Plasmodium falciparum and P. vivax: factors affecting sensitivity and specificity of PCR-based diagnosis of malaria.

We have previously reported on development of a simple PCR-based method to detect Plasmodium falciparum in which patient blood samples are lysed and then filtered onto paper. The present studies, conducted in Thailand, were designed to identify factors contributing to differences between results from microscopy and PCR. To analyze microscope-positive, PCR-negative results, we demonstrated that using the lysis/filtration sample preparation method, target DNA degradation is not a significant factor. Similarly, we showed that sensitivity of PCR among patient samples did not differ using the lysis centrifugation method or organically extracted DNA. We further demonstrated that 7/13 samples which were negative by PCR for P. falciparum were positive by PCR when P. vivax-specific primers were used. Microscope-negative, P. falciparum PCR-positive samples were analyzed in two ways: the true rate of false-positivity for PCR (2%) was established by analyzing 498 samples from patients living in areas without transmission. We further showed that when microscope-negative, PCR-positive samples were amplified using an independent P. falciparum-specific PCR target sequence, 42/47 were PCR-positive. We conclude that the accuracy and reduced limit of detection of microscopy are major confounders when comparing this method to PCR.

DNA probes as epidemiological tools for surveillance of Plasmodium falciparum malaria in Thailand.

We have previously reported on development of a DNA probe-based method for diagnosing Plasmodium falciparum infection directly from patient blood samples. In the present studies, we sought to examine applicability of the method to large epidemiological surveys, comparing sensitivity, specificity, time required to obtain results, and costs with those of conventional microscopic examination. Results of DNA probe hybridization were also compared between laboratories in the US and Thailand, to assess transferability of the DNA probe technology. Five separate surveys of approximately 5000 villagers each were performed between December 1987 and June 1989 (26,176 samples total). Sensitivity ranged from 61% to 92% for both US and Thai laboratories, while specificity ranged from 98.2% to 99.9%. Agreement between the US and Thai laboratories was good, with kappa coefficients between 0.62 and 0.78 for different surveys. Between 4 and 8 person-days were required to obtain results from each set of 5000 samples by DNA hybridization, whereas microscopic examination required 150 person-days. Approximate costs were US 0.17 per sample for DNA probe analysis, and US$0.36 for microscopic examination. We conclude that the DNA probe method offers significant advantages when large numbers of samples must be surveyed for P. falciparum.

A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction.

We have developed a simple method for treating blood samples permitting direct detection of Plasmodium falciparum parasites using the P. falciparum-specific DNA probe pPF14 after polymerase chain reaction (PCR) amplification of target DNA sequences, and have compared this method with microscopic examination of thick blood smears. For PCR amplification, blood samples were lysed, then filtered onto filter paper. After drying, a piece of the filter paper was added directly to the PCR mixture for amplification. The presence of PCR products was detected using nonisotopically labeled probe. This method permits detection of less than than 10 parasites in a 20-microliters sample, and minimizes the effects of PCR inhibitors generally found in blood. Samples were collected from patients presenting at malaria clinics in Mae Sod and Mae Ramat, Thailand, and 626 were analyzed both by the PCR method and by conventional microscopy. Of these, 157 were positive both by microscopy and by PCR, while 297 were negative by both methods. PCR detected 131 samples that were negative by microscopy, and failed to detect 41 samples identified as positive by microscopy. All discordant samples were re-analyzed by microscopy and by PCR. Upon re-examination at a higher sensitivity, microscopy identified five additional positive cases, while six previously positive cases were found to be negative. This method of treating blood samples for PCR may also be useful in other diagnostic assays.