Novel Potential Biomarkers for Opisthorchis viverrini Infection and Associated Cholangiocarcinoma.

BACKGROUND/AIM: Early detection of disease is a pivotal factor for determining prognosis and clinical outcome of patients with cancer. As cholangiocarcinoma (CCA) is currently difficult to detect and most cases of such cancer present with late-stage disease at the time of initial diagnosis, we employed proteomic analysis of the bile to identify potential candidate biomarkers for Opisthorchis viverrini (OV)-associated CCA. MATERIALS AND METHODS: Proteins in pooled bile samples from patients with CCA and OV infection, with CCA without OV infection, with OV infection but no CCA, and with neither OV infection nor CCA were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel trypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: According to our analysis, three proteins, namely aristaless-like homeobox1 isoform X1 (ALX1), major histocompatibility complex polypeptide-related sequence A (MICA), and uncharacterized protein C14orf105 isoform X12 were found to be potential markers for OV infection, as they were predominantly found in all OV-infected groups. Although these proteins were detected in both OV-infected patients with and without CCA, their abundance was 2.90-, 7.06-and 3.65-fold higher, respectively, in those with CCA. In patients with CCA, potential novel biomarkers wre immunoglobulin heavy chain, translocated in liposarcoma (TLS), visual system homeobox 2 (VSX2) and an unnamed protein product. CONCLUSION: We provided novel information regarding potential biomarkers for OV infection and CCA. These two protein profiles could benefit diagnosis as well as monitoring of CCA.

The prevalence of molecular markers of drug resistance in Plasmodium vivax from the border regions of Thailand in 2008 and 2014.

The prevalence of Plasmodium vivax is increasing in the border regions of Thailand; one potential problem confounding the control of malaria in these regions is the emergence and spread of drug resistance. The aim of this study was to determine the genetic diversity in genes potentially linked to drug resistance in P. vivax parasites isolated from four different border regions of Thailand; Thai-Myanmar (Tak, Mae Hong Son and Prachuap Khiri Khan Provinces), and Thai-Cambodian borders (Chanthaburi Province). Isolates were collected from 345 P. vivax patients in 2008 and 2014, and parasite DNA extracted and subjected to nucleotide sequencing at five putative drug-resistance loci (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o and Pvk12). The prevalence of mutations in Pvdhfr, Pvdhps and Pvmdr1 were markedly different between the Thai-Myanmar and Thai-Cambodian border areas and also varied between sampling times. All isolates carried the Pvdhfr (58R and 117N/T) mutation, however, whereas the quadruple mutant allele (I57R58M61T117) was the most prevalent (69.6%) in the Thai-Myanmar border region, the double mutant allele (F57R58T61N117) was at fixation on the Thai-Cambodian border (100%). The most prevalent genotypes of Pvdhps and Pvmdr1 were the double mutant (S382G383K512G553) (65.1%) and single mutant (M958Y976F1076) (46.5%) alleles, respectively on the Thai-Myanmar border while the single Pvdhps mutant (S382G383K512A553) (52.7%) and the triple Pvmdr1 mutant (M958F976L1076) (81%) alleles were dominant on the Thai-Cambodian border. No mutations were observed in the Pvcrt-o gene in either region. Novel mutations in the Pvk12 gene, the P. vivax orthologue of PfK13, linked to artemisinin resistance in Plasmodium falciparum, were observed with three nonsynonymous and three synonymous mutations in six isolates (3.3%).

Chromosome Abnormalities and Absolute Telomere Lengths of Leukocytes from Silk Weavers with Emphasis on Potential Genotoxicity and Mutagenicity of Silk Dyes

