Usefulness of a new immunochromatographic assay using fluorescent silica nanoparticles for serodiagnosis of Thai patients with amebiasis.

A fluorescence immunochromatography (FIC) kit was developed recently using fluorescent silica nanoparticles coated with a recombinant C-terminal fragment of the surface lectin intermediate subunit (C-Igl) of Entamoeba histolytica to establish rapid serodiagnosis of amebiasis. We further evaluated the system using serum samples from 52 Thai patients with amebiasis. Of the patients, 50 (96%) tested positive using FIC. The samples were also tested using enzyme-linked immunosorbent assay (ELISA) with C-Igl as the antigen. Two samples were negative on ELISA but positive on FIC. The correlation coefficient between the fluorescence intensity using FIC and the optical density value using ELISA was 0.5390, indicating a moderate correlation between the two tests. Serum samples from 20 patients with malaria and 22 patients with Clostridioides difficile infection were also tested using FIC. The false-positive rates were 4/20 (20%) and 1/22 (4%) in patients with malaria and C. difficile infection, respectively. Combining the data from the present study with our previous study, the sensitivity and specificity of FIC were determined to be 98.5% and 95.2%, respectively. The results of the 50 samples were studied using a fluorescence scope and a fluorescence intensity reader, and the findings were compared. Disagreements were found in only two samples showing near-borderline fluorescence intensity, indicating that the use of scope was adequate for judging the results. These results demonstrate that FIC is a simple and rapid test for the serodiagnosis of amebiasis.

Natural vectors of Plasmodium knowlesi and other primate, avian and ungulate malaria parasites in Narathiwat Province, Southern Thailand.

To date, four species of simian malaria parasites including Plasmodium knowlesi, P. cynomolgi, P. inui and P. fieldi have been incriminated in human infections in Thailand. Although the prevalence of malaria in macaque natural hosts has been investigated, their vectors remain unknown in this country. Herein, we performed a survey of Anopheles mosquitoes during rainy and dry seasons in Narathiwat Province, Southern Thailand. Altogether 367 Anopheles mosquitoes were captured for 40 nights during 18:00 to 06:00 h by using human-landing catches. Based on morphological and molecular identification, species composition comprised An. maculatus (37.06%), An. barbirostris s.l. (31.34%), An. latens (17.71%), An. introlatus (10.08%) and others (3.81%) including An. umbrosus s.l., An. minimus, An. hyrcanus s.l., An. aconitus, An. macarthuri and An. kochi. Analyses of individual mosquitoes by PCR, sequencing and phylogenetic inference of the mitochondrial cytochrome genes of both malaria parasites and mosquitoes have revealed that the salivary gland samples of An. latens harbored P. knowlesi (n = 1), P. inui (n = 2), P. fieldi (n = 1), P. coatneyi (n = 1), P. hylobati (n = 1) and an unnamed Plasmodium species known to infect both long-tailed and pig-tailed macaques (n = 2). The salivary glands of An. introlatus possessed P. cynomolgi (n = 1), P. inui (n = 1), P. hylobati (n = 1) and coexistence of P. knowlesi and P. inui (n = 1). An avian malaria parasite P. juxtanucleare has been identified in the salivary gland sample of An. latens. Three other distinct lineages of Plasmodium with phylogenetic affinity to avian malaria species were detected in An. latens, An. introlatus and An. macarthuri. Interestingly, the salivary gland sample of An. maculatus contained P. caprae, an ungulate malaria parasite known to infect domestic goats. Most infected mosquitoes harbored multiclonal Plasmodium infections. All Plasmodium-infected mosquitoes were captured during the first quarter of the night and predominantly occurred during rainy season. Since simian malaria in humans has a wide geographic distribution in Thailand, further studies in other endemic areas of the country are mandatory for understanding transmission and prevention of zoonotic malaria.

Analysis of sequence diversity in Plasmodium falciparum glutamic acid-rich protein (PfGARP), an asexual blood stage vaccine candidate.

