is HCMV chronic infection. In extreme cases, there may be central nervous system / perceptual defects or ocular/auditory damage. Approximately one in 750 children in the United States are born with or develop permanent disabilities due to HCMV. This translates into approximately 8,000 children a year. Permanent birth defects associated with congenital HCMV include small head size, lack of coordination, mental disability, and lack of hearing and/or vision. In extreme cases, congenital HCMV may result in death of the neonate. At this time, no tracking exists for the correlation of heart disease development with congential HCMV infection. HCMV treatment and prevention. There is currently no vaccine available to prevent HCMV. Because the HCMV virus has evolved to possess an elaborate system of immune evasion strategies [15], current efforts to develop HCMV vaccines must focus on stimulating both the innate and adaptive immune system in order to be successful. The antiviral treatment of choice for HCMV is the nucleoside analog ganciclovir and it is given to all transplant patients. While ganciclovir may prevent hearing loss in children, it may have side effects. Another nucleoside drug, cidofovir, is also available but is less prominent in use. Both of these drugs have been associated with the emergence to resistant viral strains. A new anti-HCMV drug, maribavir, is currently in clinical trials. Of interest are findings that an oral ganciclovir regime reduces myocarditis in immune competent adults in certain cases [16]. The best way to prevent HCMV transmission is through behavior modification which emphasizes hygiene. Accordingly, women who are pregnant or who may become pregnant are recommended by the CDC to take the following precautions [13]; (i) wash hands often, especially after contact with saliva or diapers of young children, (ii) never kiss children below the age of six years of age on the mouth or cheek, and (ii) do not share food, drinks, or utensils with young children.

HCMV pathogenesis in adults. Primary HCMV infection in an immunocompetent individual is usually subclinical, rarely causing illness. However, large scale infection can cause an HCMV infectious mononucleosis that has a clinical presentation very similar to that caused by Epstein Barr virus. HCMV mono may involve fever, myalgia, lymphadenopathy, splemomegaly, and hepatomegaly [17]. Other rare symptoms of HCMV include tonsillopharyngitis, pneumonitis, myocarditis, arthritis, ulcerative colitis, and meningitis. Transplant patients may also present with retinitis, esophagitis, and gastritis. HCMV infection in transplant patients also predisposes them to infection by other opportunistic pathogens. As noted above, recent evidence indicates that HCMV infection is also a likely risk factor for heart disease in transplant patients. HCMV infection in immunocompromised individuals. While neonates and newborns may be considered immunocompromised individuals, HCMV remains the major pathogen of risk for solid organ transplant patients. More than half of all transplant cases show evidence of HCMV infection. The associated morbidity of these infections is a major cause of rejection. For this reason, anti-HCMV drugs must be administered throughout solid organ transplants. While HCMV infection of solid organ transplant recipients are usually acute primary infections, CMV infection following stem cell transplants are frequently due to reactivation of latent virus. HCMV is a high risk pathogen for individuals who have an impaired adaptive immune response, which is why neonates and newborns are at such high risk. Prior to the establishment of highly active anti-retroviral therapy (HAART), HCMV infections in human immunodeficiency virus (HIV) positive patients were a serious opportunistic infection. At that time, retinitis caused by HCMV was the primary cause of blindness in AIDS patients. Today, patients with chronic HIV infection may develop pulmonary arterial hypertension [18], though the impact of HAART on its incidence remains controversial. The role that HCMV might play in this process is unknown at this time. However, the progressive loss of CD4+ T cells renders these individuals at risk for opportunistic infections, such as new or reactivating HCMV. Perspective. HCMV is a major human pathogen which places both the mother and child at risk. Once infected, the individual remains infected for life and may spread the virus to others. Although effective antiviral drugs exist for HCMV, the problem is that most infections are asymptomatic. Existing research efforts are focusing on the development of an HCMV vaccine. Currently, the best way to prevent HCMV infection is to limit exposure to bodily fluids. Importantly, HCMV is associated with congenital birth defects. Recent evidence supports a molecular basis for the role of HCMV in vascular cell proliferation and arterial hypertension. It may now be the time to consider coronary heart disease as another potential congenital consequence of neonatal HCMV infection.

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Referencias
1. Adam E, Melnick JL, Probtsfield JL, Petrie BL, Burek J, Bailey KR, McCollum CH, DeBakey ME (1987) High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis. Lancet 2:291-293. 2. Wu TC, Hruban RH, Ambinder RF, Pizzorno M, Cameron DE, et al. (1992) Demonstration of cytomegalovirus nucleic acids in the coronary arteries of transplanted hearts. Am J Pathol 140:739-747. 3.Valantine HA, Gao SZ, Menon SG, Renlund DG, et al. (1999) Impact of prophylactic immediate posttransplant ganciclovir on development of transplant atherosclerosis: a post hoc analysis of a randomized, placebo-controlled study. Circulation 100:61-66. 4. Daus H, Ozbek C, Saage D, Scheller B, Schieffer H, et al. (1998) Lack of evidence for a pathogenic role of Chlamydia pneumoniae and cytomegalovirus infection in coronary atheroma formation. Cardiology 90:83-88. 5. Adler SP, Hur JK, Wang JB, Vetrovec GW (1998) Prior infection with cytomegalovirus is not a major risk factor for angiographically demonstrated coronary artery atherosclerosis. J Infect Dis 177:209-212. 6. Gerna G, Zipeto D, Percivalle E, Parea M, Revello MG, et al. (1992) Human cytomegalovirus infection of the major leukocyte subpopulations and evidence for initial viral replication in polymorphonuclear leukocytes from viremic patients. J Infect Dis 166:1236-1244. 7. Ryckman BJ, Rainish BL, Chase MC, Borton JA, Nelson JA, et al. (2008) Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells. J Virol 82:60-70. 8. Streblow DN, van Cleef KW, Kreklywich CN, Meyer C, Smith P, et al. (2007) Rat cytomegalovirus gene expression in cardiac allograft recipients is tissue specific and does not parallel the profiles detected in vitro. J Virol 81:3816-3826.

9. Smith MS, Bentz GL, Alexander JS, Yurochko AD (2004) Human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence. J Virol 78:4444-4453. 10. Daley E, Emson C, Guignabert C, de Waal Malefyt R, Louten J, et al. (2008) Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 205:361372. 11. Cheng J, Ke Q, Jin Z, Wang H, Kocher O, et al. (2009) Cytomegalovirus infection causes an increase of arterial blood pressure. PLoS Pathog 5:e1000427. 12. Lanari M, Lazzarotto T, Papa I, Venturi V, Bronzetti G, et al. (2001) Neonatal aortic arch thrombosis as a result of congenital cytomegalovirus infection. Pediatrics 108:E114. 13. (2008) Knowledge and practices of obstetricians and gynecologists regarding cytomegalovirus infection during pregnancy--United States, 2007. MMWR Morb Mortal Wkly Rep 57:65-68. 14. Gartner LM, Morton J, Lawrence RA, Naylor AJ, OHare D, et al. (2005) Breastfeeding and the use of human milk. Pediatrics 115:496-506. 15. Tortorella D, Gewurz BE, Furman MH, Schust DJ, Ploegh HL (2000) Viral subversion of the immune system. Annu Rev Immunol 18:861-926. 16. Fernandez-Ruiz M, Munoz-Codoceo C, Lopez-Medrano F, Fare-Garcia R, Carbonell-Porras A, et al. (2008) Cytomegalovirus myopericarditis and hepatitis in an immunocompetent adult: successful treatment with oral valganciclovir. Intern Med 47:1963-1966. 17. Gandhi MK, Khanna R (2004) Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments. Lancet Infect Dis 4:725-738. 18. Sitbon O, Lascoux-Combe C, Delfraissy JF, Yeni PG, Raffi F, et al. (2008) Prevalence of HIV-related pulmonary arterial hypertension in the current antiretroviral therapy era. Am J Respir Crit Care Med 177:108-113.

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Archives of Clinical Microbiology: New Vision


Ahmed S. Attia Ph.D.
Editor in Chief Department of Microbiology and Immunology Faculty of Pharmacy, Cairo University, Cairo, Egypt. ahmed.s.attia@gmail.com

Welcome back to Archives of Clinical Microbiology, one of the newest journals in the field of clinical microbiology and infectious diseases. After publishing our first issue earlier this year we have faced some challenges that required changes both in personal and approaches. First, we would like to extend our thanks and best wishes to our first Editor in Chief, Dr. Aamir Shahzad, who have done outstanding efforts in launching Archives of Clinical Microbiology and building our prestigious editorial board. However, for personal reasons he decided to step down but he is still committed for helping us grow and expand. On May of 2010, I humbly accepted the responsibility as the new Editor in Chief of Archives of Clinical Microbiology. Since then we were able to significantly expand our users database of editors, reviewers, authors, and readers and we are continuing to grow. We have received a substantial number of manuscript submissions that have gone through a rigorous, efficient, fair and relatively quick peer review process. The first fruit of this growing operation is between your hands today in the form of the second issue of Archives of Clinical Microbiology (Vol.1 No.2). Our authors are coming from different parts of the world to continue our commitment to remove geographical and economical boundaries in the face of publishing high quality work. Staring now on, we are committed to produce to our readers a new issue of Archives of Clinical Microbiology on a regular basis.

Archives of Clinical Microbiology will continue to work as a hybrid journal by giving the authors the option to make their articles freely accessible to anyone worldwide by paying the very low article processing charges or the articles remain accessible through subscription only. To further encourage authors to submit their work to Archives of Clinical Microbiology, for the following two issues we are running a promotional competition to waive the article processing charges for one article per issue that would be published in the open access section. The selection of the free article will be done (after peer review) by highly qualified committee that will look in the completeness and the innovation of the accepted articles. I wish youll enjoy our latest issue of Archives of Clinical Microbiology and we hope to see your work in our next issues.

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Interleukin (IL)-1a, IL-7 and IL-13 Profiles among Nigerians with Trypanosoma brucei gambiense Infection
1

Isaac, C., 1;*Nmorsi, O.P.G., 1Igbinosa, I.B., 2Umukoro, D.O. 3Imarenezor, E.P.K 2,4 Ukwandu, N.C.D.

1. Tropical Disease Research Unit, Department of Zoology, Ambrose Alli University, Ekpoma, Nigeria 2. Family Medicine Department, Delta State University, Abraka. 3. Department of Microbiology, Ambrose Alli University, Ekpoma, Nigeria 4. Department of Medical Microbiology, Ambrose Alli University, Ekpoma, Nigeria Corresponding author: Prof. O.P.G. Nmorsi E-mail: nmorsiopg@yahoo.com

Background Human African trypanosomiasis (HAT) is an endemic parasitic disease exclusively located in intertropical Africa where it is transmitted by the Glossina vector. The diseases processes and in deed the control of this infection partly require an understanding of cytokine immune responses to the disease. The aim of this study therefore is to determine the profiles of IL- 1a, IL-7 and IL-13 among HAT positive volunteers. Methods and Findings This investigation was carried out in Abraka, a HAT endemic area in Nigeria. Sera from 35 seropositive volunteers were categorized as weakly, moderately and strongly positive. Also, 16 of these 35 seropositive subjects demonstrated parasite in the blood and as a result were further grouped into early (n=4) and late (n=12) stages of HAT based on the demonstration of trypanosomes and the count of white blood cell in the cerebrospinal fluid (CSF). Sera of seropositive volunteers were subjected to interleukins (IL-) (IL-1a, IL-7 and IL-13) assay. Similarly, serum and the CSF of the early and late stages of HAT were also subjected to interleukin test. In the serum, IL-1a levels were significantly higher among the strongly positive volunteers than the control subjects (p<0.05). Conversely, depressed levels of IL- _ was seen for HAT late stage (p<0.001). IL-7 level was significantly lower in strongly positive individuals than the control volunteers. Also, late stage IL-7 level was significantly depressed (p<0.05). There was no significant difference in IL-13 levels among HAT infected individuals and their non infected counterparts. Conclusion We suggest that the depressed levels of CSF IL-1a and IL-7 should serve as markers of HAT late stage in Abraka, Nigeria.

Introduction
Sleeping sickness or human African trypanosomiasis (HAT) is an endemic parasitic disease exclusively located in intertropical Africa where it is transmitted by the Glossina vector (1). HAT if not detected and treated at the early stage could progress to the late stage (2). The control of African trypanosomiasis requires among others the contribution of variant specific glycoproteinspecific B- and T-cell responses (3,4) as well as the macrophage/ monocyte phagocyte system (5,6). Cytokines orchestrate a type I and/or a type II immune response, which influences the outcome of African trypanosomiasis (7,8,9,10). Trypanosome-derived products have been shown to activate the generation of pro-inflammatory mediators including IL-1a by macrophages (11). Chronic secretion of macrophage-derived mediators is in part responsible for the pathogenic aspects of HAT (12,13). In laboratory infected mice with T.b. brucei, an increase in the level of IL-1a has been reported (7), including upregulation of granulocyte and macrophage due to IL-1a administration in mice (14). Synergistic role of IL-1a led to enhanced cytokine production and haematopoietic recovery (15,16). IL-7, a type 1 cytokine is documented to be induced by IL- 1a and TNF- a (17), has been implicated in the pathology of trypanosomiasis where CD8+ predominance was reported (8). Also, Th2 cells produced anti-inflammatory cytokines due to B-cell
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predominance following an increase in total cell number in spleen and lymph nodes of protozoan-infected mice treated with IL-7 (18). Altered genes encoding IL7R alpha and liquid IL-7 in the CSF compartment have been observed in individuals with disease of the central nervous system (19). The role of type II cytokines (IL-4, IL-10 and IL-13) in resistance to African trypanosomiasis is speculative based on contrasting reports that it could exert deleterious (20), protective (9,21) or no effect on the outcome of trypanosomiasis disease (22). It was shown that IL-13, an anti-inflammatory cytokine, did not influence the susceptibility of Trypanosoma infection in mice and were not the main trigger to alternative macrophage activation (10). IL-13 has been suggested to be a good indicator for the course of CSF infections (23). Despite the relative importance of cytokines in the control of HAT and their divergent roles in the immunopathology of this disease, there is paucity of information in relation to the status of IL-1a, IL-7 and IL-13 in T.b. gambiense-infected individuals in our locality. This investigation therefore seeks to determine the profiles of these cytokines in HAT positive volunteers in Abraka, an endemic focus in Nigeria.

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Materials and methods


Study area
This study was carried out in three communities (Umeghe, Urhouka and Ugono) of Abraka, a HAT endemic focus of Nigeria. The three Abraka communities surveyed are communities. Aforementioned communities lie between latitude 547-615N and longitude 5.42-6E with population of over 5,000, who are predominantly farmers.

Results
Figure 1 shows IL-1a profile among categories of seropositive human subjects. The differences in the levels of IL-1a for weakly positive and moderately positive individuals compared with the control subjects were not significant (p>0.01). Serum IL-1a levels were significantly higher in strongly positive volunteers than control subject (p<0.05). The serum IL-1a concentrations for the categories of seropositive subjects were not significant (p>0.01).

Ethical considerations

Ethical permission was obtained from the Delta State Ministry of Health and Eku Baptist Hospital. Before the commencement of the investigation, the nature, objectives and benefits of the investigation were thoroughly explained to inhabitants of these communities. Informed consents were obtained from 474 consented volunteers who were screen for Trypanosomiasis infection using card agglutination test for trypanosomiasis (CATT) kit (24).

Population recruited

Of the 474 screened, sera of 35 seropositive subjects with T.b. gambiense infection were evaluated for interleukins (IL-1a , IL-7 and IL-13) levels. Also, 16 volunteers from the 35 seropositive subjects, demonstrated trypanosomes in their serum.

Figure 1: iL-1a proFiLe among categories oF seropositive voLunteers

Staging of HAT

Sera obtained from venous blood were used to categorize the level of infection by double serial dilution as: weakly positive (1:2-1:4) (n=9), moderately positive (1:8-1:16) (n=12) and strongly positive (1:32) (n=14) according to the manufacturers instruction (Intituut voor Tropische Geneeskunde, Antwerpen, Belgium). Of the 35 seropositives, 16 volunteers demonstrated parasite in the blood and were further categorized into early and late stage of HAT. Volunteers categorized as early stage, demonstrated the parasite in the blood only while late stage showed the parasite both in the blood and CSF. The staging of HAT was done on the recommendation of (25). Most of the seropositive volunteers were observed to suffer from malaise, anaemia, headache, pyrexia, weight loss and weakness following the medical history and physical examination carried out on the volunteers.

Table 1 presents the IL-1a concentrations in serum and CSF of HAT volunteers with early and late stages of infection. The difference in serum IL-1a concentrations between early and late stages of infection was statistically significant (p<0.001). Significant depression of CSF IL-1a concentrations was observed for HAT late stage (p<0.001).

Exclusion criteria

tabLe 1: iL-1a proFiLe in serum and csF oF Hat earLy and Late stages

We excluded volunteers with overt diseases like viral hepatitis B, HIV or sickle cell anaemia using standard procedures.

Interleukin assay

Sera and CSF were obtained from 35 HAT positive subjects and then assayed for IL-1a, IL-7 and IL-13 using standard enzyme linked Immunosorbent assay (ELISA) according to the manufacturers instruction (Abcam Plc, United kingdom). Also, interleukin evaluation was carried out on 10 control group who were selected on the basis of being HAT negative and also not infected with the aforementioned diseases.

The levels of IL-7 for weakly positive and moderately positive volunteers were not significantly depressed (p>0.05). However, IL-7 level in strongly positive volunteers was significantly lower than the control subjects (p<0.001). There was no significant difference in the serum IL-7 concentration among seropositive subjects (p>0.01) (Figure 2). Table 2 shows IL-7 concentrations in serum and CSF of individuals with HAT early and late stages of infection. The mean difference in serum IL-7 concentrations between early stage and late stage of infection was statistically significant (p<0.05).The CSF IL-7 concentration was significantly higher in volunteers with early stage of HAT than late stage (p<0.05). The mean differences of IL-13 levels between weakly positive, moderately positive and strongly positive volunteers compaThis article is available from: http://www.acmicrob.com

Data analysis

Data obtained were subjected to statistical analysis, namely Welch t-test and Tukey-analysis of variance (ANOVA) using Instat statistical package.

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tabLe 3: iL-13 proFiLe in serum and csF oF Hat earLy and Late stages Figure 2: iL-7 proFiLes among categories oF seropositive voLunteers

tabLe 2: iL-7 proFiLe in serum and csF oF Hat earLy and Late stages

(VSG) from T. brucei, elicit IL-1a. This suggests that IL-1a may be implicated in the immunopathogenesis of HAT infection. Also, we report depressed levels of CSF IL-1a in HAT late stage. We observed that the profile of IL-13, an anti-inflammatory cytokine was unaltered in the CSF of HAT late stage. This data therefore suggests that the imbalance between IL-13 and IL-1a could be due to the inability of IL-13 to immunomodulate IL-1a in HAT late stage. This partly contradicts the hypothesis of a switch from dominant type I to a predominant type II cytokine (26, 27). However, we hypothesize that other anti-inflammatory cytokines such as IL-10 may be implicated in the down regulation of this cytokine in the CSF (28). We observed depressed levels of IL-7 with severity of infection. These findings contradicts the report that human chronic trypanosomiasis was associated with elevated IL-7 production (8). It has been documented that natural killer cells are recognized as major effectors of innate resistance to protozoan infections through the control of the growth of this pathogens by indirectly producing cytokines (29). In a report, IL-7 deficient patients were observed to be deficient of T and natural killer (NK) cells (30). This study therefore suggests that depressed IL-7 concentration with disease progression may be associated with the pathogenic conditions in HAT positive patients which could be due to a regulatory role of anti-inflammatory cytokine in the CSF as documented by (28). We reported unaltered IL-13 levels in seropositive volunteers and serum/CSF IL-13 levels in the early and late stages of HATinfected human subjects. We therefore suggest that IL-13 is not a mediator to host immune response of HAT infection. This observation is in consonance with the report that IL-13 in trypanosome susceptible mice was not the main trigger of alternative macrophages because IL-13 signaling occurred independently of an anti-inflammatory cytokine (IL-4), corroborating the natural propensity of animals to develop alternatively activated macrophages (10, 31). The clearance of trypanosome parasites through innate immunity involves macrophage/monocyte phagocyte system (5,6). This investigation showed elevated IL-1a among the strongly positive volunteers. Conversely, depressed levels of IL-7 were observed in HAT late stage. These data suggests that IL-1a and IL-7 could be major mediators in the immunopathology of T. b. gambiense infection. Additionally, we propose that depressed levels of CSF IL-1a and IL-7 should serve as markers of HAT late stage in our locality.
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red with HAT negative volunteers were not significant (p>0.05) (Figure 3). The differences in the levels of IL-13 among the categories of seropositive volunteers were also not significant (p>0.01). Table 3 shows IL-13 concentrations in serum and CSF of volunteers with early and late stages of HAT. The mean difference in serum IL-13 concentrations between HAT early stage and late stage of infection was not statistically significant (p>0.05).There was also no significant difference in the mean CSF IL-13 concentration between HAT early stage and late stage (p>0.05).

Figure 3: iL-13 proFiLe among categories oF seropositive voLunteers

Discussion
Our data demonstrated elevated level of serum IL-1a among the strongly seropositive volunteers. Similarly, serum IL-1a levels of HAT late stage patients were increased compared to early stage. In contrast, we also showed depressed concentration of CSF IL1a in HAT late stage. The elevated levels of serum IL-1a supports the report of (11) in which derived variant surface glycoprotein
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References
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15. Pang G, Couch L, Batey R, Clancy R, Cripps A (1994) GM-CSF, IL-a, IL-1a, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1_ and TNF-_. Clin Exp Immunol 96: 437-443. 16. Kovacs CJ, Kerr JA, Daly BM, Evans MJ, Johnke RM (1998) Interleukin 1 alpha (IL1a) and macrophage colony-stimulating factor (M-CSF) accelerate recovery from multiple drug-induced myelosuppression. Anticancer Res 18(3A): 1805-1812. 17. Weitzmann NM, Cenci S, Ritas L, Brown C, Pacifici RC (2000) Interleukin-7 stimulates osteoclastogenic formation by up-regulating T-cell production of soluble osteoclastogenic cytokines. Immunobiol 96(5): 1873-1878. 18. Gessner A, Will A, Vieth M, Schroppel K, Rollinghoff M (1995) Stimulation of Bcell lymphopoiesis by interleukin-7 leads to aggravation of murine leishmaniasis (1995). Immunol 84(3): 416-422. 19. Landmark F, Duvefelt K, Lacobaeus E, kockum I, Wallstrom E et al (2007) Variation in Interleukin-7 receptor alpha chain (IL-7R) influences risk of multiple sclerosis. Nat Genet 39(9): 1108-1113. 20. Uzonna, JE, Kaushik RS, Gordon JR, Tabel H (1998) Immunoregulation in experimental murine Trypanosoma congolense infection: anti-IL-10 antibodies reverse trypanosome-mediated suppression of lymphocyte proliferation in vitro and moderately prolong the lifespan of genetically susceptible BALB/c mice. Parasite Immunol 20:293-302. 21. Inoue N, Inoue M, Kuriki K, Yamaguchi H, Nagasawa H et al (1999) Interleukin 4 is a crucial cytokine in controlling Trypanosoma brucei gambiense infection in mice. Vet Parasitol 86:173-184. 22.Schopf, LR, Filutowicz H, Bi XJ, Mansfield JM (1998) Interleukin-4-dependent immunoglobulin G1 isotype switch in the presence of a polarized antigen-specific Th1-cell response to the trypanosome variant surface glycoprotein. Infect Immun 66:451-461. 23. Mukodzi S, Muller C, Ahrens N, Kuno S, Stenger R-D (2002). CSF cytokines (IL-6, IL-12, and IL-13): the right marker for shunt reimplanation after infections? Critical Care, 6(Suppl.1): P64. 24. Magnus E, Vervoot T, Van Meirvenne NA (1978) A card agglutination test with stained diagnosis of T.b.gambiense. Annales de la socit belge de Mdecine Tropicale 58: 169-76. 25. World Health Organization (1998). Control and Surveillance of African trypanosomiasis. Report of a WHO expert committee. World Health Organization. Geneva, Switzerland. Technical Report. Series. No., 8881, 114. 26. Goerdt S, Orfanos CE (1999) Other functions, other genes: alternative activation of antigen-presenting cells. Immunity, 10: 137-142. 27. Namangala B, Nol W, De Baetselier P, Brys L, Beschin A (2001) Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomiasis. J Infect Dis 183: 1794-1800. 28. de Waal, Malafyt, R. and Moore, K.W. (1998). Interleukin-10. In: Thompson AW., Ed. The cytokine handbook. 3rd ed. San Diego: Academic Press. pp. 333-364. 29. Scharton-Kersten, MT, Sher A (2002) Roles of natural killer cells in innate resistance to protozoan infections. Curr Opin Immunol 9(1): 44-51.
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30. Ahmed A, Saha B, Patwardhan A, Shivprasad S, Nandi D (2009) The major players in adaptive immunity.1. Humoral immunity. Resonance 14(5): 455-471. 31. Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM (2000) M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 164:6166-6173.

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Phytochemical Screening And Hepatoprotective Properties Of The Aqueous Root Bark Extract Of Sarcocephalus latifolius (Smith) Bruce (African Peach)
YESUFU, HB1. , BASSI, PU2. KHAN, IZ1. ABDULRAHAMAN, FI1, MOHAMMED, GT3
1. Chemistry Department, Faculty of Sciences, University of Maiduguri, P.M.B 1069, Borno State.,Nigeria. 2. Department of Clinical Pharmacology and Therapeutics, College Of Medicine2 University Of Maiduguri, P.M.B 1069, Borno State,Nigeria 3 Department of Pharmaceutics and Pharmaceutical Microbiology,Faculty of Pharmcy, University of Maiduguri PMB 1069, Borno state, Nigeria Correspondance: Dr Peter U Bassi MBBS,MSc,FMCP(Nig) Dept. of clinical Pharmacology and Therapeutics College of Medical Sciences PMB 1069, University of Maiduguri Borno State, Nigeria Email: pubassi@gmail.com Tell: +2348034945067

Objective: The hepatoprotective properties of the aqueous extract of Sarcocephalus (S). latifolius (Smith) Bruce was studied by investigating its effect on some serum biochemical parameters in rats treated with Carbon tetrachloride (CCl4). Methods: Twenty Wister albino rats weighing 200-230g were divided into five groups of five rats each (Group A, B, C, D and E) by random selection.Rats in groups B, C and D were orally administered 100mg/kg, 200mg/kg and 300mg/kg body weight aqueous extracts daily for 20 days, while group A (control) was injected with 5ml/kg body weight normal saline for the same period. The animals in groups B, C, D and E were injected intraperitoneally with 3mg/kg body weight of CCl4; on the last day and observed for 24hours after which they were sacrificed by decapitation and blood collected for biochemical and haematological analysis. Result: Phytochemical analysis of the aqueous extract showed the presence of flavonoids, alkaloids, carbohydrates, tannins and saponins. Alanine transaminase (ALT), Aspartate transaminase (AST), total and conjugated bilirubin levels were significantly decreased (P<0.05) across all the groups treated with extract and the toxin (CCl4). The decrease was dose dependent. Conclusions: The decrease observed in the biochemical parameters, suggest that the aqueous extract of S. latifolius possess hepatoprotective properties. Key words: Sarcocephalus latifolius, aqueous extract, phytochemicals, liver function, hepatoprotective, rats.

Introduction
The plant African peach (S.latifolius) is of the family Rubiaceae. It is a multistemmed tree or shrubs up to 12m found in undisturbed fringing forest and close savannah woodland (1). Its generic name is derived from the Greek word sarco (fleshy) and cephalus (headed) in reference to the flowers. The specific epithet is derived from the latin word lati (broad) and folius (leaved) (2). A hermaphrodite tree flowering from April-June and fruits ripen from July-September (3). The grey baboon (Papio anubis) disperses its seed (4). Earlier reports on the various medicinal uses of this plant have been reported by many traditional medicine practitioners to be effective in the treatment and management of many ailments such as febrile illnesses, stomach disorder, cough, malaria fever and jaundices (5). Others includes constipation, dysmenorrhoea, abscesses, vomiting and threatened abortions (6).Clinically S.latifoliues has been shown to paralyses Trichostrongylus columbriformis larvae in a concentration dependent manner (7), lower blood glucose level in normal and alloxan induced rats (8).It has also demonstrated high anti fungal activity against Macrophomina phaseolina, the causal agent of dry root decay in pawpaw(9).

Materials and Methods


Sample collection and preparation: The plant material was
collected from Gyella, Mubi South Local Government Area of Adamawa State and authenticated by Prof. S.S. Sanusi of the Department of Biological Sciences, University of Maiduguri, Borno State, Nigeria. The sample was washed, air-dried and pulverized using mortar and pestle. The pulverized sample (250g) was boiled with distilled water (2 litres) for one hour. The extract obtained was concentrated in vacuo at 40C and stored at 40C until used.

Phytochemical Analysis

Phytochemical test was carried out to determine the presence of carbohydrates, tannins, saponins, alkaloids, steroidal glycosides and flavonoids by simple qualitative standard methods (10), (11),(12).

Animals

White albino rats of mixed sexes weighing 200-230g were obtained from the Department of Human Anatomy Animal House, University of Maiduguri. They were housed in standard cages and fed with growers mash (Sander Nigeria Ltd) and water ad libitum.
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Experimental Procedure

The rats were weighed and assigned into five groups of five animals each. Group A was orally fed with a single dose of normal saline (5ml/kg body weight) daily and served as control. Animals in group B, C and Dwere orally fed with 100mg/kg, 200mg/kg and 300mg/kg aqueous root bark extract respectively. This was administered to the rats daily for 21 days using feeding tube. Group E was introduced on the last day of treatment and injected intraperitoneally alongside other groups (, B, C, D and E) with equal dose of (3mg/kg body weight) carbon tetrachloride (CCl4) to induce liver toxicity. The rats were sacrificed by humane decapitation 24hrs after injecting the toxin (CCl4). Blood was allowed to clot for 30 minutes and then centrifuged at 2500 rpm for 15 minutes and serum harvested (13). Liver function tests for alanine transferase (ALT), aspartate transaminase (AST), total and direct bilirubin were done calorimetrically using reagent kits (Randox lab. Ltd) (14).

Treatment group

Extract Dose

Mean no. of inhibition in 24Hrs 56.67+ 4.04 216.330.58c 214.671.15c 176.671.15b 126.671.15a

Percentage protection 0.00 0.67 18.33 44.33


Amino Trans-

Control CCl4 Extract + CCl4 Extract + CCl4 Extract + CCl4

3mg/kg 100mg/kg 200mg/kg 300mg/kg

Table 2: Effect of S. latifolius aqueous root bark extract on mean Aspartate ferase (AST).

Mean + SD based on five observations. Results with different superscript (a, b, c) on the same row are significantly different (P< 0.05).

Statistical Analysis

Data collected from the biochemical parameters assayed were summarized as Mean S.E.M. Difference between individual groups was assessed by the student t-test. P value less or equal to 0.05 was considered statistically significant (15).

Results
Phytochemical analysis of the ethanol extract of S.latifolius, showed that tannins are present at high concentrations, while flavonoids, alkaloids, carbohydrates are present at moderate concentration and saponins, Cardiac glycosides, and Terpenoids/ steroids were present in low concentration. The results obtained are shown in Table 1. The aqueous root bark extract of S.latifolius was found to significantly (P<0.05) inhibit the effect of CCl4 on the liver and the protection was dose dependent. For aspartate transaminase (Table 2) percentage protection were 0.67, 18.33 and 44.33% for groups treated with 100mg/kg, 200mg/kg and 300mg/kg of the aqueous extract and injected intraperitoneaS/No. 1. 2. 3. 4. 5. 6. 7. 8. 9. Phytochemicals Carbohydrates Phenolic Compounds (Anthraquinone) Tannins Phlobatannins Saponins Cardiac glycosides Terpenoids/Steroids Flavonoids Alkaloids Aqueous Extract Results ++ +++ + + + ++ ++

lly with toxin (CCl4). For alanine transferase (Table 3) percentage protection were 0.5, 5.0 and 8.1% for group treated with 100mg/kg, 200mg/kg and 300mg/kg of the aqueous extract and injected intraperitoneally with toxin (CCl4). Percentage protection for total bilirubin (Table 4) were 3.67, 18.60 and 37.00% for groups treated with 100mg/kg, 200mg/kg and 300mg/kg of the aqueous extract and injected intraperitoneally with toxin (CCl4). Percentage protection for conjugated bilirubin (Table 5) were 19.0, 20.4 and 47.6% for groups treated with 100mg/kg, 200mg/ kg and 300mg/kg of the aqueous extract and injected intraperitoneally with toxin (CCl4).