Objectives: This study is aimed to assess the possible genotoxicity and mutagenicity of silk dyes on silk weavers. Methods: Peripheral blood leukocytes were obtained from 24 silk weavers and 24 age- and sex-matched controls in northeastern Thailand. After mitogen stimulation in culture, chromosome abnormalities were examined using Giemsa banding and the absolute telomere length (aTL) was measured with SYBR green qRT-PCR. To confirm genotoxic and mutagenic effects of silk dyes, leukocytes from one each of healthy male and female volunteers were cultured with various concentrations of 3 dark red silk dyes under the presence of mitogen. Chromosome abnormalities and the telomere length were determined as above. Results: The proportion of normal metaphase in the silk weaving workers was significantly lower than that in controls. The frequency of chromosome aberrations was higher in the silk weavers than in control group. Polyploidy was detected only in the silk weavers. The aTL was significantly shorter in the silk weavers than in control group (p < 0.05). When leukocytes from normal volunteers were stimulated with mitogen under the presence of various concentrations of 3 silk dyes, suppressed the mitotic index (MI) and normal metaphase, whereas the proportion of prophase and the incomplete chromosome forming increased significantly. All dyes induced polyploidy. Dye #CA5 induced structural changes in male leukocytes, whereas #30 induced the changes in female leukocytes. The #CA5 increased aTL of normal leukocytes in a dose-dependent manner. Conclusions: All dyes, especially #CA5, have high genotoxicity and mutagenicity to induce chromosome aberrations and telomeric instability. Taken all those results together, regular health checking of silk weavers who have been exposed to those dyes is critically necessary to prevent various chemical-induced carcinogenesis.

Plasmodium vivax rhomboid-like protease 1 gene diversity in Thailand.

BACKGROUND: Plasmodium vivax infection remains a major public health problem, especially along the Thailand border regions. We examined the genetic diversity of this parasite by analyzing single-nucleotide polymorphisms (SNPs) of the P. vivax rhomboid-like protease 1 gene (Pvrom1) in parasites collected from western (Tak province, Thai-Myanmar border) and eastern (Chanthaburi province, Thai-Cambodia border) regions. METHODS: Data were collected by a cross-sectional survey, consisting of 47 and 45 P. vivax-infected filter paper-spotted blood samples from the western and eastern regions of Thailand, respectively during September 2013 to May 2014. Extracted DNA was examined for presence of P. vivax using Plasmodium species-specific nested PCR. Pvrom1 gene was PCR amplified, sequenced and the SNP diversity was analyzed using F-STAT, DnaSP, MEGA and LIAN programs. RESULTS: Comparison of sequences of the 92 Pvrom1 831-base open reading frames with that of a reference sequence (GenBank acc. no. XM001615211) revealed 17 samples with a total of 8 polymorphic sites, consisting of singleton (exon 3, nt 645) and parsimony informative (exon 1, nt 22 and 39; exon 3, nt 336, 537 and 656; and exon 4, nt 719 and 748) sites, which resulted in six different deduced Pvrom1 variants. Non-synonymous to synonymous substitutions ratio estimated by the DnaSP program was 1.65 indicating positive selection, but the Z-tests of selection showed no significant deviations from neutrality for Pvrom1 samples from western region of Thailand. In addition McDonald Kreitman test (MK) showed not significant, and Fst values are not different between the two regions and the regions combined. Interestingly, only Pvrom1 exon 2 was the most conserved sequences among the four exons. CONCLUSIONS: The relatively high degree of Pvrom1 polymorphism suggests that the protein is important for parasite survival in face of changes in both insect vector and human populations. These polymorphisms could serve as a sensitive marker for studying plasmodial genetic diversity. The significance of Pvrom1 conserved exon 2 sequence remains to be investigated.

Viability and morphological changes of Acanthamoeba spp. cysts after treatment with Effective microorganisms (EM).

Acanthamoeba is a free-living opportunistic protozoan parasite that is found in diverse environments. It can cause keratitis, mostly related to inappropriate use of contact lenses, as well as life threatening diseases including encephalitis, disseminated sinusitis, and skin ulcers. This study investigated morphological changes and fine structures of the cyst form of Acanthamoeba spp. after treatment with effective microorganisms (EM™) using light and scanning electron microscopies. Acanthamoeba cysts treated with 1:2, 1:4, 1:6, and undiluted EM™ showed higher percentages of non-viable cysts than those treated with 1:8, 1:10, 1:100, 1:200, and 1:400 EM™ and at 5 days post-treatment developed from cystic stage to trophozoite stage. Acanthamoeba cysts treated at concentrations of 1:2, 1:4, 1:6, and undiluted EM™ exhibited cytoplasmic clumping and shrinkage of amoeba cells away from cyst walls. The effective EM™ concentration lethal to Acanthamoeba spp. cyst could provide information to monitor the environmental control system.

