Malaria diagnosis by nested PCR

Abstract

In 1998, 256.697 malaria cases were confirmed in Colombia, 132,712 of which were caused by Plasmodium falciparum, 121. 161 by, and 2,824 mixed infections. Consequently, the Malaria Program of the National Reference Laboratory initiated an evaluation of the nested polymerase chain reaction (PCR) technique for malaria diagnosis. This technique amplifies variable sequences present in the 18s small subunit ribosomal RNA (ssrRNA) genes of P falciparum and P vivax. The infected blood samples were collected in Quibdó (Chocó) from 102 febrile patients who requested malaria diagnosis; the presence of parasites was established by both PCR and microscopical examination of thick blood smears. In order to evaluate the nested PCR, the following criteria were adopted: precision, accuracy, detection treshhold, sensitivity, specificity, positive and negative predictive values and Kappa index. General sensitivity was 100%, with a sensitivity for P vivax of 96.7% and for P falciparum of 100%. General specificity and specificity for both species wore 100%. The general positive predictive value was 90.0%, for P vivax 77.0% and for P falciparum 66.1%. The general negative predictive value was 90.3%. 90.6% for P vivax and 94.3% for P falciparum. The general concordance (or Kappa index) was 1 and the species concordance was 0.96. The detection treshhold of parasite DNA was 10 fg/ul. One sample diagnosed as P. vivax by microscopy was determined as a mixed infection of P falciparum and P vivax by nested PCR. Nested PCR can be employed as a diagnostic tool in the following roles: (1) a screening method for large scale epidemiological trials, (2) in evaluations of the malaria control program in Colombia, (3) in detection of early or low levels of persistent parasiternia, possibly indicative of drug-resistant malaria, and (4) as a reference test for planned diagnostic trials.