Treatment group Control CCl4 Extract + CCl4 Extract + CCl4 Extract + CCl4

Extract Dose 3mg/kg 100mg/kg 200mg/kg 300mg/kg

Mean no. of inhibition in 24Hrs 17.67+ 1.15 127.331.15c 126.671.15c 121.002.00b 117.331.53a

Percentage protection 0.0 0.5 5.0 8.1

Table 3: Effect of S. latifolius aqueous root bark extract on mean Alanine Amino Transferase (ALT) Mean + SD based on five observations. Results with different superscript (a, b, c) are significantly different (P< 0.05)

Treatment group

Extract dose

Mean no. of inhi- Percentage probition in 24hrs tection 50.00+ 1.00 90.001.00d 86.701.53c 73.300.58b 56.701.53a 0.00 3.67 18.60 37.00

Control CCl4 Extract + CCl4 Extract + CCl4 Extract + CCl4

3mg/kg 100mg/kg 200mg/kg 300mg/kg

Table 1: Phytochemical Screening Results of S. latifolius root bark Key: + ++ +++ Present in low concentration. Present in moderate concentration Present in high concentration. absent.

Table 4: Effect of S. latifolius aqueous root bark extract on mean Total Bilirubin (TB) _ 10 Mean + SD based on five observations. Results with different superscript (a, b, c d) are significantly different (P<0.05).

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Treatment group Control CCl4 Extract + CCl4 Extract + CCl4 Extract + CCl4

Extract dose 3mg/kg 100mg/kg 200mg/kg 300mg/kg

Mean no. of inhi- Percentage probition in 24Hrs tection 30.00+ 1.00 70.001.00c 56.701.15b 55.700.58b 36.700.58a 0.0 19.0 20.4 47.6

Table 5: Effect of S. latifolius aqueous root bark extract on mean Conjugated Bilirubin (CB) _ 10 Mean + SD based on five observations. Results with different superscript (a, b, c) are significantly different (P<0.05)

100mg/kg body weight of aqueous extract plus CCl4, showed severe congestion, necrosis, calcification of hepatocytes, mononuclear cell infiltration, with areas of vacoulation and interstitial haemorrhage; while rats treated with 300mg/kg body weight of the aqueous extract plus CCl4 shows minimal blood congestion, reduction in steatosis, fatty degeneration and peripheral hyalinazation of the hypatoctes of the liver tissue. This adds credence that physiologic recovery preceded obvious histological changes and the results may suggest that diets supplemented with S.latifolius will improve hepatoprotection against oxidative liver damage. Total and conjugated bilirubin levels were significantly increased in the CCl4 treated rats as compared to control. Administration of the extracts (100mg/kg, 200mg/kg and 300mg/kg body weight) leads to a significant reduction (P < 0.05) in their levels. Report from Tirkey et al (25) also showed a marked rise in bilirubin levels after CCl4 administration. Bilirubin, is a major product of haemoglobin breakdown which rises when there is liver injury or damage; leading to the discolouration of the skin and eyes known as jaundice (26). Elevation of total bilirubin which results from decreased uptake and conjugation of bilirubin by the liver is caused by liver cell dysfunction, while increased levels of direct or conjugated bilirubin is due to decreased secretion from the liver or obstruction of the bile ducts (26). Reduction of CCl4 induced increases in total and conjugated bilirubin by S. latifolius extract further show its protective effect against CCl4 induced liver toxicity. The extract perhaps protects the liver cell from damage, thereby enhancing bilirubin uptake and conjugation by the liver and subsequent secretion into the bile ducts. These reports from our study show that the ethanol extract of S. latifolius possess antihepatotoxic activity as demonstrated by its reduction of CCl4 induced elevations in the levels of ALT, AST, total and conjugated bilirubin. This hepatoprotective effect suggests that the plant may also possess antioxidant properties that helped to combat the CCl4-induced oxidative stress in the liver. The ability of natural compounds to attenuate carcinogen induced hepatotoxicity is believed to be related to their intrinsic antioxidant properties (27). Phytochemical result from this study revealed the presence of flavonoids, which has been reported to protect against toxicity induced by environmental toxicants (28) such as CCl4. Alkaloids present in plants are known to have numerous beneficial pharmacological effects(10,29). Some bioflavonoids have been reported to possess antioxidant properties which help to combat free radical induced oxidative stress (24, 29). The chemoprotective activities of flavonoids are related to their ability to inhibit peroxidative damage caused by environmental toxicants. Several secondary metabolites have been shown to have wide ranges of antimicrobial activities (30). In a study, by Kouassi Maximin et al( 2007), S. latifolius was one of sixty-four extracts assayed from twenty-one plants used in the Malian traditional medicine that were found to be significantly active against the intracellular forms of Leishmania major (31). Similarly Abreu et al (2001), showed that S.latifolius displayed antiplasmodial activity against P. falciparum (32).
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Discussion
Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in experimental studies of liver diseases (16). The first metabolite of CCl4; trichloromethyl free radical, is believed to initiate the biochemical processes leading to oxidative stress, which is the direct cause of many pathological conditions such as diabetes mellitus, cancer, hypertension, kidney damage, liver damage and death(17 19). These activated radicals bind to micromolecules and reduce lipids peroxidative degradation of polyunsaturated fatty acids. This leads to the formation of lipid peroxides which in turn gives products like malonylaldehyde that cause damage to membranes (20). This lipid peroxidative degradation of biomembranes is one of the main causes of hepatotoxicity by CCl4. This is usually evidenced by a rise in the serum marker enzymes of the liver namely ALP, AST and ALT. In this study, the aqueous root bark extracts of S.latifolius, demonstrated protection against liver toxicity induced by CCl4 in a dose dependent manner. The protection observed could be linked to the type of phytochemicals present. The terminal event in the attack on the liver by CCl4 is the production of highly reactive radicals leading to lipid peroxidation and inhibition of calcium pump of the microsome giving rise to liver lesions (21). However, antioxidants inactivate free radical reactions initiated by CCl4 by either blocking the initiation of free radicals or scavenge free radicals and terminate free radical damage(22). Photochemical such as flavonoids are potent antioxidant because of their ability to scavenge hydroxyl radicals, superoxide and lipid peroxy radicals (23, 24). In the study, an elevation in the levels of the end products of lipids peroxidation of the liver of the rats treated with CCL4 was observed. The increase in AST and ALT suggests enhanced lipid peroxidation giving rise to liver damage and failure of the antioxidant defense mechanism to prevent formation of excessive free radicals. The group treated with S.latifolius root bark inhibits the changes (p<0.05) at high concentrations (300mg/kg > 200mg/kg >100mg/kg body weight respectively), compared to CCl4 treated group, hence it is possible that the mechanism of hepatoprotection of S latifolius is due to its antioxidant effect. Histopthological studies showed normal integrity of the hepatocytes in the control group. The liver of rats treated with
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Interestingly, phytochemical screening of the current investigation has revealed that extracts from the root extract possess at least three to four of the following classes of secondary metabolites: flavonoids, terpenoids, tannins, alkaloids and saponin, hence may not only be hepatoprotective but useful as chemotherapeutic agents and need to be tested for protection against hepatic pathogens.

11. Trease, G.E. and Evans, W.C. (2002). A Text book of Pharmocognosy,(15th edition). Elsevier Company, Philadelphia, USA. Pp.191-418. 12. Mishra, A. K, Mishra, A., Kehri H. K., Sharma, B. And Pandy, A.K. (2009). Inhibitory activity of Indian spice plant Cinnamomum zeylanicumextract against Alternaria solaniand Curvularia lunata, the pathogenic dematiaceous moulds. Annals of clinical Microbiology and Antimicrobials, 8:9, doi: 10.1186/1476 0711 -8-9. 13. Turner, R.A. (1965). Screening methods in Pharmacology (1st edition), Academic press, New York. 299 p. 14. Reitman, S. and Frankel, S. (1957). Determination of Glutamate-Pyruvate transaminase (ALT) and Aspartate Aminotransfrase (AST). J. Clin. Path. 28:56. 15. Mead, R. and Cumow, R.N.(Editors) (1983). A simple experiment. In: Statistical Methods in Agriculture and Experimental Biology.Chapman Hall, London. Pp 33-46. 16. Uma M.M, Rao P.G.M (2005). Antihepatotoxic effects of Cotton seed oil in Rats Ind J Pharmaco 37:180. 17. Guven, A., GuvenA. and Gulmez, M. (2003). The effect of kefir on the activities of GSH-PX, GST, CAT, GSH, and LPO levels in carbontetrachloride induced mice tissues. J. Vet Med B Infect Dis Vet Public Health. 50:412-416. 18. Ahmed, F.F., Cowan, D. L. and Sun, A Y. (1987). Detection of free radical formation in various tissue after acute carbontetrachloride administration in gerbil. Life Sci. 41:24692475. 19. Pohl, L., Schuliek, R. and George, J. (1984). Reductive oxygenation mechanism of metabolism of carbontetrachloride phosgene by cytochrome p-450. Mol Pharmacol. 25: 318324. 20. Ulicna O, Greskshek M, Vancovao O, Zlator I, Bocek P (2003). Hepatoprotective effect of Rooibos tea (Aspalathus linearis) on CCl4 induced liver damage Physiol Res 52: 461-466. 21. Herfindal, E.T. and Gourley, D.R. (2002). Text book of therapeutics. Drug and Disease Management (7th edition) Williams and Wilkins, Philadelphia, U.S.A. Pp. 74-79. 22. Gamzi, S.C., Vinay, K.and Tuekar, C. (1999). Robbins pathologic basis of disease (6th edition) W.B. Saunder Company. Philadelphia, U.S.A. Pp. 846-868. 23. Litito, S.B and Frei, B (2006). Comsumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon ? Free Radic. Biol. Med. 41(12):1727-1746. 24. Farombi, E. O., Akanni, O. O. and Emerole, G. O. (2001). Antioxidant and Scavanging activities of flavonoid extract (kolaviron) of Garcinia kola seeds in vitro. Pharmaceutical Biology.47:3163-3168. 25. Tirkey, Pukhwal, S., Kuhad, A. and Chopra, K. (2005). Hesperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbontetrachloride in rat liver and kidney. BMC Pharmacol. 5: 2-6 doi 10.1186/1471221052. 26. Sanjiv, C. (2002). The liver book: A comprehensive guide to diagnosis, treatment and recovery. Atria Jimcafe Company. 27. Farombi, E. O. (2003). locally derived natural antioxidant substances in Nigeria:

Conclusion
The results of this study show that the aqueous extracts of S. latifolius root park have hepatoprotective actions and suggest that flavonoids present in S. latifolius root bark may have a major role in this action. Future studies are planned to carry out quantitative analyses of the phytochemical parameters in order to have a more accurate picture about the possible mechanism of action of the components of these extracts. In addition, assaying for more enzymes such as gamma-glutamyltransferase (GGT), alkaline phosphatase and lactate dehydrogenase 5 (LDH5) isoenzymes would provide more clarification on the hepatoprotective effects of this plant.

References:
1. Hutchison, F. and Datziel, M.D. (1963). Flora of West Tropical Africa (2nd edition) Whitefriars press, London. Pp. 164-165. 2. Arbonnier, M. (2002). Trees, shrubs and lianas of West African dry zones. (2nd Edn.). Cirad, Margraf, Netherlands. P. 463. 3. Keay, R.W.J. (1st Edition.1989). Trees of Nigeria. Clarendon press, Oxford, United Kingdom of America. Pp. 413-421. 4. Lieberman, D. (1979).Seed dispersal by baboons in the Shai Hills, Ghana. Ecology, 60(1): 65-75. 5. Gammaniel, K., Wambebe, C., Amupitan, J., Hussaini, I.M., Amos, S., Awodoan, A., Dunah, A., Ekuta, W.J., Akeju, E.M.O., Usman, H. and Eweren, N. (1997). Active column fractions of N. latifolia of P. berghei and rabbit ileum. J Pharm R Dev. 2: 44-47. 6. Abbiw, D.K. (1990). Useful plants of Ghana. Intermediate technology and the Royal botanical garden. Kew, London. Pp. 127-196. 7. Asuzu, U.I and Njoku, C.J. (1996). The anthelminitic effects of Alstonia boonei bark and Nauclea latifolius leaf aqueous extracts on Trichostrongylus infective larvae. Fitoterapia. 67(3):220-272. 8. Gidado, A., Ameh, D.A., and Atawodi, S.E., (2005). Hypoglycemic activity of Nauclea latifolia in lowering glucose level in rats. Afri J Biotech., 4:91- 93. 9. Oluma, H.O.A and Elaigwu, M. (2006). Antifungal activity of some medicinal plants against macrophomina phaseolina (tassi) gold. Nig J Bot., 19 (1): 124-125. 10. Harborne, J.B. (1973). Phytochemical methods: A guide to modern techniques of plant analysis. Chapman & Hall, London. Pp. 279-282.

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Potential role as new chemotherapeutic agents. In: Molecular and Therapeutic Aspects of Redox Biochemistry. Theeshan Bahorun and Ameenah Gurib-Fakim (eds.), pp 208-225. OICA International (UK) Limited. 28. Mishra, A.K , Mishra,A, Kehri,H.K, Sharma,B and Pandey, A.K(2009). Inhibitory activity of Indian spice plant Cinnamomum zeylanicum extracts against Alternaria solani and Curvularia lunata, the pathogenic dematiaceous moulds. Ann Clin Microbiol Antimicrob. 2009; 8: 9(7). doi: 10.1186/1476 0711-8-9. 29. Chioma A. A, Uchenna B. U, and Ogechi N,(2008). Effect of ethanol extract of Pyrenacantha staudtii leaves on carbontetrachloride induced hepatotoxicity in rats. Nig Soc Experimatal Bio: 20(1), PP. 17-22 30. Cordell G.A, Quinn-Beattie M.L, Farnsworth N.R(2001). The potential of alkaloids in drug discovery. Phytother Res.; 15:183205. doi: 10.1002/ptr.890 31. Kouassi M. A, Jean-Robert I, Karine N.I, Drissa D, Jacques M and Kurt H. (2007). Antileishmanial activities associated with plants used in the Malian traditional medicine. J Ethnopharm;110(1): 99-104 32. Abreu P, Pereira A (2001). New Indole alkaloids from Sarcocephalus latifolius. Nat Prod Lett;15(1):43 8.

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Hospital-Acquired Bloodstream Infections in Cancer Patients between 2005 and 2007 in a Turkish University Hospital.
Kurtaran B1;*, Candevir A1, Tasova Y1, Kibar F2, Inal AS1, Yavuz S3, Kara O3, Gurkan E4, Paydas S3, Aksu HSZ1, Gulsah Seydaoglu5
1Cukurova University, Faculty of Medicine, Department of Infectious Diseases 2Cukurova University, Faculty of Medicine, Department of Microbiology 3Cukurova University, Faculty of Medicine, Department of Medical Oncology 4Cukurova University, Faculty of Medicine, Department of Haematology 5Cukurova University, Faculty of Medicine, Department of Biostatistic Corresponding Author: Behice Kurtaran, Cukurova University, Faculty of Medicine, Department of Infectious Diseases, Balcali, TURKEY, e-mail: behicekurtaran@gmail.com

Objective: This study is aimed to determine the local profile of blood culture isolates and changes in the susceptibility patterns to guide the antibiotic therapy in oncology and haematology units. Methods: Microbiologically documented hospital-acquired bloodstream infections were reviewed between 2005 - 2007 as a part of infection control surveillance in haematologyoncology department of a university hospital. Results: 194 microorganisms were isolated in 170 bloodstream infections episodes. Among these episodes, 79,1% (n=31), 80,9% (n=51) and 70,5% (n=48) were monomicrobial in years 2005, 2006 and 2007, respectively. Among the isolated 194 microorganisms, the ratio of the gram-negative bacteria were slightly increasing throughout these three years; 68,9%, 70,4% and 77%, whereas the gram-positive bacteria were decreasing 31,1%, 23,9% and 19,2%, respectively and Candida species were the cause of 4 episodes (5,6%) in 2006 and 3 episodes in 2007 (3,8%) (p>0.05). The majority of the cases were primary bloodstream infections. The most prevalent secondary cause of bacteremia was urinary tract infections in year 2005 and 2007 and pneumonia in 2006. Extended spectrum beta lactamases (ESBL) rate among E.coli and Klebsiella spp. isolates were 69,6%, 40% and 79,2% in years 2005, 2006 and 2007 respectively. Vancomycin resistance was high; 15/20 among Enterococcus species in the three years. The most effective agents against gram negative bacteria were aminoglycosides and carbapenems. Conclusion: As a conclusion, gram negative microorganisms especially the Enterobacteriaecea are the major cause of bacteremia in haematology and oncology patients. Due to the high resistance rates, antibiotic therapy should be selected strictly. Key Words: Bloodstream infection, cancer, hospital-acquired

Introduction
Infections are inevitable complications which are result of neutropenia and impaired immune response after chemotherapeutic use in cancer patients. Most of these infections are hospital-acquired in nature, because patients with cancer have prolonged and repeated contact with hospital environment and are exposed to numerous sources of infection which may involve invasive procedures. Additionally, they generally suffer from immunosuppressive chemotherapy and radiation. Aggressive antineoplastic chemotherapy is a risk factor for infections in immunocompromised patients with cancer (1-4). Bloodstream infections in cancer patients account for approximately more than 20% of hospital-acquired infections (5-7). Hospital-acquired bloodstream infections in cancer patients can be caused by a variety of microorganisms, but the most common pathogens are bacteria followed by fungi. The prognosis of nosocomial bloodstream infections is influenced by many factors, including the microorganism causing the sepsis, the source of infection, the absolute neutrophil count, the bone marrow status, and the presence or absence of shock.(4-6). Bloodstream infections are often associated with high mortality rate.

Currently, gram-positive bacteria are isolated more often than gram negative bacteria in bloodstream infections in cancer patients (8). However: in developing countries this shift is not true because of the limited sources for infection control measures (8-11). Additionally, by prompt and appropriate empirical antibiotic therapy, mortality was shown to be decreased and over the decades it became mandatory. One of the major principles of the management of infections in patients with cancer is to recognize the variability from one time period to another. Regular local data relating causative microorganisms leading to hospitalacquired bloodstream infections are very important to control infections in these countries, such as Turkey. For this reason, local surveillance data should be maintained regularly for early and appropriate empirical therapy. The objective of this article is to report a 3 years study of hospital-acquired bloodstream infection surveillance results in an oncology unit that is based on National Nosocomial Infections Surveillance (NNIS) system methods. These data might serve for comparative studies with other oncology ICUs and also help to develop a more efficient surveillanThis article is available from: http://www.acmicrob.com

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ce system, allowing for detailed infection control measures for these high-risk patients.

(range 20-84 years) in haematology unit and 52 years (range 2673 years) in oncology unit in 2007. Of the 170 episodes, 79,1% (n=31), 80,9% (n=51) and 70,5% (n=48) were monomicrobial in years 2005, 2006 and 2007, respectively. A total of 194 microorganisms were isolated, 39 episodes of documented bloodstream infection in 30 patients in 2005, 63 episodes in 50 patients in 2006 and 68 episodes in 51 patients in 2007. Of the 194 episodes, while the ratio of the gram-negative bacteria were slightly increasing in three years; 68,9%, 70,4% and 77%, gram-positive bacteria were decreasing 31,1%, 23,9% and 19,2%, and Candida species were causative agents in 4 episodes (5,6%) in 2006 and 3 episodes in 2007(3,8%) (p>0.05) (Table 1, figure 1). The majority of the cases were primary bloodstream infections. The most prevalent secondary cause of bacteremia was urinary tract infections in year 2005 (15,6%) and in year 2007 (11,6%) and pneumonia (8,5%) in 2006 (Table 2). In our haematology and oncology unit, source of bloodstream infection was assessed and no significant difference was shown in between. But in 2005 two patients were monitorized in the same service and the source of infection was not identified. However, bloodstream infection rate (bloodstream infection episode/total number of hospitalized patients) increased from 7,3% to 13,1% in haematology unit, decreased from 5,9% to 3,8% in oncology unit between 2006 and 2007. This data is shown in Table 3. High rates of resistance in both gram negative and positive pathogens were prominent. Extended spectrum beta lactamases (ESBL) percentages among E.coli and Klebsiella isolates were 69,6%, 40% and 79,2% in years 2005, 2006 and 2007 respectively. Vancomycin resistance was also high; 7/8, 3/5 and 5/7 among Enterococcus species in three years. The methicillin resistant Staphylococcus aureus (MRSA) rates were 2/3 in the year 2005, 2/5 in 2006 and 1/5 in 2007. The most effective agents for gram negative bacteria were aminoglycosides and carbapenems. The resistance rates among the predominant pathogens Enterobactericaea are shown in Table 4.

Material and methods


The study was conducted at haematology-oncology department of Cukurova University Hospital. Both hematological and solid organ malignancies were followed up by this department. Before 2006 our haematology and oncology patients were hospitalized in the same ward and after 2006 these two units were separated. Antibiotic policies have been derived by infectious diseases, haematology and oncology professionals since 2000 according to Infectious Diseases Society of America (IDSA) guidelines for febrile neutropenia (12-13). Surveillance data of the infection control committee reviewed blood culture positive infections in these units for the years 2005-2007. An episode of bacteremia was defined as a clinically significant isolate(s) in blood cultures with compatible clinical findings of infection. An episode was considered as monomicrobial only when one organism was isolated; the recurrent isolation of the same organism from blood over a four-week period was considered as a part of the same episode. When more than one organism was isolated from a patients blood cultures within 48 hours, the episode was called as polymicrobial. The diagnosis of bloodstream infection was based on the Center for Disease Control (CDC) criteria and performed by infection control doctor and nurses. Bactec (BD Becton Dickinson) and VITEC 2 (BioMerioux) systems were used for culture, identification and susceptibility testing. Standard accredited methods were used for organism isolation and identification, and sensitivity testing was performed using a comparative plate disc diffusion method (8). Resistance to an antibiotic was defined as an intermediate or resistant according to the disc sensitivity test result. Glycopeptide resistance amongst the enterococci was confirmed using Etest methodology. Statistical analysis was performed using the statistical package SPSS v 15.0. Comparisons were done using the Chi-square test or trend analyses. Descriptive statistics were used and data were expressed as frequency (n), percent (%), median and range.

Results
In the haematology-oncology unit, a total of 2486 patients were hospitalized in a three year period (411, 1086 and 989 in years 2005, 2006 and 2007, respectively). There were 170 episodes of documented bloodstream infections (bacteremia/fungemia) in total of 131 patients over the three year period. The rate of bloodstream infections (episodes / total hospitalized patients in oncology and haematology unit) was 6,8%. Male:female ratio was 1,3:1. The median age in haematology and oncology unit who had bloodstream infection was 42 years (range 17-73 years) in 2005, 39 years (range 18-75 years) in 2006 and 47 years
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Discussion
Malignant hematologic diseases require intensive treatment regimens. Neutropenia following chemotherapy in this patient population is common, often profound and prolonged, with an increased risk of infection (14). Neutropenia is recognized as an independent risk factor for central line related blood stream infection (15). Our study revealed that Gram negative bacteria were predominant. This has been an observation among similar studies done
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Table 1. Microorganisms causing blood-stream infections in haematology-oncology units.

Table 2. Sources of blood stream infections (BSI) in haematology-oncology units.

2005
n Gram-negative E. coli K. pneumoniae A. baumannii P. aeruginosa E. cloacae Burkholderia spp. C. indologenes K. oxytoca S. malthophilia P. mirabilis Rhizobium radiobacter Shigella sonnei Other nonfermantative gram (-) bacillus Other gram negative organisms Total Gram-positive Enterococcus spp S. aureus Coagulase negative staphylococcus S. pneumoniae S. mitis Other Streptococcus spp Total Fungi C. albicans C. krusei C. kefyr C. lusitaniae Total 45 100,0 1 71 31 68,9 50 3 14 8 3 2 31,1 17,8 6,7 4,4 17 5 5 4 1 2 2,2 4,4 1 1 2,2 4 1 1 1 1 1 16 6 3 2 % 35,6 13,3 6,7 4,4 n 22 7 6 5

2006
% 31,0 9,9 8,5 7,0 5,6 1,4 1,4 1,4 1,4 1,4 7 5 1 n

2007
% 32,0 9 7,7 2,6 3,8 Primary blood stream infection Urinary infection Pneumonia Soft tissue infection 9 6,4 1,3 Total 25 7 6 2 3 35*

2005
Frequency Percent 77,8 58**

2006
Frequency Percent 81,7 65***

2007
Frequency Percent 83,3

7 2 1

15,6 4,4 2,2

4 6 3****

5,6 8,5 4,2

9 3 1****

11,6 3,8 1,3

45

100,0

71

100,0

78

100,0

* 4 catheter induced BSI infections ** 5 catheter induced BSI infections *** 6 catheter induced BSI infections **** Decubitis infection, cellulitis cases

1,4

5,1

24 7,0 7,0 5,6

15 7 5 2

19,2 9 6,4 2,6

Despite the 4-fluoroquinolones are not part of a prophylactic regimen on either unit, fluoroquinolone resistance was a significant problem among strains of Enterobacteriacea isolated from blood. Resistance was, however, seen in nearly 37% isolates of E.coli, K. pneumoniae and other coliforms and in 22% of P. aeruginosa isolates of. MRSA bacteremia is not endemic in either unit and therefore active MRSA infection control measures are worthwhile. It is interesting to note that documented fungemia is low in last three years. While it is recognized that blood cultures have a low sensitivity for detection of fungemia, in studies using rRNA PCR and sequencing methodology showed no evidence of fungemia (16). Over the last decades there has been a shift from gram negative causative agents to gram positive pathogens especially coaguFigure 1. The percentage of microorganisms causing bloodstream infection according to years.

2,2

1 2

1,4 2,8 1 70,4 4,2 60 1 1 1 1,4 100,0 78 100 1,3 77 1,3 1,3 1,3

in patients in the developing countries (9-11). The reasons for this could be the relatively lower use of indwelling catheters and other portal devices. Another reason can be the lack of the application of prophylactic antibiotic regimens in neutropenic patients. Our institute does not use empirical antibiotics for prevention of bacterial infections among cancer patients.

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Table 3. Source of blood stream infections (BSI) in haematology-oncology units in 2006 and 2007.
Unit Primer BSI* 2006 Haematology1 Oncology2 Total 30 22 52 2007 46 14 60 Catheter related BSI 2006 0 6 6 2007 2 3 5 Urinary tract Respiratory tract related BSI related BSI 2006 0 4 4 2007 5 4 9 2006 4 2 6 2007 3 0 3 2 1 3 Soft tissue related BSI 2006 2007 1 0 1

with P. aeruginosa have been associated with increased mortality in some studies. Acinetobacter spp. have emerged as prominent multidrug-resistant bacteria in several intensive care units all over the world, and their occurrence in the setting of malignancy could be disastrous (20-21). Intravascular devices are considered the main sources of primary BSI (22). Our data showed that only 10.1% of BSIs were considered CVC-related. CVC-related infection is one of the major complications in haematology and oncology patients, and usually requires removal of the infected CVC to cure the infection (23). Intravascular catheters are often used for chemotherapy and fluid replacement, which frequently result in catheter-induced infections. A determination of the disinfection method to be employed is often possible when the potential risks of infection associated with the use of certain patient care materials in the hospital environment are considered (24). During the last two decades, the incidence of CVC-related Gramnegative bacteremia and septicemia caused by Stenotrophomonas maltophilia have increased (25-26). S. maltophilia, as well as other non-fermentative Gram-negative bacilli, may contaminate the infusate and enter the catheter. In our patient population in 2007 S. maltophilia was the fifth most prevalent Gram-negative pathogen responsible from bloodstream infection. Considerably higher rates of resistance in both gram negative and positive pathogens were striking in our study. This may be because of the antibiotic policies, severe underlying diseases, chemotherapeutics used, prolonged hospital stay and ineffective infection control measures. Strict infection control policies for this very susceptible patient group seem to be essential as with the other parts of the hospital.