RED BLOOD CELL DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES AND SUSCEPTIBILITY TO PLASMODIUM VIVAX INFECTION IN THAIS.

Plasmodium vivax is the most prevalent malaria infection in Thailand. P. vivax uses Duffy Antigen Receptor for Chemokines (DARC) or Duffy antigen (Fy) as a receptor for entry into reticulocytes. Polymorphism of DARC exon 2 gene (FYA/FYB) in 40 P. vivax-infected subjects were investigated using nested PCR of blood samples spotted on filter paper collected during August 2013 to November 2013 from various malaria clinics in Thailand. Distribution of DARC genotypes was FYA 62.5%, FYB 20% and FYAB 17.5%, consistent with that of Hardy-Weinberg equation. Mutation G17A was found in both FYA and FYB alleles, resulting in Gyl48 and Asp48 of Fya and Fyb antigen, respectively. Mean parasitemia among the three groups is not statistically different. To the best of our knowledge, this is the first such study in Thailand.

Intervals to Plasmodium falciparum recurrence after anti-malarial treatment in pregnancy: a longitudinal prospective cohort.

BACKGROUND: Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment failure is common. The objective of this study was to examine the duration of persistent parasite carriage following anti-malarial treatment in pregnancy. METHODS: The data presented here are a collation from previous studies carried out since 1994 in the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed using the same unique methodology detailed in the Materials and Methods section. Screening for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick sample collected. Parasite DNA was extracted from the blood-spot samples using saponin lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2 and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or indeterminate. Factors associated with time to microscopy-detected recrudescence were analysed using multivariable regression techniques. RESULTS: From December 1994 to November 2009, 700 women were treated for P. falciparum and there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR genotyping. Most of the recurrences, 85% (770/909), occurred after treatment with quinine monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean number of days to recurrence was significantly shorter in women with recrudescent infection, 24.5 (95%: 23.4-25.8), compared to re-infection, 49.7 (95%: 46.9-52.7), P<0.001. The proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63 from the start of treatment was 29.1% (124/428), 13.3% (57/428) and 5.6% (24/428). Recrudescent infections≥100 days after treatment occurred with quinine and mefloquine monotherapy, and quinine+clindamycin and artesunate+atovaquone-proguanil combination therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax infection increased the geometric mean interval to recrudescence by 1.28-fold (95% CI: 1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased 0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence) and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia. CONCLUSIONS: Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial treatment. Long intervals to recrudescence are more likely with the use of artemisinin-containing treatments and also observed with intercalated P. vivax infections treated with chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration of follow-up, preferably until delivery or day 63, whichever occurs last.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax.

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/µl (25sporozoites/µl) for P. falciparum DNA and between 0.5 and 5pg/µl (25-250sporozoites/µl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.

Salivary Glands Proteins Expression of Anopheles dirus A Fed on Plasmodium vivax- and Plasmodium falciparum-Infected Human Blood.

Mosquitoes are able to adapt to feed on blood by the salivary glands which created a protein that works against the haemostasis process. This study aims to investigate the salivary glands proteins expression of 50 adult female An. dirus A mosquitoes, a main vector of malaria in Thailand, each group with an age of 5 days which were artificial membrane fed on sugar, normal blood, blood infected with P. vivax, and blood infected with P. falciparum. Then mosquito salivary gland proteins were analyzed by SDS-PAGE on days 0, 1, 2, 3, and 4 after feeding. The findings revealed that the major salivary glands proteins had molecular weights of 62, 58, 43, 36, 33, 30, and 18 kDa. One protein band of approximately 13 kDa was found in normal blood and blood infected with P. vivax fed on day 0. A stronger protein band, 65 kDa, was expressed from the salivary glands of mosquitoes fed with P. vivax- or P. falciparum-infected blood on only day 0, but none on days 1 to 4. The study shows that salivary glands proteins expression of An. dirus may affect the malaria parasite life cycle and the ability of mosquitoes to transmit malaria parasites in post-24-hour disappearance observation.