Glutamic acid-rich protein of Plasmodium falciparum (PfGARP) binds to erythrocyte band 3 and may enhance cytoadherence of infected erythrocytes. Naturally acquired anti-PfGARP antibodies could confer protection against high parasitemia and severe symptoms. While whole genome sequencing analysis has suggested high conservation in this locus, little is known about repeat polymorphism in this vaccine candidate antigen. Direct sequencing was performed from the PCR-amplified complete PfGARP gene of 80 clinical isolates from four malaria endemic provinces in Thailand and an isolate from a Guinean patient. Publicly available complete coding sequences of this locus were included for comparative analysis. Six complex repeat (RI-RVI) and two homopolymeric glutamic acid repeat (E1 and E2) domains were identified in PfGARP. The erythrocyte band 3-binding ligand in domain RIV and the epitope for mAB7899 antibody eliciting in vitro parasite killing property were perfectly conserved across isolates. Repeat lengths in domains RIII and E1-RVI-E2 seemed to be correlated with parasite density of the patients. Sequence variation in PfGARP exhibited genetic differentiation across most endemic areas of Thailand. Phylogenetic tree inferred from this locus has shown that most Thai isolates formed closely related lineages, suggesting local expansion/contractions of repeat-encoding regions. Positive selection was observed in non-repeat region preceding domain RII which corresponded to a helper T cell epitope predicted to be recognized by a common HLA class II among Thai population. Predicted linear B cell epitopes were identified in both repeat and non-repeat domains. Besides length variation in some repeat domains, sequence conservation in non-repeat regions and almost all predicted immunogenic epitopes have suggested that PfGARP-derived vaccine may largely elicit strain-transcending immunity.

Structural organization and sequence diversity of the complete nucleotide sequence encoding the Plasmodium malariae merozoite surface protein-1.

The merozoite surface protein-1 (MSP1) is a prime candidate for an asexual blood stage vaccine against malaria. However, polymorphism in this antigen could compromise the vaccine's efficacy. Although the extent of sequence variation in MSP1 has been analyzed from various Plasmodium species, little is known about structural organization and diversity of this locus in Plasmodium malariae (PmMSP1). Herein, we have shown that PmMSP1 contained five conserved and four variable blocks based on analysis of the complete coding sequences. Variable blocks were characterized by short insertion and deletion variants (block II), polymorphic nonrepeat sequences (block IV), complex repeat structure with size variation (block VI) and degenerate octapeptide repeats (block VIII). Like other malarial MSP1s, evidences of intragenic recombination have been found in PmMSP1. The rate of nonsynonymous nucleotide substitutions significantly exceeded that of synonymous nucleotide substitutions in block IV, suggesting positive selection in this region. Codon-based analysis of deviation from neutrality has identified a codon under purifying selection located in close proximity to the homologous region of the 38 kDa/42 kDa cleavage site of P. falciparum MSP1. A number of predicted linear B-cell epitopes were identified across both conserved and variable blocks of the protein. However, polymorphism in repeat-containing blocks resulted in alteration of the predicted linear B-cell epitope scores across variants. Although a number of predicted HLA-class II-binding peptides were identified in PmMSP1, all variants of block IV seemed not to be recognized by common HLA-class II alleles among Thai population, suggesting that diversity in this positive selection region could probably affect host immune recognition. The data on structural diversity in PmMSP1 could be useful for further studies such as vaccine development and strain characterization of this neglected malaria parasite.

Cryptic Plasmodium inui and Plasmodium fieldi Infections Among Symptomatic Malaria Patients in Thailand.

