Episodic positive selection in the Cam734 haplotype and low prevalence of the A144F mutation in Plasmodium falciparum chloroquine resistance transporter gene among Thai isolates.

Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.

Episodic positive selection in the Cam734 haplotype and low prevalence of the A144F mutation in Plasmodium falciparum chloroquine resistance transporter gene among Thai isolates

@#Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.

Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand.

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype.

A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients.

Pneumocystis jirovecii pneumonia (PCP) is a leading cause of morbidity and mortality in immunocompromised patients. Despite the sensitivity of the commonly used PCR for diagnosing P. jirovecii with primers pAZ102-H/pAZ102-E and pAZ102-X/pAZ102-Y derived from mtLSU rRNA (conventional PCR), some PCP patients who had demonstrable organisms by staining methods failed to give positive PCR results. Herein, we devised a more sensitive PCR assay derived from the same gene target to circumvent these false-negative tests. Single brochoalveolar lavage (BAL) samples were collected from human immunodeficiency virus (HIV)-infected (n = 66) and non-HIV (n = 36) immunocompromised patients presenting with fever, dyspnoea, cough and pulmonary infiltrates. Pneumocystis jirovecii was diagnosed with Giemsa-stained smear, immunofluorescence assay, conventional single-round and nested PCR, and new single-round and nested PCR in 46 (45.1%), 53 (52.0%), 69 (67.6%), 74 (72.6%), 87 (85.3%) and 91 (89.2%) patients, respectively. The new PCR could detect P. jirovecii DNA in BAL fluids two to three orders of magnitude more dilute than conventional PCR. Sequence analysis revealed one to three nucleotide substitutions within the primers for conventional PCR among clinical isolates. Although both conventional and new PCR assays were highly specific for diagnosing P. jirovecii, the new PCR yielded more positive results than conventional PCR among BAL samples that were negative by both Giemsa stain and immunofluorescence assay. Hence, the new PCR offered a more sensitive detection of P. jirovecii infection and colonization than conventional PCR.

Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays.

Saliva and urine from malaria-infected individuals contain trace amounts of Plasmodium DNA, and therefore, could be used as alternative specimens for diagnosis. A nested PCR targeting the mitochondrial cytochrome b gene (Cytb-PCR) of four human malaria species and Plasmodium knowlesi was developed and tested with 693 blood samples from febrile patients living in diverse malaria-endemic areas of Thailand, and compared with microscopy and nested PCR targeting small-subunit rRNA (18S-PCR). Cytb-PCR was 16% and 39.8% more sensitive than 18S-PCR and microscopy, respectively, in detecting all of these malarial species in blood samples. Importantly, 34% and 17% of Plasmodium falciparum and Plasmodium vivax mono-infections, respectively, detected by microscopy were, in fact, mixed P. falciparum and P. non-falciparum infections. Analysis of matched blood, saliva and urine from 157 individuals showed that microscopy and Cytb-PCR of saliva yielded no significant difference in detecting P. falciparum and P. vivax. However, Cytb-PCR of saliva was more sensitive than microscopy for diagnosis of mixed-species infections. A combination of Cytb-PCR of saliva and of urine significantly outperformed microscopy (p 0.0098 for P. falciparum, p 0.006 for P. vivax, and p 0.0002 for mixed infections). Furthermore, Plasmodium malariae and P. knowlesi could also be identified in saliva and urine with this method. Therefore, the Cytb-PCR developed herein offers a high potential for the use of both saliva and urine for malaria diagnosis, with a sensitivity comparable with or superior to that of microscopy.

Molecular analysis of Cryptosporidium species isolated from HIV-infected patients in Thailand.