*BSI unknown source 1In 2006 493 patients, in 2007 435 patients were hospitalized in the haematology inpatient clinic. 2In 2006 593 patients, in 2007 554 patients were hospitalized in the oncology inpatient clinic.
Table 4. Rates of antibiotic resistance among Enterobactericaea in years 2005, 2006 and 2007.
2005 Frequency Amikacin Cefepime Ceftazidime Ciprofloxacin Gentamicin Imipenem Meropenem Piperacillintazobactam Ticarcillinclavulanate Trimetoprim-sulfamethoxazole Tobramycine 1/19 9/23 9/23 9/23 7/23 2/23 2/23 6/23 4/7 11/23 Percent 5,3 39,1 39,1 39,1 30,4 8,7 8,7 26,1 57,1 47,8 Frequency 5/30 16/30 21/30 12/30 16/30 12/30 7/30 16/30 20/30 13/30 2006 Percent 16,7 53,3 70,0 40,0 53,3 40,0 23,3 53,3 66,7 43,3 Frequency 6/48 24/50 25/50 15/46 13/49 5/50 6/50 16/49 25/44 27/50 2007 Percent 12,5 48,0 50,0 32,6 26,5 10,0 12,0 32,7 56,8 54,0

Conclusion
9/23 39,1 16/30 53,3 21/50 42,0

lase negative staphylococci (CoNS), S. aureus and viridans streptococci. Therefore, in some European countries gram negative bacilli reemerged as etiological agents in cancer patients where as gram positive predominance continued in USA. These data, including changing epidemiology and resistance patterns of microorganisms, presents the necessity of continuous surveillance of blood stream infections in haematology-oncology units (17-19). The occurrence of methicillin-resistant S. aureus (MRSA) was high (5/13) in our study. Because of high rates of methicillinresistance, utilization of empirical vancomycin at our institute must be throughly scrutinized. The indiscriminate use of vancomycin has promoted resistance and this is evident by the high occurrence of vancomycin-resistant Enterococcus isolates (15/20 of isolates). The high occurrence of non-lactose fermenters (10-15%) especially Pseudomonas aeruginosa and Acinetobacter baumannii was of concern. Both of these bacteria are associated with a high degree of resistance to antibiotics. Blood stream infections
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As a conclusion, gram negative bacteria especially the Enterobacteriaecea are the major cause of bacteremia in the haematology and oncology patients in our institute. Regarding the high rates of resistance among gram negative and positive microorganisms, combination therapy of aminoglycosides with cephalosporins and piperacillin-tazobactam, and monotherapy with carbapenems seems to be appropriate as recommended by IDSA febrile neutropenia guidelines. Linezolid therapy should also be kept in mind for resistant gram positive infections. This study also demonstrates the importance of surveillance of infection in haematology and oncology patients to detect trends in infection and the emergence of multi-resistant organisms, e.g. vancomycin resistant enterococci (VRE). In addition, the present study describes the predominance of primary BSI, most of them from unknown sources; the importance of the urinary and respiratory tract as the main source for secondary BSI. The emerging trends in antibiotic resistance and their implications for empirical therapy indicate that institutions caring for cancer patients should have active ongoing microbiological surveillance studies with the objective of monitoring infections due to antibiotic-resistant pathogens, in order to improve their current antimicrobial regimens.
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References
1. Viscoli C. The evolution of the empirical management of fever and neutropenia in cancer patients. J Antimicrob Chemother 1998; 41(Suppl. D): 65-80. 2. Oppenheim BA. The changing pattern of infection in neutropenic patients. J Antimicrob Chemother 1998; 41 (Suppl. D): 7-11. 3. Klastersky J. Science and pragmatism in the treatment and prevention of neutropenic infection. J Antimicrob Chemother 1998; 41(Suppl. D): 13-24. 4. EORTC International Antimicrobial Therapy Project Group. Three antibiotic regimes in the treatment of infection in febrile granulocytopenic patients with cancer. J Infect Dis 1978; 137: 14-29. 5. EORTC International Antimicrobial Therapy Cooperative Group. Efficacy and toxicity of single daily doses of amikacin and ceftriaxone versus multiple daily doses of amikacin and ceftazidime for infection in patients with cancer and granulocytopenia. Ann Intern Med 1993; 119: 584-593. 6. Cometta A, Zinner S, de Bock R, Calandra T, Gaya H, Klastersky J, et al. Piperacillin tazobactam plus amikacin versus ceftazidime plus amikacin as empiric therapy for fever in granulocytopenic patients with cancer. Antimicrob Agents Chemother 1995; 39: 445-452. 7. Cometta A, Calandra T, Gaya H, SH Zinner, R de Bock, A Del Favero et al. Monotherapy with meropenem versus combination therapy with ceftazidime plus amikacin as empiric therapy for fever in granulocytopenic patients with cancer. Antimicrob Agents Chemother 1996; 43: 1108-1115. 8. Working Party Report of the British Society for Antimicrobial Chemotherapy. A guide to sensitivity testing. J Antimicrob Chemother. 1991; 27(Suppl. D): 1-50. 9. Chen CY, Tang JL, Hsueh PR, Yao M, Huang SY, Chen YC, et al. Trends and antimicrobial resistance of pathogens causing bloodstream infections among febrile neutropenic adults with hematological malignancy. J Formos Med Assoc 2004;103:526-32. 10. Velasco E, Byington R, Martins CS, Schirmer M, Dias LC, Gon_alves VM. Bloodstream infection surveillance in a cancer centre: A prospective look at clinical microbiology aspects. Clin Microbiol Infect 2004;10:542-9. 11. Paul M, Gafter-Gvili A, Leibovici L, Bishara J, Levy I, Yaniv I, et al. The epidemiology of bacteremia with febrile neutropenia: Experience from a single center, 19882004. Isr Med Assoc J 2007;9:424-9. 12. Hughes WT, Armstrong D, Bodey GP, et al. 1997 guidelines for the use of antimicrobial agents in neutropenic patients with unexplained fever. Infectious Diseases Society of America. Clin Infect Dis 1997;25:55173. 13. Hughes WT, Armstrong D, Bodey GP, Bow EJ, Brown AE, Calandra T, et al. 2002 guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Infectious Diseases Society of America. Clin Infect Dis 2002;34:730-751. 14. Pizzo PA. Fever in immunocompromised patients. N Engl J Med 1999; 341: 893900. 15. Wurzel CL, Halom K, Feldman JG, Rubin LG. Infection rates of Broviac-Hickman catheters and implantable venous devices. Am J Dis Child 1988; 142:536-540. 16. Jugo J, Kennedyy R, Crowe MJ, G. Lamrocky, McClurg RB, Rooney PJ, et al. Trends in bacteraemia on the haematology and oncology units of a UK tertiary referral hospital. Journal of Hospital Infection 2002; 50: 48-55. 17. Sigurdardottir K, Digranes A, Harthug S, Nesthus I, Tangen JM, Dybdahl B et al. A multi-center prospective study of febrile neutropenia in Norway: Microbiologycal findings and antimicrobial susceptibility. Scan J Inf Dis 2005; 37: 455-464. 18. Baskaran ND, Gan GG, Adeeba K, Sam IC. Bacteremia in patients with febrile neutropenia after chemotherapy at a university medical center in Malaysia. Int J Inf Dis 2007; 11 (6): 513-17. 19. Akova M. Emerging problem pathogens: A review of resistance patterns over time. Int J Inf Dis 2006; 10: 3-8. 20. Cherif H, Kronvall G, Bj_rkholm M, Kalin M. Bacteraemia in hospitalised patients with malignant blood disorders: A retrospective study of causative agents and their resistance profiles during a 14-year period without antibacterial prophylaxis. Hematol J 2003;4:420-6.
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21. Spanik S, Kukuckova E, Pichna P, Grausova S, Krupova I, Rusnakova V, et al. Analysis of 553 episodes of monomicrobial bacteraemia in cancer patients: Any association between risk factors and outcome to particular pathogen? Support Care Cancer 1997;5:330-3. 22. Sherertz RJ. Practical healthcare epidemiology: surveillance for infections associated with vascular catheters. Infect Contr Hosp Epidemiol 1996;17:746-52. 23. Hanna H, Afif C, Alakech B, Boktour M, Tarrand J, Hachem R et al. Central venous catheter related bacteremia due to gram-negative bacilli: significance of catheter removal in preventing relapse. Infect Control Hosp Epidemiol 2004; 25: 646649. 24. Goren D, Fen T. Nosocomial infections in haematology and oncology clinics: preventive measures: Review. Turkiye Klinikleri J Med Sci 2005; 25:706-723. 25. Lai CH, Chi CY, Chen HP, Chen TL, Lai CJ, Fung CP, et al. Clinical characteristics and prognostic factors of patients with Stenotrophomonas maltophilia bacteremia. J Microbiol Immunol Infect 2004; 37: 350358. 26. Lai CH, Wong WW, Chin C, Lin HH, Chen WF, Yu KW, et al. Central venous catheter-related Stenotrophomonas maltophilia bacteraemia and associated relapsing bacteraemia in haematology and oncology patients. Clinical Microbiology and Infection 2004; 12 (10): 986-991.

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Varicella Pneumonia in an Immunocompromised Patient: a case report


Jamali S MD1, Paydary K MD1, SeyedAlinaghi SA MD1, Rasoulinejad M MD1
1. Iranian Research Center for HIV/AIDS (IRCHA), Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: SeyedAhmad SeyedAlinaghi, MD Mailing address: Iranian Research Center for HIV/AIDS (IRCHA), Tehran University of Medical Sciences, Tehran, Iran. Phone: +98(21)66947984. Fax: +98(21)66947984. Email: s_a_alinaghi@yahoo.com

Summary of the Case


A 29 year-old immunosuppresed woman who is a known case of Crohns disease, was referred to the Infectious Disease Department of Imam Khomeini hospital in February 2010. She complained of progressive dyspnea, severe cough and fever in the last two weeks. She had a history of contact with a patient with active varicella zoster infection. Within the last 6 months, she received combination therapy for Crohns disease composed of azathioprin, mesalamine and prednisolone. Subsequently and simultaneous with fever, the diffuse upper body rashes began to appear accompanying nausa, dyspnea, cough and hemoptysis in the last two weeks before attendance. The chest radiograph revealed predominant noduloreticular pattern of the posterior and inferior zones. The diagnosis of varicella zoster infection has been established via skin biopsy from the abdominal wall. Intravenous acyclovir was started and she started to improve over the next few days. Acyclovir has been administered for one week. As her symptoms eventually began to improve, she was discharged on day 14 and subsequently remained symptom free.

Keywords:
Varicella pneumonia, immunosuppressed

Introduction:
Although varicella pneumonia is rare, but it is the most seriously evoked complication of varicella infection in adults. The incidence rate of respiratory complications of varicella infection in adults are almost 25-fold more prevalent than in children. Although varicella in adults accounts for only 2% of all chickenpox cases, it may eventually lead to death [1-3]. To date, only a few case series and other retrospective analysis have been conducted to describe the very complicating features of this condition. We present a case of varicella pneumonia in an immunocompromised woman presented with dry cough, hemoptysis and positive radiological findings.

weeks before attendance. She had also noted epigastric tenderness with intermittent diarrhea in the last week. At the same time, she suffered from severe fatigue, weakness and pruritis. On examination, she looked unwell with diffuse purpuric maculopapular and somewhat crusted rashes over her face, neck and torso. She was agitated and feverish. Abdomen was quite tender in the epigastric region. Organomegally was not detected on examination. Lower limb forces were decreased and reported as 3/5 on examination, while upper limb forces were normal (5/5) in both sides.

Case Presentation:
A 29-year-old, non-pregnant, non-smoker female who is a known case of Crohns disease was referred to the Infectious Disease Department of Imam Khomeini hospital in February 2010. She complained of progressive dyspnea, severe cough and fever in the last two weeks. She had no history of a prior chickenpox; however, she mentioned a history of contact with a patient with active varicella zoster infection. Within the last 6 months, she received combination therapy for Crohns disease composed of azathioprin (100 mg daily), mesalamine (Asacol; 2400 mg daily) and prednisolone (10 mg daily). Subsequently and concurrent with fever, the diffuse upper body rashes began to appear accompanying nausea, dyspnea, cough and hemoptysis in the last two

FIGURE 1: Diffuse maculopapular and crusted rashes over the face and neck.

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The chest radiograph revealed predominant noduloreticular pattern of the posterior and inferior zones. The diagnosis of Varicella zoster infection has been established via skin biopsy from the abdominal wall. Since the patient suffered of anemia and thrombocytopenia, bone marrow aspiration and biopsy has been performed and reported as normal. Viral markers including HIV (Enzyme immunoassay), HBS Ag and Anti HCV were not reactive. Her lab data is shown in the following table.

Discussion:
Varicella pneumonia has been estimated to occur as one in 400 cases of chickenpox infection [4]. There is no sex predilection and immunosuppressive conditions may predispose patients to get varicella pneumonia. In fact, the course of varicella seems to be more aggressive in immunosuppressed hosts such as HIV infected and patients with organ transplant, than normal persons [5-7]. Our patient had taken high doses of anti-inflammatory drugs for the last 6 months, and this may account for high severity of both her skin rashes and respiratory condition. Respiratory complications seem to occur through the bloodstream rather than the local extension; therefore, suppression of cell-mediated immunity could have provoked development of varicella pneumonia in susceptible individuals [5, 8]. Radiologic findings often include patchy or diffuse consolidation and nodular shadowing, while pleural effusion and hilar lymphadenopathy are of less prevalence [9]. Bronchial vesicular lesions have also been reported in bronchofiberscopy of patients with varicella pneumonia. It has been shown that chickenpox may be associated with a restrictive pattern of lung disease in acute and recovery phases. In addition to pneumonia, arthritis, encephalitis and secondary bacterial infections of the skin include other varicella complications [10-12]. In the present patient, liver dysfunction, anemia, thrombocytopenia and glomerulopathy were documented. It is not obvious whether chronic inflammatory diseases such as Crohns disease could have facilitated the development of above mentioned conditions. For example, one possible explanation for observed glomerulopathy is that prolonged inflammatory condition due to inflammatory bowel disease might have resulted in the formation of immune complexes and their subsequent precipitation in glomerular membranes. Moreover, reduced force of the lower limbs could have been related to the transient electrolyte dysregulation such as hypokalemia; however, this explanation has never been documented. These conditions might have evolved as a result of respiratory dysfunction and acute inflammatory state. Although no randomized control trials have been conducted to provide evidence for the use of acyclovir in varicella pneumonia, it has become standard therapy for patients with or at risk of developing complications. The necessity of its use via IV route is also appreciable in complications such as pneumonia, particularly in immunocompromised patients. Moreover, to prevent potentially refractory respiratory failure in such patients, maximum ventilatory support is recommended. To avoid development of such severe complications in immunosuppressed patients, varicella zoster immunoglobulin is offered for those with a history of contact with a varicella infection resource [1, 5, 8]. To avoid development of varicella zoster infection complications such as life-threatening pneumonia especially in adults, a high degree of suspicion is needed. Clinical presentations of these patients in combination with accurate paraclinical assessments, particularly simple radiographies, may aid the clinicians to timely diagnose varicella pneumonia and limit the involvement via initiation of appropriate antiviral regimens, in this case acyclovir.

FIGURE 2: Chest X-ray revealed noduloreticular pattern in both lungs. TABLE: Lab data.
Urinary analysis and Urine Culture Blood Macroscopic Full Blood Count* 6500 Lymph=10% White cells/ Blood Cell ml Neutr=90% Platelet count 44000 cell/ml Serum Enzymes and Liver Function Rheumatologic Markers Test CPK 438 lu/l Alt 346 lu/L 396 lu/ L 538 lu/L

Protein

Trace

LDH

1921 lu/l

Ast

Urine No Growth Hemoglobin Culture

11.1 gr/dL

ANA

0.5 U/ml

Alp

* Reticulocyte count was reported 0.6%.

Soon after the diagnosis of chickenpox has been confirmed, intravenous acyclovir 10mg/kg was started 8 hourly and she started to improve over the next few days. Intravenous acyclovir has been administered for one week. Her chest radiographic findings and hematological markers returned to normal within a week, the fever resolved and skin lesions began to fully crust and disappear. The patient was discharged on day 14 and subsequently remained symptom free.

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References:
1. Lee S, Ito N, Inagaki T (2004) Fulminant varicella infection complicated with acute respiratory distress syndrome and disseminated intravascular coagulation in an immunocompetent young adult. Int Med 43:1205-1209. 2. Gucuyener K, Citak EC, Elli M (2002) Complications of varicella zoster. Indian J of Pediatr 69(2):195-196. 3. Kaneko T, Ishigatsubo Y (2004) Varicella pneumonia in adults. Int Med 43(12):1105-1106. 4. Mandell GL, Douglas GR, Bennett JE (2000) Principal and Practice of Infectious Diseases. 5th Ed. Philadelphia, Churchill Livingston. 1582 p. 5. Mohsen AH, McKendrick M (2003) Varicella pneumonia in adults. Eur Respir J 21:886891. 6. Popara M, Pendle S, Sacks L, Smego RA, Mer M (2002) Varicella pneumonia in patients with HIV/AIDS. Int J Infect Dis 6:68. 7. Fehr T, Bossart W, Wahl C, Binswanger U (2002) Disseminated varicella infection in adult renal allograft recipients: four cases and review of the literature. Transplantation 73:608611.

8. Ho BCH, Tai DYH (2002) Severe adult chickenpox infection requiring intensive care. Ann Acad Med Singapore. 33:84-8. 9. Nee PA, Edrich PJ. Chickenpox pneumonia: case report and literature review (1999) J Accid Emerg Med 147-150. 10. El-Daher N, Magnussen R, Betts RF (1998). Varicella pneumonitis: clinical presentation and experience with acyclovir treatment in immunocompetent adults. Int J infect dis 2:147-151. 11. Frangides CY, Pneumatikos I (2004). Varicella-zoster virus pneumonia in adults: report of 14 cases and review of the literature. Eur J Internal Med 15:364-370. 12. Jaeggi A, Zurbruegg RP, Aebi C (1998) Complications of varicella in a defined central european population. Arch dis child 79: 472-477.

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Strongyloides Colitis presented with acute abdomen


Dr.Safaa AlKhawaja1, Dr. Zahra Jafar2, Dr. Khalil AlAradi3
1. Consultant, Infectious Disease Physician Salmaniya Medical Center, Bahrain skhawaja@health.gov.bh P.O. Box 12, Ministry of Health, Bahrain (Corresponding Author) 2. Senior Resident, Department of Internal Medicine 3. Senior Resident, Department of Internal Medicine

Abstract:
Strongyloides colitis is a rare presentation of strongyloidiasis. The infection is easily curable but carries a high mortality rate if misdiagnosed or untreated. Here, we report a case of severe Strongyloides colitis in a young Bangladeshi man, who presented with acute abdomen and peritonitis. A low index of suspicion was the main source of diagnostic delay. We believe that the high morbidity and mortality rates after misdiagnosis of this curable infection warrant efforts to increase the awareness of the disease with high index of suspicion, particularly among patients coming from endemic areas.

Keywords:
Strongyloides stercoralis, peritonitis, colitis

This report covers a rare case of strongyloides enterocolitis that presented initially with malabsorption, then progressed to colonic perforation and secondary peritonitis in a young patient with no known risk factors for hyperinfection apart from malnutrition.

Introduction
Strongyloidiasis is caused by a Strongyloides stercoralis infection. It is endemic in tropical and subtropical regions and also occurs sporadically in temperate areas [1]. Manifestations of this infection can range from asymptomatic eosinophilia in the immunocompetent host to disseminated disease with septic shock in the immunocompromised host, which carry high mortality rate [2]. Human infection occurs when infective (filariform) larvae penetrate intact skin. Once infected, most people have an asymptomatic, chronic infection of the gastrointestinal tract, with less developed waxing and waning of gastrointestinal symptoms. The unique ability of Strongyloides stercoralis to complete its life cycle within the human host enables the worms to increase their burden dramatically through a cycle of autoinfection, which can end up with disease persistence. A high intestinal worm burden can be manifested as severe malabsorption [3-4] and possibly as hyperinfection syndrome, which usually occurs in immunocompromised hosts but has also been reported in few case series among patients with no known risk factors for immunocompromised state [5-6]. The majority of symptomatically infected patients experience waxing and waning gastrointestinal symptoms that persist for years due to the presence of the adult worms in the small bowel, which induce duodenitis [2].

The Case
A thirty-four-year old, Bangladeshi, male patient, who had been working in Bahrain for eight months as security guard, was presented to emergency department with a history of malaise after fatigue of five days duration. He had history of experiencing chronic abdominal pain with occasional vomiting for more than three years. On examination, the patient was afebrile and hemodynamically stable, but pale and cachectic. He weighed 39 kg and had bilateral ankle edema. Abdominal examination revealed a distended abdomen with ascitis; all other systemic examinations were within the normal range. Initial laboratory investigations showed severe anemia with hemoglobin of 3.1 gm/dl (microcytic hypochromic picture) and a normal WBC count with no esinophilia. Serum iron was very low (1mol/l). A liver function test showed hypoalbuminemia, with albumin level of 6 g/ dl. Serum urea, creatinine levels were normal and liver enzymes were not elevated, as were the initial chest radiographs. Other investigations, including folate, B12 and hemoglobin electrophoresis, were all normal, and there was no proteinurea. One day after hospitalization, he started to develop high-grade fever and worsening of his abdominal pain. Abdominal examination showed tenderness all over, with a maximum at the epigastric region and rebound tenderness. His white cell count increased to 24,000/cu.mm, with 26% bands and 0% esinophil. A septic workup was collected and

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the patient was started on antibiotics (Piperacillin-Tazobactam and Metronidazole). Repeated chest radiographs revealed gas under his diaphragm, which raised the suspicion of a perforated viscous. Subsequently, a CT-scan of his chest and abdomen revealed significant intra-peritoneal air and fluid collection with edema and transmural air in colonic wall (Figure 1 & Figure 2) with normal lung parenchyma.

Histopathologic examination of the resected colonic segment revealed a dilated colon with multiple circular deep ulcers. Microscopy showed heavy infiltration of eosinophils, lymphocytes, plasma cells, and neutrophils in the mucosa, with heavy infiltration of Strongyloides stercoralis larva (Figure 3).

FIGURE 3: Photomicrograph of longitudinal section of Strongyloides stercoralis larva (arrow) in the colonic mucosa surrounded by eosinophils (400x magnificatn) FIGURE 1: Abdominal CT scan showing significant free intra-peritoneal air and fluid collecon A preoperative stool analysis was sent and it showed Strongyloides stercoralis filariform larva. Three sets of blood cultures were all sterile. The patient was diagnosed with severe strongyloides colitis, and accordingly he was treated with Albendazole 400 mg twice daily for a total of 14 days. Serology for Strongyloides was positive with high titer. The patient was discharged after 17 days of hospitalization with a plan for closure of colostomy after three months. Later, after the patient had a closure of colostomy, another stool analysis was completed. This time it was negative for occult blood and Strongyloides.

Discussion
Our patient stated that he had had chronic abdominal pain for few years prior to this acute presentation, which indicates chronic strongyloidiasis. His presentation with fatigue, lower-limb edema and ascitis was likely due to severe malabsorption which manifested as hypoalbuminemia and iron-deficiency anemia and might be also related to gastrointestinal bleeding secondary to worm infestation and ulceration. The presentation of strongyloidiasis with malabsorption has been reported in few case series in the past [3], but not to the severity of malabsorption presented in our patient who had severe anemia (hemoglobin 3.1 gm/ dl) and severe hypoalbuminemia of 6 g/dl. Our patient then developed severe strongyloides colitis that ended with colonic perforation and secondary peritonitis, which is very rare presentation of strongyloidiasis, particularly in the absence of known risk factors for an immunocompromised state apart from malnutrition, and with the absence of lung involvement by the Strongyloides which is commonly seen in hyperinfection syndrome [7-8]. Diagnosis of strongyloidiasis can be done by stool microscopy and/or serology. The sensitivity of single-stool microscopy is usually around

FIGURE 2: Abdominal CT scan showing significant intra-peritoneal air and transmural air and edema of the colonic ll The patient was given a transfusion of four units of blood and was prepared for surgery. A laparotomy revealed a single sealed perforation of 1 cm diameter at the hepatic flexure of the colon. The area around the colon was severely inflamed, but there was minimal peritoneal contamination. The involved segment of the colon was resected with a proximal colostomy and distal mucous fistula. Wide bore drains were left in the pelvis and the right paracolic gutter. Postoperatively, the patient was managed in the ICU. The patient improved, and the intra-abdominal drains were removed before his transfer to the general ward after six days of ICU admission.

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30% [9]. In our patient, all stool analyses were positive for Strongyloides larva, which indicates a high intestinal worm burden. Serologic testing is both sensitive and specific, with estimates of 82% 95% sensitivity and 84%92% specificity [10-13]. It can be an invaluable diagnostic tool, but it is expensive and not widely available. A biopsy from affected part of the gastrointestinal tract may demonstrate the larva [14]; in our patient there was heavy infiltration of Strongyloides larva in the resected colon.

References
1 Wirk B, Wingard JR (2009) Strongyloides stercoralis hyperinfection in hematopoietic stem cell transplantation.. Transpl Infect s 11:143-148. 2. Lim S, Katz K, Krajden S, Fuksa M, KeystonJ et al. (2004) Complicated and fatal Strongyloides infection in Canadians: risk factors, diagnosis and management. CJ 171 :479-484 3. Milner, Irvine R A, Barton C J, Bras G, Richards R (1965) Intestinal malabsorption in Strongyloides stercolaris infection. Gut 6: 574-581. 4. Scowden E B, Schaffner W, Stone W J (1978) Overwhelming Strongyloidiasis: An unappreciated opportunistic infection. Medicine 57: 527-5. 5. Keiser P B, Nutman T B (2004) Strongyloides stercoralis in the immunocompromised population. Clin Microbiol Rev 17:208-2. 6. Owor R, Wamukota W M(1976) A fatal case of strongyloidiasis with Strongyloides larvae in the meninges. Trans R Soc Trop Med Hyg 70:497-499. 7. Lin AL, Kessimian N, Benditt JO (1995) Restrictive pulmonary disease due to interlobular septal fibrosis associated with disseminated infection by Strongyloides stercoralis. Am J Respir Crit Care Med 151:205-209. 8. Nozais JP, Thellier M, Datry A, Danis M (2001) Disseminated strongyloidiasis. Presse Med 30:813-818. 9. Al-Hasan M, McCormick, Ribes JA ( 2007) Invasive enteric infections in hospitalized patients with underlying strongyloidiasis. Am J Clin Pathol. 128:622-627. 10. Loutfy M R, Wilson M, Keystone JS, Kain KC (2002) Serology and eosinophil count in the diagnosis and management of strongyloidiasis in a non-endemic area. Am J Trop Med Hyg 66:749-752 11. Carroll SM, Karthigasu KT, Grove DI (1981) Serodiagnosis of human strongyloidiasis by an enzyme-linked immunosorbent assay. Trans R Soc Trop Med Hyg 75:706-7 12.Neva FA, Gam AA, Burke J (1981) Comparison of larval antigens in an enzyme-linked immunosorbent assay for strongyloidiasis in humans. J Infectis 144: 427-2. 13.Savage D, Foadi M, Haworth C, Grant A (1994) Marked eosinophilia in an immunosuppressed patient with strongyloidiasis. J Intern Med. 236:473-475. 14. Arsic-Arsenijevic V, Dzamic A, Dzamic Z, Milobratovic D, Tomic D (25) Fatal Strongyloides stercoralis infection in a young woman with lupus glomerulonephritis. J Nephrol 18:7879

Conclusion
Strongyloidiasis is common in certain areas of the world where there is a high risk for potential exposure. Accordingly, a high index of suspension and testing for strongyloidiasis by stool microscopy and serology is mandatory for individuals who come from endemic area and who present with vague or unexplained gastrointestinal symptoms, or for those who present with malabsorption. Testing should also be considered for patients who are intending to start immunosuppressive therapy, because the risk for disseminated strongyloidiasis in this case is very high.

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The role of Herbal Medicine use in HIV/AIDS treatment


RO Orisatoki+ & OO Oguntibeju*
+School of Public Health, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, SK , Canada S7N 5E5 *Department of Biomedical Sciences, Faculty of Health & Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa. + Corresponding Author: Rotimi O Orisatoki, rotioris@yahoo.com Telephone number: (306) 966-1649 Facsimile number: (306) 966-7920

Abstract
Herbal medicine use is becoming very popular in many countries especially in the western world, where public health safety has become a concern, especially its concomitant use with orthodox medicine. The devastating impact of HIV/AIDS pandemic coupled with the severe shortage of health personnel has compelled patients to develop coping mechanisms by adopting alternative sources of primary health care, one of which has been the use of herbal therapies. An integration of herbal medicine into the current medical curriculum will enable future physicians to communicate better with their patients on this evolving healthcare system. This review briefly examines the role of herbal medicine in HIV/AIDS treatment and management. It is hoped that this review will provide important and relevant information that will help policy makers to put in place control measures against the abuse of herbal therapy.

Introduction
The use of herbal medicine is increasingly becoming more popular in many countries [1]. This practice has continued to be a main source of health care in the rural communities especially in developing countries, since modern medicine has not been able to reach the majority of the populace. Also, herbal medicines are still being commonly sold by practitioner and their agents without any restriction with most of the health care providers receiving little or no formal training in this area. This lack of proper training may be associated with the inability of herbal practitioners or their agents to answer questions patients have about its efficacy either as a supplement to orthodox medicine or as a therapy to treat or prevent disease. Their inability to answer questions may partly be linked to the fact herbal medicine involve a sophisticated theory or system, with the knowledge that is often passed on, verbally or otherwise, from generation to generation [2,3,4]. Notwithstanding, there have been a remarkable increase in the popularity of herbal preparations especially in developed countries, which has stimulated considerable public health concern among physicians who are sometimes uncertain about the safety of herbs especially when used concomitantly with regular orthodox medications [5]. HIV/AIDS pandemic is currently the most socio-economic challenge that is facing the world at large as it affects mostly the young and economically productive population [6]. A study has shown that majority of people living with HIV/AIDS are susceptible to fungal and bacterial opportunistic infections that result from immunosuppression and treatment of such infections is therefore one of the areas that traditional health services for the control of the disease is prevalent[6]. The World Health Organization (WHO) estimates that 4 billion people (80% of the Worlds population) use herbal medicines for some aspect of pri-

mary healthcare [7]. Treatment of diseases using traditional remedies is an age old art which has been confined into the backstage due to access to western biomedicine, adequate education, employment opportunities and economic growth [8].

Regional use of herbal medicine


Herbal medicine in the Caribbean Afro-Caribbean pharmacopoeia is the body of knowledge and practices around medicinal plants with its origins in the cultures of African slaves brought to the Caribbean [9]. Herbal baths are common in Haitian culture for both spiritual and medicinal practices and represent the second most important category of administration after ingestion in the region [10]. There is significant use of herbal remedies in the Caribbean and recent studies in Trinidad show relatively high prevalence of use for symptomatic relief in asthma and therapeutic management in diabetes mellitus [11-12]. No herbal treatment of HIV/AIDS was seen in all literature review done by the authors. Herbal use in North America The increasingly diverse US immigrant populations have led to the growing use of medicinal herbs. A survey conducted by the National Center for Complementary and Alternative Medicine in 2004 revealed that use of herbal therapy or of other natural products was most common of the complementary and alternatives medicines and the commonest reason for use of herbal medicines by Americans was that they believed they would improve health when used in combination with conventional medical treatments [13].

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Herbal use in Africa Herbs have a long history in African traditional medicine, however there has been increasing attention and interest in its use in recent times [3]. Traditional herbal medicine has continued to be a main source of health care in the rural communities and heavy reliance on it by the majority of the sub-Sahara Africa population has led to the generally accepted conclusion that it is most preferred form of treatment of HIV-related symptoms [14]. Herbal Use in Asia Medicinal herbs are a major component of Traditional Chinese Medicine (TCM). It is estimated that over 600 different herbs have been used to treat various human diseases including those caused by viral infection, accounting for approximately one-fifth of the entire Chinese pharmaceutical market and are regarded as the state cultural treasure by the Chinese government[15]. Studies on the anti-HIV activities and mechanisms of TCMs are very limited and are expected to accelerate. Herbs native to Japan were classified in the first pharmacopoeia of Japanese traditional medicine in the ninth century [16]. Ayurveda is a herbal medical system primarily practiced in India. It includes diet and herbal remedies, while emphasizing the body, mind and spirit, in disease prevention and treatment [17]. Herbal use in Europe Complementary or unconventional treatments are used by many doctors and other therapists throughout Europe. The major forms are acupuncture, homoeopathy, manual therapy or manipulation, and phytotherapy or herbal medicine. The relative popularity of therapies differs between countries, but public demand is strong and growing. Regulation of practitioners varies widely: in most countries only registered health professionals may practice, but in the United Kingdom practice is virtually unregulated. Germany and some Scandinavian countries have intermediate systems. Legal reforms are in progress in the Netherlands and the United Kingdom. European institutions are starting to influence the development of complementary medicine [18]. In Germany, herbal medications are dispensed by pharmacists, subjected to same criteria for efficacy, safety and quality as are other drug products. Despite the progress in orthodox medicine, interest in alternative medicine, including herbal medicine is the increase. A great variety of plants are used for medicinal treatments, either the dried plant or a specific part of it ( root, leaves, fruit, flowers, seeds), is formulated into suitable preparations-compressed as tablets or made into pills, used to make infusions (teas), extracts, tinctures or mixed with excipients to make lotions, ointments, creams [19-20].

promoting the use of traditional medicines with antiretroviral treatments [23]. Two principal African herbal compounds used for HIV/AIDS treatment in sub-Saharan Africa include Hypoxis hemerocallidea (African potato-an immunostimulant) and Sutherlandia. These two herbal remedies are currently recommended by the South African Ministry of Health for HIV management [24]. We believe that medical practitioners and researchers ought to be informed about the use of herbal medicine for both HIV-related illnesses and other co-morbid conditions as part of their history taking and clinical assessments. Failure to do so may cause health-care workers to inadvertently overlook the full spectrum of potential herb-drug interactions that may be experienced by an AIDS patient. For instance, a study in Canadian found that more than 53% of HIV outpatients taken traditional herbs did not report its use to their treating physician [25]. Also, antiretroviral therapy recipients have been reported to use herbs to alleviate some of the negative side effects of antiretroviral (ARV) drugs such as nausea and diarrhea [21]. In North America, commonly used herbal dietary supplements have been found to impede on ARV drug effectiveness. Specifically, garlic supplements (Allium sativum) and St Johns Wort (Hypericum perforatum) have been shown to have detrimental effects on the plasma concentrations of saquinavir and indinavir. [26]. The most common reasons patients gave for using herbs include general wellbeing, relaxation, pain, stress, spiritualism and healing. It was reported that 9% of outpatients believed that it was possible to treat HIV solely with the use of herbs, while others use it to improve energy level, to supplement dietary intake and to enhance response. However, in a US study, the most common treated conditions using herbal medicine include anxiety/fear, depression, pain and neuropathy [25, 27]. Fogarty et al. (2007) also found that 44.3% of an Australian HIV patient sample reported mixed use of marijuana for therapeutic and recreational purposes [28]. Although modern medicine may exist side-by side with such traditional practice, herbal medicines have often maintained their popularity for historical and cultural reasons, so such people see traditional medicine as a complementary health care not alternative to modern medicine [21]. However, there are some challenges to collaboration between traditional herbal medicine and orthodox medicine especially in the developing countries mainly due to shortage of mutual trust and appreciation between the two health system, limited availability of training in basic preventive medicine, palliative care for traditional herbal healer, lack of meaningful referral between conventional health providers and traditional herbal healers, exclusion of traditional healing methods from the training curricula of doctors and traditional healers fear of losing their treatments secrets to scientists and researchers [14]. A patients level of knowledge about HIV disease, a belief that ART is effective and prolongs life and recognition that poor adherence may result in viral resistance and treatment failure all impact negatively upon a patients ability to adhere. Beliefs about the medications (including traditional) themselves also play a role in adherence. Patients who report low confidence in the efficacy of the medications and perceive minimal benefits resulting from ART are less likely to be adherent. It is estimated from other studies that at least 30% of patients on ART will use any form of traditional complementary and alternative medicine [29]. Because the exact level of risk and/or benefits resulting from traditional herbal medicine-antiretroviral- drug co-therapy amongst AIDS

Herbal medicine in the treatment of HIV/AIDS


Traditional herbal use has been reported to be common among individuals with moderate and advanced HIV disease [21]. In Africa, traditional herbal medicines are often used as primary treatment for HIV/ AIDS and for HIV-related problems including dermatological disorders, nausea, depression, insomnia and weakness [22]. The use of traditional herbal medicine by AIDS patients after HIV diagnosis was noted in a study in Uganda [21]. Despite a paucity of evidence on effectiveness and the possibility of serious side effects, some African ministries of health currently promote traditional medicines for the treatment of HIV and associatedsymptoms. In the case of South Africa, the Ministry of Health is actively

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patients is largely unknown, the concerns raised about herb-ARV drug interactions, communication between patients and physicians about herbal medicine is valuable in enabling physicians to address issues of potential herb-drug interactions and ensuring appropriate medical care [30]. The range of care for patients encompassed a broad range of treatment options. South Africans living with HIV/AIDS are now encouraged to make their own informed choices about the types of treatment they wish to seek, including antiretroviral (ART), exercise, nutrition as well as traditional and complementary medicines (TCAM) [31]. The devastating impact of HIV/AIDS pandemic in the region (Southern Africa) coupled with the severe shortage of health personnel might have forced the inhabitants to develop coping mechanisms by adopting alternative sources of primary health care, one of which has been the use of herbal therapies [32].