BACKGROUND: Some nonhuman primate Plasmodium species including P. knowlesi and P. cynomolgi can cross-transmit from macaque natural hosts to humans under natural infection. This study aims to retrospectively explore other simian Plasmodium species in the blood samples of symptomatic malaria patients in Thailand. METHODS: A total of 5271 blood samples from acute febrile patients from 5 malaria endemic provinces and 1015 blood samples from long-tailed and pig-tailed macaques from 3 locations were examined for Plasmodium species by microscopy and species-specific polymerase chain reaction. The Plasmodium mitochondrial cytochrome oxidase 1 (COX1) gene was analyzed by amplicon deep sequencing as well as Sanger sequencing from recombinant plasmid clones to reaffirm and characterize P. inui and P. fieldi. RESULTS: Besides human malaria, P. knowlesi, P. cynomolgi, P. inui and P. fieldi infections were diagnosed in 15, 21, 19, and 3 patients, respectively. Most P. inui and all P. fieldi infected patients had simultaneous infections with other Plasmodium species, and seemed to be responsive to chloroquine or artemisinin-mefloquine. P. inui was the most prevalent species among macaque populations. Phylogenetic analysis of the COX1 sequences from human and macaque isolates reveals the genetic diversity of P. inui and suggests that multiple parasite strains have been incriminated in human infections. CONCLUSIONS: Both P. inui and P. fieldi could establish infection in humans under natural transmission. Despite occurring at a low prevalence and mostly co-existing with other Plasmodium species, P. inui infections in humans have a wide distribution in Thailand.

Analysis of Acanthamoeba genotypes from public freshwater sources in Thailand reveals a new genotype, T23 Acanthamoeba bangkokensis sp. nov.

A survey of Acanthamoeba in 100 public freshwater sources in 28 provinces across Thailand has identified 9 genotypes comprising T2/6, T3-T5, T9, T11, T12, T18 and a novel 'T23' among 131 isolates. Sequencing of the near complete 18S rRNA gene of Acanthamoeba of all isolates has shown that the most predominant genotype T4 found in 87 isolates (66.4%) contained 4 subtypes, i.e. T4A, T4B, T4C and T4F, while all isolates assigned to genotype T2/6 belonged to subtype B. Among intron-bearing genotypes, most isolates harbouring genotype T3 contained S516 introns, characterised by 3 distinct variants whilst all genotypes T4A and T5 were intronless. Identical 18S rRNA sequences of Acanthamoeba were identified across regions of the country and four isolates in this study shared the same sequences with those from remote nations, suggesting that some strains have reproductive success in diverse ecological niche. Nucleotide diversity of genotypes T2/6B, T3, T4, T9 and T11 in this study was significantly less than that among global isolates outside Thailand, implying that limited sequence diversity occurred within local populations. A remarkably higher level of nucleotide diversity in genotype T11 than those of other genotypes (0.041 vs. 0.012-0.024) could be due to cryptic subtypes. Recombination breakpoints have been detected within genotypes and subtypes as well as within isolates despite no evidence for sexual and parasexual cycles in the genus Acanthamoeba. Tajima's D, Fu & Li's D* and F* statistics revealed significantly negative deviation from neutrality across genotypes and subtypes, implying purifying selection in this locus. The 18S rRNA gene of the novel genotype 'T23' displayed 7.82% to 28.44% sequence differences in comparison with all known genotypes. Both Bayesian and maximum likelihood phylogenetic trees have placed genotype T23 as sister to the clade comprising genotypes T10, T12 and T14, all of these possess cyst structure belonging to morphological group III. Hence, Acanthamoeba bangkokensis sp. nov. is proposed for this novel genotype. It is likely that more genotypes of Acanthamoeba remain to be discovered while the evolution of the 18S rRNA gene of this pathogenic-free living amoeba seems to be ongoing.

Immunoglobulin G responses to variant forms of Plasmodium vivax merozoite surface protein 9 upon natural infection in Thailand.

Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target. Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients' sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.

Plasmodium cynomolgi Co-infections among Symptomatic Malaria Patients, Thailand.

Among 1,180 symptomatic malaria patients, 9 (0.76%) infected with Plasmodium cynomolgi were co-infected with P. vivax (n = 7), P. falciparum (n = 1), or P. vivax and P. knowlesi (n = 1). Patients were from Tak, Chanthaburi, Ubon Ratchathani, Yala, and Narathiwat Provinces, suggesting P. cynomolgi is widespread in this country.

