Asfaw Debella, Dawit Abebe, Frew Tekabe, Hailu Mamo, Almaz Abebe, Bekure Tsegaye, Gonfa Ayana, Ambaye

Degefa, Paulos Negussie, Eshetu Yimer, Feyessa Challa, Eshetu Lemma, Alemtsehaye Tefera, Yared Mekonnen, Negede Afework, Kissie Mudie, Ashinef Tadele, Tsegaye Kidanemariam, Berhanu Muchie, Negussie Dadi, 2011. Ethiop Med J, Vol. 49,Supplement 1

TOXICITY STUDY AND EVALUATION OF BIOCHEMICAL MARKERS TOWARDS THE IDENTIFICATION OF THE CAUSATIVE AGENT FOR AN OUTBREAK OF LIVER DISEASE IN TAHTAY KORARO WOREDA, TIGRAY
Asfaw Debella1 , Dawit Abebe1 , Frew Tekabe, Hailu Mamo2, Almaz Abebe1 , Bekure Tsegaye1, Gonfa Ayana1, Ambaye Degefa1 , Paulos Negussie1 , Eshetu Yimer1, Feyessa Challa1, Eshetu Lemma1, Alemtsehaye Tefera, Yared Mekonnen1, Negede Afework1,Kissie Mudie1 , Ashinef Tadele,1 Tsegaye Kidanemariam1, Berhanu Muchie2, Negussie Dadi2,

ABSTRACT
Background: A team of experts of the Faculty of Medicine, Addis Ababa University reported the emergence of unidentified fatal liver disease in Tahtay Koraro Woreda, Tigray in the mid of December 2005. The EHNRI has been then instructed to investigate the possible etiological agent that are likely to be responsible in triggering the health problem and a field survey team consisting of experts were went to the affected area to investigate the situations surrounding the disease. Objectives: This investigation was conducted to determine the possible etiological agent(s) for the stated health problem in the affected village. Method: Acute toxicity study was performed on animal model for the various samples used in human consumption, which was followed by histopathological examination of the liver of the sacrificed laboratory animals. In order to facilitate the elucidation of the causative agent for the alleged health problem further tests for clinical markers and antigens were also performed on the serum collected from affected persons. Result: Neither death nor toxic symptoms manifestations were observed on laboratory animals when feeding the consumable samples for a period of two weeks, however histopathological examination of the liver of the sacrificed animals that were given the unprotected pond water and Tela samples from the affected village as a drink revealed severe hepatoic necrosis. Biochemical test results of the serum samples revealed raised level of some clinical markers that are highly significant for detecting liver abnormality of toxic origin. Serological test for surface antigen ruled out the possible causes of infectious origin such as viral hepatitis. Conclusion: The overall results confirmed that the causative agent for the outbreak of the liver disease was of toxic origin rather than due to infectious agent and this was found to be associated with consumption of contaminated water as well as Tela. Key words: Liver disease, etiological agent, acute toxicity, histopathogy, clinical markers

INTRODUCTION
A fatal liver disease with unidentified etiological agent having a characteristic symptoms of abdominal cramp, febrile syndrome, jaundice, bleeding tendencies and edematous states was reported from Tsaeda Emba village of Tahtay Kararo District, Tigray by a team of experts from the Medical Faculty of Addis Ababa University (1). Based on the clinical and epidemiological investigation conducted by the expert group, it was suggested to be a fatal toxic hepatitis. Other possible causes like viral hepatitis, leshmaniasis and schistosomiasis were ruled out by appropri1 2

ate investigations. Autopsy studies from a patient who succumbed due to this disease revealed venoocclusive liver disease and intravenous thrombosis suggesting toxic hepatitis (2). Because of liver's unique function in processing chemicals and metabolic products that enter the blood stream a number of predisposing factors contributes for liver disease. Many of the undesired chemicals are difficult for the kidneys to excrete out of the body unless they are processed by the liver. In due process of metabolism, unstable toxic products may be produced, which can attack and injure the liver. Exposure to a certain amount of offending