While many years of herbal use in traditional settings can be used as a testimony that a particular herbal ingredient is effective or safe, several problems need to be addressed. It is now known that ingredients that form part of herbal preparations are incorporated into modern practice and are now used in developed countries as part of health promotion or disease prevention strategies. One of the most difficult issues to contend with in translating traditional herbal practices into conventional western medicine is the individualization of prescription containing multiple herbal and other ingredients. Whether backed by medical science or simply by years of use, traditional treatments remain popular and as more research is carried out, some may play a complementary role in modern medicine. There is an urgent need for educational intervention with regard to herbal medicine in the training of our physicians. We propose that an integration of herbal medicine into the current medical curriculum so that future physicians would be better prepared to communicate with their patients on this healthcare modality. Continuing education programme are also recommended so that practicing physicians would have the opportunity to upgrade their knowledge in this rapidly expanding area of significant public health concern.

Conclusion
The inclusion of traditional herbal healers in the health care system especially in primary healthcare team in developing countries could improve quality of life and safety standards and their use as a complimentary therapy could play a role in the palliative care of people living with HIV/AIDS.

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References
1. Bannerman R (1993) Traditional Medicine and Healthcare. Geneva. WHO 2. Zhang X (2000) Integration of traditional and complimentary medicine into national health care systems. J Manipulative Physiol Ther 23: 139-140. 3. David M (1997) Advising Patients who seek alternative medical therapies. Ann Intern Med 27(1): 61-69. 4. Stephen M, Margaret L, Robert M (2003) Complementary and alternative medical practices: training, experiences, and attitudes of a primary care medical school faculty. J Am Board Fam Pract 16: 318-326. 5. Joint United Nations programme on HIV/AIDS (UNAIDS)/World Health Organization (WHO) (2007) AIDS epidemic update Geneva Dec. 6. Kisangau D, Lyaruu H, Hosea K, Joseph C (2007) Use of traditional medicines in the management of HIV/AIDS opportunistic infections in Tanzania: a case in the Bukoba rural district. J Ethnobiol Ethnomed 10: 24-27 7. World Health Organization (2002) Traditional medicine; growing needs and potential. WHO Policy perspectives on medicines. WHO, Geneva 1-6 8. Langlois-Klassen D, Kipp W, Rubaale T (2008) Whos talking? Communication between health providers and HIV-infected adults related to herbal medicine for AIDS treatment in Western Uganda. Soc Sci Med 67(1): 165-76. 9. Laguerre M (1987) Afro-Caribbean Folk Medicine. South Hadley, MA: Bergin and Garvey Publishers, pp.71-72 10. Volpato G, Godinez D, Beyra A, Barreto A (2009) Use of medicinal plants bt Haitian immigrants and their descendants in the Province of Camaguey, Cuba. J Ethnobiol Ethnomed 23: 23-26. 11. Clement Y, Williams A, Aranda D, Chase R, Watson N, et al. (2005) Medicinal herbal use among asthmatic patients attending a specialty care facility in Trinidad. BMC Compl Altern Med 5: 3-5 12. Mahabir D, Gilliford MC (1997) Use of medicinal plants for diabetes in Trinidad and Tobago. Rev Panam Salud Publica 1(3): 174-179. 13. National Center for Health Statistics (2004) Complementary and alternative medicine use among adults. http://www.nccam.nih.gov/news/2004/052704.htm 14. Bamidele J, Adebimpee O, Oladele E (2009) Knowledge, attitude and use of alternative medical therapy amongst urban residents of Osun State SouthWestern Nigeria. Afri J Trad Compl Altern Med 6 (3):281-288. 15. Li L (2000) Opportunity and challenge of traditional Chinese medicine in face of the entrance to World Trade Organization. Chin Inform Trad Chin.Med 7:7-8 16. Saito H (2000) Regulation of herbal medicines in Japan. Pharmacol. Regul 41:515-519.

17. Morgan K .Medicine of the gods: Basic principles of Ayurvedic medicine. http://www. compulink.co.uk/~mandrake/ayurveda.htm 18. Fisher P, Ward A (1994) Complementary medicine in Europe. BMJ 309:107-11. 19. Zudek EM (1994) Traditional Native healing: alternative or adjunct to modern medicine? Can Fam Phys 40:1923-1931. 20. Tapsell LC (2006) Health benefits of herbs and spices: the past, the present, the future. Med J Austr 21:10-13 21. Langlois-Klassen D, Kipp W, Jhangari GS, Rubaale T (2007) Use of traditional herbal medicine by AIDS patients in Kabarole District, Western Uganda. Am J Trop Med and Hyg 77: 757-763. 22. Hodgson T, Rachanis C (2002) Oral fungal and bacterial infections in HIV-infected individuals: an overview in Africa. J Oral Dis 8: 80-87. 23. Manfredi R, Chiodo F (2000) The effects of alternative treatments for HIV disease on recommended pharmacological regimens, Int J Antimicrobial Agents 2000; 13: 281-285. 24. SADC (2002) SADC ministerial consultative meeting on Nutrition and HIV/AIDS. Johannesburg January 20 25. Furler M, Einarson T, Walmsley S, Millson M, Bendayan R (2003) Use of complementary and alternative medicine by HIV-infected outpatients in Ontario, Canada. AIDS Patient Care STDs 17(4): 155-168. 26. Piscitelli S, Burstein A, Welden N, Gallicano K, Falloon J (2000) Indinavir concentrations and St. Johns wort. Lancet 255: 547-548. 27. Dhalla S, Chan KJ, Montaner JS, Hogg RS (2006) Complementary and alternative medicine use in British Columbia- a survey of HIV positive people on antiretroviral therapy. Compl Therap Clin Pract 12(4): 242-248. 28. Forgarty A, Rawstorne P, Prestage G, Crawford J, Grierson J, Kippax S (2007) Marijuana as therapy for people living with HIV/AIDS: social and health aspects. AIDS Care 19 (2): 295-301. 29. Peltzer K, Preez NF, Ramlagan S, Fomundam H (2008) Use of traditional complementary and alternative medicine for HIV patients in KwaZulu-Natal, South Africa. BMC Public Health, 14 (7) :21-26 30. Astin J (1998) Why patients use alternative medicine: results of a national study. JAMA 279:1548-1553. 31. Drew A, Myers S (1997) Safety issues in herbal medicine: implications for the health professions Med J Aust. 166(10): 538-541. 32. Hollenburg D, Zakus D, Cook T, Xu X (2008) Re-positioning the role of traditional, complementary and alternative medicine as essential health knowledge in global health: do they still have a role to play? World Health Population 10 (4): 62-75.

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Multi-system organ failure following administration of yellow fever vaccine: a case report
Julie Ehret Leal, PharmD1, Amy N. Thompson, PharmD, BCPS2,Joseph D. Thomas, MD3, Walter A. Brzezinski, MD4
1Clinical Specialist, Department of Pharmacy Services; Medical University of South Carolina 2Assistant Professor, Department of Clinical Pharmacy and Outcome Sciences; South Carolina College of Pharmacy 3Department of Internal Medicine; Medical University of South Carolina 4Associate Professor, Department of Internal Medicine; Medical University of South Carolina Corresponding author: Julie Ehret Leal, PharmD 135 Rutledge Ave, Suite 820R, Charleston, SC 29412 Phone: (843) 876-0888 Fax: (843) 876-0767 Email: ehret@musc.edu No sources of support were received No disclaimers

Abstract
Title: Multi-system organ failure following administration of yellow fever vaccine: a case report Background: Yellow fever vaccine-associated viscerotropic disease (YEL-AVD), is an extremely rare and life-threatening condition that occurs shortly after administration of the vaccine. It is characterized by multi-system organ failure including hemorrhagic fever, shock, renal failure, and hepatic failure. Case: A 77-year-old Caucasian male experienced multi-system organ failure within seven days of receiving the yellow fever vaccine. The patients signs and symptoms included fever, acute renal failure, hepatitis, lymphocytopenia, thrombocytopenia, hypotension, and respiratory failure. Conclusions: Since serious adverse events are possible following administration of yellow fever vaccine, physicians should administer the vaccine only to those truly at risk for exposure to the virus.

Introduction
Yellow fever is a viral hemorrhagic disease transmitted to humans via the bite of infected female mosquitoes. There are an estimated 200,000 cases of yellow fever in the world annually, resulting in 30,000 deaths [1]. Most of these cases occur in the tropical regions of Africa and South America where the presence of infected mosquitoes is most prominent [1]. Yellow fever infections are generally mild and self-limiting, but severe cases can occur, which are characterized by hemorrhagic fever, shock, renal failure, and hepatic failure [2]. Since there are no known treatments for yellow fever other than supportive care, severe cases often result in death. Yellow fever vaccine is a live, attenuated virus given as a one-time injection with a recommended re-vaccination interval of 10 years [3]. It is a safe and effective vaccine producing only mild systemic adverse effects, such as low-grade fever, headache, and myalgias in 10 to 30% of vaccine recipients [3] However, in select individuals, yellow fever vaccine-associated viscerotropic disease (YEL-AVD) is a rare illness characterized by signs and symptoms very similar to the actual yellow fever disease, but caused by vaccine virus replicating in multiple organs. This often leads to multi-system organ failure and death. As of July 2009, more than 40 confirmed and suspected cases have been reported worldwide [3]. In the United States, the incidence of YEL-AVD is very low, reported as 0.4 cases per 100,000 doses administered [1]. The following is a case of a 77-year-old male who experienced multisystem organ failure and subsequent death shortly after receiving the live, attenuated 17D strain yellow fever vaccine.
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Case
A 77-year-old Caucasian male with a past medical history of hypertension, diabetes mellitus type II, and Factor V Leiden deficiency presented to his primary care physician approximately six days following administration of yellow fever vaccine with complaints of unsteadiness, urinary urgency, and generalized weakness and malaise for the previous two days. The patient received the vaccine in preparation for travel to South America. Physical exam was remarkable only for gait unsteadiness and pale skin color. Multiple labs were drawn, including a comprehensive metabolic panel, complete blood count with differential, prothrombin time and international normalized ratio, thyroid function tests, prostate specific antigen, and urinalysis. The only abnormalities were a slight elevation in serum creatinine from a baseline of 1.0 to 1.4 mg/dL, a urinalysis that revealed moderate blood, and a left shift on complete blood count with greater than 90% neutrophils in the setting of a normal white blood cell count. A diagnosis of urinary tract infection was made and the patient was sent home with a prescription for ciprofloxacin 500 mg twice daily for one week. The following day, the patient presented to the emergency department with complaints of fever, nausea, vomiting, back and abdominal pain, and worsening lethargy. Physical assessment was notable for a temperature of 100.5 degrees. Labs revealed hemoglobin and hemotocrit of 11.7 gms/dL and 35.9% respectively, sodium of 127 mmol/L, potassium of 3.3 mmol/L, serum creatinine of 1.4 mg/dL, and neutrophils of 97%. An infectious process was suspected at this time, and the patient was admitted to the hospitals general medicine service for further treatment. Upon admission, blood and urine cultures were obtained and
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empiric broad-spectrum antibiotic coverage was initiated with vancomycin, ceftriaxone, ampicillin, and acyclovir. Over the next 24 hours, the patient was consistently febrile, with temperatures in excess of 103 degrees. He quickly progressed to hemodynamic instability and sepsis syndrome exhibited by tachypnea, tachycardia, hypotension, and mental status changes. He was admitted to the intensive care unit the next day, intubated, and placed on cardiopulmonary support with a working diagnosis of sepsis syndrome of unknown etiology. In order to rule out an infectious process in the central nervous system, a successful lumbar puncture was performed at this time, which was negative. Doxycycline was added to his antibiotic regimen as well as norepinephrine and vasopressin for pressure support. He received continuous intravenous fluids with normal saline for volume resuscitation. The patient began to experience oliguric renal failure requiring continuous veno-venous hemofiltration (CVVH) beginning on hospital day 4 with a serum creatinine of 3.3 mg/dL with a peak serum creatinine of 4.5 mg/ dL eleven days later. Acute hepatitis as evidenced by elevated transaminases was noted beginning on hospital day three and continued throughout hospital course (peak AST of 312 IU/L on hospital day six and peak ALT of 106 IU/L on hospital day 10). Interestingly, total bilirubin throughout hospital course remained within normal limits. On hospital day 5, coagulopathy was noted with an International Normalized Ratio of 5.1, which was unresponsiveness to fresh frozen plasma. The patients neurological status declined to a point where he no longer responded to painful stimuli. The patient also experienced acute muscle injury as evidenced by a creatine kinase of 4334 IU/L. CT scans, MRI images, and chest X-rays were obtained and did not elucidate any signs of infection or causes of the patients multi-system organ failure. The patient was tested for Ehrlichia, leptospira, Rocky Mountain spotted fever, herpes simplex virus, cytomegalovirus, and human immunodeficiency virus, which were all negative. At this point, yellow fever vaccine-associated viscerotropic disease (YEL-AVD) was suspected. On this same day, cultures and viral PCRs were sent to the Centers for Disease Control and Prevention (CDC) for analysis, which came back as negative for infection and detection of yellow fever virus, respectively. A serum dilution-plaque reduction neutralization test (PRNT) to detect yellow fever neutralizing antibodies was greater than 160 on the day of admission and climbed to greater than 10,000 on hospital day 5. The patient continued to require cardiopulmonary support despite broadspectrum antibiotic, antiviral, and antifungal coverage. He required constant volume resuscitation, which resulted in diffuse anasarca with a weight gain of greater than 55 kilograms. After more than a week of cardiovascular support, permission was granted by the family to withdraw care, and the patient passed away. Autopsy was offered to the patients family, but was declined.

(YEL-AVD). YEL-AVD occurs as a result of an atypical, widespread inflammatory response upon administration of the vaccine. Initial symptoms of the disease usually occur two to five days following administration of the vaccine and include fever, nausea, vomiting, and jaundice. Signs of multi-organ failure appear quickly, most notably elevated hepatic enzymes, respiratory failure, blood dyscrasias, and renal failure. In some cases, the attenuated virus can be found in body tissues. Fatality rates with YEL-AVD, even with aggressive treatment, are approximately 53% based on currently available literature [1]. The specific factors that increase a patients risk for developing YEL-AVD are not fully known, making it difficult to predict possible reactions. Previously published reports indicate that advanced age may be a risk factor for developing YEL-AVD. Martin and colleagues reported four cases of YEL-AVD in patients greater than 63 years of age [3]. In each of these cases, the patients presented within five days of receiving the vaccine with major complaints of fever, myalgias, and confusion, similar to the presentation of our patient. Vaccine-like yellow fever virus was isolated in three out of the four patients in their report. Of the four patients described, three of them died. Another similar case was reported by Chan and colleagues in a 56-year-old male [4]. While the case presented in this paper is a geriatric patient, it is important to note that older patients are not the only population at risk for developing YEL-AVD. Gerasimon and Lowry reported a case of an otherwise healthy, 22-year-old female who presented to an emergency department with a four day history of vomiting and diarrhea [5]. Records indicate that she had received the yellow fever vaccine six days prior as standard protocol for military deployment. She rapidly decompensated while hospitalized, and was transferred to the intensive care unit for respiratory failure. Four days after being admitted to the hospital, she went into cardiac arrest and died. Several similar case reports describing YEL-AVD with subsequent death in young patients have been published, with ages ranging from 4 years of age to 26 years of age [6-9]. The patient presented in this case report fits the criteria for YEL-AVD. It is important to note that he had no known drug or food allergies that could have contributed to the described events. Since an autopsy was not performed, a major limitation of confirming YEL-AVD as the true diagnosis was the unavailability of organ tissue for biopsy to confirm presence of the attenuated virus. However, based on the patients presentation and hospital course, it is strongly believed that he succumbed to the rare and serious effects associated with receiving the yellow fever vaccine.

Discussion
The live, attenuated yellow fever vaccine was first introduced in the 1930s and has protected millions of susceptible people from contracting the illness. However, when the Vaccine Adverse Events Reporting (VAERS) system was established by the CDC and the United States Food and Drug Administration (FDA) in 1990, rare cases of multi-system organ failure shortly following administration of the vaccine began to appear. First described in the literature in 2001, this condition has since been termed yellow fever vaccine-associated viscerotropic disease

Conclusions
Current recommendations from the CDC state that anyone nine months of age or older traveling to or living in areas with risk of yellow fever transmission, especially in South America and Africa, should be vaccinated with yellow fever vaccine to reduce the risk of contracting the illness [1]. However, because severe adverse events are possible following vaccination, physicians should administer the vaccine only to persons truly at risk of exposure to yellow fever.

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Appendix 1.

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References
1. Yellow fever (2009) World Health Organization. Available : http:// www.who.int/mediacentre/factsheets/fs100/en/. Accessed 18 Mar 2010. 2. Yellow fever fact sheet (2007) Centers for Disease Control and Prevention. Available : http://www.cdc.gov/ncidod/dvbid/yellowfever/ YF_FactSheet.html. Accessed 18 Mar 2010. 3. Gershman M, Schroeder B, Staples JE (2010) Chapter 2: The pre-travel consultation, travel-related vaccine-preventable diseases. In: Travelers Health Yellow Book. Available: http://wwwnc.cdc.gov/travel/yellowbook/2010/chapter-2/yellow-fever.aspx Accessed 18 Mar 2010. 4. Martin M, Tsai TF, Cropp B, Chang GJ, Holmes DA, et al (2001) Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases. Lancet 358: 98-104. 5. Chan RC, Penney DJ, Little D, Carter IW, Roberts JA, Rawlinson WD (2001) Hepatitis and death following vaccination with 17D-204 yellow fever vaccine. Lancet 358: 121-122.

6. Gerasimon G, Lowry K (2005) Rare case of fatal yellow fever vaccineassociated viscerotropic disease. Southern Med Journ 98(6): 653-656. 7. Struchiner CJ, Luz PM, Dourado I, Sato HK, Aguiar SG, et al (2004) Risk of fatal adverse events associated with 17DD yellow fever vaccine. Epidemiol Infect 132: 939-946. 8. Vasconcelos PFC, Luna EJ, Galler R, Silva LJ, Coimbra TL, et al (2001) Serious adverse events associated with yellow fever 17DD vaccine in Brazil: a report of two cases. Lancet 358: 91-97. 9. Doblas A, Domingo C, Bae HG, Bohorquez CL, Ory F, et al (2006) Yellow fever vaccine-associated viscerotropic disease and death in Spain. Journ of Clinical Virology :156-158. 10. Belsher JL, Gay P, Brinton M, DellaValla J, Ridenour R, et al (2007) Fatal multiorgan failure due to yellow fever vaccine-associated viscerotropic disease. Vaccine 25: 8480-8485.

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Genetic diversity of Aeromonas spp. isolates from animal food origin demonstrated by Random amplified Polymorphic DNA analysis.
Indu Sharma*1, A. K Pramanik 2 and A. Kumar3
1. Corresponding author: drsharma7652@gmail.com Department of Microbiology, Assam University, Silchar-788011. India 2. Department of Veterinary Public Health, West Bengal university of Animal and Fishery Sciences, Kolkata-700037, India. 3. Department of Veterinary Public Health, IVRI, Izat nagar, India.

Abstract
A total of 332 samples of animal food origin comprising 104 poultry, 137 fish, 51 pork and 40 goat (chevon) were screened for genetic diversity of Aeromonas spp. from animal food origin. In the present study, out of 20 random primers of OPH series, OPH-1, OPH-2 and OPH-3 were found to produce the most reproducible and scorable amplicon profiles. The amplicon sizes ranged from 250 bp to 2.9 Kb with a common >250 bp fragments in all the isolates and the overlaid dendogram revealed similarity ranged upto 75% in all the samples Keywords:, Aeromonas, RAPD, Food animals

Introduction
Aeromonas spp. has become increasingly recognized as enteric pathogens. These organisms cause acute diarrhoea in children [1] and adults [6] and sporadic diarrhoea or dysentery in those older than 60 years, which can be severe and even life threatening [4-5]. However, today, these are also responsible for causing gastroenteritis outbreaks in humans and travelers diarrhea [19]. The spectrum of infectious diseases caused by Aeromonas species includes gastrointestinal infections as well as extra intestinal infections such as cellulites, wound infections, septicemia, urinary tract infection and hepatobiliary and ear infections [15]. Virulence of Aeromonas spp. is multifactorial and incompletely understood. Factors contributing to virulence include toxins, proteases, hemolysins, lipases, adhesins, agglutinins, and various hydrolytic enzymes [7,2]. These virulence factors are useful in distinguishing between potentially pathogenic and non-pathogenic strains. Some investigators observed that Aeromonas induced gastroenteritis is due to an enterotoxin which is cytotoxic in nature [16,14] while others reported aerolysin to be the main virulence factor involved in intestinal disorders. About 6.5% of diarrhoeal cases in the southern part of India have been attributed to Aeromonas [10], which indicates an urgent need for information on the casual role of this pathogen in other parts of the country. Aeromonas associated gastroenteritis is probably under diagnosed due to the lack of recognition of its significance, confusion over its taxonomy and the difficulty for a laboratory to routinely identify isolates with virulence-associated properties, such as enterotoxin production and entero-invassiveness [8]. According to the International Commission on Microbiological specifications for Food 1996, many classical procedures for the detection of Aeromonas spp. were found to be laborious and time consuming or not allowing quantitative assessment of these organisms, thus indicating the need for a reliable, universal and standard method.

Materials and methods Bacterial strains and culture conditions.


A total of 332 samples of which 38 isolates were identified by the 16S rRNA technique were included in the study. The isolates were grown on ADA (Ampicillin Dextrin Agar (HI-MEDIA Laboratories, Mumbai, India) at 37C for 18-24 hours. Of the 38 isolates, 35 (92.10%), 2 (5.26%) and 1 (2.63%) were recognized as A. hydrophila, A. sobria and A. caviae. All the strains were dominantly environmental isolates in our collection. The details of the samples collected have been summarized in the following Table one. TABLE 1: Details of samples procured from retail shops of Meghalaya and Assam
SL.NO. 1. 2. 3. 4. TYPE OF FOOD ANIMALS POULTRY FISH PIG GOAT MATERIALS COLLECTED INTESTINE GILLS, INTESTINE MEAT MEAT MEAT TOTAL NO. OF SAMPLES COLLECTED 104 137 51 40 332

Biochemical studies.
The following biochemical tests were done in all strains according to conventional protocols: esculin hydrolysis, citrate utilization, motility, indole production, and acid production from rhamnose, sorbitol, lactose, D-sucrose, and salicin. Test samples were incubated under the same conditions as used for bacterial growth. All tests were carried out in duplicate, and appropriate positive and negative controls were included.
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Molecular typing of isolates by PCR.


The use of PCR assay for molecular typing revealed that all the 38 isolates belonged to Aeromonas spp. and were positive for 16S rRNA (100%), ahh1 (60.52%), asa1 (42.10%), A. hydrophila aerA (13.15%) and AHCYTOEA/aerA (5.26%). In the present investigation it was noted that aerolysin producing toxin genes were the most prevalent irrespective of their geographical locations and the most common single gene carried among all the isolates examined was ahh 1 (60.52%). The results recorded in this study are in agreement with those reported by Wang et al. 2003 [18] in the multiplex PCR.

Results Biochemical studies:


Biochemical characterization of the isolates revealed that heterogenecity existed between the isolates. All the 38 isolates screened for primary characterization were tested positive for oxidase and catalase test. The Gram stained Aeromonas isolates showed Gram-negative bacilli appearing as short rods. Further confirmation test which characterized the Aeromonas isolates upto species level were performed using standard protocols. Some variability was observed with regard to VP test and gelatin liquefaction. However, biochemical reactions were found to deviate from the ideal phenotype of each isolate. Of the 38 isolates, 35 (92.10%), 2 (5.26%) and 1 (2.63%) were recognized as A. hydrophila, A. sobria and A. caviae.

Isolation of genomic DNA:


Colonies of Aeromonas spp. grown on Ampicilin Dextrin agar at 37C for 24 hr. were scraped off and suspended in 0.85% NaCl. After centrifugation, the pellet was resuspended in a lysis buffer and incubated after adding protease K. The DNA was extracted using the Promega (USA), DNA isolation Kit according to the procedure established by the manufacturer. The quality and quantity of the DNA were determined spectrophotometrically at 260 nm. The extracted DNA was stored at 2-8oC for further use.

RAPD analysis:
In the present study, out of 20 random primers of OPH series, OPH-1, OPH-2 and OPH-3 were found to produce the most reproducible and scorable amplicon profiles. The amplicon sizes ranged from 250 bp to 2.9 Kb with a common >250 bp fragments in all the isolates (Fig. 1 and 2). The dendogram from the overlaid graphs arising from the RAPD profiles of Aeromonas isolates with primer OPH-1, OPH-2 and OPH-3 revealed that similarity ranged upto 75% in all the samples. Four clusters were observed comprising of C18 and FG16; FI9 and C3; FM4 and PM2. Sample PM2 exhibited 12% similarity, C18, FG16, FI9 and C3 exhibited 28% similarity where as sample FM4 exhibited 44% similarity. Also, PM2 was found to be completely different from other clusters (Fig 3).
MW (bp) 1 2 3 4 5 6 7 8

Random amplification of polymorphic DNA:


Primers for RAPD [12] used in the study are presented in Table 2. Amplification was performed using a thermocycler (iCycler, BIORAD, USA). The 50 l reaction contained 10 x NH4 buffer, 4.0 mM Mgcl2, 200 M of dNTPs, 2U Taq DNA polymerase and 20 ng of genomic DNA. Amplification conditions were 35 cycles of 10 sec. at 94C, 30 sec. at 37C, 60 sec. at 72C and a final extension of 5 min. at 72C. The amplification products were analyzed in 1% agarose (Promega, USA) gel. The gel was prepared by dissolving agarose in 1X Tris acetate (TAE) buffer (Genei, Bangalore). Same buffer was used for electrophoretic run. A total of 5l of each amplicons and 1l marker DNA (100bp DNA ladder mix; MBI Fermentas, USA) were mixed separately with 1l of 6X gel loading dye (MBI Fermentas, USA) and loaded in the wells of the gel. Electrophoresis was carried out in Mini plus horizontal (GENE Mate gel system, UK) electrophoretic apparatus at a constant voltage of 60 V for 1 hour and 20 min. or until the second dye marker had run 3/4th of the gel. Then the gel was stained in ethidium bromide (Pharmacia Biotech, Sweden) @ 0.4 g/ml in distilled water solution for 10-15 min. and was visualized in gel documentation system (Gel Logic 100 Imaging System, Biostep) and photographed. Table No. 2: Primers used for RAPD amplification
SL.No. 1. Primer H1 Sequence 5 TGC CGA GCT G 3 Reference

1100 400 200 100

FIG.1: RAPD of Aeromonas isolates with primer OPH-1. Lane 1 & 8: 100 bp DNA Ladder mix. Lanes 2-7: Test organisms.

MW (bp)

2 3 4 5 6 7

8 9

800 [12] 200 100

2.

H2

5 AGT CAG CCA C 3

[12]

3.

H3

5 CGC GCC GG 3

[12]

FIG.2: RAPD of Aeromonas isolates with primer OPH-2. Lane 1 & 9: 100 bp DNA Ladder mix. Lanes 2-8: Test organisms.

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positivity of different characteristics [9, 2], the use of three tests for the identification of Aeromonas spp. with about 90% accuracy has been recommended. These tests include aesculin hydrolysis, VogesProskauer and gas from glucose. Strains which are aesculin negative, VP positive and gas from glucose positive can be identified as A. hydrophila, where as A. caviae are negative for gas from glucose and aesculin hydrolysis and do not ferment glucose but positive for VP. In contrast A. sobria ferments glucose and hydrolysis aesculin and maybe VP negative or positive. But in contrary to this, Martnez-Murcia et al., 2005 [11] reported that none of the biochemical tests evaluated in their present study were able to separate two species. The RAPD assay is an effective way of distinguishing isolates of various pathogenic anaerobic bacteria for epidemiological investigation and also for tying of isolates from different disease outbreaks [17]. In the present study, out of 20 random primers of OPH series, OPH-1, OPH-2 and OPH-3 were found to produce the most reproducible and scorable amplicon profiles. The amplicon sizes ranged from 250 bp to 2.9 Kb with a common >250 bp fragments in all the isolates (Fig. 1 and 2). Similar results were also observed by OhIci et al. 2000 [12]. They reported close genetic matrix similarity among the tropical strains using the OPH series primer (H1, H2 and H3). All the representative isolates in the present investigation revealed similarity upto 75% (Fig 3). This was in conformity with the similarity reported by other investigators [12-13]. Such differences may well be related to the source, frequency and type of Aeromonas isolates encountered in different geographical areas. The present study has established that when an appropriately chosen set of primers is employed, the RAPD analysis provides an alternative, rapid, reproducible and powerful genomic typing for the Aeromonas species. In conclusion, the RAPD revealed that amplicon sizes ranged from 250 bp to 2.9 Kb with a common >250 bp fragments in all the isolates and 75% similarity was observed in all the representative isolates. Presence of four clusters were observed comprising of C18 and FG16; FI9 and C3; FM4 and PM2.

FIG.3: Dendogram from the overlaid graph arising from RAPD primers OPH-1, OPH-2 and OPH-3 of selected representative isolates of Aeromonas isolates. (FM4-Fish meat; FI9-Fish intestine; C3 & C18-Chicken; FG16-Fish gills and PM2-Pork meat).

Discussion
Characteristic biochemical reactions revealed that all the A. hydrophila isolates hydrolyzed aesculin thereby producing brownish black colour of the medium and the A. sobria isolates exhibited negative results by not hydrolyzing aesculin. Furthermore, production of acid from Arabinose was observed both in A. hydrophila and A. sobria isolates but the A. caviae isolate exhibited negative acid production. Some investigators [3] identified Aeromonas upto the genomospecies level by the use of citrate and production of acid from which it enabled them to separate the members of A. hydrophila complex and further reported that these pathogenic genomospecies should be regarded as an important threat to public health. Since Aeromonas genus is a heterogeneous group, some differences have been observed. Since the identification of Aeromonas spp. often requires the use of non-conventional biochemical assays that are time consuming and often require long incubation period before the final results can be recorded, attempts have been made to find out the minimal identifying characteristics for the use in clinical laboratories. Based on observations recorded in the present investigation and that of reported

Acknowledgement
The first author is thankful to the university authorities for providing financial assistance in the form of scholarship.

