Anti-Plasmodium vivax merozoite surface protein 3 ϒ (PvMSP3 ϒ) antibodies upon natural infection.

Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3ß. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4-81.7% for IgG1 and 63.6-77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies.

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.

Study on the dielectrophoretic characteristics of malaria-infected red blood cells.

Malaria is a tropical disease caused by parasites in the genus Plasmodium, which still presents 241 million cases and nearly 627,000 deaths recently. In this work, we used the dielectrophoresis (DEP) to characterize red blood cells in a microchannel. The purpose of this work is to determine the difference between the normal and the malaria-infected cells based on the DEP characteristics. The samples were infected cells and normal red blood cells, which were either prepared in culture or obtained from volunteers. Diamond-shaped and curved micropillars were used to create different degrees of DEP in the gap between them. The DEP crossover frequencies were observed with the diamond-shaped micropillars. The cell velocity under negative dielectrophoresis (nDEP) at a low frequency was examined with the curved micropillars. The measured lower crossover frequencies were remarkably different between the malaria-infected cells and the normal cells, whereas the higher crossover frequencies were similar among the samples. The velocity under nDEP was lower for the infected cells than the normal cells. The results imply that the malaria infection significantly decreases the capacitance but increases the conductance of the cell membrane, whereas a change in cytoplasmic conductivity may occur in a later stage of infection.

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.

A discrete dielectrophoresis device for the separation of malaria-infected cells.

Malaria is a serious disease caused by Plasmodium parasites that infect red blood cells (RBCs). This paper presents the continuous separation of malaria-infected RBCs (iRBCs) from normal blood cells. The proposed method employed the discrete dielectrophoresis (DEP) in a microfluidic device with interdigitated electrodes. Our aim is to treat a sample having high concentration of cells to realize high throughput and to prevent the clogging of the microchannel with the use of the discrete DEP. The discrete DEP force for deflecting cells in the device was controlled by adjusting the magnitude, frequency, and duty cycle of the applied voltage. The effectiveness of the proposed method was demonstrated by separating the malaria-infected cells in samples having a cell concentration of 106 cells/µl. From experimental results, we determined the enrichment that is needed to enhance the detection in the case of low parasitemia. The enrichment of the infected cells at the device output was 3000 times as high as that of the input containing 1 infected cell to 106 normal cells. Therefore, the proposed method is highly effective and can significantly facilitate the detection of the infected cells for the identification of Malaria patients.

Case Report: An Eyelid Nodule Caused by Candidatus Dirofilaria hongkongensis Diagnosed by Mitochondrial 12S rRNA Sequence.

A 59-year-old female living in Rayong Province, eastern Thailand, presented with painless, right upper eyelid nodule for 3 months. Upon removal of the eyelid mass, a well-circumscribed, firm globular mass with diameter about 1 cm was found. Histopathological examination revealed an immature female dirofilarial worm reminiscent of Dirofilaria repens, characterized by prominent sharp longitudinal ridges at external surface of the cuticle. Analysis of the mitochondrial 12S rRNA sequence showed that the worm belongs to Candidatus Dirofilaria hongkongensis. It is likely that some infections previously reported as D. repens based on histological examination may have actually been due to Candidatus D. hongkongensis.

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.

Myositis Caused by Trachipleistophora hominis in a Person With Human Immunodeficiency Virus: The First Case in Thailand.

BACKGROUND: To date, cases of extraintestinal micro-sporidiosis have been increasingly reported in both otherwise healthy and immunocompromised individuals. Among them, microsporidial myositis is very rare. To the best of our knowledge, this is the first report of microsporidial myositis caused by Trachipleistophora hominis in a patient with human immunodeficiency virus (HIV) in Thailand. CASE REPORT: A Thai man with HIV presented with fever and muscle pain at both anterior thighs and left arm for 3 months. Muscle biopsy was performed, and pathology exhibited neutrophil infiltration and focal aggregations of microsporidial spores. The 18S ribosomal RNA sequence revealed the species of this microsporidium as T hominis, and albendazole of 800mg/day was initiated. He gradually improved, and was discharged home 6 weeks after hospitalization. CONCLUSIONS: To the best of our knowledge, this is the first report of microsporidial myositis caused by Trachipleistophora hominis in a person with HIV in Thailand.