Ethiopian Health and Nutrition Research Institute, Addis Ababa, Ethiopia, P. O. Box 1242, Addis Ababa. Drug Administration and Control Authority, Quality Control and Toxicology Laboratory, Addis Ababa

chemical substance of organic or inorganic origin at any time can cause liver damage, in some cases, there may be rapid improvement of the injured liver after the exposure is discontinued (3). In addition to toxic chemical metabolites, viral hepatitis also cause considerable illness or death in the human population due to acute infection of the liver, liver cirrhosis and cancer (4). Hepatic failure is also a seldom cause of death due to leptospirosis infection, a zoonotic disease that is transmitted by ingestion of food and water contaminated by the urine of infected vector or through the skin lesions or mucous membranes exposed to contaminated water or soil (5). A number of tests can be employed to determine liver abnormality, liver function tests (LFT) is one of the techniques used to assess injury to the liver rather than hepatic function. Although, blood test may reflect problems arising outside the liver, such as post hepatic hemolysis (elevated bilirubin level) or bone disease (elevated alkaline phosphatase). Often abnormal LFT indicate that something is wrong with the liver, as well as provide clues to the nature of the problem. In contrast, normal LFT do not necessarily tell that the liver is normal because patients with cirrhosis, end stage liver and bleeding esophageal varices can have normal LFT. Of the routine LFT, only serum albumin, bilirubin and prothrombin time (PT) provide useful information on how well the liver is functioning (6). The most commonly used markers of hepatocyte injury are aspartate aminotransferase (AST), formerly serum glutamic-oxaloacetic transaminase (SGOT) and alanine aminotransferase (ALT), formerly serum glutamate-pyruvate transaminase (SGPT). In hepatitis C, liver cell death occurs by apoptosis (programmed cell death) as well as by necrosis. Hepatocytes dying by apoptosis presumably synthesize less AST and ALT as they wither away. That is why at least one third of patients infected with hepatitis C virus have persistently normal serum ALT levels despite the presence of inflammation on liver biopsy. Patients with cirrhosis often have normal or only slightly elevated serum AST and ALT levels. Thus, AST and ALT lack some sensitivity in detecting chronic liver injury (7, 8). Toxic hepatitis caused by toxic chemical metabolites can cause abnormalities in certain clinical markers, the most significant clinical laboratory abnormalities are elevations in gamma-glutamyl transferase, alkaline phosphatase, aspartate aminotransferase activi-

ties, and an increase in bile acid concentration. Hyperbilirubinemia, hypoproteinemia, hyperammonemia, an inflammatory leukogram are also commonly seen (9). In view of the aforementioned facts of hepatic abnormality and the reported health problem association with the liver, this study was initiated to facilitate the elucidation of the causative agent(s) that is most likely to be responsible to the outbreak of liver disease in the affected village. The correlation of the major symptoms characterized in the affected village with chemical intoxicants and the localized nature of the problem highlights the need to focus more on toxicological studies on the consumable samples collected from the affected village and, for comparison, in a nearby village where there was no report of the problem. In addition, tests for the levels of biochemical markers that facilitate the determination of liver abnormality particularly toxic hepatitis were also performed on the blood samples collected from affected persons. It is hoped that the findings of this investigation will facilitate an immediate intervention to control the outbreak in the affected village and prevent the occurrence of similar episode in other areas.

