In-vitro cultivation: a sensitive method for detecting Blastocystis hominis

Currently, the detection of human infection with Blastocystis hominis is usually based on the examination under a light microscope of faecal samples, either directly, as 'simple smears', or after some form of concentration. Whether short-term, in-vitro cultivation would increase the sensitivity of such detection remains a matter of controversy. Over 900 fresh stool specimens, from soldiers in the Royal Thai Army, were each checked for the parasite using three methods: simple smears; formalin-ethyl-acetate concentration; and cultivation in Jones' medium. Although 334 of the samples were found to be culture-positive, the parasites were only detected in 142 of the simple smears, and faecal concentration led to an even lower sensitivity (64 positive samples). In-vitro cultivation does seem worthwhile in the detection of B. hominis carriage in field studies.     

Transmission of Enterocytozoon bieneusi genotype a in a Thai orphanage

A cross-sectional study of Enterocytozoon bieneusi infection in children who lived in an orphanage in Bangkok, Thailand was conducted in April 2003. Two hundred ninety stool specimens were collected and examined under light microscopy after staining with gram-chromotrope. Confirmation of E. bieneusi was done using transmission electron microscopy. Of 290 samples, 12 (4.1%) were positive for E. bieneusi. Genotypic characterization of 10 E. bieneusi showed that all were genotype A, which might indicate the same source of infection. Multivariate analysis showed that orphans who were 12-23 months old, girls, and living in one particular house were independently associated with E. bieneusi infection. Our study suggests that E. bieneusi infection in this orphanage might be transmitted person to person.     

Central Role of Hemoglobin Degradation in Mechanisms of Action of 4-Aminoquinolines, Quinoline Methanols, and Phenanthrene Methanols

We have used a specific inhibitor of the malarial aspartic proteinase plasmepsin I and a nonspecific cysteine proteinase inhibitor to investigate the importance of hemoglobin degradation in the mechanism of action of chloroquine, amodiaquine, quinine, mefloquine (MQ), halofantrine, and primaquine. Both proteinase inhibitors antagonized the antiparasitic activity of all drugs tested with the exception of primaquine. An inhibitor of plasmepsin I, Ro40-4388, reduced the incorporation of radiolabelled chloroquine and quinine into malarial pigment by 95%, while causing a 70% reduction in the incorporation of radiolabelled MQ. Cysteine proteinase inhibitor E64 reduced the incorporation of chloroquine and quinine into malarial pigment by 60 and 40%, respectively. This study provides definitive support for the central role of hemoglobin degradation in the mechanism of action of the 4-aminoquinolines and the quinoline and phenanthrene methanol antimalarials.     

Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from Thailand

We investigated the association between the Plasmodium vivax dihydrofolate reductase (Pvdhfrtas) and the P. vivax dihydropteroate synthase (Pvdhps) genotype and in vitro sensitivity to the antifolates pyrimethamine, WR99210, chlorcycloguanil, sulfadoxine, and dapsone. Drug responses of 32 P. vivax isolates were assessed in two in vitro systems: schizont maturation inhibition and a yeast expression system. The geometric mean of 50% inhibition concentration (IC(50)) values for pyrimethamine, chlorcycloguanil, WR99210, sulfadoxine, and dapsone were 85 +/- 88, 784 +/- 662, 95 +/- 87, 2,424 +/- 2,784, and 1,625 +/- 1,801 nM, respectively, for the schizont maturation assay. Five different Pvdhfr alleles and four Pvdhps alleles were observed: 26 of 32 quadruple mutant alleles of Pvdhfr (F57I,L/S58R/T61M/S117T), four triple mutants (S58R/T61M/S117T, K49C/S58R/S117N), and two double mutant isolates (S58R/S117N). All isolates carried Pvdhps 585V. Twenty four isolates carried double mutant Pvdhps (A383G/A553G), six an additional mutation, S382A,C/A383G/A553G, and two a single mutation, A383G. Increasing geometric mean IC(50) values were observed with increased number of Pvdhfr mutations from double to quadruple. Results suggest that quadruple mutant alleles confer decreased sensitivity to pyrimethamine but retain sensitivity to WR99210.     

Relationship between Antimalarial Drug Activity, Accumulation, and Inhibition of Heme Polymerization in Plasmodium falciparum In Vitro

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.     

Molecular epidemiology of drug resistance markers of Plasmodium falciparum malaria in Thailand

To determine differences in the distribution of drug resistance mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genes of P. falciparum isolates in Thailand, a study was conducted using polymerase chain reaction-restriction fragment length polymorphism to detect mutations in P. falciparum isolates obtained from three areas with different levels of in vivo mefloquine (MQ) resistance. All isolates carried mutant allele T76 of the pfcrt gene and wild-type allele D1246 of the pfmdr1 gene except for one isolate, which showed the wild-type K76 allele. This isolate was obtained from Chanthaburi Province, an area with high MQ resistance. Relatively low rates of the mutant alleles D1042 and Y86 of the pfmdr1 gene were found among Thai isolates of P. falciparum. However, a statistically significant difference in the distribution was noted. Most of the mutant isolates were found among isolates from areas with moderate or low MQ resistance. Only one isolate with mixed mutant and wild-type N1042 and D1042 and two mutants of Y86 were found among the isolates from areas with high MQ resistance. The findings provide limited support for the hypothesis that mutant alleles of pfmdr1 may be associated with increased sensitivity to MQ.     

Transmission of intestinal blastocystosis related to the quality of drinking water

A cross-sectional study was performed to evaluate the risk factors of Blastocystis hominis infection in the Thai army population of the 11th Infantry Division, Chachoengsao Province, Thailand. 201 army personnel and their family members were enrolled in this study. Intestinal parasitic infections in this population were assessed by stool examination using simple smear, formalin/ether technique and Kato-thick smear. Approximately one third of the specimens were positive for one or more intestinal parasites. With the prevalence of 21.9%, B. hominis was the most common intestinal parasite found in this population. Our data indicated that blastocystosis in this army population was significantly linked to the quality of drinking water. After being adjusted for potential confounders, consuming neither filtered nor boiled water was independently associated with blastocystosis.     

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.     

Prevalence and Risk Factors for Blastocystis Infection among Children and Caregivers in a Child Care Center,Bangkok, Thailand

In September 2009, a cross-sectional study was conducted to evaluate parasitic infections in a child care center in Khlong Toei, Bangkok, Thailand. Of 503 children and staff members, 258 (51.3%) stool samples and questionnaires were obtained. The most common parasitic infection was Blastocystis sp. (13.6%). Blastocystis sp. subtype 3 was predominantly found (80.0%), followed by subtypes 2 (12.0%) and 1 (8.0%). The prevalence of Blastocystis infection varied among different age groups. The prevalence of Blastocystis infection in non-HIV-infected children aged < 10 and 10–19 years were 14.5% and 10.3%, respectively, which were not significantly different. All 31 HIV-infected children were not infected with Blastocystis sp. The most likely reason could be the result of properly using prevention measures for this specific group.     

Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand

In January 2005, a survey of intestinal parasitic infections was performed in a primary school, central Thailand. Of 675 stool samples, Blastocystis was identified with a prevalence of 18.9%. Genetic characterization of Blastocystis showed subtype 1 (77.9%) and subtype 2 (22.1%). Study of the water supply in this school was performed to find the possible sources of Blastocystis. Blastocystis from one water sample was identified as subtype 1, which had a nucleotide sequence of small subunit (SSU) ribosomal RNA (rRNA) gene that was 100% identical to that of Blastocystis infected in schoolchildren. Our information supports the evidence of water-borne transmission in this population.