Cryptosporidium isolates from diarrhoeal stools of human immunodeficiency virus (HIV)-infected patients in Thailand were genetically analysed by sequencing the variable region in the 18S rRNA gene. Twenty-nine isolates from four children and 25 adults attending King Chulalongkorn Memorial Hospital in Bangkok during 1996 and 2000 were analysed. All patients suffered from chronic watery diarrhoea and had low CD4+ lymphocytes (mean +/- SD=105.5 +/- 133.2 cells/microl). Four Cryptosporidium species were identified, i.e. C. parvum (genotype 1), C. meleagridis, C. muris and C. felis occurring in 24, 3, 1 and 1 isolates, respectively. Oocysts of C. muris were significantly larger than oocysts of other species; C. felis was the smallest in these populations (P < 0.01). Sequences of the ITS1, 5.8S rRNA and ITS2 regions of C. muris and C. meleagridis identified in this study displayed unique sequences from those of other known species. Based on a limited number of isolates analysed, only C. meleagridis and C. muris were found in HIV-infected children, whereas the genotype 1 of C. parvum predominated in HIV-infected adults.

Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodium vivax.

We analyzed 22 clinical isolates of Plasmodium vivax from Thailand and 17 from Brazil to investigate the extent of sequence variation in the thrombospondin-related adhesive protein of Plasmodium vivax (PvTRAP), a homologue of P. falciparum TRAP (PfTRAP) which has been considered to be a promising vaccine candidate. In total 54 haplotypes were identified from 73 distinct gene clones. Coexistence of different PvTRAP in circulation occurred in 10 and 13 isolates from Thailand and Brazil, respectively. Forty out of 48 substituted nucleotides are non-synonymous changes. Most of the substituted residues reside in the von Willebrand factor type A-domain (region II), a sulfated glycosaminoglycan-binding domain (region III) and a proline-rich region (region IV). All nucleotide substitutions are dimorphic. Two haplotypes from Thailand contain an inserted sequence encoding aspartic acid-serine-proline in the proline-rich region. Sequence analysis has revealed that nucleotide diversity in PvTRAP is low although Brazilian isolates display a higher degree of variation than those from Thailand. Phylogenetic construction using the neighbor joining method has shown that most of the Thai and the Brazilian isolates appear to be mainly clustered into distinct groups. Significantly greater than expected values of the mean number of non-synonymous (d(n)) than synonymous (d(s)) nucleotide substitutions per site were observed in regions II and III of PvTRAP. Analysis of the published PfTRAP sequences has shown a similar finding in regions II and IV suggesting that positive selection operates on the regions. Hence, different regions in PvTRAP and PfTRAP could be under different pressures in terms of immune selection, structural and/or functional constraints.

Intragenic recombination in the 3' portion of the merozoite surface protein 1 gene of Plasmodium vivax.

To date, little has been known about the extent of sequence variation in the C-terminal part of the Plasmodium vivax merozoite surface protein 1 (PvMSP1) which has been considered to be a potential vaccine candidate. Here, we examined the variation in the region encompassing interspecies conserved blocks (ICBs) 8 and 10 of PvMSP1 by DNA sequencing of 14 Thai isolates and three Brazilian isolates. Eighteen different alleles were detected. Three new sequence types had been identified in polymorphic region between ICB8 and CB9: one was possibly a result of intragenic recombination between the Belem and Salvador I alleles and the others displayed unique repeats. A striking variation was observed in a stretch of 38 codons in polymorphic block between conserved block CB9 and ICB10, resulting in eight different sequence types, probably generated by interallelic recombination at a single or multiple sites. There is no apparent linkage between these two polymorphic sites. On the other hand, a single or stretches of nucleotide substitutions are dimorphic like in Plasmodium falciparum MSP1 (PfMSP1) in the remaining parts, creating microheterogeneity of sequences. The C-terminal 19 kDa-encoding region was extremely conserved with a single dimorphic exchange at a known position. Thus, this study provides evidence of intragenic recombination occurring in the 3' portion of PvMSP1 and suggests that the 3' portion of PvMSP1 is more diverse than that in PfMSP1.

Heterogeneity in cyst morphology within isolates of Acanthamoeba from keratitis patients in Thailand.