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References
1. Agger WA (1986) Diarrhoea associated with Aeromonas hydrophila. Pediatr Infect Dis 5(Suppl.): 105-108. 2. Abbott LS, Cheung WKW, Bystrom-Kroske S, Malekzadeh T, Janda MJ (1992) Identification of Aeromonas strains to the genospecies level in the clinical laboratory. J Clin Microbiol 30(5): 1262-1266. 3. Borrell N, Figueras JM, Guarro J (1998) Phenotypic identification of Aeromonas genomospecies from clinical and environmental sources. Can J Microbiol 44(2):103-108. 4. Champsaur H, Andermont A, Matheiu D, Rottman E, Auzepy P (1982) Cholera-like illness due to Aeromonas sobria. J Infec Dis 145: 361-366. 5. Echeverria P, Blacklow NR, Sanford LB, Cukor GC (1981) Travellers diarrhoea among American peace corps volunteers in rural Thailand. J Infec Dis 143: 767-771. 6. Gracey M, Burke V, Robinson J (1982) Short reports- Aeromonas spp. in travellers diarrhoea. British Med J 289:658. 7. Janda JM, Abott SL (1996) Human pathogens. In: Austin B et al., eds. The genus Aeromonas. London, Wiley. pp. 151-173. 8. Janda JM, Guthertz LS, Kokka RP, Shimada T (1994) Aeromonas species in septicemia: laboratory characteristics and clinical observations. Clinical Infectious Diseases 19: 7783. 9. Joseph SW, Colwell RR, McDonell MT (1987) Taxonomy, ecology, isolation and identification: Aeromonas taxonomy. Experrientia 43: 349-550. 10. Komathi AG, Ananthan S, Alavandi SV (1998) Incidence and enteropathogenecity of Aeromonmas species associated with childhood gastroenteritis in Chennai (Madras), India. Jpn J Med Sci Biol 51: 1-12. 11. Murcia-Martinez JA, Soler L, Saavedra JM, Chacon RM, Guarro J, et al. (2005) Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from A. bestiarum. Int Microbiol 8: 259-269.

12. OhIci B, Oliver G, Powell R (2000) Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed field gel electrophoresis analysis. Dis Aquat Org 39: 109-119. 13. Redondo NP, Jarero RJ, Figueroa ALJ (2004) Antibiotic resistance and presence of plasmids in: Aeromonas hydrophila, Vibrio fluvialis and Vibro furnissi isolated from Carassius auratus auratus. Vet Mex 35(1): 1-10. 14. Stelma GN, Johnson CH, Spaulding P (1986) Evidence for the direct involvement of -hemolysin in Aeromonas hydrophila enteropathogenecity. Curr Microbiol 14: 71-77. 15. Vila J, Ruiz J, Gallardo F, Vargas M, Soler L, et al. (2003) Aeromonas spp. and travellers diarrhoea: Clinical features and antimicrobial resistance. Emerg Infec Dis 9(5): 552-555. 16. Wadstrom T, Ljungh A, Wretling B (1976) Enterotoxin, haemolysin and cytotoxic protein in Aeromonas hydrophila from human infections. Acta Pathol Microbiol Scand 8: 112-114. 17. Wang G, Tyler DK, Munro KC, Johnson MW (1996) Characterization of cytotoxic, haemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique haemolysin. J Clin Microbiol 34(12): 3203-3205. 18. Wang G, Clark GC, Liu C, Pucknell C, Munro KC, et al. (2003) Detection and characterization of the haemolysin genes in Aeromonas hydrophila and A. sobria by multiplex PCR. J Clin Microbiol 41(3): 1048-1054. 19. Yamada S, Matsushita S, Dejsirilert S, Kudoh Y (1997) Incidence and clinical symptoms of Aeromonas associated travellers diarrhea in Tokyo. Epidemiol Infect 119:121-126.

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Extended spectrum -lactamase in clinical isolates of Escherichia coli in a tertiary care centre.
1*Dr. Kaiser Ahmed, 2**Dr. Manzoor Thakur, 3**Dr. Bashir Fomda, 4**Dr. Gulnaz Bashir, 5**Dr. Peer Maroof, 6#Dr. SM Kadri

1* Demonstrator; Government Medical College ,Srinagar , Kashmir, India 2**Additional Professor, Sheri Kashmir Institute of Medical Sciences (SKIMS), Srinagar , Kashmir, India 3**Associate Professor, Sheri Kashmir Institute of Medical Sciences (SKIMS), Srinagar , Kashmir, India 4**Asistant Professor, Sheri Kashmir Institute of Medical Sciences (SKIMS), Srinagar , Kashmir, India 5**Senior Resident , Sheri Kashmir Institute of Medical Sciences (SKIMS), Srinagar , Kashmir, India 6#Faculty Member , Regional institute of Health and Family Welfare ,DHS, Srinagar , Kashmir, India GMC (Government medical college,srinagar), SKIMS (Sheri-kashmir institute of medical sciences). #Corresponding author: SM Kadri, PO Box 1143, Gpo, Srinagar , Kashmir, India. E-mail. kadrism@gmail.com

Abstract
Background: The Extended Spectrum -Lactamase (ESBL) producing organisms are increasing rapidly and becoming a major problem in the area of infectious disease. The present study was conducted to know the prevalence of ESBL producing Escherichia coli (E. coli ) isolated from different clinical specimens received in the Department of Microbiology at the Sheri-Kashmir institute of medical sciences, (SKIMS) and to observe the drug resistance pattern of these ESBL producing E. coli. Methods: Various isolates of E. coli were obtained from patients admitted or attending Out Pateint Department (OPD) over a period of 2 years from 1st August 2005 to 31st July 2007. In this study, 221 E. coli were subjected to screening by using cefotaxime, ceftazidime and ceftriaxone 30mg discs. Among them, 211 were positive for potential ESBL production which were further subjected to confirmatory tests by the following Phenotypic methods; double disc synergy test (DDST), phenotypic confirmatory disc diffusion test (PCDDT) and E-test. Findings: 55.9 % (118/211) of E.coli isolates were positive for ESBL production from different clinical specimen, highest number being from urine (72.9 %). The highest number of ESBL production were from inpatients (71.2 %) followed by outpatient (28.8 %).The resistance pattern of ESBL positive isolates showed higher resistance to 3rd and 4th generation cephalosporins (97.5 % to 99.2 %), quinolones (93.1 % to100 %) and aminoglycosides (65.2 %). They showed high degree of sensitivity to imipenem (98.3 %), nitrofurantoin (91.5 %), gatifloxacin (64.1 %) and amikacin (78.2 %). Conclusions: The high prevalence of ESBL production among E. coli was observed which should alert the physician as it is associated with indiscriminate use of 3rd and 4th generation cephalosporins. The presence of ESBL in outpatient is of main concern as it can be responsible for community acquired ESBL and can spread fast in our community. The multidrug resistance pattern of ESBL isolates was observed.

Introduction
Resistant bacteria are emerging worldwide as a threat to the favourable treatment outcome of common infections in community and hospital settings [1]. Among antibiotics ,- lactams are the most widely used agents accounting for over 50 % of all systemic antibiotics in use [2]. Mechanisms by which clinical isolate of Gram negative bacteria resist -Lactam antibiotics are through production of Lactamases, modification of cell wall and modification of target sites with reduced affinity for -Lactam antibiotics [3-4]. Among these, the production of -lactamase appears to be of primary concern and one of the most rapidly developing and clinically significant antimicrobial resistance mechanism [3-4]. The first plasmid mediated -lactamase in Gram negatives was reported in 1965 from an E. coli isolate from a patient in Athens, Greece, named Temnoniera (hence the designation TEM.). Another common plasmid mediated -lactamase found in Klebsiella pneumonia (K.pneumoniae.) and E. coli is SHV-1 (named after the sulfhydryl variableactive site) [5]. Newer -lactam antibiotics (extended spectrum eta-lactam) that would not be susceptible to these enzymes became widely used in the
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1980s [3]. The first report of plasmid-encoded -lactamase capable of hydrolysing the extended spectrum cephalosporins was published in 1983[5] which was first isolated in Germany. ESBLs spread rapidly to Europe, US and Asia and are now found all over the world [2]. Being plasmid mediated, they are easily transmitted among members of enterobacteriaceae thus facilitating the dissemination of resistance not only to -lactams but to other commonly used antibiotics such as quinolones and aminoglycosides [2]. E. coli is one of the most common isolate in our hospital settings and as -lactam antibiotics are mainstay of treatment, the increasing number of E .coli isolates exhibit ESBLs; and as such -lactam group of antibiotics will be almost ineffective in few years to come. E. coli is most common organism after Klebsiella to exhibit ESBLs. There is paucity of information about antimicrobial resistance especially on ESBLs from Kashmir. The present study was undertaken, keeping in view of the above facts, to know the prevalence and resistance patterns of clinical isolates of ESBL producing E. coli by employing DDST and PCDDT. In addition, E-test method of detection was performed on selected ESBL positive strain.
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Methods: Clinical isolates;


A total of 221 E. coli isolates from different clinical specimens during the period of August 2005 to July 2007, were screened for potential ESBL activity. These strains were isolated from different clinical specimens like urine, blood, sputum, pus and other body fluids which were received in the bacteriological division of microbiology. All the samples were processed and identified as per the standard bacteriological division of microbiological protocols and procedures [6]. E .coli ATCC 25922 was used as a negative ESBL control. Based on routine antibiotic disc sensitivity tests, isolates that exhibited resistance to any one of the third generation cephalosporins: ceftazidime, cefotaxime, ceftriaxone were shortlisted to test for the production of ESBL.

and E-Test. Double disk potentiation test showed eleven E.coli isolates positive for ESBL production. i.e.; 9.3% (11/118) as indicated in figure 1. PCDDT detected 99.2% (117/118) E. coli positive for ESBL production (figure 1). Ten isolates among 117 potential ESBL producers were also confirmed by DDST. Twenty selected isolates were confirmed by E-test method. All of them tested positive for ESBL production with MICs ranging from 42.66 g/ml to 320 g/ml (Figure 1).

Antibiotics;

The following antibiotic sensitivity discs were used for primary screening: ceftazidime 30 g, cefotaxime 30 g and ceftriaxone 30 g. In addition, Augmentin disk containing 20 g of amoxicillin plus 10 g of clavulanic acid and ceftazidime (30 g) + cavulanic acid (10 g) were used for confirmatory tests. E-test strips of ceftazidime and ceftazidime + clavulanic acid were used for selected ESBL isolates.

Screening for ESBLs by DDST: [7,9-14]

FIGURE 1: depicts % detection of ESBL enzymes in E. coli by confirmatory tests. The highest number of ESBL positive isolates were from inpatients (71.2 %) followed by (28.8 %) from outpatient (Table 1). The distribution of ESBL positive isolates among wards was as follows; 11.8 % from nephrology, 8.4 % from gastroenterology and general medicine each while the least number was isolated from haematology 1.7 % (Table 1). TABLE 1: Ward wise distribution of ESBL positive Escherichia coli isolates.
Ward Cardiology CVTS Neurosurgery Accident & Emergency General Surgery Gastroenterology Urology Nephrology General Medicine Endocrinology Plastic Surgery Neurology Neonatology Haematology Oncology * SICU ** OPD TOTAL ESBL Positive N 5 4 0 4 6 10 5 14 10 8 2 3 5 2 3 3 34 118 (%) (4.24) (3.39) (0.00) (3.39) (5.08) (8.47) (4.24) (11.86) (8.47) (6.78) (1.69) (2.54) (4.24) (1.69) (2.54) (2.54) (28.81) ESBL Negative N 2 1 1 5 3 6 7 9 9 4 9 3 5 2 1 1 25 93 (%) (2.20) (1.10) (1.10) (5.40) (3.20) (6.50) (7.50) (9.70) (9.70) (4.30) (9.70) (3.20) (5.40) (2.20) (1.10) (1.10) (26.90)

E. coli isolates that exhibited resistance to third generation cephalosporins were screened to detect ESBL producers. Cefotaxime 30 g disc was placed at a distance of 15 mm edge to edge from a centrally placed disc containing 20 g of amoxicillin + 10 g of clavulanic acid. Plates were incubated at 35C for 18-20 hours and the pattern of zones of inhibition was noted. Isolates that exhibited a distinct shape/size with potentiating towards amoxicillin + clavulanic disc were considered potential ESBL producers.

PCDDT: [7, 9-11]

ESBL production was confirmed among potential ESBL producing isolates by phenotypic tests. Third generation cephalosporins with and without clavulanic acid were used as follows;- ceftazidime (30 g) and ceftazidime (30 g) +clavulanic acid (10 g). Disk diffusion assay was carried out as per guidelines of Clinical Laboratory Standards Institute (CLSI), 2005 and difference in zone diameters between disk with and without clavulanic acid were recorded [29]. A difference of >5 mm between the zone diameters of ceftazidime and ceftazidime + clavulanic acid disk is taken to be phenotypic confirmation of ESBL production.

E-test of selected ESBL isolates. [7-8]

E-test of selected ESBL isolates was carried out. Ratio of ceftazidime MIC and ceftazidime + clavulanic acid MIC equal to or greater than 8 indicated the presence of ESBL.

Results:
Two hundred and eleven of 221 isolate (95.5 %) were positive for potential ESBL producers and these isolates were further subjected to confirmatory tests. One hundred and eighteen E. coli isolates tested positive for ESBL production by three cofirmatory tests ; DDST, PCDDT

*SICU (Surgical intensive care unit) ; ** OPD (Outpatient department).

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Urine (72.9 %) was the main source of ESBL producing isolates followed by pus (9.3 %) and then blood (7.6 %) (Table 2). TABLE 2: showing isolation of ESBL positive E.coli from various clinical Specimens.
Positive Specimen Urine Pus Pleural fluid Blood Bile Asitic fluid Endotracheal tip Sputum N 86 11 2 9 6 1 1 1 % 72.9 9.3 1.7 7.6 5.1 0.8 0.8 0.8 N 59 16 0 8 9 1 0 0 Negative % 63.4 17.2 0 8.6 9.7 1.1 0 0

Ciprofloxacin Ofloxacin Gatifloxacin Levofloxacin Moxifloxacin Nitrofurantoin Cotrimaxozole Imipenem Meropenem

Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant

8 108 4 102 41 23 0 2 0 10 54 5 29 65 58 1 8 1

6.9 93.1 3.8 96.2 64.1 35.9 0 100 0 100.0 91.5 8.5 30.9 69.1 98.3 1.7 88.9 11.1

9 75 5 76 41 19 2 1 2 2 15 13 16 51 3 17 7 0

10.7 89.3 6.2 93.8 68.3 31.7 66.7 33.3 50.0 50.0 53.6 46.4 23.9 76.1 15.0 85.0 100 0

*CSF; cerebrospinalfluid

The prevalence of ESBL producing E.coli among potential producers of ESBL was 55.9 % (118/211) which was confirmed by DDST, PCDDT and E-test. Third generation cephalosporins showed 97.5 % to 99.2 % resistance while cephalosporin + clavulanic acid combination reported only 20 % resistance in ESBL producers (Table3). Fourth generation cephalosporin, cefpime showed 100% resistance. In quinolones; levofloxacin and moxifloxacin reported 100 % resistance followed by ofloxacin and ciprofloxacin 96.2 % and 93.1 % respectively. In aminoglycosides, gentamicin showed 65.2 % resistance. Cotrimaxozole resistance was seen in 69.1 % isolates. Imipenem, nitrofurantoin, meropenem, gatifloxacin and amikacin showed 98.3 %, 91.5 %, 88.9 %, 64.1 % and 78.2 % sensitivity respectively (Table 3). In Non-ESBL producers 100 % resistance seen among third and fourth generation cephalosporins while in quinolones; ciprofloxacin had 89.3 % and ofloxacin 93.8 % resistance. In aminoglycosides, gentamicin had 78.5 % resistance. Cotrimaxozole had 76.1 % resistance (Table 3). TABLE 3 : Antibiotic susceptibility of ESBL Producers and Non-producers of E. coli
ESBL A ntibiogram N *Ce_Cephalosporins **Ca_Cephalosporins ***Ci_Cephalosporins #C+S_Cephalosporins Cefpime Amikacin Gentamicin Sensitive Resistant Sensitive Resistant Sensitive Resistant Sensitive Resistant Resistant Sensitive Resistant Sensitive Resistant 1 117 1 117 3 115 76 19 14 43 12 31 58 Positive % 0.8 99.2 0.8 99.2 2.5 97.5 80.0 20.0 100 78.2 21.8 34.8 65.2 N 0 93 0 93 0 93 38 31 15 32 22 14 51 Negative % 0 100 0 100 0 100 55.1 44.9 100.0 59.3 40.7 21.5 78.5

*cefotaxime, **ceftazidime, ***ceftriaxone, #cefotaxime plus sulbactum.

Discussion:
ESBLs are clinically important because they destroy cephalosporins, given as first line agents to many severely ill patients. Delayed recognition and inappropriate treatment of severe infections caused by ESBL producers with cephalosporins has been associated with increased mortality [8,30]. In this study, 95.5 % of the E. coli isolates were positive using cefotaxime, ceftazidime and ceftriaxone 30 g each discs as initial screening agent which were further subjected to confirmatory tests .The high percentage of resistance was seen to 3rd and 4th generation cephalosporins; ceftazidime (99.2 %), cefotaxime (99.2 %), ceftriaxone (97.5 %) and cefpime (100 %) and these findings matched with various studies conducted in India and elsewhere [3,18-19]. The 9.3 % ESBL positive strains confirmed by DDST is very much low when compared to other study [3]. The reasons for discordance are various factors like precise placement of discs, correct storage of the clavulanic discs and performance of appropriate control tests are critical to the sensitivity of DDST. Double disk test can lack sensitivity because of the problems of optimal discs spacing [3,9,11] but our study result was comparable with a study conducted in a teritiary care at Medical College of Virginia [14]. PCDDT result obtained is similar to what has been found in other studies [11,20]. E-test confirmation test method detected ESBL production in twenty selected ESBL positive isolates confirmed by other confirmatory tests. Their ESBL status and MIC was determined and comparable with other studies done [21-22]. The highest number of ESBL positive isolates were from inpatients followed by outpatients which matched with a study reporting ESBL producing bacteria 87 % from inpatients and 12.7 % from outpatients [23].This high percentage of ESBLs from outpatient should alert the physician. Urine was the main source of ESBLs production in all the specimen followed by pus and blood which was almost similar to study by Bithikia et al [3]and Rafay et al [18]. Much higher (58 %) prevalence of ESBL producers in urinary isolates of gram negative bacilli was observed in India by Mathur et al [24].The out-patient presence of ESBL is
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of main concern as it is now come to the alert of the physician that ESBL is spreading fast in the community and responsible for communityacquired ESBLs and the highest number being from urine specimens [1,10-11]. The prevalence of ESBL positive E. coli of 55.9 %(118/211) was confirmed by DDST, PCDDT and E-test .The above prevalence of ESBL positive isolates is in concordance with various studies and vary among different geographical areas, countries and institutions [3,19,24,15]. Amit-Jain et al [19] detected marked geographic variation. Incidence in India was 47.5 % and other countries 36 % to 55 %. A study from Northern India [19] in 2000 by Amit Jain reported an incidence of 58.06 % for ESBL producing E. coli which is almost equal to our current study conducted. The frequency of ESBL-producing organisms differ significantly in accordance with geographic location. Although frequency of ESBL producing E.coli in Europe, North Latin America and Western Pacific is reported at 1-8 %, its prevalence in the Asia Pacific region and South Africa is reported at more than 20 % [25]. The lower prevalence of ESBL in western countries compared to others can be explained by implementation of infection control measures, restricted and judicious use of oxyimino-cephalosporins, implementation of appropriate ESBL detection methods recommended by CLSI, importance of hand hygiene reinforcement, recommended isolation precautions for patients colonized or infected with ESBL producers, continuous education programs and research studies which all are known to contribute to decreasing the spread of ESBL producing organisms. The antibiogram of both -lactam and non--lactam group of antibiotics showed multidrug resistance pattern (Table3). A study by Tankhiwale [26] reported 82 % resistance to cotrimaxozole. Nitrofurantoin constituted a reasonable option for treatment (62.5 % sensitivity). Amikacin was drug of choice as 85.7 %isolates were sensitive to this drug. Both these drugs showed similar results in our study. Cephalosporin plus sulbactam showed 20 % resistance which was higher than reported in a study [16].A study by Villanvena [9] reported multidrug resistance pattern similar to our study but reported 100 %

sensitivity to imipenem and meropenem in contrast to our study showing 11.1 % resistance in meropenem and 1.7 % resistance to imipenem as such these two drugs can no longer be considered a drug of choice for ESBL producing E.coli and further these finding corroborated with other studies [16,18,23-24,26]. ESBL producing organisms hydrolyse -lactam antibiotics, so antibiotic choice for infections with such organism is seriously reduced. Further, the plasmids bearing the genes encoding ESBLs frequently also carry genes encoding resistance to aminoglycosi- des and trimethoprim/ sulfamethoxazole [27]. In the current study multidrug resistance is observed to 3rd and 4th generation cephalosporins, quinolones and aminoglycosides. ESBLmediated resistance is not always obvious in vitro to all cephalosporins. Many ESBL producers are multiresistant to non--lactam antibiotics such as quinolones and aminoglycosides, thereby narrowing treatment options. Some producers achieve outbreak status spreading among patients and locals, perhaps owing to particular pathogenicity traits [30]. Once an ESBLproducing strain is detected, the laboratory should report it as resistant to all pencillins, cephalosporins and aztreonam, even if they test as susceptible. Other anti-microbial agents can be reported as they are tested [28]. The high prevalence of ESBL in E.coli exists in our institute can be traced to the indiscriminate use of 3rd and 4th generation cephalosporins. The presence of ESBL in outpatient is of main concern as it can be responsible for spreading ESBL from hospital to general population. Therfore, judicious use of 3rd and 4th generation cephalosporins will be effective means of controlling and decreasing the spread of ESBL.

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References
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Investigations of Recurrent outbreaks of unknown fever, establish rural dengue activity in West Midnapore, a costal district in West Bengal, India.
A.Sarkar1, D.Taraphdar1 and S.Chatterjee2*
1 Research Fellow , ICMR virus unit, Kolkata 2 Assistant Research Officer, ICMR virus unit, Kolkata Running Head: Studies of Dengue virus activity in West Bengal, India. *Corresponding Author: Dr. Shyamalendu Chatterjee, ICMR virus unit, GB- 4, 1st Floor ID & BG Hospital, 57 Dr. S. C. Banerjee Road, Beliaghata, Kolkata-700010. INDIA Phone: (91-33) 2353-7425 Fax: (91-33) 2353-7424 Email: shyamalenduchatterjee@gmail.com Mob. No. +919433347772

Abstract
Title: Investigations of Recurrent outbreaks of unknown fever, establish rural dengue activity in West Midnapore, a costal district in West Bengal, India. Background: In the year 2002, an investigation was conducted for the diagnosis of an unknown fever outbreak in the District of West Midnapore, West Bengal; on request of the Director of Medical Education, Govt. of West Bengal. In the year 2004, again unknown fever outbreak took place in some other area of same district and that too was investigated on request of the State Medical Department. The recurrence of same episode in the year 2007 was investigated by the local medical team and the collected serum samples were sent to us for laboratory based conformation. Methods and findings: During the first episode (2002), a total of 1158 fever cases were reported with 7 deaths, which spread all over the district since 2nd week of May till 15th July, 2002. In the second episode (2004), 792 cases were reported and 2 deaths were recorded. A total of 781 acute sera samples could be collected during these three consecutive out breaks. The samples were collected from the patients with clinical features of fever with head ache, body ache, nausea, retro orbital pain, abdominal pain and rashes of duration 2-7 days, which is closely related with the symptoms of dengue infection. Only 195 convalescent sera were made available. Acute samples were tested for the presence of dengue specific IgM antibodies by ELISA method. There was a significantly higher incidence of fever cases in children belonging to the age group up to 10 years. No virus could be isolated from the acute sera collected from fever cases. The results of serological survey showed the presence of IgM antibodies to Dengue virus in only in 446 (57%) of the acute cases. Amongst the195 convalescent sera, four fold rise of HAI Antibody titre to Dengue virus was observed only in 77 (39%). Conclusion: Analysis of the epidemiological and serological findings of different years revealed that the out breaks were due to Dengue infection. Children up to 10 years of age in this district were mostly affected during these out breaks. Key words: Midnapore, West Bengal, Dengue Virus

Introduction
Dengue viruses are the members of the genus Flavivirus of the family Flaviviridae, consists four antigenically distinct Sero types, which do not offer cross protection. Dengue is an important mosquito borne disease in the world in terms of morbidity, mortality and economic cost [1], especially in the tropics, with more than 2/5th of the world population living in areas at risk for dengue [2-4]. Infection with any one of the four types leads to a mild, self limiting febrile illness (dengue fever, DF) A more severe form of the disease, dengue haemorrhagic fever (DHF)/ dengue shock syndrome (DSS), is responsible for high mortality rate, especially in children [5]. Dengue has now become a regular occurrence worldwide including America, Africa, Asia and the South Pacific, where the vector of the disease is wide spread. The incidence of DF is on the increase and spreading to geographic regions not previously affected. Movement of Dengue between different geographic areas
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and also from urban to rural is an important element in the epidemiology of the disease. From being a sporadic illness, epidemics of dengue have now become a regular occurrence all over the world. In India Dengue was first isolated in 1946 and many epidemics have since been reported [6-9]. The clinical features of dengue virus infection range from nonapparent infection through dengue fever (DF) and the more severe dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) [10-12]. DHF/DSS, although common in South East Asia, has also been reported in India during Dengue epidemics. DHF was first reported in Calcutta, West Bengal, in 1963 [13] again in 1964 [14] and subsequently in different states of India [15-22]. Laboratory diagnosis of a recent dengue virus infection may be done by detection of the virus in patients blood, either by virus isolation in
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susceptible cell cultures [3] or by detection of the viral RNA by reverse transcriptase-polymerase (RT-PCR) chain reaction based techniques [23-24]. These processes are very specific and should be performed within 48 hrs following the onset of illness in a well equipped laboratory, as the virus disappears after that period. However, detection of IgM antibodies by ELISA method and detection of rise of antibody titres with the convalescent samples by Haemagglutination inhibition test are well accepted serological methods for the diagnosis of dengue infection. This paper reports the detailed investigation of repeated outbreak of unknown fever in the year 2002, 2004 and 2007; in the district West Midnapore and establishes dengue activity in rural areas on the basis of laboratory investigations.

After a fort night, Local health workers could collect only 74 paired blood samples at the convalescent stage from the two affected villages. These samples were sent to the ICMR virus unit, maintaining the cold chain. Clinical manifestation All the patients had the clinical features of continued fever, headache, pain all over the body, anorexia, epigastric pain, nausea and/or vomiting, extreme weakness, bleeding manifestations like, epistaxis, haematemesis, haematuria etc. only in one case, paetichial rash was observed. Regarding seven death cases, one case died of paralytic illeus, three cases died of haematemesis, two cases died of haematuria and one case was brought dead to the hospital with a history of rapid up rise of fever followed by sudden collapse. Investigation in the year 2004 This year, village Kesia, and Laxmanpur, of Garbeta block were affected. These two villages are about 90 km from the District Headquarter and adjacent to Salboni of West Midnapore. During investigation, it was revealed that the total populations of these two villages are 4059. Date of first case report was 16th May 2004, total number of fever cases up to 3rd June were 792. All the age groups were more or less affected. During this visit in the year 2004,139 and 115 samples were collected from Kesia and Laxmanpur respectively, in the manner adopted in the year 2002. The two patients who died in these villages had suffered from high fever with headache and cough associated with severe respiratory distress. During the time of investigation the outbreak was in declining state. There was no evidence of sex differentiation among cases. The distribution of clinical presentation among the cases and the age wise distribution of fever cases were collected from the health centre. Clinical manifestations All the cases had continued high fever, rashes all over the body with cough and mild respiratory symptoms. Majority of them had headache, nausea / vomiting, malaise and drowsiness with duration of 3 days to 5 days. Joint pain and backache were the common features in all the cases. A total of 63 convalescent paired sera were collected after 15 days by the local health authority with the help of medical Technicians and health workers. Sample collection in the year 2007 This year, again an outbreak of unknown fever took place in a mild form in all over the district. The clinical manifestation was almost same with the previous years. No death was recorded in this year. The health authority of the district West Midnapore took the initiative for collection of blood samples. A total of 239 blood samples from all over the district were collected by the doctors of Sadar Hospitals, block hospitals and the primary health centers. These samples were sent to us for necessary diagnosis.

Methods
Location and Meteorological information Midnapore was the biggest district in the state of West Bengal, having a sea port from the ancient time, with historical back grounds. Presently the district has been divided into East and West Midnapore. The district West Midnapore, our study area, is situated at an altitude of 23 meters above sea level on the bank of Bay of Bengal with an average rain fall of 1656 mm, mostly between the months of May and September. In the summer the temperature ranges from 42C to 50C with high humidity.

Sample collections and investigations


Investigation in the year 2002 Cases started occurring initially in the Guiadaha village of Gorbeta block followed by Salboni block. These two villages were worst affected and are situated 30-40 km from the district head quarter, surrounded by paddy fields and agricultural lands. Source of drinking water was tube wells. Village Guiadaha has a total population of 1541.It was a typical rural village with kachha houses and kachha road. The village has reported 311 cases since its onset, at the local primary health centre. During investigation, it was revealed that several people suffered from two episodes of fever, headache, nausea, abdominal pain and retro orbital pain at an interval of 4 days to 6 days. In most of the houses visited, stagnant and stored water were observed for regular uses, which facilitated the breeding of the mosquitoes. Mosquitoes were abundant at the village and day biting mosquitoes were also present. From 1st to 17th July, the numbers of daily attendants of fever cases in the OPD of the hospital were collected. During this visit a total of 288 blood samples were collected from the acute cases from two villages. Of which we could collect 63 blood samples from the OPD and from the indoor patients of the hospitals and rest of the 110 blood samples were collected by the local health workers and the medical technicians of the health centre on door to door visit. The Salboni village has a total population of 5819. It is semi urban in settings. This village has reported 540 cases up to 14th July, 2002. In both the villages there were many cases, who suffered but did not report to any health facility. Only 115 blood samples could be collected from acute cases from the affected village.

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A total of 58 convalescent paired sera were collected by the local health authority with the help of medical Technicians and the health workers. All the sera collected during different years, were transported on wet ice to the ICMR virus unit, Calcutta, where they were stored at -800C until tested. The possibilities of bacterial and prokaryotic etiology in the collected sample were excluded through investigations at the local hospitals. All the sera were tested within 1 month to 2 months from the date of collection.

Serology The acute samples were tested for the presence of Dengue IgM antibody by IgM capture ELISA (MAC-ELISA) method, using a kit (Prepared by National Institute of virology, Pune, India), following the prescribed protocol. O.D was measured at 450 nm using an ELISA reader (Titertek Multiskan Plus, Lab systems Finland, Type- 314). Haemagglutination inhibition test A total of 195 convalescent samples could be collected from the affected areas and were tested along with the respective acute sera for any rise of HAI antibody in the convalescent state. The HAI test was carried out according to the method of Clarke and Casals [19]. For confirmation of the etiologic agent, the convalescent sera along with the acute samples were subjected to HAI test against Dengue and JE antigen. Samples were considered positive, if they had a fourfold rise of titer in the convalescent paired sera in comparison with the corresponding acute samples.

Laboratory investigation
Virus isolation Attempts were made to isolate the virus from the acute samples, if any, by using C6/36 cell lines. Two hundred micro liters of selected samples (2 days fever cases with dengue like symptoms) were spread over the monolayer of C6/36 cell line and allowed to adsorb for 120 minutes in an incubator at 28C under 5% CO2 concentration. After adsorption, the excess sample materials were discarded and minimum essential media (MEM; GIBCO BRL, US) supplemented with 2% fetal bovine serum (FBS; GIBCO BRL, US) and Penicillin Streptomycin antibiotics (PenStrep; Gibco) were added in 24 well tissue culture plate (Tarsons) and were incubated again in the same condition as before. It was observed regularly for the appearance of cytopathic effect (CPE) up to 7-8 days.

Results
Sample collections and investigations The total number of fever cases attended the OPD of Guiadaha hospital from 1st July to 17th July has been presented (Fig 1). There was a steady increase in number of patients. On 15th July, 2002; highest no. of Patients attended the OPD.

FIG 1.