MATERIAL AND METHODS

Sample collection for laboratory analysis: Blood samples from 59 patients and samples of cooked meal, mainly Injera were collected from Tsada Amba village. In addition to these water, cereal samples of millet, sorghum, maize and Teff, and Tela samples were also collected from Tsaeda Emba and Jek Emba villages (case and control villages), respectively. The collected samples were stored and transported to Addis Ababa using cool box containing icepack. All the samples were used for various analysis. Acute toxicity studies: Fifty five adult male and female albino mice weighing 25 to 30g were used for the experiment. All animals were housed in standard cages in uniform lighting (12 hrs. dark and 12 hrs. light cycle) at room temperature. They were deprived of food for 24 hours while giving water ad libitum before the commencement of the experiment. Cereal flours, water and Tela (distilled or freeze dried to remove the alcohol and then dissolved in distilled

water during testing and normal un-distilled Tela was also used during testing) samples collected from the case and control villages were then fed or given as a drink (for water) or administered via oral gavage (for Tela) to ten groups of laboratory mice. The 11th group was used as a control under normal feeding condition (1 group = 5 males and 5 females mice). The mice were then continuously observed until toxic signs or death occurred for a period of 24 hours. In cases where no death occurred the observation was continued for 15 more days. After 15 days, two mice from each group were randomly taken and sacrificed to examine for possible histopathological change on their liver which was then followed by qualitative detection of pyrrole, the toxic metabolite (s) of PA through chemical analysis (6, 10, 13). Gross morphologic and histopathologic evaluation of the liver: The animals were sacrificed by strangulation and laparatomized. The liver is then taken out and seen for any gross abnormality, weighed and fixed in 10% neutral buffered formalin solution and kept for histopathological evaluation. Serological and Biochemical tests: Tests for clinical markers (GOT, GPT, ALP, GGT, TBilli, LDH, Amylase, Glucose, Creatinine, Urea, Total protein, Albumin, CEA, Alfa feto protein, CA19-9, Ferritin) on serum were performed using 17 different types of kits obtained from Rouche diagnostics, Germany; Bicon, Germany and Human, Germany. Tests for leptospirosis was performed using LeptoTek-Dridot kit Biomerieux bv, Netherlands. Surface antigen test for HBV on sera was performed using Hepanostika HBsAg Uni-Form II, kits Biomerieux bv, Netherlands.

end of 2nd weeks of feeding (or giving as a drink) revealed severe hepatic necrosis for distilled Tela and mild necrosis for the water from unprotected pond and the well that is fitted with a hand pump at Tsaeda Emba village ( histological slides are not available to be included ). Qualitative chemical analysis of the necrotic liver of the mice (mice experimental group that were given the water and Tela samples collected from case village as a drink) also revealed the presence pyrrole which is the toxic metabolite of pyrrolizidine alkaloid. Nevertheless, no liver abnormality was detected on the liver of the sacrificed mice that were initially fed on the grain samples of both the affected and control areas and the water samples from the control village (Jek Emba) that was given as a drink (Table 1). Serological and biochemical tests: Biochemical test results of the blood samples collected from sick persons presented themselves for examination at Tsaeda Emba village revealed raised alkaline phosphatase (ALP) level in most of the cases, i.e. indisproportionately to GOT (23 cases, 39%) and GPT (3 cases, 5%). Increased levels of -glutamyl transferase (GGT), carinoembryonic antigen (CEA), lactate dehydrogenase (LDH) and total biluribin were observed in 11 (19%), 12 (20%), 18 (31%)and 14 (24%) patients, respectively. The elevation of amylase in 34 cases (58%) also indicates the involvement of pancreatic problem in addition to the liver. The high Tela intake in the locality may also contributes to the raised level of amylase. Whereas elevation of Alfa Feto Protein (AFP), ferritin, total protein, direct biluribin and CA 19-9 that possibly indicate the development of hepatoma were not observed in the majority of cases (Table 2). Serological tests for Hepatitis B virus surface antigen (HBsAg) were positive in 16 cases out of 59 patients (Table 2). The higher percentage of HBsAg (27%) positivity in the tested samples is found to be coupled with elevated level of some clinical markers indicate the progression of the liver disease. Three samples were also found to be positive to leptospirosis antigen out of the 28 tested sera samples (Table 2).