Structural diversity, natural selection and intragenic recombination in the Plasmodium vivax merozoite surface protein 9 locus in Thailand.

The merozoite surface protein 9 (MSP9) of malarial parasite forms co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP1) prior to erythrocyte invasion. Interruption of this process could hamper subsequent asexual erythrocytic development of malaria parasites; therefore, these proteins are considered potential vaccine candidates. In Plasmodium vivax, MSP9 (PvMSP9) contains both conserved and polymorphic repetitive domains that were immunogenic upon natural malaria exposure and conferred protection in vaccination studies in animal models. To investigate the extent of sequence diversity at this locus, 104 P. vivax isolates from 4 major malaria endemic areas of Thailand were analyzed. Results revealed that pvmsp9 contained 3 repeat domains (R1-R3) flanked by conserved domains. Repeat domains exhibit extensive sequence and length variation, in which 14, 39 and 16 haplotypes for domains R1-R3, respectively, circulated in this country. Sequence diversity in pvmsp9 among P. vivax isolates from each endemic area displayed population structure. The extent of sequence diversity in pvmsp9 isolates from the provinces of Tak, Chanthaburi, Ubon Ratchathani and Prachuap Khiri Khan in northwestern, eastern, northeastern and southwestern areas, respectively, was almost comparable and was remarkably higher than that from Yala/Narathiwat population in southern Thailand. Evidence for intragenic recombination in this locus was observed within each P. vivax population except among isolates from Yala and Narathiwat. Synonymous nucleotide diversity significantly exceeded nonsynonymous nucleotide diversity in domains R2 and R3, indicating purifying selection. However, micro-scale signatures of positive and negative selections occurred in both conserved and repeat domains, implying two opposing forces, probably from functional or structural constraint and host immune pressure, could have influenced diversity at this locus. The immunodominant T and B cell epitopes so far identified were invariant or highly conserved across isolates. Further analysis of global isolates is warranted for vaccine design based on this protein.

Insights into the molecular diversity of Plasmodium vivax merozoite surface protein-3γ (pvmsp3γ), a polymorphic member in the msp3 multi-gene family.

Plasmodium vivax merozoite surface protein 3 (PvMSP3) is encoded by a multi-gene family. Of these, PvMSP3α, PvMSP3ß and PvMSP3γ, are considered to be vaccine targets. Despite comprehensive analyses of PvMSP3α and PvMSP3ß, little is known about structural and sequence diversity in PvMSP3γ. Analysis of 118 complete pvmsp3γ sequences from diverse endemic areas of Thailand and 9 reported sequences has shown 86 distinct haplotypes. Based on variation in insert domains, pvmsp3γ can be classified into 3 types, i.e. Belem, Salvador I and NR520. Imperfect nucleotide repeats were found in six regions of the gene; none encoded tandem amino acid repeats. Predicted coiled-coil heptad repeats were abundant in the protein and displayed variation in length and location. Interspersed phase shifts occurred in the heptad arrays that may have an impact on protein structure. Polymorphism in pvmsp3γ seems to be generated by intragenic recombination and driven by natural selection. Most P. vivax isolates in Thailand exhibit population structure, suggesting limited gene flow across endemic areas. Phylogenetic analysis has suggested that insert domains could have been subsequently acquired during the evolution of pvmsp3γ. Sequence and structural diversity of PvMSP3γ may complicate vaccine design due to alteration in predicted immunogenic epitopes among variants.

Polymorphism and natural selection in the merozoite surface protein 3F2 (PVX_97710) locus of Plasmodium vivax among field isolates.