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Virus isolation CPE was not developed and No virus could be isolated from any of the acute sera collected from fever cases. Serology Out of 781 acute blood samples, collected during these three years, Dengue Virus specific IgM antibodies were observed only in 446 (57.10%) samples. Year wise collection of acute samples and detection of Dengue IgM antibody in them, are presented (table1). TABLE 1:
Year wise distribution of number of samples collected, tested and Dengue IgM positive cases in the district of West Midnapore: Year 2002 2004 2007 TOTAL Total number of samples collected and tested 288 254 239 781 Total number of IgM positive to Dengue 142 156 148 446

In all the three years, the maximum IgM positive cases were observed in the age group of 0-10 (28.69%), as compared to the other age groups (Fig 2).

Among the 781 patients samples tested, 453 were male and 328 were female. Out of 453 male, 217 (47.90%) were positive. On the other hand, 185 (56.40%) of the 328 samples collected from the female cases, were positive to Dengue IgM antibody. The sex wise distribution of suspected cases of Dengue and Dengue IgM positive cases have been presented (table 2). TABLE 2:
Year & Sex wise distribution of Dengue positive cases in the district of West Midnapore, during the study period: Year 2002 2004 2007 Male No. tested 167 147 139 No. positive 78(47%) 58(40%) 81(58%) No. tested 121 107 100 Female No. positive 64(53%) 54(51%) 67(67%)

FIG 2. A total of 77 samples out of 195 convalescent sera revealed fourfold rise of antibody titre, in comparison with the acute sera, against Dengue antigen. Apart from that, only 27 sera had very low antibody titre against JE, both in acute and the convalescent stage. Rest of the convalescent samples did not produce any reaction either against dengue or against JE antigen. The results of the HAI test against Dengue and JE antigen have been given (table 4). TABLE 4:
Result of serological investigations of the paired sera to Flavivirus infection collected during the study period: Paired patient sera Year No. tested 74 63 58 Rise of antibody to JE 9 11 7 Den 22(30%) 29(46%) 26(45%)

There was a decrease in the number of the samples collected, during the three outbreaks, as compared to the year 2002. The age wise distribution of Dengue suspected cases and Dengue IgM positive cases have been presented (table 3). TABLE 3:
Age wise distribution of Dengue positive cases in the district of West Midnapore, during the study period: Year 0-10 2002 2004 2007 TOTAL 41 44 44 11-20 36 40 39 Age group (in years) 21-30 29 30 30 31-40 15 18 16 41-50 14 15 13 42(9.41%) 51+ 7 8 7 22(4.93%)

2002 2004 2007

Discussion
The epidemiology of Dengue is of continuing health importance, as the incidence of DF and DHS/DSS is increasing worldwide and is appearing in areas where it was previously unreported. In India, Den-1 and Den-4 was first reported in 1964 [25-26] and Den-3 in 1968 [27]. DHF was first reported in Calcutta, West Bengal in 1963 and in 1964 [13-14] Ever since, several reports of DF and DHF outbreaks have come from different cities in India and these include reports from Ludhiana [28], Lucknow [29], Chennai [30], Mangalore [31], Assam/ Nagaland [32]

129(28.92%) 115(25.78%) 89(19.95%) 49(10.98%)

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and Vellore [33]. In Delhi, although frequent outbreaks of DF have been occurring since 1967, major outbreaks of DHF and DSS have been reported in the year 1999 and 2001 [34-35]. All these outbreaks of DF and DHF/DSS have been recorded from the cities and the urban areas. No such outbreak has yet been recorded from the rural area like the district of West Midnapore, West Bengal. In the year 2002, all most one fifth of the total populations of the two villages were affected. Out of seven deaths, five cases had the symptoms of dengue haemorrhagic fever and one had the history of shock syndrome. More over the history of two episodes of fever at an interval of 4 days to 6 days in the same locality indicates the possible circulations of two types of Dengue viruses. This has in turn facilitated the haemorrhagic manifestations. The detection of IgM antibody to dengue virus by ELISA test, in 446 samples out of 781 cases, amply proves that the febrile illness was due to the dengue virus infection in the recent past (Table-1). According to the sex wise distribution of the suspected cases (Table-2), there was a significant higher incidence of dengue IgM Sero positivity in females and not in males. This may be explained by the fact that the female individuals usually resides at home at the day time and get exposure to the mosquitoes (Aedes aegypti), as it is a domestic and peridomestic in nature. A significant higher incidence of dengue IgM Sero positivity were observed in the age group of 0-10 years followed by 11-20 years(Table 3),(Fig 2), which is similar to the observations made by many workers at different times and from different parts of India. The results obtained in the convalescent sera by HAI test, revealed 4 fold rise of antibody titre in 77 samples, as compared with the acute sera. Only 27 convalescent sera produced group B flavivirus reaction and had a very low titre of antibody to JE in them, indicate their exposure to JE elsewhere in the remote past (Table 4). The activity of JE in this district has already been established [36]. So on the basis of the house hold condition, epidemiological information, detection of IgM antibody to Dengue virus in the acute sera and rise of dengue antibody titre in the convalescent sera confirms the etiologic agent of the febrile illness was dengue virus. It is worthy to mention that the selected acute sere/samples did not produce CPE in the tissue culture system and no virus could be isolated due to the fact that, the initiation of antibody (IgM) response mounted the activity of the virus during the course of infection at the late stage. On the other hand, samples might have lost the viability of the Dengue virus during transport and repeated freeze thawing conditions, as this may led to lose of viral titre or RNA, making it difficult to isolate the virus from the selected samples.

This scenario on the outbreak of dengue in three years at intervals, in the district of West Midnapore, happens to be the same as it has taken places in different cities in India. More over many of the ecological conditions in this area do not differ substantially from those where the disease has been prevalent. However, Dengue infections have been recorded mainly from the urban areas of the countries, all over the world. Due to the rapid and increased urbanization, Dengue has spread all over the world only in the urban area. But the reports of DF and DHF from the rural area either absent or scanty. This is an important observation that DF and DHF have first emerged in the rural area as the major public health problem in the district of West Midnapore, West Bengal, India; that needs to be taken notice of. Hence, it constitutes a new report from a rural and costal district like West Midnapore, West Bengal; which needs continuous surveillance and molecular studies on the circulating serotypes and their genotypes for addressing the probabilities of DSS/DHF incidence in future.

Acknowledgements
The author expresses his sincere gratitude to all the staff members of ICMR Virus Unit, for their constant help and assistance to carry out the laboratory investigations and compilation of data. I also gratefully acknowledge the help I received from N.I.V, Pune for providing me the Elisa kit and antigen to carry out the work smoothly. The enthusiastic repeated help obtained from the staff members of the office of Chief Medical Officer of Health, Medical Officers and Medical Technologists of the District Hospital, Sub divisional hospital and the primary health center at different places in the District West Midnapore during the investigations, is gratefully acknowledged.

Fundings:
We received all sorts of financial help from the Indian Council of Medical Research, New Delhi, India to carry out the work in the ICMR VIRUS UNIT, KOLKATA, INDIA.

Competing interests:
the first two authors are Research Fellows at the ICMR Virus Unit and the communicating author is an Assistant Research Officer of ICMR Virus Unit. The authors have no financial and competing interests that are affected by the material in the manuscript.

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References
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20. Broor S, et al (1997). Recent Dengue epidemic in Delhi, India. In: Saluzzo JF, Dodet B, eds. Factors in the emergence of arbovirus diseases. Amsterdam: Elsevier. pp. 123-127. 21. Padbidi VS, Mahadev PVM, Thakare JP (1996) Virological & entomological investigation of an outbreak of dengue fever in Dhuk district, Maharashtra. Indian J. Med Microbiol. 14: 25-32. 22. Ram S, Khurana S, Kausshal V (1998) Incidence of dengue fever in relation to climatic factors in Ludhiana, Punjab. Indian J. Med. Res. 108: 128-133. 23. Lanciotti RS, et al (1992) Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase polymerase chain reaction. J. clin. Microbiol 30: 545-551. 24. Henchel EA, et al (1991) Sensitivity and specificity of a universal primer set for the rapid diagnosis of dengue virus infections by polymerase chain reaction and nucleic acid hybridization. Am J Trop Med Hyg 45: 418-428. 25. Carey DE, Myers RM, Reuben R (1964) Dengue types 1 and 4 viruses in wild- caught mosquitoes in south India. Science 143:131-132. 26. Myers RM, et al (1964) The isolation of type 4 virus from human sera in south India. Indian J Med. Res 52 : 559-565. 27. Myers RM, et al (1970) Dengue outbreak in Vellore, southern India, in 1968, with isolation of four dengue types from man and mosquitoes. Indian J Med. R 58: 24-30. 28. Kaur H, et al (1997) Dengue Haemorrhagic fever outbreak in October-November 1996 in Ludhiana, Punjab, India. Indian J Med. R 106:1-3. 29. Agarwal R, et al (1999) A clinical study of the patients with dengue Haemorrhagic fever during the epidemic of 1996 at Lucknow, India. South East Asian J. Trop. Med. Public Health 30: 735-740. 30. Kabilan L, et al (2003) Dengue disease spectrum among infants in the 2001 dengue epidemic in Chennai, Tamil Nadu, India. J. Clin. Microbiol 41: 3919-3921. 31. Padbidri VS, et al (1995) The 1993 epidemic of dengue fever in Mangalore, Karnataka state, India. South east Asian J. Trop. Med. Public Health 26: 699-704. 32. Barua HC, Mahanta J (1996) Serological evidence of DEN-2 activity in Assam and Nagaland. J.Comm. Dis. 28: 56-58. 33. Cherian T, et al (1994) An epidemic of dengue haemorrhagic fever & dengue shock syndrome in and around Vellore. Indian J Med Res 100: 51-56. 34. Dar L, et al (1999) The first major outbreak of dengue haemorrhagic fever in Delhi, India. 40: 418-427. 35. Kurukumbi M, et al (2001) Sero epidemiological and active surveillance of dengue fever/ dengue haemorrhagic fever in Delhi. Indian J Med Sci 55: 149-156. 36. Chatterjee S, et al (2004) Sero surveillance for Japanese encephalitis in children in several districts of West Bengal, India. Acta Paediatr 93: 390-393.

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Helicobacter pylori infection: efficacy of probiotics and role of genome wide association studies
Baljinder Kaur*, Praveen P. Balgir, Balvir Kumar and Neena Garg
Department of Biotechnology, Punjabi University, Patiala-147 002, Punjab, India. *Corresponding author: Email: baljinderbt@hotmail.com; Tel.: +91 175 3046263; Fax: +91 175 3046262

Abstract
Helicobacter pylori is a Gram-negative human gastric pathogen which has a possible etiologic role in peptic and gastric ulcers, gastric adenocarcinoma and more rarely, lymphoma of the mucosa-associated lymphoid tissue. The pathogenicity of H. pylori is intimately associated with the expression of virulence factors such as urease, cagA, iceA, vacA and bacterial adhesion and maintenance factors. The cagA, iceA and vacA virulence factors of H. pylori show great deal of genetic diversity with respect to their ancestor strain, host and environment factors that collectively influence the pattern and severity of the disease. Antimicrobials (triple-therapy regimen) noted to be effective in empirical management of pathogen while emergence of drug resistance remains a major obstacle in eradication of this gastro-duodenal pathogen. In this regard probiotics have great potential of treating H. pylori infection because they exhibit antagonistic activities against many pathogens including H. pylori. Focus of this review is to highlight importance of probiotic micro-organism in improving host defense mechanism. Review also highlights the need of further investigation involving genome wide association studies to unravel the interactions between host, probiotics and bacterial pathogen that would determine fate of pathogen in a complex environment of gastro-duodenal region. Key words: Helicobacter pylori, Peptic ulcers, Probiotics, Bacteriocins, GWAS

Introduction to gastric pathogen Helicobacter pylori


Helicobacter pylori is of major concern today because of its causal relationship with gastroduodenal diseases. In 1984, this Gram-negative, spiral-shaped, microaerophilic bacterium that colonizes the mucosal layer of the gastric epithelium was isolated [1]. H. pylori bacteria was originally called Campylobacter pyloridis, renamed as Campylobacter pylori and then later reclassified to Helicobacter pylori because its morphological, structural and genetic characteristics has indicated that it should be placed in a new genus [2]. The bacteria are prevalent worldwide and more than half of the world population is infected with H. pylori [3-4]. H. pylori infection is common in the Indian subcontinent. Chances of exposure are widespread and infection occurs early in life under the age of five. About 79-83% of the population is exposed to H. pylori during the first two decades of life [5-6]. Serological surveys indicate a sero-prevalence of 22-57% in children under the age of five, increasing to 80-90% by the age of 20 and remaining constant thereafter [7-8]. Gender preference was not seen in case of H. pylori infection. Data of both developing and developed countries depicts direct relation of disease prevalence with age. High age-specific prevalence of H. pylori infection in developing countries reflects the lower socioeconomic level of those areas. Infection with H. pylori is highly associated with chronic active gastritis, peptic ulcers, gastric adeno-carcinoma and more rarely, lymphoma of the mucosa-associated lymphoid tissue (MALT) [9-10]. In 1994, the International Agency for Cancer Research, an arm of the World Health Organization, classified H. pylori as a potential human carcinogen [11].
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Pathogenicity of H. pylori
H. pylorihas a very strong affinity for epithelial lining of stomach and duodenum, where it attaches and subsequently disrupts microvilli and tight junctions between adjacent cells. Eroded epithelial lining allows acid and bacteria to get through it and establishes pathogen in the mucous layer. Almost all H. pylori strains produce urease that decomposes urea and forms ammonia, which is harmful to gastric mucosa [12]. Moreover, low acidity is beneficial for organisms to colonize the stomach. H. pylori urease is an important virulence factor that consists of four subunits, A, B, C and D. Subunit B, a peptide with 569 amino acids and encoded by the ureB gene, shows the strongest antigenicity and protection among all H. pylori proteins [13]. Interestingly, the fragment E of ureB (ureBE) expressed in E. coli is also able to elicit effective immune response in mice [14]. H. pylori also secretes proteases and phospholipases that damages mucosal lining. Lipopolysaccharide of the pathogen origin attracts inflammmatory cells to the mucosa. Neutrophils of the host release myeloperoxide in response to pathogen attack. A bacterial platelet-activating factor promotes thrombotic occlusion of surface capillaries. Taken together, these changes damage the protective mucosal layer in gastro-duodenal region. Exposed epithelial cells are highly prone to damaging effect of acid-peptic digestion and ultimately, inflammation of the gastric mucosa occurs which may lead to peptic ulceration [12]. A number of studies conducted in different populations of the World indicated the involvement of cagA, iceA, vacA, babA-blood group antigen binding adhesion factor [15-21], hp0169 and comB4 [22] virulence
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factors in establishing gastroduodenal H. pylori infection (Table 1). The pathogenicity of the organisms is also attributed to maintenance factors such as motility, and adaptive enzymes [23]. H. pylori attaches to inner surface of the gastric epithelium and occasionally inside epithelial cells [24]. It produces adhesins which bind to membrane-associated lipids and carbohydrates that help in adherence to epithelial cells (Figure 1 and 2). For example, the adhesin babA binds to the Lewis b antigen displayed on the surface of stomach epithelial cells [25]. H. pylori strains possess a gene called cagA, which is a marker for the cag region, a pathogenicity island of about 35 kb. The cag pathogenicity island (PAI) has about 30 genes, part of which code for a complex type

IV secretion system (Figure 3) that facilitate translocation of bacterial factors such as urease, lipopolysaccharides (LPS), broken fragments of peptidoglycan and porins through epithelial layer and subsequent activation of macrophages of gastric mucosa. Stimulated macrophages secrete IL-1, IL-8, IL-12, TNF- and induce expression of INF- by TH1 cells. IL-8 and INF- disrupt epithelial barrier function. Antigen presenting cells (macrophages) with exposed bacterial antigens attached to class II MHCs induce clonal expansion in B-cells for production of anti H. pylori antibodies and activate Tc cells through TH2 derived IL-2 that ultimately lead to destruction of infected cells. Apoptotic pathway was activated when macrophage derived immune effector molecules viz. TNF- and IL-1 stimulated fas expression in sensitized cells. The

TABLE 1. Virulence and maintenance factors of Helicobacter pylori. Factors vacA cagA babA iceA oipA (hp0638) picB Urease hp0169 comB4 rocF MUC5AC Function Virulence Factors Encodes a protein cytotoxin that induces vacuolation in eukaryotic cells Stimulates the production of interleukin-8; a part of it also code for type IV secretion system. Binds to Lewis b antigen displayed on the surface of stomach epithelial cell Up-regulated upon contact of H. pylori with the gastric epithelium Induces IL-8 secretion by epithelial cell induces IL-8 expression in gastric epithelial cells Neutralizes acid Adhesion and Maintenance Factors Essential for H. pylori stomach colonization as it encodes for a collagenase Essential for colonization, as it encodes a putative ATPase which is a part of DNA transformation associated type-IV transport system Encodes arginase that facilitates production of ammonia and favours NO production in stimulated macrophages Primary receptor for H. pylori in human stomach 22 22 35 151 References 11, 15-21 15-21, 26-29, 34 15-21, 25 15-21 200 36 12, 13

FIGURE 1. Pathological changes in gastric epithelium upon H. pylori infection.


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FIGURE 2. Constitution of type IV secretory apparatus and pathophysiology of H. pylori infection.

FIGURE 3. Genetic organization of cag pathogenicity island (PAI).

low GC content of the cag PAI relative to the rest of the Helicobacter genome suggests that the island was acquired from other bacterial species by horizontal gene transfer [26]. Infection with cagA+ strains enhances the risk for development of duodenal ulcers and adeno-carcinoma of the distal stomach [27]. ThecagPAI stimulates the production of interleukin 8 [28, 29] via. intracellular NOD1 receptor and the nuclear factorB pathway. The elevated level of chemokines in host is
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responsible for an increased gastric inflammationwhich subsequently leads to disease development. Further, host chemokine response also enhances pathogen clearance by activation of apoptosis mechanism. Essentially, all H. pylori strains carry vacA gene that encode a protein cytotoxin that induces vacuolation in a wide variety of eukaryotic cells [11]. The gene encoding this cytotoxin is present in all strains but exhibitsa mosaicism in the terminal(s) and median(m) regions. Thereare
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several alleles corresponding to varying amounts of toxin produced: s1m1 corresponds to the highest production, followed by s1m2, while strains with the s2m2 allele do not produce any toxin [30]. Two novel proteins; a secreted collagenase, encoded by hp0169 and a putative ATPase encoded by comB4, being part of a DNA transformation-associated type IV transport system of H. pylori are found to be absolutely essential for colonization [22]. Patients infected with cag+ H. pylori strains have a stronger inflammatory response in the stomach and are at a greater risk of developing peptic ulcers or stomach cancer than those infected with strains lacking this island [31]. Pathological changes in gastric epithelium upon infestation with H. pylori are diagrammatically illustrated in Figure 1 and 2. After H. pylori attaches itself to gastric epithelium, the type IV secretion system expressed in part by the cag PAI injects its own peptidoglycan fragments as the inflammation inducing agent into the gastric epithelial cells. The injected peptidoglycan is recognized by Nod1 receptor, which then stimulates expression of cytokines that promote inflammation [32]. H. pylori also injects cagA protein into gastric epithelium, where it disrupts epithelial membrane barrier and other cellular activities [33]. Once inside the cell the cagA protein is phosphorylated on tyrosine residues by a host cell membrane-associated tyrosine kinase (src). Pathogen has been shown to activate the epidermal growth factor receptor (EGFR), a membrane protein with a tyrosine kinase domain. Activated EGFR alters signal transduction and gene expression in host epithelial cells that may contribute to pathogenesis. It has also been suggested that a C-terminal region of the cagA protein can regulate host cell gene transcription independent of protein tyrosine phosphorylation [27, 34]. iNOS expression and production of NO in macrophage are upregulated with H. pylori infection both under invivo and invitro conditions. Even major pathogenicity protein i.e. urease (ureA+), activate iNOS and increase NO production. Urease is an essential survival factor for H. pylori during gastric colonization, is implicated on NO dependent damage and carcinogenesis [35]. Other virulence proteins including vacA, cagPA-1 and picB show a selective and significant decrease in stimulated iNOS mRNA, protein and NO2- production with the ureAstrain compared with wild type H. pylori. H. pylori induces a weak immune response which fails to eradicate the pathogen. Translation of iNOS mRNA and NO production by H. pylori stimulated macrophages is also inhibited by the polyamine spermine derived from ornithine decarboxylase (ODC) and is dependent on the availability of iNOS substrate i.e. L- arginine [36]. siRNA knockdown studies of two inducible genes cationic amino acid transporter (CAT2) and ODC in gastric macrophage, indicated that addition of spermine or knockdown of CAT2 inhibits L-arginine uptake, lowers iNOS protein levels and NO production, whereas knockdown of ODC has the opposite affect. High ODC activity of macrophage was reported in chronic gastritis which results in formation of polyamine spermine. Increased polyamine spermine concentration in turn decreases iNOS expression and NO generation in H. pylori stimulated macrophage that is essential for survival of pathogen during chronic diseases (Figure 4). The constitutive expression of rocF arginase also facilitates the production of ammonia and also favors the production of nitric oxide in stimulated macrophages [35]. Taken together, these findings indicate that in case of chronic gastritis up regulation of ODC in gastric macrophages impairs host defense against H. pylori by suppressing iNOS derived NO production [35, 36]. The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H.pylori infection, activation of caspase-8,-9and -3along with the expression of the proapoptotic Bcl-2
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FIGURE 4. Modulation of enzyme activities (arginase, CAT2, iNOS, ODC) and NO production during acute and chronic gastritis.

family proteins Bad and Bid was studied in human gastric epithelial cells [37]. According to Shibayama et al. [37], H.pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway is independent of cagA or vacuolating toxin. Even a membrane fraction of H.pylori is sufficient to activate this pathway. In chronic cases H. pylori infection could lead to peptic/gastric ulcers and adenocarcinoma. Two related mechanisms could promote cancer by H. pylori infection. According to first mechanism, there is enhanced production of free radicals (ROIs) near H. pylori and a concomitant increased rate of host cell mutation. Second mechanism the perigenetic pathway involves enhancement of the transformed host cell phenotype by means of alterations in cell proteins such as adhesion proteins [38]. It has been proposed that H. pylori induce inflammation and locally high levels of TNF- and/or interleukin 6 (IL-6). According to the proposed perigenetic mechanism, inflammation-associated signaling molecules such as TNF- can alter gastric epithelial cell adhesion and lead to the dispersion and migration of mutated epithelial cells without the need for additional mutations in tumor suppressor genes such as genes coding for cell adhesion proteins [39]. There is a great deal of genetic diversity between various strains of H. pylori, so is the outcome of disease. Genotypic diversity of 78 strains of
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H. pylori was dissected at the Institute of Post Graduate Medical Education and Research, Calcutta, India [40]. Study revealed that DNA sequence motifs of vacAm1 (middle region) alleles are distinct from those of East Asia and the West alleles, whereas the cagA sequences of Calcutta and Western strains are closely related. Another virulent factor of H. pylori, the iceA shows a weak association with disease development. Gene iceA2 is associated with most of the Indian strains, whereas the alternative but unrelated gene iceA1 occupies the same gene locus in approximately 200 strains of H. pylori studied from other geographic regions. Around 20% of the Calcutta strains carry an internal deletion in gene iceA1 which is absent in all the Western strains employed in the study. Thus, the occurrence of internally truncated iceA1 gene seems to be unique to Calcutta strains. Two mobile DNA elements that were rare in East Asian strains are also common in Calcutta strains. In a separate study, genetic diversity of H. pylori strains of Indian subcontinent was also compared with those of European cultures by multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) followed by phylogeographic analysis of the haplotypes [41]. The distribution of cagPAI genes within these strains was analyzed using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. All the Indian H. pylori isolates analyzed reveal a European ancestry and belong to H. pylori sub-population, hpEurope. Although gastric colonization withH. pyloriinduces histologic gastritis in all infected individuals, but the disease remains asymptomatic as only a minority of patients develop any apparent clinical signs of this colonization. It is estimated that H. pylori-positive patients have a 10 to 20% lifetime risk of developing ulcer disease and a 1 to 2% risk of developing distal gastric cancer [42-43]. The risk of development of these disorders in the presence ofH. pylori infection depends on a genetic make up of bacterial strain, host, and environmental factors that mostly influence the pattern and severity of gastritis [44]. Gastritis can be a brief and sudden illness (acute gastritis) or a long lasting inflammation (chronic gastritis) of gastric tissues that is a result of H. pylori infection. Both gastric and duodenal ulcers are strongly related toH. pylori infection. Colonization withH. pylorivirtually always leads to infiltration of both antrum and corpus regions of the gastric mucosa with neutrophilic and mononuclear cells. A close correlation exists between the level of acid secretion and the distribution of gastritis. There is a reduction in acid secretion capacity of the stomach during infection which often results from loss of parietal cells (as in atrophic gastritis) or inhibition of parietal cell function by vagotomy or acid-suppressive drugs, in particular, proton pump inhibitors (PPIs) [42]. Parietal cell function suffers badly due to secretion of local inflammatory molecules such as cytokines, including interleukin-1 (IL1). Condition becomes more severe with the augment of nonspecific dyspeptic symptoms, such as fullness, nausea, vomiting and inflammation of both the proximal and distal stomach mucosa and pangastritis. This phase is often associated with hypochlorhydria, which may be followed by spontaneous clearance and resolution of gastritis [45-47]. Approximately 50% of patients with H. pylori-associated peptic ulcer disease suffered ulcer recurrence within 1 year [48-49]. Eradication ofH. pyloridramatically changes the natural course of ulcer and almost completely prevents its recurrence [48-51].

there is no conclusive evidence regarding transmission of H. pylori infection via. any of these products [55-60]. Three routes of transmission from the stomach of an infected individual to another have been described in literature. These include (i) the iatrogenic route (via. tubes or endoscopes), which is least common, (ii) faecal-oral transmission (the most common) [5-6] and (iii) the oral-oral transmission. There has been no identified association of the infection with sexual transmission [11]. As the H. pylori infection remains asymptomatic in early years of infection, thus the progression of disease is generally studied by taking X-ray of the oesophagus, stomach, and duodenum; endoscopy, stool and biopsy testing [61]; blood antibody test, stool antigen test against H. pylori, 13C-urea breath test [62]. A strong correlation exists between a history of ulcer and H. pylori infection in the family. Environmental and genetic factors might have influence on susceptibility to the infection. In addition, the high prevalence of H. pylori infection in subjects with no family history of ulcer suggests how the living conditions, socioeconomic factors and cultural background of the subjects are important in mounting the prevalence and transmission of H. pylori infection. Ahmed et al. [63] assessed the relationship between subjects with a history of gastric or duodenal ulcer and the risk of infection in their offspring in population of South India, which is considered the population being considered at high risk of stomach cancer. It was observed that the transmission of H. pylori may be influenced by the presence of ulcer or that H. pylori strains causing peptic ulcer may be more infective than other strains as published in earlier studies.

Cure of ulcers
The most proven effective treatment is a 2-week course of treatment called triple therapy. It involves taking two antibiotics to kill the bacteria and either an acid suppressor or a stomach-lining shielder. Two types of acid-suppressing drugs might be used: H2 blockers (e.g. Cimetidine, Ranitidine, Famotidine, Nizatidine) and proton pump inhibitors (eg: Omeprazole, Lansoprazole, Pantoprozole, Rabeprazole, Esomeprazole, Leminoprazole) [64]. Bismuth subsalicylate, a component of Pepto-Bismol, is used to protect the stomach lining from acid. It also kills H. pylori. Sucralfate, a basic aluminium sulphate sucrose complex, is another ulcer-preventing agent having anti-pepsin and antiacid properties. It reacts with gastric juice to form a sticky paste which protects the mucosa by coating, and also binds to ulcer-affected sites [65].

Emergence of drug resistance in H. pylori


The prevalence of H. pylori related chronic gastritis, duodenal and gastric ulcer is quite high in Eastern India [20]. Situation becomes worse due to emergence of drug resistance in H. pylori. Various Indian studies highlight the occurrence of drug resistance in H. pylori strains isolated from biopsy or stool samples of the patients and as a major obstacle in eradication of this gastro-duodenal pathogen. According to Mukhopadhyay and coworker about 90% of Calcutta strains of H. pylori were metronidazole resistant [40]. The drug sensitivity profile of H. pylori isolated from different parts of India, namely, Hyderabad, Mumbai and Lucknow was studied at National Institute of Cholera and Enteric Diseases, Kolkata, India [66]. Most of the strains (85%) have shown resistance to metronidazole and 7.5% strains to tetracycline, which is quite high compared to other reports in India. All Kolkata strains are however, highly sensitive to clarithromycin, furazolidone and amoxicillin. Bacterial genotype also has a great influence on the efficiency of
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Routes of transmission and detection of peptic ulcers


H. pylori has a narrow host range and is found exclusively in humans and some nonhuman primates [49, 52-54]. H. pylorihas been detected in saliva, vomitus, gastric refluxate, and feaces of animals. However
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proton pump inhibitor-based triple-therapy regimen. The efficiency of a multidrug formulation consisting of a proton pump inhibitor and two antibiotics viz. omeprazole, clarithromycin and amoxycillin was analysed in patients of Eastern India [67]. Bacterial vacA m1 allele was most represented genotype among patients with eradication failures (68%) than in those with successful eradication (39%) (P<0.05). No significant association of vacAs1 (signal sequence allele) or cag pathogenicity island status with persistence was detected. Persistent infection and recurrence after eradication therapy is a great problem in H. pylori infection [20]. Thus, current antibiotic-based triple therapies are not practical for global control due to the high cost, genotypic variation in H. pylori strains, problems with patients compliance and the emergence of antibiotic-resistant strains [68]. Vaccination against H. pylori has therefore been considered as the best approach to control H. pylori infection and administration of oral bacterial antigens. In a mice model, the efficacy of the vaccine raised against H. pylori urease was tested. This in combination with a mucosal adjuvant protected mice against Helicobacter infection that could be further extended to humans to provide protection against this gastric pathogen [69-70].

ing phagocytic cell functioning [71,82]. IFN--activated macrophages inhibit growth of pathogenic bacteria like Mycobacterium as a result of TfR downregulation [71, 81]. Thus, deprivation from essential growth factors represents an integral part of host defense functions. Importance of probiotics is evidenced by their ubiquitous occurrence in natural food products, their GRAS (Generally Recognized as Safe) status, and their ability to exert health benefits beyond basic nutrition. Probiotic organisms display numerous antagonistic activities. These antagonistic effects are mainly exhibited due to production of organic acids, but also of other compounds such as bacteriocins and antifungal compounds [83-87]. Applications of bacteriocin starter cultures and bacteriocin thereof in various food systems were already addressed in a number of review articles [88-90]. Health claims of various probiotic strains include normalization of gastro-intestinal [91-92] and vaginal ecosystem [93, 94], improvement of specific and non-specific immune responses [82], detoxification of carcinogens and suppression of tumors and cancers [95-97], reduction of blood pressure in hypertensive patients [98] and cholesterol [99]. Importance of probiotic lactic acid bacteria in treatment of milk allergies [100] and improvement of mineral absorption capacity of the intestine are also well documented [101].

Probiotics and gut physiology


The mammalian gastro-intestinal tract contains a complex and diverse society of both pathogenic and nonpathogenic (probiotic) bacteria. Probiotics are thought to supplement the microbial gut community, maintain epithelial barrier function and promote general immune homeostasis [71]. More recent commercial efforts focus on food supplementation with live probiotic cultures in the form of fermented milk products with either a single strain (L. acidophilus La1, commercial name LC1; B. longum BB536, commercial name ProCult3, and others) or mixed cultures of various lactobacilli (L. acidophilus, L. plantarum, L. rhamnosus, L. fermentum), bifidobacteria (B. infantis, B. bifidum, B. longum), yeasts (Saccharomyces sp.) and other microbes (Streptococci). Interestingly, the stimulatory capacity of probiotics have been tested in farm animals where their contribution to improve overall health status, immune system functions, reducing risk of infection and improvement in the yield of poultry and meat products is highly appreciated [72]. Evidences support the stimulatory capacity of the probiotic microorganisms, but the final verdict is not out, yet. Questions as to which species, strains or mixtures thereof are most beneficial, and the molecular basis for these effects, require more detailed studies [73-74]. A series of review articles have been published in the past year outlining the efficacy of probiotics and prebiotics in human health [75-80].