RESULTS
Findings from acute toxicity studies: Toxicity studies of the water, Tela and cereal flour samples collected from the case and control villages were performed in laboratory mice in order to simulate the consequent symptomatology and determine possible lethality. The result revealed no death to mice nor manifestation of toxic symptoms (except for the distilled or freeze dried brew Tela from the case area) for testing periods of 2 weeks (Table 1). However, histopathological examination of the liver of the sacrificed mice carried by a pathologist at the

Table 1. Acute toxicity of mice fed on samples and pathological results of the scarified mice Mice (1 group = 10 mice, 5 male and 5 female) Group 1 Group 2 Group 3 Group 4 Group 5 Toxic effects and % lethality in 2 weeks period Toxic signs No No No No No % death Nil Nil Nil Nil Nil

Collection site

Pathological findings

Rem

Case area Control area Well fitted with a hand pump, case area Unprotected pond, case area Well fitted with a hand pump, control area Unprotected spring, control area Distilled (or lyophilized), case area (residue in H2O Un-distilled, case area Distilled (or lyophilized), control area (residue in H2O Un-distilled, control area Normal feed

Normal Normal Vascular edema, congestion Mild hepatic necrosis Vascular edema, congestion Mild hepatic necrosis Normal

PA metab po

Group 6

No

Nil

Normal

Group 7

Depressed, anorexic, decreased locomotor activity No No

Nil

Vascular edema, congestion Severe hepatic necrosis

PA meta t

Group 8 Group 9

No No

Normal Normal

Group 10 Group 11

No No

No No

Normal Normal

Note: 2 mice from each group that were randomly selected were sacrificed after 2 weeks feeding period for histopathological examination and test for pyrrole (pyrrolizidine alkaloid metabolite)

Table 2 Serological and biochemical tests result data

Tests GPT 9 21 13 21 11 44 33 19 21 23 9 63 13 34 22 34 23 27 15 33 18 16 ALP 254 322 454 339 698 683 886 726 705 349 720 295 497 247 261 789 811 848 337 815 931 221 276 GGT 16 131 73 41 114 30 75 48 60 23 88 45 172 60 76 47 26 18 76 155 88 34 41 T Bili 0.63 0.4 0.72 0.89 0.78 0.77 0.38 0.46 0.33 0.44 0.98 2.02 1.12 1.14 0.82 0.62 0.64 0.96 0.96 0.89 1.37 1.12 D Bili 0.56 0.18 0.53 0.24 0.49 0.24 0.04 0.02 0.41 0.23 0.31 0.97 0.48 0.26 0.11 0.19 0.25 0.08 0.27 0.51 0.30 0.31 LDH 488 922 1202 476 606 518 796 775 496 565 1949 304 1567 538 509 689 690 567 980 438 520 460 472 Amyl 230 296 170 294 132 233 544 166 266 212 327 206 119 234 566 1694 221 266 255 194 381 166 344 Glu 59 97 87 87 58 63 63 49 67 84 61 80 82 61 65 74 61 57 62 83 59 58 69 Crea 0.8 0.87 0.93 0.79 0.59 0.63 0.63 0.67 0.64 0.49 0.65 0.9 0.82 0.88 0.86 0.44 0.65 0.68 1.04 0.83 0.74 0.91 0.91 Urea 18.9 34.6 35.3 32.9 26.1 10.5 20.7 23.2 17.5 14.5 22.4 28.9 38.7 33.8 25.2 30.8 23.3 32.0 28.9 39.1 32.5 23.6 32.2 TProt 7.3 6.0 7.4 7.7 7.0 6.3 7.8 7.7 7.9 5.6 7.3 8 9.3 7.5 8.6 6.5 6.6 7.0 7.9 7.6 6.3 7.7 6.9 Alb 5.1 3.6 4.2 5.0 4.8 3.5 5.3 5.4 4.8 3.9 4.1 3.5 4.2 4.4 5.4 5.1 4.6 4.7 4.1 3.8 3.8 4.5 4.2 CEA 4.77 2.7 2.84 1.3 2.42 1.32 2.91 2.96 4.15 1.89 2.32 2.55 2.41 2.55 1.96 3.21 1.97 1.99 2.79 0.721 6.13 1.33 3.5 AFP 1.21 <0.5 0.89 1.38 <0.5 0.52 <0.5 1.27 0.55 <0.5 <0.5 1.6 1.07 2.45 1.45 0.52 <0.5 0.57 1.45 <0.5 <0.5 <0.5 1.66 CA 19-9 13.77 8.3 26 5.09 15.69 10 115.8 21.35 10.03 13.40 <0.6 5.67 12.66 3.95 13.60 10.3 9.57 5.62 2.38 1.05 10.54 5.88 4.85 Ferrit 45.55 163.9 4.84 6.55 8.87 16.9 64.5 46.4 7.88 1.41 21.92 292.7 292.4 142.6 114 15.66 24.45 27.07 305.4 80.87 111.5 74.83 132.9 Pos Pos