Plasmodium vivax, the chronic relapsing human malaria parasite with the most widespread distribution, possesses proteins associated with the merozoite surface that could be targets for host immune responses and potential vaccine candidates. Of these, the merozoite surface protein 3 of P. vivax (PvMSP3) is an attractive vaccine target as well as a genetic marker for epidemiological surveillance. PvMSP3 comprises a group of protein members encoded by a multigene family. Although some protein members, i.e. PvMSP3α and PvMSP3ß, have been targets for molecular and immunological investigations, the most abundantly expressed protein member during late asexual erythrocytic stages, PvMSP3F2 (PVX_97710), remains unexplored. To address domain organization and evolution of this locus, the complete coding sequences of 31 P. vivax isolates from diverse malaria endemic areas of Thailand were analyzed and compared with 10 previously reported sequences. Results revealed that all PvMSP3F2 sequences differed but could be divided into 5 repeat-containing domains flanked by 6 non-repeat domains. Repeat domains II and IV at the 5' portion and domain X at the 3' portion exhibited extensive sequence and length variation whereas repeat domains VI and VIII located at the central region were relatively conserved. Despite a repertoire of PvMSP3F2 variants, predicted coiled-coil tertiary structure and predicted B-cell epitopes seem to be maintained. Evidence of intragenic recombination has been detected among field isolates in Thailand that could enhance sequence diversity at this locus. Non-repeat domains I and IX located at the 5' end and at the 3' portion, respectively, seem to have evolved under purifying selection. Evidence of positive selection was found in non-repeat domains III, V and VII where a number of predicted HLA class I epitopes were identified. Amino acid substitutions in these predicted epitopes could alter predicted peptide binding affinity or abolish peptide epitope property, suggesting that polymorphism in these epitopes conferred host immune evasion. Further studies on PvMSP3F2 are warranted, particularly on interaction with host immune system and the potential role of this PvMSP3 protein member as a vaccine target.

Multiple Novel Mutations in Plasmodium falciparum Chloroquine Resistance Transporter Gene during Implementation of Artemisinin Combination Therapy in Thailand.

Mutations in the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) are associated with drug susceptibility status of chloroquine and other antimalarials that interfere with heme detoxification process including artemisinin. We aim to investigate whether an increase in duration of artemisinin combination therapy (ACT) in Thailand could affect mutations in Pfcrt. The complete coding sequences of Pfcrt and dihydrofolate reductase (Pfdhfr), and size polymorphisms of the merozoite surface proteins-1 and 2 (Pfmsp-1 and Pfmsp-2) of 189 P. falciparum isolates collected during 1991 and 2016 were analyzed. In total, 12 novel amino acid substitutions and 13 novel PfCRT haplotypes were identified. The most prevalent haplotype belonged to the Dd2 sequence and no wild type was found. A significant positive correlation between the frequency of Pfcrt mutants and the year of sample collection was observed during nationwide ACT implementation (r = 0.780; P = 0.038). The number of haplotypes and nucleotide diversity of isolates collected during 3-day ACT (2009-2016) significantly outnumbered those collected before this treatment regimen. Positive Darwinian selection occurred in the transmembrane domains only among isolates collected during 3-day ACT but not among those collected before this period. No remarkable change was observed in the molecular indices for other loci analyzed when similar comparisons were performed. An increase in the duration of artesunate in combination therapy in Thailand could exert selective pressure on the Pfcrt locus, resulting in emergence of novel variants. The impact of these novel haplotypes on antimalarial susceptibilities requires further study.

Variation in intronic microsatellites and exon 2 of the Plasmodium falciparum chloroquine resistance transporter gene during modification of artemisinin combination therapy in Thailand.

