Genetic diversity in the transmission-blocking vaccine candidate Plasmodium vivax gametocyte protein Pvs230 from the China-Myanmar border area and central Myanmar.

BACKGROUND: Sexual stage surface antigens are potential targets of transmission-blocking vaccines (TBVs). The gametocyte and gamete surface antigen P230, a leading TBV candidate, is critical for red blood cell binding during exflagellation and subsequent oocyst development. Here, the genetic diversity of Pvs230 was studied in Plasmodium vivax parasite isolates from the China-Myanmar border (CMB) and central Myanmar. METHODS: Plasmodium vivax isolates were collected in clinics from malaria-endemic areas of the CMB (143 samples) and Myanmar (23 samples). The interspecies variable part (IVP, nucleotides 1-807) and interspecies conserved part (ICP, 808-2862) of Pvs230 were amplified by PCR and sequenced. Molecular evolution studies were conducted to evaluate the genetic diversity, signature of selection, population differentiation, haplotype network, and population structure of the study parasite populations and publicly available Pvs230 sequences from six global P. vivax populations. RESULTS: Limited genetic diversity was observed for the CMB (π = 0.002) and Myanmar (π = 0.001) isolates. Most amino acid substitutions were located in the IVP and cysteine-rich domain of Pvs230. Evidence of positive selection was observed for IVP and purifying selection for ICP. Codon-based tests identified specific codons under natural selection in both IVP and ICP. The fixation index (FST) showed low genetic differentiation between East and Southeast Asian populations, with FST ranging from 0.018 to 0.119. The highest FST value (FST = 0.503) was detected between the Turkey and Papua New Guinea populations. A total of 92 haplotypes were identified in global isolates, with the major haplotypes 2 and 9 being the most abundant and circulating in East and Southeast Asia populations. Several detected non-synonymous substitutions were mapped in the predicted structure and B-cell epitopes of Pvs230. CONCLUSIONS: We detected low levels of genetic diversity of Pvs230 in global P. vivax populations. Geographically specific haplotypes were identified for Pvs230. Some mutations are located within a potential B-cell epitope region and need to be considered in future TBV designs.

Effective High-Throughput Blood Pooling Strategy before DNA Extraction for Detection of Malaria in Low-Transmission Settings.

In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 µl in 1 sample, was optimal, and the parasite density as low as 2 p/µl for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.

Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+)-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N)/d(S) ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.