Severe Keratitis Caused by Acanthamoeba Genotype T12 in Thailand: A Case Report.

Acanthamoeba keratitis is predominantly caused by genotype T4. We report a case of severe keratitis caused by Acanthamoeba in a 39-year-old man who had prior accidental exposure to a corrosive chemical. The patient developed central full thickness ring infiltration and epithelial defect with hypopyon that required keratoplasty. The acanthamoebae isolated from the patient exhibited thermotolerance phenotype with the capability to grow well at ambient temperature and at 42°C. Analysis of a near complete 18S rRNA gene of this isolate revealed a distinct sequence that can be unequivocally assigned to genotype T12, a rare genotype incriminated in corneal infections.

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.

Genotyping of Entamoeba nuttalli strains from the wild rhesus macaques of Myanmar and comparison with those from the wild rhesus macaques of Nepal and China.

Entamoeba nuttalli found in macaques is phylogenetically the closest species to Entamoeba histolytica and is potentially pathogenic. In this study, the prevalence of Entamoeba infections was examined in wild rhesus macaques by examining 73 and 90 fecal samples collected from two sites, Popa Taung Kalat (PTK) and Pho Win Taung (PWT), in Myanmar. The positive rates of E. nuttalli detected using PCR were 49% and 31% in PTK and PWT, respectively, but no infections of E. histolytica and E. moshkovskii were found. Entamoeba dispar was detected in 6% of samples only from PWT. Positive rates of E. chattoni and E. coli were both 70% in PWT and 67% and 79% in PTK, respectively. Six E. nuttalli strains from PTK and eight from PWT were obtained in the culture with xenic medium and then, one and two strains, respectively, were axenized and finally cloned. The genotypic analysis of serine-rich protein genes revealed two genotypes each in both sites. The genotypes found in five of six strains from PTK were similar to those from the strains found in Nepal, whereas the remaining one from PTK and two from PWT were similar to those obtained from macaques in China. The sequence of the 18S rDNA of strains with these four genotypes was identical to that of the strains from China. Six loci of tRNA-linked short tandem repeats were analyzed for further genotyping of the strains. Although there were two types in locus A-L in PTK isolates, one of each type for PTK and PWT was found in the other loci, including locus A-L in PWT strains. These results demonstrated that the E. nuttalli strains from Myanmar are closer to the strains from macaques in China rather than those from macaques in Nepal.

Analysis of D-A locus of tRNA-linked short tandem repeats reveals transmission of Entamoeba histolytica and E. dispar among students in the Thai-Myanmar border region of northwest Thailand.

Intestinal parasitic infections, including those caused by Entamoeba species, are a persistent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Entamoeba species and to analyze their genotypic diversity. Stool samples were collected from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive samples for E. histolytica and E. dispar were exclusively obtained from a few school classes, whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nuttalli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. dispar (n = 47) to investigate their diversity and potential modes of transmission. Five genotypes of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A were divergent, but several unique genotypes were significantly prevalent in limited classes, indicating that intra-classroom transmission has occurred. As it was unlikely that infection would have been limited within school classes if the mode of transmission of E. histolytica and E. dispar had been through the intake of contaminated drinking water or food, these results suggest a direct or indirect person-to-person transmission mode within school classes. Positive rates for three Entamoeba species were 2-fold higher in students who had siblings in the schools than in those without siblings, suggesting that transmission occurred even at home due to heavy contacts among siblings.

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.

Draft Genome Sequences of Chlamydiales Bacterium STE3 and Neochlamydia sp. Strain AcF84, Endosymbionts of Acanthamoeba spp.

Chlamydiales bacterium STE3 and Neochlamydia sp. strain AcF84 are obligate intracellular symbionts of Acanthamoeba spp. isolated from the biofilm of a littoral cave wall and gills from striped tiger leaf fish, respectively. We report the draft genome sequences of these two environmental chlamydiae affiliated with the family Parachlamydiaceae.