A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria

SC Parija, Rahul Dhodapkar, Subashini Elangovan, DR Chaya

Department of Microbiology, Jawaharlal Institute of Postgraduate, Medical Education and Research, Pondicherry

Introduction
Malaria presents a diagnostic challenge to the medical community worldwide. Its occurrence is noted in more than 90 countries.

It is estimated that there are more than 50 million cases and 1.1-2.2 million deaths due to malaria every year. In India, in the year 2005, there were approximately 1.8 million cases of malaria reported of which 44.5% were caused by Plasmodium falciparum.

 Due to the serious nature of P. falciparum infections, prompt and accurate diagnosis of the condition is essential for effective management.

Malarial parasite
Life cycle - In man (Schizogony) - In female anopheles mosquito (sporogony)

Clinical feature i) Fever 3 distinct stages The cold stage The hot stage The sweating stage

Anaemia occurs due to i) Destruction of parasitised and non- parasitised red blood cells ii) Hypersplenism iii) Autoimmune lysis of coated infected and uninfected RBC iv) increased fragility of RBC v) Decreased RBC production from bone- marrow supression. Splenomegaly

Complications of malaria More common due to P.falciparum (malignant malaria )  Cerebral malaria  Acute renal failure  Liver damage  Dehydration  Gastrointestinal symptoms  Blackwater fever  Hyperpyrexia  hypoglycemia

Laboratory diagnosis of malaria Peripheral blood smear- Thick smear 40 times sensitive than thin smear - can detect as few as 5 parasites/ l - Thin smear essential for species identification of malarial parasite Time of collection of sample- late in febrile paroxysm

Staining JSB stain, fields or geimsa stain

Rough estimate can be made by counting the number of parasites observed per thick film fields 1+ = 1-10 parasites per 100 thick film fields ++ = 11- 100 parasites per 100 thick film fields +++ = 1-10 parasites per thick film field ++++ = more than 10 parasites per thick film field.

QBC system Malarial parasites are concentrated by centifuging the blood in a special capillary tube. - The tube is coated with acridine orange and an anticoagulant - When examined by fluorescence microscopy, the acridine orange stained malarial parasite flouresce green yellow against a dark red back ground .

Kawamoto AO interference filter - Thick and thin films are stained by adding a drop of AO stain - The nuclei of the malarial parasite and white cells fluoresce bright green and cytoplasm of parasite fluoresces red.

Diagnosis using rapid immunologic strip tests - HRP 2 test ( Histidine rich protein 2) - produced by P.falciparum. - released from the parasitised cell into circulation includes i)ParaSight F test- sensitivity is reported as 84.2 96.6% ii) ICT Pf test - pLDH test (OptiMAL test) enzyme pLDH is produce by all human malarial parasite species during their growth in red cell

Materials and Methods

The study was conducted in Department of Microbiology, JIPMER, Pondicherry, a tertiary care institute in South India. Specimens were collected from patients presenting clinically with fever with chills and rigor and other suggestive symptoms of malaria.

 Sample collection Oral consent was taken from the patients prior to the collection of specimens. Approximately 5ml of venous blood was collected from each patient during the peak of fever and transported to the laboratory.

 Thick and thin blood smears Thick and thin blood smears were prepared as per the standard method described elsewhere. - The smears were stained with Leishman's stain. - The slides were examined by an experienced microscopist, and the average time spent on each slide varied depending on parasite density.

- Thick smears were reported negative after examination of 200-300 oil immersion fields (oif) with no parasites observed; - a thin smear was given negative when no parasites were observed in 200 oif.

 Quantitative buffy coat technique (QBC) - Quantitative buffy coat technique (BD Diagnostics) was employed for the detection of malarial parasites in blood. Specially designed microhematocrit tubes coated with acridine orange were provided by the manufacturer.

- Approximately, 55-60l of blood was loaded into the tubes and stopper and float were applied at either ends; the tubes were centrifuged at 12000RPM in a preprogrammed centrifuge as per the manufacturer's instructions

 The interpretation was done using a standard microscope fitted with Para Lens ultraviolet microscope adaptor and a 60 objective connected to fiber optic ultraviolet light module. The parasites were seen in buffy coat layer and the interface between RBC and WBC regions.

