In this paper, real-time PCR of sample pools, followed by species-specific PCRs and expert microscopy, has been shown to be an effective surveillance tool. Almost 2% of individuals screened by mass blood examination were positive by PCR, but were microscopy negative when the slides were examined by the local microscopist. Expert microscopists detected parasites in eight of the nine PCR-positive subjects. Thus, a classical mass blood examination would have missed a substantial number of infections.
In this study, expert microscopy was performed on slides from individuals in the positive pools before the second round PCR was done. This enabled some results to be reported the same day the pooled PCR results were obtained. This raises the possibility that PCR and pooling can be used to reduce microscopy time. Local microscopists usually spend only about 10 minutes examining a slide, but, as described above, the sensitivity of local microscopy is low. The sensitivity of expert microscopy is much higher, partially because about 30 minutes are required for an examination. If an expert microscopist were to examine all 475 slides, it would take ~237.5 hours. However, using the results of the pooled PCR as a guide, the experts had to examine 32 slides (eight pools), taking ~16 hours. This is much less than the time spent by the local microscopist examining all 475 slides (~80 hours). After pooled PCR and expert microscopy, results were reported as quickly as 48 hours after blood was drawn. Thus, a combination of PCR pooling and expert microscopy might be an appropriate surveillance tool.
The study has two limitations. First, expert microscopy was only performed on PCR-positive pools, so the true sensitivity and specificity of real-time PCR cannot be calculated. Second, only about half of the village residents participated in the survey, so the true malaria prevalence may be different.
Of particular interest was the observation that none of the infected individuals contained gametocytes in their blood. This was also true in the previous study in Trat (Rogawski et al., unpublished results). This has implications for understanding malaria transmission. In a previous study in Mae Hong Son , infectivity was closely associated with the presence of gametocytes in the blood. The results presented here suggest that the prevalent asymptomatic infections may not be major infectious reservoirs at the time of the survey. However, it is possible that many of these asymptomatic infections represent early stage infections. Thus, the true value of PCR-based surveillance might be in early detection and prevention of disease and subsequent mosquito infectivity. On the other hand, it is also possible that many of these infections might clear parasitaemia, just as patient 88 appeared to do.
In summary, this paper shows that pooling and real-time PCR, combined with expert microscopy is rapid and cost-effective and is superior to local microscopy in detection of infections. It might serve an important role in efforts to eliminate malaria in low-transmission settings.