The amino acid substitution at residue 76 of the food vacuolar transmembrane protein encoded by the chloroquine resistance transporter gene of Plasmodium falciparum (Pfcrt) is an important, albeit imperfect, determinant of chloroquine susceptibility status of the parasite. Other mutations in Pfcrt can modulate susceptibility of P. falciparum to other antimalarials capable of interfering with heme detoxification process, and may exert compensatory effect on parasite growth rate. To address whether nationwide implementation of artemisinin combination therapy (ACT) in Thailand could affect sequence variation in exon 2 and introns of Pfcrt, we analyzed 136 P. falciparum isolates collected during 1997 and 2016 from endemic areas bordering Myanmar, Cambodia and Malaysia. Results revealed 6 haplotypes in exon 2 of Pfcrt with 2 novel substitutions at c.243A > G (p.R81) and c.251A > T (p.N84I). Positive selection was observed at amino acid residues 75, 76 and 97. Four, 3, and 2 alleles of microsatellite (AT/TA) repeats occurred in introns 1, 2 and 4, respectively, resulting in 7 different 3-locus haplotypes. The number of haplotypes and haplotype diversity of exon 2, and introns 1, 2 and 4 were significantly greater among isolates collected during 2009 and 2016 than those collected during 1997 and 2008 when 3-day ACT and 2-day ACT regimens were implemented nationwide, respectively (p < 0.05). By contrast, the number of haplotypes and haplotype diversity of the merozoite surface proteins 1 and 2 of these parasite populations did not differ significantly between these periods. Therefore, the Pfcrt locus of P. falciparum in Thailand continues to evolve and could have been affected by selective pressure from modification of ACT regimen.

Sequence diversity and positive selection at the Duffy-binding protein genes of Plasmodium knowlesi and P. cynomolgi: Analysis of the complete coding sequences of Thai isolates.

Plasmodium knowlesi and P. cynomolgi are simian malaria parasites capable of causing symptomatic human infections. The interaction between the Duffy binding protein alpha on P. knowlesi merozoite and the Duffy-antigen receptor for chemokine (DARC) on human and macaque erythrocyte membrane is prerequisite for establishment of blood stage infection whereas DARC is not required for erythrocyte invasion by P. cynomolgi. To gain insights into the evolution of the PkDBP gene family comprising PkDBPα, PkDBPß and PkDBPγ, and a member of the DBP gene family of P. cynomolgi (PcyDBP1), the complete coding sequences of these genes were analyzed from Thai field isolates and compared with the publicly available DBP sequences of P. vivax (PvDBP). The complete coding sequences of PkDBPα (n=11), PkDBPß (n=11), PkDBPγ (n=10) and PcyDBP1 (n=11) were obtained from direct sequencing of the PCR products. Nucleotide diversity of DBP is highly variable across malaria species. PcyDBP1 displayed the greatest level of nucleotide diversity while all PkDBP gene members exhibited comparable levels of diversity. Positive selection occurred in domains I, II and IV of PvDBP and in domain V of PcyDBP1. Although deviation from neutrality was not detected in domain II of PkDBPα, a signature of positive selection was identified in the putative DARC binding site in this domain. The DBP gene families seem to have arisen following the model of concerted evolution because paralogs rather than orthologs are clustered in the phylogenetic tree. The presence of identical or closely related repeats exclusive for the PkDBP gene family suggests that duplication of gene members postdated their divergence from the ancestral PcyDBP and PvDBP lineages. Intragenic recombination was detected in all DBP genes of these malaria species. Despite the limited number of isolates, P. knowlesi from Thailand shared phylogenetically related domain II sequences of both PkDBPα and PkDBPγ with those from Peninsular Malaysia, consistent with their geographic proximity.

The Plasmodium vivax merozoite surface protein 3ß sequence reveals contrasting parasite populations in southern and northwestern Thailand.

BACKGROUND: Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand. METHODOLOGY: We employed the P. vivax merozoite surface protein 3ß (PvMSP3ß) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3ß gene and examined its genetic diversity and molecular evolution between the parasite populations. PRINCIPAL FINDINGS: Of 58 isolates containing single PvMSP3ß alleles, 31 sequence types were identified. The overall haplotype diversity was 0.77 ± 0.06 and nucleotide diversity 0.0877±0.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3ß (HD=1.0). In contrast, the southern parasite population displayed a single PvMSP3ß allele (HD=0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas. CONCLUSION: Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax control measures such as drug policy and vaccine development.

Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

BACKGROUND: Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. METHODS: A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. RESULTS: The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. CONCLUSIONS: The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.