HBs Ag

ntinued From Table 2

Tests GO T GPT ALP GGT T Bili D Bili LDH Amyl Glu Crea Urea TProt Alb CEA AFP CA 19-9 Ferrit HBsA g

.N

25 20 27 36 41 31 20 28 61 51 44 23 24 23 71 34 45 61 18 22 28 28 33

7 5 17 22 9 15 13 13 20 20 13 11 10 12 21 20 41 35 17 21 5 17 10

197 184 574 695 223 643 819 225 464 613 736 134 183 193 1298 723 561 422 92 909 178 840 486

26 19 33 17 23 24 53 20 19 27 33 60 19 57 234 190 250 94 17 21 24 21 14

1.20 1.16 1.04 0.79 0.71 0.45 0.67 0.43 0.97 0.37 1.04 1.46 0.95 0.57 3.18 1.62 1.62 1.27 1.47 1.02 1.40 1.18 1.11

0.98 0.39 0.74 0.54 0.12 0.21 0.33 0.13 0.43 0.42 0.61 0.13 0.01 0.27 1.57 0.69 0.27 0.21 0.44 0.50 0.90 0.58 0.45

577 458 566 653 372 552 520 388 939 718 651 411 376 613 657 500 675 646 377 569 446 683 717

108 156 180 155 164 222 230 349 269 357 218 267 153 107 215 479 260 290 174 263 218 162 349

80 86 49 61 53 50 60 54 70 68 58 62 46 67 69 164 68 65 80 66 71 80 83

0.83 0.67 0.59 0.60 0.84 0.63 0.67 0.81 0.66 0.60 0.45 0.72 0.70 0.88 0.68 0.94 0.52 0.60 0.70 0.69 0.81 0.68 0.66

26.2 17.9 22.3 27.7 30.2 22.2 27.2 22.9 20.5 16.1 8.0 15.7 19.3 29.3 14.5 28.0 31.0 25.6 25.5 28.0 47.5 27.7 30.2

6.6 6.6 6.7 6.7 7.7 6.7 7.8 7.7 7.3 7.0 6.8 7.0 6.2 6.4 6.6 7.6 7.3 7.1 7.3 7.5 7.6 8.0 7.3

4.6 4.3 4.4 4.8 5 4.5 5 4.5 5.1 4.7 5.0 4.6 4.6 3.6 3.5 4.7 5.3 4.8 5.0 5.1 5.1 5.8 5.0

2.53 3.79 1.71 3.84 2.95 1.56 4.37 3.13 3.67 1.53 2.49 3.23 3.68 2.47 3.91 4.51 2.84 4.54 4.05 2.88 3.61 3.16 1.83

1.29 1.46 <0.5 <0.5 1.06 <0.5 0.71 2.36 2.91 <0.5 0.93 3.29 0.97 0.90 1.37 1.77 1.07 <0.5 1.92 0.94 206.4 3.31 <0.5

10.58 38.02 <0.6 <0.6 21.37 3.48 11.47 19.60 23.42 5.26 50.67 36.54 14.0 <0.6 8.95 7.79 9.63 <0.6 6.93 0.6 9.53 39.82 5.27