 Antigen detection using pLDH and aldolase - Commercially available antigen detection kit detecting plasmodium LDH and aldolase (Malariagen Pf/Pv Antigen Rapid test, Aspen Laboratories, India) were used. - The test was conducted using anticoagulated venous blood; the sample was added to the test strip using a calibrated dropper provided with the kit, and the strip was placed in a microwell containing buffer.

- The result was read after 15 min as per the manufacturer's instructions. - It was interpreted as positive for P. falciparum if T1 and T2 bands were seen along with control (C) band; if only T2 and C were seen, it was interpreted as positive for Plasmodium vivax.

Results
- A total of 411 samples were observed over the period of study. - Of these a total of 82 samples were positive by thick smear and 45 were positive by thin smear. - QBC was positive in 66 samples and 62 samples were positive by the antigen detection test .

Sensitivity

specificity

Positive predictive value 100

Negative predictive value 89

Thin smear

54.8

100

QBC

78.94

98

90

95

Antigen detection

75

100

100

94.2

- Total incidence of malaria was found to be 19.95% (82/411). - Most of the cases were detected to be due to P. falciparum ; there were only 2 cases due to P. vivax detected by thin smear and antigen detection.

Discussion
- Rapid detection and effective treatment is a pre-requisite for reducing the morbidity and mortality due to malaria. Leishman's or Giemsa stained thick smears are considered to be the 'Gold standard' in diagnosis.

- However, the interpretation of thick smears is laborious and results depend on the quality of microscope, staining, technique with which blood film is prepared and also the concentration and motivation of microscopist.

- Newer techniques like QBC and Antigen detection assays are rapid, simple and easy to interpret. - In the present study, they compared different methods available for rapid diagnosis with Leishman-stained thick smears. - The sensitivity of Leisman-stained thin smear was found to be lowest (54.8%); however, this method had a high specificity and positive predictive value (100%) .

- QBC, which utilizes the principle of acridine orange staining with differential centrifugation, had a sensitivity of 78.94%, specificity, PPV and NPV were found to be 98%, 90%, and 95%, respectively. Although the sensitivity of QBC has been reported to be as high as 99.7% by Benito et al , relatively low sensitivity of QBC was observed in their study.

- One of the reasons for this could be that as the hospital is present in an endemic region for malaria the levels of parasitemia could have been low.

- Despite this, the specificity of the test as yielded by this study was in concurrence with that of other observers. Although the test is proclaimed to be highly sensitive and easy to use, adjustment to the technique was found to be difficult by the first-time users who were involved in the study. Also the detection of parasites other than P. falciparum was not very sensitive as the two cases of P. vivax detected by thin smear and Malarigen were found to be negative by QBC

- There were six cases found to be positive by QBC, which were subsequently found to be negative for thick and thin smear and Malarigen test. - This can be explained by the fact that certain artifacts in blood like Howell Jolly bodies or platelet fragments might resemble the ring forms of P. falciparum.

- Malarigen for detection of malaria antigen had a sensitivity, specificity and PPV of 75%, 100% and 100%, respectively. - This test was based on detection of pLDH and aldolase with the help of monoclonal antibodies. The values obtained for this kit-based procedure were much lower than those observed for other tests based on similar principle.

- This low sensitivity could be attributed to low parasitemia levels as observed by Iqbal et al . who observed 75% sensitivity at parasitemia < 100/l. - The specificity was comparable to other observers using the tests based on similar principle. However, the test was found to be user friendly and interpretation was more objective as compared to smear and QBC.

Conclusion
Since malaria is endemic in certain regions of India, we need to employ more sensitive tests, which are also rapid to detect low levels of parasitemia in population. Therefore, they recommend QBC to be used in setups where appropriate facilities are available; however, in situations where adequate laboratory back up is not available, simpler and easy to use techniques like antigen detection can be employed despite having lower sensitivity.