We isolated Acanthamoebae from the first two keratitis patients identified in Thailand in 1988 and 1990. The patients developed decreased vision, severe photophobia, severe eye pain and foreign body sensation after minor corneal trauma. The lesions included generalized superficial punctate keratitis, stromal corneal ulcer with keratic precipitate and uveitis in one case, and corneal ulcer with abscess in the other. Both cases were diagnosed by isolation of characteristic trophozoites and cysts of Acanthamoeba from corneal tissue by non-nutrient agar culture method. Based on cyst morphology, A. castellanii and A. polyphaga were detected in one case, and A. castellanii and A. triangularis in the other. Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA-RFLP) revealed that each patient harboured a single parasite population. One shared mtDNA-RFLP with an authentic strain of A. castellanii, and the other gave a new unique pattern. Thus species identification of Acanthamoeba based on cyst morphology per se can be arbitrary, and mtDNA-RFLP may be more appropriate for accurate species/strain differentiation amongst morphologically heterogeneous populations of Acanthamoebae.

Allelic recombination and linkage disequilibrium within Msp-1 of Plasmodium falciparum, the malignant human malaria parasite.

The C-terminal, cysteine-rich 19kDa domain of merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a target of the host's humoral immunity and thus a malaria vaccine candidate. Although variation in the 19kDa domain is limited among parasite isolates, tertiary structure-dependent intramolecular associations between the 19kDa domain and other parts of MSP-1 are suggested to be involved in immune evasion by allowing competitive binding of protective and non-protective antibodies directed to their epitopes, which are conformationally in close proximity but separated at the primary structure. Since allelic recombination can account for the major variability of the Msp-1 gene, we examined whether linkage disequilibrium occurs between polymorphic loci in the 5'- and the 3'-region, the latter encoding the 19kDa domain. From 184 Thai field isolates, we selected 69 isolates with a single allelic type in six variable blocks of Msp-1 as determined by PCR-based allelic typing. All the isolates showed no evidence of recombination in blocks 6 to 16, whereas recombination was apparent in blocks 2 to 6. Sequencing of the 3'-region revealed two potential recombination sites in block 17. Strong linkage disequilibrium was seen between polymorphic loci in the 5'- and 3'-regions. The strength of this disequilibrium did not correlate with distance between loci. We discuss the possible role of epistatic selection on particular association types (haplotypes) of Msp-1.

Increased sensitivity of routine laboratory detection of Strongyloides stercoralis and hookworm by agar-plate culture.

The efficacy of agar-plate culture has been evaluated for the detection of Strongyloides stercoralis and hookworm, compared with direct smear, the formalin-ether sedimentation technique and the filter-paper method. Of 1085 stool samples from the routine laboratory service at King Chulalongkorn Memorial Hospital in Bangkok, 241 samples harboured S. stercoralis, 153 hookworm and 2 Rhabditis hominis. The recovery rate of S. stercoralis by agar-plate culture is significantly superior to the other methods (P < 0.005). The ratios of positive results from the methods used to the total number of S. stercoralis-positive cases were as follows: 1:1.03 by agar-plate culture, 1:1.85 by the filter-paper method, 1:1.98 by the sedimentation technique and 1:10.48 by direct stool smear. A similar trend of the efficacy ratio of each method was obtained for hookworm detection. The characteristic furrows left by hookworm larvae, and larvae and adults of S. stercoralis could be used for preliminary species identification. Daily search for furrows on agar plates for up to 6 consecutive days resulted in an increased sensitivity for diagnosis of both S. stercoralis and hookworm infections.

First outbreak of human trichinellosis caused by Trichinella pseudospiralis.

This is the first report of an epidemic of human infection with Trichinella pseudospiralis. An outbreak of trichinellosis affecting 59 individuals, of whom one died, occurred in southern Thailand during 1994-1995. The source of this epidemic was raw pork from a wild pig that was distributed to villagers by a local hunter. The most striking clinical features among 50 individuals who could be followed were muscular swelling, myalgia, and asthenia persisting for > 4 months. These were associated with significant elevations of creatine phosphokinase and lactate dehydrogenase levels. All patients had Trichinella-specific IgG antibodies in an enzyme-linked immunosorbent assay. Muscle biopsies, performed in six cases, showed nonencapsulated, actively migrating Trichinella larvae. Experimental infection of mice with larvae from human biopsies revealed nonencapsulated muscle larvae consistent with T. pseudospiralis. The identification of muscle larvae from a human specimen by random amplified polymorphic DNA analysis confirmed the causative agent to be T. pseudospiralis. Patients seemed to respond best to treatment with albendazole.

