Role Of Nested Polymerase Chain Reaction (PCR) In The Clinico-Epidemiological Diagnosis Of Malaria In Nepal

Abstract

Malaria poses a diagnostic challenge to laboratories of both developed and developing countries. Microscopic examination of Giemsa stained blood smears is the most commonly used method of malaria diagnosis in Nepal with recent introduction of Quantitative Buffy Coat (QBC) at B.P. Koirala Institute of Health Sciences (BPKIHS). Eighty-five randomly selected samples among 3182 patients referred to Clinical Laboratory Services (CLS) at BPKIHS for QBC malaria testing from June 18, 2002 to July 17, 2003 were analyzed by QBC malaria test, Giemsa stained thin smear microscopy and nested PCR. Out of 85 samples, 48 (56.47%), 17 (20.00%) and 24 (28.24% ) were positive by malaria QBC test, microscopy and PCR, respectively. Among 24 Plasmodium genus specific PCR positive samples, 12 (50.00%) were P. falciparum, 7 (29.16%) were P. vivax, 4 (25.00%) were mixed infection with P. falciparum, and P. vivax. One sample (4.17%) was positive for malaria by genus specific PCR but was negative for all species specific PCR. Three P.falciparum and one P. vivax microscopic positive samples were found to have mixed infection with both P.falciparum and P. vivax by PCR. Although QBC was able to pick up all microscopy positive cases, a large numbers of QBC positive cases were negative by both microscopy and PCR suggesting that QBC may have a high false positive rate. The results of this study indicated that malaria was seasonal in Nepal, was much more common among males and maximum numbers of malaria positive cases were found in the age group of 15-30 years. As expected, PCR detected much more cases than the microscopy technique and more importantly many more mixed infections were identified by the PCR in this study. However, the nested PCR was a very labor intensive procedure. Since, the existing microscopy has low sensitivity, and the QBC has very high false positive rate, PCR would be the most reliable alternative for clinical and epidemiological diagnosis of malaria in Nepal. However, its routine use in the clinical laboratory in Nepal in the present form does not appear to be feasible because of its high cost, long processing time and requirement to run several PCR cycles for each samples. However, it could be a valuable tool for quality control as it can be used in selective samples to verify the microscopy or QBC test results.