41.73 65.06 1.86 22.94 39.96 3.20 30.14 20.87 23.66 12.31 2.70 165.8 85.7 204.1 134.0 78.07 48.39 85.61 48.21 47.58 123.3 43.29 43.88 Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

ntinued From Table 2 G OT 29 26 40 74 55 27 32 68 41 20 37 17 26 GPT 9 8 14 32 27 12 9 21 12 5 15 1 10 ALP 216 265 264 1180 301 666 360 229 122 130 156 496 221 GGT 17 65 20 502 93 23 22 100 46 74 27 25 41 T Bili 0.98 1.94 1.98 1.51 1.18 1.7 1.51 1.94 2.60 1.81 1.93 1.71 1.82 D Bili 0.32 0.85 0.64 0.53 0.53 0.65 0.80 0.69 0.76 1.02 1.07 0.86 1.04 LDH 485 361 446 795 556 620 457 1072 891 301 653 342 384 Amyl 301 287 270 421 274 319 260 263 256 148 399 156 231 Glu 62 52 61 66 65 74 50 124 100 73 69 78 67 Tests Crea Urea 0.69 0.97 1.12 0.70 1.04 0.82 0.87 1.09 1.12 0.67 0.98 0.59 0.94 35.5 15.7 19.3 29.3 14.5 30.8 27.0 27.8 24.9 10.2 19.9 6.2 15.4 TProt 7.9 7.0 6.2 6.4 6.6 7.6 7.3 8.9 8.6 7.0 9.8 6.5 7.8 Alb 4.9 5.5 5.8 4.0 4.9 5.5 4.8 3.1 5.6 4.0 4.1 4.5 5.2 CEA 3.38 3.20 3.52 4.14 4.77 5.25 6.87 2.27 1.82 2.99 3.00 3.39 2.35 AFP 2.21 0.92 1.70 <0.5 1.06 2.41 11.82 1.16 2.15 0.99 2.05 <0.5 2.4 CA 199 2.27 5.36 3.14 34.57 18.27 6.66 4.42 4.82 5.75 2.08 9.60 <0.6 <0.6 Ferrit 49.09 77.48 160.2 43.31 90.96 18.69 49.83 879.4 582.2 161.7 735.5 38.51 137.1 HBs Ag

Li pto

Pos Pos Pos Pos

Normal value ranges for biochemical markers (source kits leaflets)

PARAMETER T.PROTEIN ALBUMIN CEA AFP CA 19-19 FERRITIN CHOLESTEROL UREA CREATININ

RAMETER @ C

NORMAL VALUE Men: up to 38 U/l ; Women: up to 32 U/l Men: up to 41 U/l ; Women: up to 31 U/l Men: 80-306 U/l ; Women: 64-306 U/l Men: 8-61 U/l ; Women: 5-36 U/l Adults up to 1.1 to 1mg/dl (18.8mmole/l) Adults up to 0.25 to 1mg/dl (4.3mmole/l) Adults 225-450 U/l < 220 U/l 75-115 mg/dl

OT-AST

S/ N 10 11 12 13 14 15 16 17 18

NORMAL VALUE Children from 3 years & adults: 6.6-8.7g/dl (66-87 g/l) 3.8-5.1 g/dl (38-51 g/l) < 3.4 < 5.8 < 39.0 Male: 30-400 ; Female: 13-150 Desirable cholesterol level: < 200 mg/dl; Borderline high cholesterol: < 200-239 mg/dl; High cholesterol: > 240 mg/dl Men: up to 38 U/l; Women: up to 32 U/l Men: 0.7-1.20 mg/dl (62-106mmole/l) Women: 0.50-0.90 mg/dl (44-80mmole/l)

T-ALT

GT

BILIRUBIN

BILIRUBIN

AMYLASE

UCOSE (Fasting)