Mechanism of probiotic action


Lactic acid bacteria have acquired the status of probiotic starter culture in the food industry because of their GRAS status and increased consumer awareness of the potential health risks derived not only from food borne pathogens, but also the artificial chemical preservatives used to control them. Their growth lowers pH that inhibits the growth of most of the other microorganisms, the biochemical conversions involved in growth enhance the flavor, improve organoleptic and nutritional properties [102] and many strains produce antagonistic compounds such as organic acids, hydrogen peroxide, diacetyl and bacteriocins [103]. Bacterial species primarily used as probiotic cultures in the International as well as National food industries are Lactobacillus acidophilus (La2, La5, Johnsonii, NCFM, DDS-1, SBT-2062), L. bulgaricus (Lb12), L. lactis (La1, A164, BH5), L. plantarum (299v, Lp01), L. rhamnosus (GG, GR-1, 271, LB21 ), L. reuteri (SD2112), L. fermentum (RC-14 ), Bifidobacterium longum (BB536, SBT-2928), B. breve (Yakult), B. bifidum (Bb-12 ), B. esselnsis (Danone{Bio Activia}), B. lactis (Bb-02), B. infantis (Shirota, Immunitass, 744, 01 ) [104]. Gratia was the first to discover that the antimicrobial property of bacterial cells is exhibited by synthesizing proteinaceous toxins that inhibit the growth of similar or closely related bacterial strain(s) [105]. Later on series of bacteriocin producers have been identified and their importance in food fermentations was tested. Isolated pediocins and their producer strains such as Pediococcus acidilactici, P. pentosaceous, P. damnosus are also potential candidate for the development of novel antimicrobial and therapeutic agents [106-109]. Probiotics may exert some of their protective functions through modulation of immune activity and epithelial functions in both the large and small intestine. In vitro models suggested that immune and epithelial cells can discriminate between different microbial species through activation of Toll-like receptors [110]. Resta-Lenert and Barrett showed that live Streptococcus thermophilus and Lactobacillus acidophilus could inhibit the adhesion and invasion of enteroinvasive E. coli into human intestinal cell lines [111]. In vitro investigations also revealed the ability of Lactobacillus rhamnosus GG to prevent cytokine-induced apoptosis in intestinal epithelial cell models through the inhibition of TNF-induced activation of the proapoptotic p38/mitogen-activated protein kinase [112]. Epithelial cells release interleukin-8 in response to pathogenic bacteria but not to probiotic strains [113]. Bacterial DNA of pathogenic strains evoke phosphorylation of the extracellular signal-regulated
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Summary of health benefits exhibited by probiotics


Probiotic microorganisms in the intestine compete with pathogenic microorganisms, thereby preventing pathogenic colonization and invasion. Although most microorganisms are able to synthesize organic molecules required for their survival and maintenance, some molecules e.g. amino acids, fatty acids, nucleotides, enzyme cofactors etc. are used directly or metabolized from nutrients available in the host gut. Abundance of such nutrients within distinct host microenvironments led to loss of genes required for their biosynthesis in many microorganisms [71, 81]. This dependency on essential host nutrients represents a major force for pathogen selection of distinct host habitats. An instructive example for nutritive hostpathogen competition is represented by the mutual requirement for iron. Iron is an essential micronutrient for growth, basic metabolism and maintenance of most of the living organisms. Probiotic bacteria produce interferon gamma (IFN-) that stimulates immune system of the host by improv Under License of Creative Commons Attribution 3.0 License

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kinase pathway and turn on activator protein-1 [114], and of probiotic strains modulate nuclear factor-B (NF- B) pathway in response to TNF- as indicated in Figure 1 and 2 [115]. Selected probiotics can stimulate host dendritic cells (DCs) regulatory functions by targeting specific pattern-recognition receptors and pathways and confer protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis [116]. The preventive effect of probiotic-pulsed DCs required a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase, MyD88-, TLR2- and NOD2-dependent signaling and induction of CD4+, CD25+ regulatory cells in an IL-10-independent pathway. A study demonstrated the role of probiotics to counteract stressinduced changes in intestinal barrier function, visceral sensitivity and gut motility in a strain specific manner. Effects are mediated through direct bacterial-host cell interaction and/or via soluble factors. Probiotics may elicit these beneficial responses through various mechanisms viz. competition with pathogens for essential nutrients, induction of epithelial heat-shock proteins, restoration of tight junction protein structure, up-regulation of mucin genes, secretion of defensins, and regulation of the NF-B signalling pathway. In addition, cannabinoid receptors reduce the perception of intestinal pain [117]. A study has already investigated the adhesion and colonization dynamics of lactobacilli in vivo in humans [118]. Exogenously applied lactobacilli are generally able to only temporarily colonize the gastrointestinal tract (GIT). This phenomenon was linked to colonization resistance or the niche exclusion principle, where each niche in the GIT is colonized by well-adapted species [119].

Genes and proteins supporting probiotic function


Caco-2 or HT-29 human-derived adenocarcinoma cells are important models to study adherence of probiotics to Human epithelial cell [120]. In a genome-wide microarray-based genotyping, Pretzer et al. [121] identified mannose-specific adhesin in L. plantarum WCFS1. This glycoprotein is encoded by lp_1229 gene (renamed as msa). With the availability of in silico approaches for genome wide analysis it became possible to identify genes encoding proteins that facilitate adherence of bacteria to intestinal cells as well as for novel proteins mediating probiotic adherence to GIT. Such techniques are less labour-intensive, fast and highly precise. It saves a lot of time for initial screening, though validation of the results demand wet lab experiments. An in silico study showed that the genes encoding a fibronectin-binding protein (FbpA), a mucin-binding protein (Mub), and a surface layer protein (SlpA) all contribute to the ability of L. acidophilus NCFM to adhere to Caco-2 cells, confirming that adhesion is determined by multiple factors. Sortase and sortase dependent proteins (SDPs) such as srtA and the lspA, are also important adhesion factors of L. salivarius UCC118 [122]. Surprisingly, two peculiar cytoplasmic proteins of L. johnsonii NCC533; an elongation factor Tu (EF-Tu) and a heat shock protein GroEL have an important role in the binding of NCC533 to Caco-2 and HT-29 intestinal epithelial cells and mucins [123, 124]. Both the proteins of NCC533 have been located at the cell surface, although no secretion or cell wallbinding motifs are present to explain this observation. S-layer proteins (like SlpA and CdpA) of lactobacilli have been commonly implicated in the adherence of lactobacilli to Caco-2 cells [125-126].

components or as a result of adherence [12]. There is substantial evidence that probiotics modulate H. pylori colonization of the gastric mucosa through production of lactic acid, bacteriocins or antibiotics. Different reports support this hypothesis and have proven the efficiency of probiotic microorganism in treatment of H. pylori infection (as given in Table 2). In vitro studies showed that Lactobacillus johnsonii La1, L. salivarius, L. acidophilus, and Weissella confusa inhibit the attachment of H. pylori to intestinal HT-29 cells [61, 146] or to MKN 45 gastric cell lines [128,135]. Probiotics interfere in adhesion of H. pylori to epithelial cells and their capacity to attenuate H. pylori-induced gastritis in man is addressed in a reveiw by Felley and Michetti [147]. Previous reports have suggested a role of probiotics in treatment and prevention of H. pylori infection through probiotic-induced inhibition of H. pylori growth and adherence to epithelial cells and co-activation of host immune system [129, 147, 148]. Both in vitro and in vivo mouse model of Ushiyama et al. demonstrates inhibitory effect of Lactobacillus gasseri on H. pylori colonization [149]. In a double-masked, randomized, controlled clinical trial, 326 school children from a low socioeconomic area of Santiago, Chile, with H. pylori infection were treated with both live and heatkilled strains of Lactobacillus johnsonii, Lactobacillus paracasei and/or carrier once daily for 4 weeks. A 13C-urea breath test demonstrated a significant decrease in H. pylori colonization in children receiving live L. johnsonii but not the other groups [150]. Both of these studies support the complementary effect of probiotics in the management of H. pylori infection. The sharing of glycolipid specificity is a pre-requisite for the Lactobacillus strains to have a therapeutic effect on Helicobacter pylori eradication [134]. The MUC5AC glycoprotein has been identified as the primary receptor for H. pylori in the human stomach [151].
TABLE 2. Probiotic cultures with a potential to treat Helicobacter pylori infections. Strain L. salivarius WB1004 L. acidophilus (johnsonii) La1 L. acidophilus L. acidophilus CRL 639 L. gasseri OLL2716 L. GG Bacillus subtilis L. reuteri Weissella confusa Lactobacilli and Bifidobacteria L. casei strain Shirota L. casei strain Shirota and L. acidophilus L. casei subsp. DG L. brevis L. rhamnosus R0011 and L. acidophilus R0052 Bacillus clausii L. reuteri and L. paracasei L. salivarius References 128 61 129 130 131 132 133 134 135 136, 137 138 139 140 141 142 143 144 145

Prevention of H. pylori infection


The adhesion of H. pylori to epithelial cells is important in determining the outcome in H. pyloriassociated diseases [127]. In the gastric mucosa, H. pylori possibly interact with epithelial cells through secretory

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Probiotics have been suggested to increase efficacy of H. pylori eradication therapy by preventing antibiotic-associated side effects and thus increasing compliance. Cremonini et al. [152] randomized 85 patients with H. pylori undergoing eradication with triple therapy to one of four groups: Lactobacillus casei subspecies rhamnosus, Saccharomyces boulardii, L. acidophilus plus Bifidobacterium lactis, or placebo. In all probiotic-supplemented groups, there was a significantly lower incidence of antibiotic-associated diarrhea and taste disturbance relative to placebo. Nevertheless, there was no difference in H. pylori eradication or compliance rates between the various groups. The effects of multi-species probiotic combination on H. pylori infection in terms of adhesion, epithelial cell damage, apoptosis and inflammatory responses in Caco-2 cells were evaluated by Myllyluoma et al. [153]. All probiotics used in the study inhibited H. pylori adhesion. L. rhamnosus GG, L. rhamnosus Lc705, P. freudenreichii subsp. shermanii Js, and the combination inhibited H. pylori-induced cell membrane leakage. L. rhamnosus GG, L. rhamnosus Lc705, and the combination initially improved epithelial barrier function but increased the H. pyloriinduced barrier deterioration after incubation for 24 to 42 h. L. rhamnosus GG, L. rhamnosus Lc705, and P. freudenreichii subsp. shermanii Js inhibited H. pylori-induced IL-8 release, whereas L. rhamnosus GG, L. rhamnosus Lc705, and B. breve Bb99 suppressed PGE2 release. None of these anti-inflammatory effects persisted when the probiotics were used in combination. The combination thus increased the levels of IL8, PGE2, and LTB4 released from H. pylori-infected epithelial cells. The proinflammatory actions of the individual components dominated the anti-inflammatory effects when the probiotic bacteria were used in combination. Therapeutic response could be optimized if probiotic strains are characterized before they are used in combination. In a clinical trial on H. pylori patients, the effect of fermented milkbased probiotic preparations on H. pylori eradication was evaluated at Sitaram Bhartia Institute of Science and Research, New Delhi [154]. The search identified 10 eligible randomized controlled trials. Data were available for 963 patients, of whom 498 were in the treatment group and 465 in the control group. The pooled odds ratio for eradication by intention-to-treat analysis in the treatment versus control group was 1.91 (P<0.0001); test for heterogeneity (Cochrans Q=5.44; P=0.488). The pooled risk difference was 0.10 (95% CI 0.05-0.15; P<0.0001) by the fixed effects model (Cochrans Q=13.41; P=0.144). The pooled odds ratio for the number of patients with any adverse effect was 0.51 (95% CI 0.10-2.57; P=0.41); random effects model; heterogeneity by Cochrans Q=68.5; P<0.0001). Fermented milk-based probiotic preparations improve H. pylori eradication rates by approximately 5-15%, whereas the effect on adverse effects is heterogeneous. The drug sensitivity profiles of H. pylori isolated from different parts of the World have indicated that the pathogen has acquired resistance to the antibiotics due to point mutations and decreased binding of the antibiotics to the ribosomes [40, 66, 155]. Thus, current antibiotic-based triple therapies are not practical for global eradication due to the genotypic variation in H. pylori strains and the emergence of antibiotic-resistant strains [68]. Very few studies have actually focused on role of bacteriocins produced by probiotic bacteria in treating H. pylori infection (Table 3). Lacticin A164 of Lactococcus lactis subsp. lactis A164 and lacticin BH5 of L. lactis BH5 are two bacteriocins of lactococcal origin with anti-Helicobacter activity that tell pathogen in a dose-dependent manner [156]. Two more anti-Helicobacter pylori bacteriocins namely bulgaricin BB18 produced by L. bulgaricusBB18 andenterocin MH3 produced by E. faeciumMH3 have recently been identified [157]. These are potential antimicrobial agents and in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora.

TABLE 3. Anti-Helicobacter pylori bacteriocins. Bacteriocin Lacticin A164 Lacticin BH5 Bulgaricin BB18 Enterocin MH3 Strain Lactococcus lactis subsp. Lactis A164 Lactococcus lactis subsp. lactis BH5 Lactobacillus delbrueckii ssp. bulgaricus BB18 Enterococcus faecium MH3 Reference 156 156 157 157

Exploring genome and transcriptome of H. pylori to identify potential drugs targets


Microarray technology is a powerful tool to get an overview of cell responses to environmental changes at the transcriptional level. A single environment change or exposure to pathogen can induce a large number of changes in the transcriptome content of a particular tissue. Differential gene expression needs a further validation at the transcriptome level (through RT-PCR) or at proteome level (through ELISA, western blotting, 2D gel electrophoresis, MALDI-TOF etc.). Statistical analysis of the microarray expression data generated in transcriptomics study needs a skilled personnel and is time consuming too. Sometimes normalization of data needs to be done in order to get final result. That is why the application of this technology to the study of heterogeneous microbial populations presents a huge methodological challenge. Generally, the biochips used with mixed cultures are not pangenomic and serve rarely for transcript detection. Most of them are devoted to the detection of microbial species in complex ecosystems through ribosomal DNA sequences or to the detection of a reduced number of DNA sequences without quantifying their expression levels [158-161]. Some articles mention the use of DNA biochips for mRNA quantification, but these chips are mostly restricted to a limited number of mRNAs [162164]. The use of pangenomic biochips to study mixed cultures has been mentioned only in few research articles [165, 166]. The transcriptional profile of gastric epithelial cell lines cocultured with H. pylori and the global gene expression of whole gastric mucosa has been described in a study by Resnick and coworkers [167]. mRNA from 10 patients with peptic ulcer disease, before and after H. pylori eradication were reverse transcribed and applied on to Affymetrix cDNA microarray chips. Differentially expressed genes were identified and subset was validated by real-time polymerase chain reaction (RT-PCR). A total of 13,817 transcripts decreased and 9680 increased after H. pylori eradication. Applying cut-off criteria (p<0.02, fold-change threshold 2.5) reduced the sample to 98 differentially expressed genes. Genes detected included those previously implicated in H. pylori pathophysiology such as interleukin-8, chemokine ligand 3, -defensin and somatostatin, as well as novel genes such as gastrokine-2 (GDDR) (TFIZ1), chemokine receptor-7 and -8, and gastrokine. A serious obstacle in transcriptome studies is massive cross-hybridization between the foreign cDNA and the genome specific DNA chip. A very simple method was proposed to considerably decrease this nonspecific hybridization, consisting of adding the microbial partners DNA [166]. Co-culture technique was followed to study gene expression changes in L. lactis to the presence of Saccharomyces cerevisiae. Although no differences between growth kinetics were observed for the pure and the mixed cultures of L. lactis, the mRNA levels of 158
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genes were significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in mixed cultures. These changes in transcript abundance were demonstrated to be regulated by the ethanol produced by the yeast and were confirmed by an independent method (quantitative reverse transcription-PCR). It is important to highlight here that co-culture technique opted by Maligoy et al. [166] can be extended further to study probiotic-H. pylori interaction in vitro and can provide useful information on the actual mechanism by which probiotic bacteria prevents colonization, and/or influence clearance of the H. pylori infection from human gastroduodenal region. Sharma and coworker presented a genome-wide map (1.67Mb) of H. pylori transcriptional start sites and operons [168]. Polycistrons are more complex and uncoupled in H. pylori as hundreds of transcriptional start sites were discovered upstream to annotated genes lying within the operons. Further, control of gene expression in H. pylori is exhibited through anti-sense transcription. 60 small RNAs including the e-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs were identified. Availability of primary transcriptome of H. pylori helps in identifying novel therapeutic targets and the information obtained could be applied to rational remodeling or tailoring of human-associated probiotic microorganism in order to enhance their anti- H. pylori activity and associated functions. Clearly, the best approach would be targeting transcriptional regulators of virulence and associated factors in H. pylori in order to provide an effective cure against this major gastric pathogen.

However, because of population stratifications, studies must take account of the geographical and racial background of participants. To date, GWAS studies have identified risk and protective factors for AIDS [171], asthma [172], breast cancer [173], cardiac arrest [174], gastrointestinal diseases viz. celiac disease [175], colorectal cancer [176,177], crohns disease [178-180], esophageal cancer [181], pancreatic cancer [182,183], type I diabetes [184], type II diabetes [185,186], heart failure [187], hypertension [188], macular degeneration [189], multiple scleresis [190], neuro-degenerative alzheimers disease [191], obesity [192], rheumatoid arthritis [193], schizophrenia [194-196], urinary bladder cancer [197] and other human disorders. A number of GWAS have been conducted to discern the genetic susceptibility as well as the host pathogen interaction underlying the etiology of gastroduodenal diseases. In their two stage study conducted amongst Japanese 1,384 ulcerative colitis patients and 3,057 controls [198], Asano and coworkers identified strong association of disease with the major histocompatibility complex (MHC) region and three new susceptibility loci: the immunoglobulin receptor gene FCGR2A : rs 1801274, a locus on chromosome 13q12: rs17085007 and the glycoprotein gene SLC26A3: rs2108225. The FCGR2A: rs1801274 is a nsSNP that is critical to receptors binding affinity for IgG and has been associated with other autoimmune diseases. Franke et al identifed two new associations at chromosomes 7q22: rs7809799 and at chromosome 22q13 in ILI7REL: rs5771069 amongst Europeans [199]. However such studies are incomplete in absence of clear cut pathogen characterization which alone can lead to determination of host pathogen susceptibility genes and the pathways involved in such interaction.

Peptide vaccines available against H. pylori


The human microbiome could be manipulated by such smart strategies to prevent and treat acute H. pylori infection and a variety of other disorders. Information obtained from metagenomics and the human microbiome will tremendously expand our knowledge of the genetic composition of microbial species associated with human gut. This information can be directly applied to engineer human-associated probiotic microorganism and enhances their associated functions. In an attempt to use Lactococcus lactis for oral delivery of vaccine against H. pylori, fragment E of ureB (ureBE) was cloned from a clinical isolate of H. pylori [169]. A prokaryotic expression vector, pAMJ399, with the ureB fragment E and the Staphylococcus aureus protein A anchor fragment (spaX), was constructed. The fusion protein was expressed under the control of the P170 promoter in Lactococcus lactis. Western blot assay of lactococcal cell wall extracts with a polyclonal chicken antiserum confirmed the immunity of the expressed recombinant protein that was located on the cell surface. These results provide the first report of a surface display system in lactic acid bacteria for the delivery of oral vaccines against H. pylori.

Conclusion and future perspectives


H. pylori is a common pathogen of gastro-duodenal region associated with chronic gastritis, peptic and gastric ulcers, gastric adeno-carcinoma and more rarely, lymphoma of the mucosa-associated lymphoid tissue. Pathogen shows a high prevalence in developing and poor countries. Emergence of drug resistance in H. pylori is a major obstacle in eradication of this gastric pathogen. Why susceptibility to this gastric pathogen varies with genetic diversity of the population? Answering this question will require a substantial commitment to future research. This will need a substantial effort to identify important genotypic variations that aid in protecting individual against H. pylori. Extensive GWAS analyses will help to get insight into mechanism of pathogen attachment, etiology of disease and altered immune responses for pathogen clearance before it establishes itself into the gastric mucosal system. Besides academics, the impact of such studies has been widespread as novel therapeutic agents and better preventive measures could be suggested to control pathogen colonization or to eradicate pathogen in the host gastrointestinal tract and to prevent outbreak of the H. pylori infection. The populations indicating higher susceptibility to H. pylori might have a differential expression of genes such as adhesion factors and alternative induction mechanism of disease could only be identified following a well planned GWAS. The potential of probiotic lactic acid bacteria in aggravating resistance to H. pylori, to kill pathogen through production of anti-H. pylori bacteriocins and/or other antagonistic factors is revealed here. The application of probiotic organisms in food preservation as well as prevention of human gastro-intestinal diseases is emerging these days. Mechanism by which probiotics confer resistance to H. pylori is still to be deciphered and requires a separate GWAS on human populations taking oral doses of probiotic organism. Moreover, GWAS helps to investigate genetics of a disease and identify novel therapeutic targets. Information thus obtained could be directly applied to tailor probiotic organisms or vaccines against H. pylori. The future perspectives of the study help to display probiotic lactic acid bacteria and/ parts thereof that may provide an effective prevention or cure against this major gastric pathogen.
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Genome-wide association study (GWAS) or whole genome association study (WGAS)


GWAS is an examination of genetic variation across a given genome, designed to identify genetic associations with observable traits. Millions of single-nucleotide polymorphisms, and thousands types of copy number variations are found in large or small segments of the human genome. These genetics variations may either directly induce phenotypic changes or tag nearby mutations that influence individual variation and susceptibility to disease, thus, are considered as pointers to the region of the genome where the disease-causing problem is likely to reside. Since the entire genome is analyzed for investigating genetic of a particular disease, GWAS allow the genetics of a disease to be investigated in a non-hypothesis-driven manner [170].
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Genetic Heterogeneity of Prevotella Strains Involved in Dentoalveolar Abscess


Tomoe Tambo1, Tomoari Kuriyama1,2, Tadahiro Karasawa3, Kiyomasa Nakagawa1, Etsuhide Yamamoto1, David W. Williams4
1 Department of Oral and Maxillofacial Surgery, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan 2 Kuriyama Dental Practice, Toyama, Japan 3 Department of Internal Medicine, Fujimi Kogen Hospital, Fujimi, Japan 4 Tissue Engineering and Reparative Dentistry, School of Dentistry, Cardiff University, Cardiff, United Kingdom Running head: Genotypes of oral Prevotella Correspondence: Dr. Tomoari Kuriyama Postal address: Department of Oral and Maxillofacial Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Ishikawa, Japan Tel.: +81 (0) 76 265 2444 Fax.: +81 (0)76 234 4268 E-mail: matumoto@oral.m.kanazawa-u.ac.jp

Abstract
This study investigated the genotype diversity of Prevotella isolates from dentoalveolar abscess and the genetic relatedness between clinical and commensal isolates within the same patients. Prevotella strains recovered from pus and dental plaque of individual patients (n=9) were examined. Each specimen was inoculated on to blood agar. Six to ten presumptive Prevotella colonies per specimen grown on the primary culture plate were selected. The colonies were genotyped using a random amplified polymorphic DNA assay and the antimicrobial susceptibility profile were also determined. Multiple genotypes per organism were evident in both pus (9 of 9 patients) and plaque (3 of 9 patients). The same genotype was shared by pus and plaque strains in 6 of the 9 patients, while no genetic agreement between pus and plaque strains occurred in the remaining 3 patients. Although it has been suggested that the microflora of infection sites is comprised of genetically homogeneous strains, these findings indicate that a wide range of genetic heterogeneity occurs in infecting Prevotella strains. Furthermore, although dentoalveolar abscess is an opportunistic infection involving endogenous oral bacteria, only certain strains from the commensal population might be involved in the infection.

Introduction
The majority of acute dentoalveolar abscesses involve dental pulp necrosis [1, 2]. Although vital dental pulp is normally sterile, deep caries and serious dental trauma often result in pulp infection with bacteria residing within the mouth. Infected regions of pulp will necrotize, and subsequently, bacteria may diffuse from the necrotic pulp into the periapical tissues including the alveolar bone. Although the tissues are colonized with a number of bacteria, the host defense systems usually maintain homeostasis between host and bacteria. However, once microbial homeostasis is disrupted, bacterial numbers increase and invade the tissues, accompanied by acute inflammation. Common clinical signs and symptoms include tooth pain and gingival swelling. Extensive facial and cervical swelling may occur in advanced cases, often accompanied by malaise and fever. Although rare, infection can cause serious complications including airway obstruction and sepsis [1, 2]. Acute dentoalveolar abscess is usually polymicrobial, primarily involving strict anaerobes and oral streptococci [1-7]. Experimental studies have demonstrated that an enhancement of bacterial virulence occurs in mixed infection of these organisms [8-12]. Consequently microbial synergy is considered a possible etiologic factor of infection [4].
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The genus Prevotella, is comprised of species that are strict anaerobic Gram-negative bacilli. Members of this genus are a major component of the human microflora at various body sites including the mouth [13], and they are often predominant in acute dentoalveolar abscess [1-7]. The Prevotella genus consists of more than 10 species, which are roughly divided into black-pigmented and non-pigmented types by the color of colonies forming on blood agar [13]. Pigmented Prevotella species frequently involved in dentoalveolar abscess include P. intermedia, P. nigrescens, P. melaninogenica and P. loescheii [7, 14]. Prevotella oralis, P. oris and P. buccae are the non-pigmented Prevotella species that are most frequently isolated from the infection [7, 14]. Both pigmented and non-pigmented Prevotella species have the potential to produce purulent infection in an experimental animal model [15]. Thus Prevotella are known to play a significant role in development of acute dentoalveolar abscess. It has widely been accepted that strains of each bacterial species involved in the infection of an individual are genetically homogeneous [16]. However, scientific evidence to support this hypothesis is only available for a limited number of infections. In infections within the mouth, a high degree of genetic similarity between Porphyromonas gingivalis strains recovered from periodontitis of same individual has been reported [17]. However, there has been no investigation to deThis article is available from: http://www.acmicrob.com

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termine the genetic diversity of strains within dentoalveolar abscess of individual patients. Furthermore, with regards to other human ins fections involving Prevotella, no study has been conducted to address this hypothesis. As dentoalveolar abscess occurs opportunistically, it can reasonably be hypothesized that the bacteria of the same genotype acts both as commensals and causative agents of infection. However, little is known concerning the genetic homogeneity of these opportunistic pathogens during commensal existence and infection. The purpose of this study was therefore to investigate the genetic diversity of Prevotella strains recovered from dentoalveolar abscess. The study also determined the genetic relatedness between clinical and commensal oral isolates from the same patients.

electrophoresis with 1.5% agarose gel (Nacalai tesque, Kyoto, Japan) at 100 V/cm for 1 h and subsequent ethidium bromide staining. The banding patterns of tested strains appearing on the gel image were visually compared. All experiments were repeated in triplicate and on three separate occasions to confirm reproducibility. For isolated strains, minimum inhibitory concentrations (MICs) for amoxicillin (Astellas, Tokyo), amoxicillin/clavulanate (2:1, GlaxoSmithKline, Middlesex, UK), cefaclor (Shionogi), ceftazidime (GlaxoSmithKline), erythromycin (Shionogi), azithromycin (Pfizer), minocycline (Takeda, Tokyo), clindamycin (Pfizer), levofloxacin (Daiichi-Sankyo, Tokyo), and metronidazole (Shionogi) were determined using the agar dilution method recommended by the Clinical and Laboratory Standards Institute [19]. Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 were used as quality control strains in each test.

Methods
Specimens. Pus and plaque specimens were obtained from a total of 31 patients with acute dentoalveolar abscess (18 males and 13 females, mean age 54.5 years) attending oral and maxillofacial surgery clinics at Kanazawa University Hospital (Kanazawa, Japan) and Noto General Hospital (Nanao, Japan) between March 2007 and December 2009. All patients had tenderness and swelling of the vestibule site around the involved tooth, and mild to moderate facial swelling. All patients were however free of any serious complications including sepsis and airway obstruction. No patient had a history of being immuno-compromised or having received medication that could significantly influence immunity. Pus was obtained from the obstructive abscess by aspiration. Dental plaque was obtained from around the gingival margin of three noninfected teeth that exhibited healthy periodontal tissue. These teeth were chosen randomly in patients and collected using a fine dental instrument immediately after obtaining the pus specimen. Informedconsent for this study was obtained from all patients. Culture and identification of isolates. Pus and plaque samples were inoculated on to Brucella HK agar (Kyokuto, Tokyo, Japan) containing 5% (v/v) sheep blood for up to 72 h at 37C under aerobic and anaerobic conditions. Each specimen was also incubated on blood agar containing paromomycin (75mg/L, Pfizer, Tokyo, Japan) and vancomycin (2.5mg/L, Shionogi, Osaka, Japan) for up to 72 h at 37C under anaerobic conditions to selectively isolate strictly anaerobic Gram-negative bacilli. Six to 10 presumptive Prevotella colonies per specimen were selected from each primary culture plate and subcultured individually on fresh agar plates. Isolates from pus and plaque were identified by conventional methods [5, 13]. Identification of Prevotella strains was achieved at species level using Rap ID ANA II system (Remel, Lenexa, KS)[14]. Typing. In cases where the same Prevotella species was recovered from both pus and plaque of individual patients, the strains were genotyped using a random amplified polymorphic DNA (RAPD) assay [18] and the antimicrobial susceptibility of these isolates was also determined. With regards to the RAPD assay, colonies of each subcultured strain were resuspended in saline. Bacterial DNA was extracted and purified using the DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan). The RAPD assay was performed using Ready-To-Go RAPD Analysis Beads (GE Healthcare Japan, Tokyo, Japan). Primer 2 (5-GGTGCGGGAA-3) and primer 3 (5-CCCGTCAGCA-3) for amplification were selected by comparing the amplification patterns of each of six primers offered by the manufacturer. The PCR was undertaken in accordance with manufacturers recommendations. Analysis of each PCR product was performed by gel

Results
The results of microbiological examination of pus and plaque are presented in Table 1. Prevotella species were isolated from pus specimens in 18 (58%) of 31 patients and recovered from the plaque in 20 (65%) of 31 patients. The pus isolates included P. buccae (10 strains), P. loescheii (4 strains), P. melaninogenica (3 strains), P. intermedia/P. nigrescens (3 strains), P. oralis (2 strains), P. denticola (1 strain), P. disiens (1 strain), and unidentified Prevotella species (4 strains). With regards to plaque, the isolates included P. buccae 14 strains), P. melaninogenica (6 strains), P. intermedia/P. nigrescens (5 strains), P. loescheii (3 strains), P. oralis (3 strains), P. denticola (3 strains), P. disiens (1 strain), P. oris (1 strain), and unidentified Prevotella species (11 strains).
Table 1. Microorganisms isolated from dentoalveolar abscess pus and the dental plaque obtained from disease-free teeth of the same individuals No. of isolates Organism Prevotella Streptococcus Fusobacterium Campylobacter Staphylococcus Peptostreptococcus Porphyromonas Eubacterium Capnocytophaga Veillonella Bacteroides Corynebacterium Gemella Haemophilus Facultative catalase negative GNB Facultative catalase positive GNB Facultative catalase negative GPB Pus 28 17 14 10 8 4 4 4 3 3 3 2 1 0 8 8 5 plaque 47 18 14 15 11 3 3 1 11 9 2 5 0 1 10 5 8

GNB, Gram negative bacilli. GPB, Gram positive bacilli; data generated from a total of 31 patients

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Table 2. Prevotella isolates from 9 patients who yielded the same species of Prevotella in pus and plaque Patient Ref. A B C D E F G H I Prevotella isolates from pus P. buccae P. buccae P. buccae, P. disiens P. buccae P. buccae P. buccae, Prevotella spp* P. loescheii, P. oralis, Prevotella spp* P. melaninogenica P. loescheii, P. melaninogenica Prevotella isolates from plaque P. buccae, Prevotella spp* P. buccae P. buccae, P. disiens P. buccae P. buccae, P. intermedia/nigrescens P. buccae, Prevotella spp* P. loescheii, P. denticola, P. melaninogenica, Prevotella spp* P. melaninogenica, Prevotella spp* P. loescheii, P. melaninogenica P. buccae, Prevotella spp*

*Unidentified Prevotella species Underlined organisms were recovered both from pus and plaque from the same patient

Table 3. Number of genotypes in Prevotella strains recovered from pus and plaque No. of genotypes Patient Ref. A B C D E F G H I Tested organisms* Pus isolates 2 2 2 2 3 3 2 3 2 Plaque isolates 4 1 3 1 1 1 4 1 1 No. of genotypes that were shared by pus and plaque isolates 2 1 2 0 1 0 0 1 1 Fig.1. Typical banding profiles generated using the RAPD genotyping method. Prevotella melaninogenica strains recovered from plaque (strains 1, 2) and (strains 3-6) from patient I. Strains 1-4 demonstrate identical RAPD profiles and therefore genotypes and these are distinct from those generated for strains 5 and 6. Antibiotic susceptibility profiles appeared to correlate with the RAPD genotyping results with strains 1-4 demonstrating equivalent MICs, as was the case for strains 5 and 6. AMPC, amoxicillin; AM/CV, amoxicillin/clavulanate; CCL, cefaclor; CTX, ceftazidime; EM, erythromycin; AZM, azithromycin; MINO, minocycline; CLDM, clindamycin; LVFX, levofloxacin; MZ, metronidazole.

