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Background: Sensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. PCR techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods. However, most of these methods are of low throughput and cannot be used for large-scale molecular epidemiological studies. A recently developed capture and ligation probe-PCR (CLIP-PCR) is claimed to have the sensitivity of molecular techniques and the high throughput capacity needed for screening purposes. This study aimed to compare several molecular methods for detecting asymptomatic and submicroscopic Plasmodium infections in healthy residents of a malaria-hypoendemic region in Southeast Asia, where malaria elimination is in sight. Method: This study compared three molecular detection methods side-by-side, namely nested PCR targeting the rRNA genes, nested RT-PCR to detect parasite rRNA, and CLIP-PCR to detect parasite rRNA in 1005 healthy individuals in northeastern Myanmar. For nested PCR and RT-PCR, parasite DNA and total RNA were extracted from ∼100 μL of blood, whereas RNA used for CLIP-PCR was from a 3 mm disk of dried blood filter paper. The sensitivity and specificity of these methods were compared with those of conventional light microscopy. In addition, RT-PCR and quantitative RT-PCR (qRT-PCR) targeting the Pvs25 gene in Plasmodium vivax were used to assess gametocyte prevalence in the samples. Results: Light microscopy detected Plasmodium infections in only 1.19% of the residents harbouring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and Plasmodium falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, 5 P. falciparum and 6 P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by qRT-PCR, indicating that a large proportion of asymptomatic individuals were gametocyte carriers. Conclusion: Nested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than sixfold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods, which may be partially due to the smaller amount of RNA input used. [ABSTRACT FROM AUTHOR]

Background: The Greater Mekong Subregion is aiming to achieve regional malaria elimination by 2030. Though a shift in malaria parasite species predominance by Plasmodium vivax has been recently documented, the transmission of the two minor Plasmodium species, Plasmodium malariae and Plasmodium ovale spp., is poorly characterized in the region. This study aims to determine the prevalence of these minor species in the China-Myanmar border area and their genetic diversity. Methods: Epidemiology study was conducted during passive case detection in hospitals and clinics in Myanmar and four counties in China along the China-Myanmar border. Cross-sectional surveys were conducted in villages and camps for internally displaced persons to determine the prevalence of malaria infections. Malaria infections were diagnosed initially by microscopy and later in the laboratory using nested PCR for the SSU rRNA genes. Plasmodium malariae and P. ovale infections were confirmed by sequencing the PCR products. The P. ovale subtypes were determined by sequencing the Pocytb, Pocox1 and Pog3p genes. Parasite populations were evaluated by PCR amplification and sequencing of the MSP-1 genes. Antifolate sensitivity was assessed by sequencing the dhfr-ts and dhps genes from the P. malariae and P. ovale isolates. Results: Analysis of 2701 blood samples collected from the China-Myanmar border by nested PCR targeting the parasite SSU rRNA genes identified 561 malaria cases, including 161 Plasmodium falciparum, 327 P. vivax, 66 P. falciparum/P. vivax mixed infections, 4 P. malariae and 3 P. ovale spp. P. vivax and P. falciparum accounted for >60 and ~30% of all malaria cases, respectively. In comparison, the prevalence of P. malariae and P. ovale spp. was very low and only made up ~1% of all PCR-positive cases. Nevertheless, these two species were often misidentified as P. vivax infections or completely missed by microscopy even among symptomatic patients. Phylogenetic analysis of the SSU rRNA, Pocytb, Pocox1 and Pog3p genes confirmed that the three P. ovale spp. isolates belonged to the subtype P. ovale curtisi. Low-level genetic diversity was detected in the MSP-1, dhfr and dhps genes of these minor parasite species, potentially stemming from the low prevalence of these parasites preventing their mixing. Whereas most of the dhfr and dhps positions equivalent to those conferring antifolate resistance in P. falciparum and P. vivax were wild type, a new mutation S113C corresponding to the S108 position in pfdhfr was identified in two P. ovale curtisi isolates. Conclusions: The four human malaria parasite species all occurred sympatrically at the China-Myanmar border. While P. vivax has become the predominant species, the two minor parasite species also occurred at very low prevalence but were often misidentified or missed by conventional microscopy. These minor parasite species displayed low levels of polymorphisms in the msp-1, dhfr and dhps genes. [ABSTRACT FROM AUTHOR]

Background Nested PCR is considered a sensitive and specific method for detecting malaria parasites and is especially useful in epidemiological surveys. However, the preparation of DNA templates for PCR is often time-consuming and costly. Methods A simplified PCR method was developed to directly use a small blood filter paper square (2 × 2 mm) as the DNA template after treatment with saponin. This filter paper-based nested PCR method (FP-PCR) was compared to microscopy and standard nested PCR with DNA extracted by using a Qiagen DNA mini kit from filter paper blood spots of 204 febrile cases. The FP-PCR technique was further applied to evaluate malaria infections in 1,708 participants from cross-sectional epidemiological surveys conducted in Myanmar and Thailand. Results The FP-PCR method had a detection limit of ~0.2 parasites/μl blood, estimated using cultured Plasmodium falciparum parasites. With 204 field samples, the sensitivity of the FP-PCR method was comparable to that of the standard nested PCR method, which was significantly higher than that of microscopy. Application of the FP-PCR method in large cross-sectional studies conducted in Myanmar and Thailand detected 1.9% (12/638) and 6.2% (66/1,070) asymptomatic Plasmodium infections, respectively, as compared to the detection rates of 1.3% (8/638) and 0.04% (4/1,070) by microscopy. Conclusion This FP-PCR method was much more sensitive than microscopy in detecting Plasmodium infections. It drastically increased the detection sensitivity of asymptomatic infections in cross-sectional surveys conducted in Thailand and Myanmar, suggesting that this FP-PCR method has a potential for future applications in malaria epidemiology studies. [ABSTRACT FROM AUTHOR]