DISCUSSION

Acute toxicity studies: :The absence of liver abnormality on the sacrificed mice or neither toxic manifestations nor lethality on mice group that were given the water and Tela collected from the control village as well as the cereal flour samples from the case and control villages revealed that these samples are devoid of intoxicants. The observation of severe hepatic necrosis and mild necrosis on the sacrificed mice from groups that were given distilled Tela, unprotected pond and the well that is fitted with a hand pump, respectively from the case village showed that these samples contained hepato toxic metabolites. The above experimental evidence suggested that, the PA originated from the weed somehow enters into the water to cause intoxication when consumed by the affected community. The observation of necrotic liver from the sacrificed mice that were initially given the hand pump water from the affected village (Tsaeda Emba) might be attributed due to leachable toxic substances that may originate from the unprotected pond, as both wells are located down hill and are in close proximity of about 3 to 4 meters. Serological and biochemical tests: The marked variation in the elevated values of the different clinical markers that determines the liver abnormality among patients indicates that the affected persons are in different stages of liver disease spectrum. Literature reports also indicate that elevation of -glutamyl transferase, alkaline phosphatase and aspartate transferase are good indicators for toxic liver injury by pyrrolizidine alkaloid toxicosis (9). The higher percentage of observed Hepatitis B virus surface antigen (HBsAg) in the tested samples may be attributed to either the existence of no report in the clinical symptoms for HBV infection or the unknown prevalence nature of HBV in the locality. The high percentage (27%) of HBsAg positive cases and the corresponding elevated level of some of the biochemical markers (ALP, LDH and Amylase) indicated that the development of hepatotoxicity is significantly higher in HBV co-infected patients compared to those without co-infection. Reports on the risk factor of HIV patients on hepatotoxic ART drugs regimen (e.g. retrovir (NRITs) and ritonavir (PIs)) co -infected with HBV also indicate that, the progression of liver disease is significantly higher due to hepatic cytolysis, accelerated liver scaring or cirrho-

sis. And patients die very fast as compared to those without HBV infection (11, 12). Though there is high percentage of HBsAg, in the tested sera of the patients the outbreak could not be attributed to HBV this is because the problem would have also spread to the adjacent villages rather than remain localized only to affected village. The positive indication of leptospirosis antigen might be attributed to the consumption of the water samples from the unprotected pond. Leptospirosis is a zoonotic disease of bacterial origin causing death due to hepatic failure (5). Since the well in Mai-habi-tselim is not protected, which means that there is free access to it by sick domestic or wild animals as well as rats that might contaminate it with their excreta. The occurrence of leptospirosis is therefore not also surprising as far as the unprotected pond is consumed by the community. Conclusion: On the basis of the report of venoocclusive disease from histopathological examination of the liver of a deceased patient from the affected village (Tseada Amba) by the expert group of Faculty of Medicine (AAU) complemented with the detection of toxic metabolites of pyrroloizidine alkaloid (pyrrole) in the necrotic liver of the sacrificed mice after the two weeks of laboratory animal feeding trials, biochemical test results and the sub-chronic and localized nature of the disease, the unidentified etiological agent of the liver disease in the affected village could be attributable to toxic substance rather than infectious agent. In order to contain the outbreak and possible emergence of such an outbreak in the affected village in the future the use of the unprotected pond and the hand pump fitted water sources should be barred and alternative safe water source should be sought to avoid further exposure to the intoxicants. Furthermore, active case detection and early warning system should be strengthened.

ACKNOWLEDGMENTS
We are very grateful to Federal Ministry of Health, Ethiopian Health and Nutrition Research Institute and Drug Administration and Control Authority for financial and technical support for the investigation. We express our deep appreciation to the community members of Tsaeda Emba and Jek Emba villages, members of the health office of Tahatay Koraro woreda and officials of the health bureau of Tigray Regional state. Our acknowledgment also goes to data collectors, FGD participants, key informants and others that were directly/indirectly involved in the investigation.

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