P. buccae P. buccae P. buccae P. buccae P. buccae P. buccae P. loescheii P. melaninogenica P. melaninogenica

* Prevotella species that were isolated both from pus and plaque specimens of same patients

results are summarized in Table 3. Multiple genotypes were evident from pus in all of the 9 patients and for the plaque of 3 patients. The pus and plaque specimens from each of 6 patients (patients A, B, C, E, H, and I) had species of the same genotype. Interestingly, although all genotypes of pus isolates were also seen in plaque isolates in 2 of the 6 patients (patients A and C), pus from the remaining 4 patients (patients B, E, H, I) harbored more than one genotype that was not detected in plaque. No genetic agreement between pus and plaque strains occurred in 3 patients (patients D, F, and G).

Of the 31 patients, 9 yielded the same species of Prevotella both in pus and plaque (Table 2). These strains were typed with the RAPD assay and examined for their antimicrobial susceptibility. All strains with same banding pattern exhibited the same or nearly the same MICs for the tested antibiotics (Fig. 1). All strains exhibiting different banding patterns were confirmed to have different antimicrobial susceptibility. The
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Discussion
In recent studies using appropriate methods of specimen collection and culture, Prevotella was found to be the most common isolate from acute dental abscess [5, 7, 20]. High prevalence of bacteria of this genus has also been noted by investigations using molecular techniques [21, 22]. In contrast, the incidence of individual Prevotella species
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varies in published studies, despite similar species being recovered. This discrepancy might be associated with geographical location of study, nature of subjects, and the employed study method [13, 23, 24]. Prevotella species are readily susceptible to clindamycin and metronidazole [14], whilst 30-80% of clinical isolates are reported to be resistant to penicillin [5, 25, 26]. Moreover, prevalence of penicillin-resistant Prevotella isolates from dental abscess appears to be increasing as indicated in previous studies by our group [27]. In the present study, although all of the strains obtained from the 9 patients were susceptible to amoxicillin (data not presented), a greater number of clinical isolates is required to address actual status of susceptibility in Prevotella strains involved in dental infection. The management of acute dentoalveolar abscess consists primarily of surgery, including drainage through incision and tooth extraction, antimicrobial therapy, and medical supportive care [1, 2]. Administered antibiotic is usually chosen empirically [1, 2], and members of the penicillin class are often recommended as the first-line agent [1, 2]. However, antibiotic selection based on data from microbiological examination is highly recommended in circumstances where the expected outcome of therapy has not been obtained, as well as in cases of severe infection, and in immuno-compromised patients [1, 2]. The hypothesis that a single genotype of a species is involved in any given bacterial infection is based on interpretation of laboratory data, with very few types of infection having been demonstrated to involve multiple genotypes. One notable exception is the recovery of multiple genotypes of Streptococcus pyogenes from pyoderma skin sores of the same patients [28]. Unexpectedly, however, the typing results of clinical strains in this study indicate that multiple subtypes of species are present at dentoalveolar abscess infection sites with (Table 3). Multiple subtypes also appeared to exist for commensal Prevotella strains. Fukui et al [29] reported that multiple subtypes of P. nigrescens were evident in 57% of the tested 14 dental plaques indicating that a wide diversity of genotypes of commensal Prevotella strains may exist within the mouth of an individual. In the case of Fusobacterium nucleatum, another opporother tunistic pathogen for dentoalveolar abscess, more than one genotype has been recovered from the healthy mouth of an individual [30]. Since the dentoalveolar abscess is an opportunistic infection, it is perhaps not surprising that heterogeneous Prevotella strains with different genetic and antimicrobial susceptibility characteristics are involved. This in itself would be a matter of concern for clinicians who could receive microbiological test results for only one strain, thereby not reflecting any distinct antibiotic susceptibility variation that could be present at the infection site. Although further study is required, the results of this study have significance in our current understanding of the bacteriology of dental purulent infections.

As dental plaque is a biofilm formed by bacteria colonizing dental surfaces, this material was considered appropriate to obtain commensal strains. In this study, plaque was collected from teeth associated with a healthy periodontal tissue, and these sites were selected since bacteria in subgingival plaque of teeth with periodontitis are associated with the infection itself [31]. In this study, pus and plaque shared the same strain genotype in 6 of 9 cases (Table 3). In 2 of the 6 patients, all strains genotypes in pus isolates were seen in plaque isolates. Since dentoalveolar abscess is an opportunistic infection, it would be expected that the same strains act as commensals and causative agents of infection within an individual. Interestingly, for the other 4 patients, whilst a shared Prevotella genotype was evident for pus and plaque specimens, genotypes restricted to the pus specimens were evident. Moreover, no genetic agreement between pus and plaque strains occurred in 3 cases. This might indeed be a reflection of a wide genetic heterogeneity for both clinical and commensal Prevotella strain populations. However, the discrepancy between the recovered genotypes from commensal and infected sites might also relate to the different selection pressures at the respective locations. Interestingly, it has been demonstrated that Prevotella isolates from dental abscess are more proteolytic compared with those from dental plaque obtained from healthy volunteers [32]. In the case of P. gingivalis, strains carrying specific genes involved in virulence have a high frequency of recovery from severe forms of periodontitis [33]. It may therefore follow that only selected strains with greater virulence suitably adapt to the altered environment of an abscess and thus become causative agents of infection, although further study is necessary to investigate this.

Acknowledgements
We wish to thank Miss Hayashi E (Asanogawa General Hospital), Dr Kato K (Kanazawa University Hospital), Dr. Yoshizawa K (Kanazawa University Hospital), Dr. Kitahara H (Kanazawa University Hospital), Dr. Terai K (Terai Dental Practice), and Dr. Hase T (Noto General Hospital) for their kind cooperation.

Funding
This study was supported with grains-in-aid for scientific research (No. 19791502), the Ministry of Education, Science, Culture, Sports and Science and Technology, Japan.

Competing interests
No competing interests to be declared.

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References
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23. Baumgartner JC, Siqueira JF Jr, Xia T, Ras IN (2004) Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. J Endod 30:141-144. 24. Sakamoto M, Ras IN, Siqueira JF Jr, Benno Y (2006) Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol 21:112122. 25. Roberts SA, Shore KP, Paviour SD, Holland D, Morris AJ (2006) Antimicrobial susceptibility of anaerobic bacteria in New Zealand: 1999-2003. J Antimicrob Chemother 57:992-998. 26. Fosse T, Madinier I, Hitzig C, Charbit Y (1999) Prevalence of -lactamase-producing strains among 149 anaerobic gram-negative rods isolated from periodontal pockets. Oral Microbiol Immunol 14:352-357. 27. Kuriyama T, Karasawa T, Williams DW, Nakagawa K, Yamamoto E (2006) An increased prevalence of -lactamase-positive isolates in Japanese patients with dentoalveolar infection. J Antimicrob Chemother 58:708-709. 28. Carapetis J, Gardiner D, Currie B, Mathews JD (1995) Multiple strains of Streptococcus pyogenes in skin sores of aboriginal Australians. J Clin Microbiol 33: 1471-1472. 29. Fukui K, Kato N, Kato H, Watanabe K, Tatematsu N (1999) Incidence of Prevotella intermedia and Prevotella nigrescens carriage among family members with subclinical periodontal disease. J Clin Microbiol 37: 3141-3145. 30. Suchett-Kaye G, Decoret D, Barsotti O (1998) Clonal analysis by ribotyping of Fusobacterium nucleatum isolates obtained from healthy young adults with optimal plaque control. J Periodontal Res 33:179-186. 31. Marsh PD, Martin MV, Lewis MAO, Williams DW (2009) Oral microbiology. 4th edition. Edinburgh: Churchill Livingstone Elsevier. pp.103-145. 32. Yanagisawa M, Kuriyama T, Williams DW, Nakagawa K, Karasawa T (2006) Proteinase activity of Prevotella species associated with oral purulent infection. Curr Microbiol 52:375-378. 33. Amano A, Nakagawa I, Okahashi N, Hamada N (2004) Variations of Porphyromonas gingivalis fimbriae in relation to microbial pathogenesis. J Periodontal Res 39:136-142.

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Antimicrobial and antioxidant efficacy of various solvent extracts of seeds and fruits rind of Caesalpinia pulcherrima Swartz.
Sumitra Chanda*, Jigna Parekh, Yogesh Baravalia, Shreya Parekh
Phytochemical, Pharmacological and Microbiological Laboratory Department of Biosciences, Saurashtra University, Rajkot- 360 005, Gujarat, India

*Corresponding author: E-mail: sumitrachanda@yahoo.com Tel. No. +919426247893

Abstract
Ethnopharmacological relevance: Caesalpinia pulcherrima Swartz. is an ornamental plant like a shrub or small tree. In traditional Indian medicine C. pulcherrima is used in the treatment of tridosha, fever, ulcer, abortifacient, emmenagogue, asthma, tumors, vata and skin diseases. Aim of the study: In the present study, the antimicrobial and the antioxidant activities of seeds and fruits rind of Caesalpinia pulcherrima Swartz were investigated. Material and methods: Hexane, chloroform, acetone, methanol and water extracts of C. pulcherrima were evaluated for their antimicrobial activity by agar well diffusion method. DPPH free radical scavenging activity, total phenolic, flavonoids and qualitative phytochemical analysis were also performed. Results: Among the different extracts, the methanol extract showed significant antibacterial and antioxidant activities. The MIC of the methanol extract of fruits rind showed narrow MIC range (1,250 - 78 g/ml) against all the investigated microorganisms. The methanol extract of the fruits rind showed good antioxidant activity (IC50 value 14 g/ml) which is very near to the standard (ascorbic acid IC50 value 11.4 g/ml). Conclusion: The obtained results suggested that the methanol extract of the seeds and fruits rind of C. pulcherrima possess antimicrobial and antioxidant properties which can be used for the discovery of new bioactive natural products that may serve as leads in the development of new pharmaceuticals that address unmet therapeutic needs. Keywords: Caesalpinia pulcherrima, Fabaceae/Leguminosae, antimicrobial activity, antioxidant activity, physico-chemical analysis, phenols

Introduction
Caesalpinia pulcherrima (Fabaceae/Leguminosae) is a shrub or small tree up to 5 m height, commonly known as Guleture is distributed throughout India [1]. This shrub is one of the most popular summer bloomers. From May through August this tropical looking shrub produces loads of spectacular flower clusters. The plant is used as emmenagogue, purgative, stimulant and abortifacient, also used in bronchitis, asthma and malarial fever [2]. The fruit was used to check bleeding and prevent diarrhea and dysentery, while the flowers were utilized to combat oxidative stresses [3]. According to folkloric uses, the stem has been used as an abortifacient and an emmenagogue [4, 5]. In traditional Indian medicine C. pulcherrima is used in the treatment of tridosha, fever, ulcer, abortifacient, emmenagogue, asthma, tumors, vata and skin diseases [6]. Antiviral, antiulcer, anti-inflammatory activity of different parts of C. pulcherrima has been reported [7- 9]. The use of traditional medicine is widespread and plants still represent a large source of natural antioxidants that might serve as leads for the development of novel drugs [10]. Traditionally common people use crude extracts of plant parts as curative agents [11, 12]. Plants

are known to produce antimicrobial agents as their defense mechanism; they can be considered as potential sources of new antibacterial agents. A number of antibiotics and chemotherapeutic agents of natural or semi synthetic origins are available today to fight against infections caused by various pathogenic microorganisms [13]. In searching for novel natural antioxidants, some plants have been extensively studied in the past few years for their antioxidant and radical scavenging components [14, 15] but there is still a demand to find more information concerning the antioxidant potential of plant species. Plant phenols are of interest because they are an important group of natural antioxidants and some of them are potent antimicrobial compounds [16]. DPPH (1, 1- diphenyl 2-picrylhydrazyl) has been used extensively as a free radical to evaluate reducing substances [17]. In view of the above considerations, it was thought of interest to investigate the antimicrobial and the antioxidant potential of Caesalpinia pulcherrima. The main goal of the present work was to assess the total phenolic and flavonoidal contents, antimicrobial activity, and antioxidant properties of various solvent extracts of the seed and fruits rind of C. pulcherrima.

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Material and methods


Plant collection The fruits of Caesalpinia pulcherrima Swartz were collected in September, 2007 from Rajkot, Gujarat, India. The identification of this plant was done by Dr. N.K. Thakrar, of the Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India, and kept under the Voucher specimen No. PSN246. Plant extraction The seeds and fruit rinds were separated and then washed under running tap water, air dried, homogenized to fine powder and stored in air-tight bottles. The powder of the seeds and fruits rind was extracted (10 g) successively with 100 ml each of n-hexane, chloroform, acetone, methanol and water with crude cold percolation extraction method. The extraction flasks were kept on a rotary shaker for 24 h at 120 rpm. Thereafter they were filtered through 8 layers of muslin cloth and centrifuged at 5000 rpm for 15 min. The supernatant was concentrated to dryness under reduced pressure and stored at 4C in air-tight bottles. Qualitative Phytochemical analysis The preliminary phytochemical analyses (Alkaloids, Flavonoids, Tannins, Phlobatannins, Triterpenes, Steroids, Saponins, and Cardiac glycosides) were performed according to the reported method [18]. Physico-chemical analysis Physico-chemical analyses were carried out according to reported method in The Ayurvedic Pharmacopoeia of India [19] and Vaghasiya et al. [20]. The following physico-chemical analyses; loss on drying at 105o C, total ash, water soluble ash, acid insoluble ash, Petroleum ether soluble extractive value, Ethanol soluble extractive value and Aqueous soluble extractive value were carried out. Determination of the total phenolic content The amount of the total phenolic content in the extracts of the different parts of C. pulcherrima was determined with Folin-Ciocalteu reagent method [21]. 0.5 ml sample (1 mg/ml) in 3 replicates and 0.1ml (0.5 N) Folin-Ciocalteu reagent were mixed and the mixture was incubated at room temperature for 15 min. Then, 2.5 ml saturated sodium carbonate was added and the resulting mixture was again incubated at room temperature for 30 min. The absorbance of the mixture was measured at 760 nm using spectrophotometer (Systronics: 106). The calibration curve was prepared using gallic acid (10 to 100 g/ml) solution in distilled water. Total phenol values were expressed in terms of gallic acid (SRL, India) equivalent (mg/g of extracted compound). Determination of the flavonoidal content The flavonoidal content in the extracts of the different parts of C. pulcherrima was determined by Aluminum chloride colorimetric method [22]. 1.0 ml sample (1 mg/ml) in 3 replicates, 1.0 ml of methanol, 0.5 ml of (1.2 %) Aluminum chloride and 0.5 ml (120 mM) potassium acetate were mixed and the resulting mixture was incubated for 30 min at room temperature. The absorbance of all the samples was measured at 415 nm using spectrophotometer (Systronics: 106). The calibration curve was prepared using quercetin (5 to 60 g/ml) solution in methanol. The flavonoidal content was expressed in terms of quercetin (SRL, India) equivalent (mg/g of extracted compound).

DPPH free radical scavenging assay The free radical scavenging activity of various solvent extracts of C. pulcherrima was measured by using 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) by the modified method of McCune and Johns [23]. The reaction mixture consisting of DPPH in methanol (0.3 mM, 1 ml) and different concentrations of the solvent extracts (1 ml) were incubated for 10 min. in dark, after which the absorbance was measured at 517 nm. Ascorbic acid (Hi Media, India) was used as positive control. The radical scavenging activity was expressed in terms of the amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (IC50). % DPPH radical scavenging = [(B A) / B] 100 Where B is the absorbance of the blank (DPPH plus methanol) and A is the absorbance of the sample (DPPH, methanol plus sample).

Antimicrobial Assay
Microorganisms tested The microbial strains used to assess the antimicrobial properties included six Gram positive, five Gram negative and four fungal strains (Table 4). The investigated microbial strains were obtained from National Chemical Laboratory (NCL), Pune, India. The microorganisms were maintained on nutrient agar/MGYP (Hi Media, India) slope at 4o C and sub-cultured before use. The microorganisms are clinically important ones causing several infections and it is essential to overcome them through some active therapeutic agents. Determination of the antimicrobial activity The antimicrobial assay of the extracts was carried out by agar well diffusion method [24, 25]. The media used for bacteria was Muller Hinton agar no. 2 (Hi Media, India) and for fungi it was Sabaroud dextrose agar (Hi Media, India). The tested drug was diluted in 100% DMSO at a concentration of 25 mg/ml and the antimicrobial activity was evaluated at a concentration of 2.5 mg/well. The agar was melted and cooled to 48 50oC and a standard inoculum (1.5 x 108 cfu/ml, 0.5 McFarland) was then added aseptically to the molten agar and poured into sterile Petri dishes to give a solid plate. Wells were prepared in the seeded agar plates. Each tested compound (100 l) was introduced in the well (8.5 mm). The plates were incubated at 37 C for bacteria and 28 C for fungi 24 h and 48 h respectively. The bacterial growth was determined by measuring the the diameter of inhibition zone. For each bacterial strain DMSO was used as negative control where pure solvents were used instead of the extract. The experiment was repeated three times to minimize the error and the mean values were presented. Commercial antibiotics (Hi Media, India) were used as positive control and were evaluated for their susceptibility against the microorganisms investigated. The antibiotics studied are given in Table 5. Determination of the Minimum Inhibition Concentration (MIC) The end point MIC is the lowest concentration of compounds at which the tested microorganisms does not demonstrate visible growth. The broth tube dilution method utilizes relatively large amounts of the test agent and is not suitable for high throughput screening. Therefore, agar well diffusion method was used in the present study [26] to determine the MIC of the methanol extract of the different parts of Caesalpinia pulcherrima. The solution of the extract was 2-fold serially diluted in DMSO, in the range of 10,000 78 g/ml. The MIC was defined as the lowest concentration at which visible zone of inhibition was observed.

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TABLE 1 Extractive yield (%) and Qualitative phytochemical analysis of C. pulcherrima Swartz Parts Used Extracts Hexane Chloroform Seeds Acetone Methanol Aqueous Hexane Chloroform Fruits rind Acetone Methanol Aqueous Extractive Yield (%) 2.97 2.35 1.02 7.00 7.00 0.66 0.82 2.22 13.27 4.74 Alkaloids Dra + + + May + +++ + + Wag + ++ + +++ ++ ++ + + ++ + + ++ +++ + +++ +++ +++ +++ + + ++ ++ +++ + + ++ + + + ++ +++ +++ +++ + + ++ +++ + + + + + + + + +++ + ++ ++ Fla Tan Phl Tri Ste Sap Car

Dra- Dragendroff reagent, May- Mayers reagent, Wag- Wagners reagent, Fla- Flavonoids, Tan-Tannins, Phl- Phlobatannins, Tri- Triterpene, Ste- Steroids, Sap- Saponins, Car- Cardiac glycosides

Results and Discussion


The presence of antibacterial substances in the higher plants is well established [27] and plants were also a good source for natural antioxidants [28]. Plants have provided a source of inspiration for novel drug compounds as plant derived medicines have made significant contribution towards human health. Phytomedicine can be used for the treatment of diseases caused by infectious diseases or those diseases caused by free radicals or it can be the base for the development of a medicine, a natural blueprint for the development of a drug. Extractive yield The extractive yield of the seeds and fruit rinds is given in Table 1. The extractive yield varied amongst the solvents used. In, both of the seeds and fruits rind, the methanol extracts showed highest extractive yield while the hexane and acetone extract of the fruits rind and the seeds respectively, showed minimum extractive yield. The extractive yield of the methanol extract of the fruits rind was 13.27% while that of the seeds was only 7%. Phytochemical analysis The hexane extract of the seeds showed only trace amount of triterpenes, cardiac glycosides and alkaloids (Wagners). The chloroform extract showed the presence of steroids and trace amount of tannins, triterpenes, cardiac glycosides and alkaloids (Dragendroff). The acetone extract showed the presence of flavonoids, tannins, triterpenes and trace amount of steroids, cardiac glycosides and alkaloids (Mayers) while the methanol extract exhibited the presence of all phytoconstituents except phlobatannins while the aqueous extract only contained phlobatannins and alkaloids (Dragendroff) in very trace amount. The hexane extract of the fruits rind showed the presence of flavonoids, tannins, triterpenes, cardiac glycosides, alkaloids (Mayers test) and steroids in very trace amount. Tannins, steroids, cardiac glycosides were present in the chloroform extract while phlobatannins, triterpenes and alkaloids (Mayers test) were present in very trace amount. The acetone extract showed the presence of all phytoconstituents except cardiac glycosides. The methanol extract showed the presence of all
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phytoconstituents except steroids (positive for Wagners test). Tannins, phlobatannins and triterpenes were present in aqueous extract while flavonoids and saponins were present in very trace amount (Table 1). Physico-chemical analysis Physico-chemical (proximate) parameters may enhance the efficacy of the crude powder against various ailments. Crude powder of the seeds and fruits rind of C. pulcherrima was subjected to various proximate parameters, the results of which are shown in Table 2. The fruits rind had the highest value of loss on drying (6.84%) indicated more moisture content. Water soluble ash and aqueous soluble extractive value were also the maximum in fruits rind (15.55 %). While total ash, petroleum ether soluble extractive yield and ethanol soluble extractive yield were the maximum in the seeds. The acid insoluble ash value was almost absent.
TABLE 2 Physico-chemical parameters of crude powder of C. pulcherrima Parameters Loss on drying Total Ash Water soluble ash Acid insoluble ash Petroleum ether soluble extractive value Ethanol soluble extractive value Aqueous soluble extractive value Plant parts Seeds Fruits rind Seeds Fruits rind Seeds Fruits rind Seeds Fruits rind Seeds Fruits rind Seeds Fruits rind Seeds Fruits rind % 5.37 6.84 4.25 3.65 2.35 2.78 0.05 3.73 0.35 9.75 8.54 11.39 15.55

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TABLE 3 Quantitative estimation of total phenols, flavonoids and DPPH free radical scavenging activity (IC50 value) of various extracts of seeds and fruits rind of C. pulcherrima Parameter Part used Hexane Seeds Total phenols (mg/g) Fruits rind Seeds Fruits rind Seeds Fruits rind ND 183.5 ND 7.5 ND 36 Chloroform 13.9 90.7 9.55 14.6 500 47 Extracts Acetone 230.3 624.6 6.7 9.8 20.5 15 Methanol 205.5 606.9 2.90 6.5 29 14 Aqueous 46.2 221.5 0.31 2.6 290 22

Flavonoids (mg/g)

DPPH (ug/ml)
ND = Not done

Total phenolic and flavonoidal content The antioxidant activity of the phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. In addition, they have a metal chelating potential [29]. Flavonoids are a group of naturally occurring compounds widely distributed, as secondary metabolites in the plant kingdom. These flavonoids have been reported to possess antioxidant and antiradical properties [30]. The total phenolic and flavonoidal content of the seeds and fruits rind are shown in Table 3, in which the extractive solvents of both parts revealed that, the total phenolic content was more than the flavonoidal content. Furthermore, the solvent extracts of the fruits rind had more phenolic content than that of the seeds; while the maximum content being in acetone extract, closely followed by methanol extract. The same trend was observed in the seeds extracts. The flavonoidal content also was more in the fruits rind as compared to that of the seeds, while maximum content being in the chloroform extract. A number of studies have focused on the biological activities of the phenolic compounds, which are the potential antioxidants and free radicalscavengers [29, 31]. In the present study, the acetone and the methanol extracts contained the maximum amount of phenolics. Hence this may be a good source for phenolic compounds. Many studies have shown that natural antioxidants in plants are closely related to their biofunctionalities such as the reduction of chronic diseases and inhibition of pathogenic bacterial growth, which are often associated with the termination of free radical propagation in biological systems [32]. The phenolic compounds are also thought to contribute directly to the antioxidant activity. Yen et al. [33] and Duh et al. [34] suggested a correlation between phenolic content and antioxidant activity. DPPH free radical scavenging activity There are different methods for the estimation of the antioxidant activity. DPPH free radical is commonly used as a substrate to evaluate the antioxidant activity; it is a stable free radical that can accept an electron or hydrogen radical to become a stable molecule. The DPPH scavenging activity of the different solvent extracts of the seeds and fruit rind was different (Table 3). Comparing the five solvent extracts
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of the seeds and fruits rind, revealed that, the scavenging activity was more in the fruits rind than the seeds extract. The IC50 value of acetone and methanol extract of the fruits rind (IC50 value - 15 and 14 g/ml respectively), was as good as that of the standard ascorbic acid (IC50 value - 11. 4 g/ml); the other 3 extracts also showed a low IC50 values. In the seeds extracts, only the acetone and the methanol extracts showed a low IC50 values. The acetone and the methanol extracts of the fruits rind showed the maximum phenolic content and also a good antioxidant activity. It is also appeared that the extracts having more phenolics exhibited greatest antioxidant activity suggesting that the high scavenging activity may be due to hydroxyl groups existing in the phenolic compounds and the chemical structure that can provide the necessary component as a radical scavenger. Similar results were reported by Nuutila et al. [35], Yen et al. [33] and Saravana Kumar et al., [36]. Antimicrobial activity The antimicrobial activity of various solvent extracts of the seeds and the fruits rind of C. pulcherrima against six Gram positive, five Gram negative and four fungal strains are shown in Table 4 and the antimicrobial activity of these organism with different antibiotics is shown in Table 5. Amongst the four solvents, the acetone and the methanol extracts of both the seeds and the fruits rind showed better antimicrobial activity. The Gram positive bacteria were more susceptible than Gram negative bacteria. All the solvents extracts of both the seeds and the fruits rind showed a feeble anti-fungal activity. The most susceptible bacteria was M. flavus. From the above results, it can be stated that irrespective of the part and the solvent used, C. pulcherrima showed better antibacterial activity than antifungal activity. The antimicrobial activity of the fifteen studied strains against standard antibiotics is shown in Table 5. The antimicrobial activity of some of the solvent extracts was comparable with that of the standard antibiotics. From the results of antimicrobial activity, the most active extract was subjected to the evaluation of minimum inhibitory concentration (MIC). The minimum inhibitory concentration of the methanol extract
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TABLE 4 Antimicrobial activity of various solvent extracts of seeds and fruits rind of C. pulcherrima Part used Extracts Se Hexane Seeds Chloroform Acetone Methanol Hexane Fruits rind Chloroform Acetone Methanol 18 16 15 14 18 18 Zone of inhibition (mm) Gram positive bacteria Bc 10 10 14 13 13 14 18 18 Bs 10 19 13 14 13 18 17 Sa 12 10 17 16 16 16 19 20 Mf 11 17 23 21 20 20 22 21 Cr 16 15 15 15 17 18 Kp 15 20 19 18 18 18 18 17 Gram negative bacteria Pv 12 16 16 17 17 17 16 St 12 12 12 12 11 11 Ea 11 11 15 13 13 12 11 11 Pa 11 16 15 14 14 20 19 Ct 13 11 Fungus Ca 12 11 12 14 Tb 10 10 10 11 11 Cn 12 13 11 11 10 11 11 11

Se - Staphylococcus epidermidis ATCC12228; Bc - Bacillus cereus ATCC11778; Bs - Bacillus subtilis ATCC6633; Sa - Staphylococcus aureus ATCC 29737; Mf - Micrococcus flavus ATCC10240; Cr - Corynebacterium rubrum ATCC14898; Kp - Klebsiella pneumoniae NCIM2719; Pv - Proteus vulgaris NCTC8313; St - Salmonella typhimurium ATCC23564; Ea - Enterobacter aerogenes ATCC13048; Pa - Pseudomonas aeruginosa ATCC27853; Ct - Candida tropicalis ATCC4563; Ca - Candida albicans ATCC2091; Tb - Trichosporon beigelii NCIM3404; Cn - Cryptococcus neoformans NCIM3542

TABLE 5 Antimicrobial activity using standard antibiotics Microorganisms S. epidermidis B. cereus B. subtilis S. aureus M. flavus C. rubrum K. pneumoniae P. vulgaris S. typhimurium E. aerogenes P. aeruginosa C. tropicalis C. albicans T. beigelli C. neoformans Antibiotics Cb 17 10 20 22 32 22 32 11 22 18 22 ND ND ND ND I 39 36 37 25 37 24 35 16 16 19 26 ND ND ND ND Ca 25 18 25 19 24 20 17 15 18 16 16 ND ND ND ND Cf 24 19 30 14 22 17 25 19 15 12 29 ND ND ND ND Cj 22 20 35 21 30 28 28 17 ND ND ND ND Ak 17 12 15 14 21 17 22 11 15 17 22 ND ND ND ND T 25 17 21 19 27 20 30 15 18 11 10 ND ND ND ND Pc 11 17 17 22 28 19 27 16 14 12 18 ND ND ND ND M 18 16 16 10 16 ND ND ND ND C 24 10 16 15 27 15 36 18 16 13 13 ND ND ND ND At 14 11 14 16 28 15 33 14 12 14 ND ND ND ND Fu ND ND ND ND ND ND ND ND ND ND ND 27 Ny ND ND ND ND ND ND ND ND ND ND ND 19 18 17 21 Ap ND ND ND ND ND ND ND ND ND ND ND 14 13 15 17 Kt ND ND ND ND ND ND ND ND ND ND ND 20 24

ND = Not done; - means no zone of inhibition observed, Cb = Carbenicillin; I = Imipenem; Ca = Ceftazidime; Cf = Ciprofloxacin; Cj = Cefaclor; Ak = Amikacin, T = Tetracycline; Pc = Piperacillin; M = Methicillin; C = Chloramphenicol; At = Azithromycin, Fu = Fluconazole; Ns = Nystatin; Ap = Amphotericin- B; Kt = Ketoconazole
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of the seeds and the fruits rind of C. pulcherrima is shown in Table 6. The MIC of C. pulcherrima ranged from 10,000 78 g/ml. In the seeds and the fruits rind extracts, K. pneumoniae showed the least MIC value (< 78 g/ml) while C. albicans showed MIC value > 10,000 g/ml.

Conclusion
Plant based antimicrobials have enormous therapeutic potential, they can serve the purpose without any side effects that are often associated with the synthetic antimicrobials. It is hoped that this study will lead to the establishment of some results, that could be used for further investigation to isolate the active compounds from the promising extracts, which could be used to formulate new and more potent antibacterial and antioxidant drugs from natural origin.

TABLE 6 Results of Minimum Inhibitory Concentration of methanol extract of C. pulcherrima MIC (g/ml) Seeds 312 2,500 1,250 5,000 5,000 < 78 312 5,000 5,000 2,500 10,000 5,000 1,250 Fruits rind 156 625 1,250 1,250 1,250 < 78 312 78 1,250 1,250 10,000 2,500 10,000

Acknowledgement
One of the authors Yogesh Baravalia is thankful to University Grants Commission, New Delhi, India for providing financial support as Junior Research Fellow.

Microorganisms S. epidermidis B. cereus B. subtilis S. aureus C. rubrum K. pneumoniae P. vulgaris S. typhimurium P. aeruginosa C. tropicalis C. albicans T. beigelli C. neoformans

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