Interallelic recombination in the merozoite surface protein 1 (MSP-1) gene of Plasmodium vivax from Thai isolates.

The merozoite of Plasmodium vivax possesses a high molecular mass surface protein called Pv-merozoite surface protein 1, PvMSP-1, which exhibits antigenic diversity among isolates. In this study, the extent of sequence variation in the polymorphic region and the flanking interspecies conserved blocks (ICBs) 5 and 6 of the PvMSP-1 gene was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty different alleles were obtained from 15 Thai isolates. Results revealed five distinct sequence types of the polymorphic region, two of which were newly identified in this study: one probably generated by intragenic recombination at a site different from that previously reported and the other by duplication of a 30 nucleotide (nt) sequence at the 3' end of the region. On the other hand, almost all nucleotide substitutions in the flanking regions, ICB5 and ICB6, were dimorphic, creating microheterogeneity in the region. Furthermore, stretches of nucleotide substitutions were found to be linked in ICB6, suggesting the potential recombination sites between these stretches. It is also noted that extensive sequence variation in the PvMSP-1 gene and coinfection with different PvMSP-1 alleles occurred among the P. vivax population in the endemic areas of Thailand.

The merozoite surface protein 2 (MSP2) gene of Plasmodium falciparum from a Thai isolate.

The merozoite surface protein 2 (MSP2) of Plasmodium falciparum was a malaria vaccine candidate. The gene encoding MSP2 of a Thai isolate was amplified by polymerase chain reaction followed by subcloning into a phagemid vector and sequencing. Sequence alignment with other previously published sequences revealed that the MSP2 allele in this isolate belonged to FC27 allelic family. The central variable sequence of the MSP2 allele in this study was related to an allele from Indonesia. The flanking sequences of the variable region were highly conserved.

Allelic variation in the circumsporozoite protein of Plasmodium falciparum from Thai field isolates.

Allelic variation in the Plasmodium falciparum circumsporozoite (CS) protein gene has been examined by sequencing the entire gene in 15 isolates from an endemic area of Thailand. The isolates contain a total of six new allelic forms of the tetrapeptide repeats and eight variants of the T cell epitope (TCE) region of the CS gene. All nucleotide substitutions in the TCE are nonsynonymous. There is no apparent association between the sequence patterns in the repeats and in the TCE. Comparison of the TCE with published sequences has shown that most variants of our isolates are not identical to those found in different geographic areas, suggesting geographic variation in genetic diversity of the CS protein. In a phylogenetic tree, the new Thai alleles did not cluster together, suggesting a considerable heterogeneity within some geographic areas. Furthermore, analyses of tetrapeptide repeats from a number of isolates and strains showed evidence of three genetic mechanisms for the generation of variation in the repeats of the CS gene: point mutation, duplication of one or more repeat units, and intragenic recombination.

Sequence conservation in the C-terminal part of the precursor to the major merozoite surface proteins (MSP1) of Plasmodium falciparum from field isolates.

The C-terminal part of the precursor to the major merozoite surface proteins (MSP1) of Plasmodium falciparum contains potential protective epitopes and two cleavage sites for processing which take place prior to erythrocyte invasion by the merozoite. Since sequences available to date are limited and derived from cultured parasites, we have examined the extent of variations of this important part of the MSP1 gene from natural populations. Our sequence analyses of 1.6-1.7 kb from blocks 13-17 of the gene obtained from 19 Thai wild isolates have identified a deletion of a codon and 18 nucleotide substitutions, all of which are dimorphic substitutions and all but one create amino acid exchanges. However, residues at two cleavage sites for the C-terminus 42 kDa polypeptide and the 19-kDa polypeptide, a subfragment of the former, are conserved. Furthermore, all 12 cysteine residues at the C-terminal 19-kDa polypeptide are perfectly conserved, allowing the formation of 2 epidermal growth factor-like structures. These results indicate that in contrast to extensive variations at the N-terminal part of MSP1, limited variations occur at the C-terminal part.