The geographical distribution of allele polymorphisms of Plasmodium vivax in different regions of Thailand.

OBJECTIVE: To investigate the genetic structure of Plasmodium vivax circumsporozoite surface protein (PvCSP) and P. vivax merozoite surface protein 1 (PvMSP1) genes from field isolates of malaria parasites from four different regions of Thailand. MATERIAL AND METHOD: The data was collected by cross-sectional survey, consisting of 273 P. vivax infected blood samples from malaria clinics in 4 different border regions of Thailand from February 2008 to February 2009. The dried blood spots were extracted for DNA and Plasmodium species confirmed by species-specific primer sets. PvCSP and PvMSP1 genes were amplified and their population genetics were analyzed by using the Heterozygosity (H) formula, F-STAT and LIAN programs. RESULT: There was considerable variation in the PvMSP1 gene within 2 fragments for which HE was 0.8303, whereas PvCSP showed low HE at 0.1418. Significant differences in allele frequencies between sites were quantified by Fst, Linkage disequilibrium (LD). The results showed PvMSP1 F2; Fst = 0.063, p = 0.07; PvMSP1 F2 RFLP pattern; Fst = 0.154, p = 0.005; PvMSP1 F3; Fst = 0.23, p = 0.005 and the overall loci showed Fst = 0.151, p = 0.005 (Fisher's exact test). All values of Index association (I(S)A) were non-significant. There was no evidence of LD within the P. vivax populations. CONCLUSION: H(E) at each locus of the PvMSP1 gene showed significant differences in allele frequencies between sites.

Dengue-2 virus carrying capacity of Thai Aedes aegypti strains with different susceptibility to deltamethrin.

Deltamethrin-resistant Aedes aegypti currently threatens the effectiveness of dengue hemorrhagic fever control operations in Thailand. Although a previous study has suggested that insecticide resistance may increase Ae. aegypti susceptibility to dengue-2 virus infection, our experimental data showed no significant association between laboratory-induced deltamethrin-resistance in a Thai Ae. aegypti isolate and its susceptibility to dengue -2 infection.

Biology of Culex sitiens, a predominant mosquito in Phang Nga, Thailand after a tsunami.

A tsunami affected area in Phang Nga province, Thailand was explored randomly as some freshwater sites had changed into brackish-water sites. A survey of four areas found Culex sitiens to be the most dominant mosquito species.This mosquito prefers to breed in putrefied water with garbage and it was found in almost every stagnant, brackish-water site in full sunlight. The larval density was more than 300 larvae/dip/250 ml water. Its biting cycle, determined by human landing catch, was nocturnal, with a single peak at 19.00-20.00 hr. The maximum rate was 108 mosquitoes per person/hour. The biology of the mosquito was studied by colonization in natural water under laboratory conditions. The mean number of eggs per raft was 158.1 ± 31.7, hatchability 96.6 ± 4.1%, development from 1st instar larvae to adult was 8.8-11.7 days, and longevity of adult males was 7.3-41.3 days and females 11.0-52.7 days. The ratio of adult males to adult females was 1:1.1 ± 0.2.

Genetic diversity in new members of the reticulocyte binding protein family in Thai Plasmodium vivax isolates.

BACKGROUND: Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes. METHODOLOGY/PRINCIPAL FINDINGS: The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3∶1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates. CONCLUSIONS/SIGNIFICANCE: The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.

The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand.

OBJECTIVE: To investigate the genetic polymorphism of Plasmodium vivax (P. vivax) PvCSP and PvMSP1 genes from field isolates at four endemic regions (North, East, West and South) of Thailand. METHODS: The 152 P. vivax infected cases from dried blood spots were DNA extracted and confirmed by species-specific primer sets using multiplex PCR method. PvMSP1 fragments F2 and F3; PvCSP were genotyped using RFLP-PCR method. RESULTS: Totally amplified DNA which was multiple genotypes for PvMSP1 F2 and PvMSP1 F3 were 12.50% and 8.55%, respectively while PvCSP was 3.95%. The overall frequency of multiple genotypes was 25%. There were 12 allele types of PvMSP1 F2 using AluI enzyme digestion and 8 size variations were found in PvMSP1 F3. The isolates from western region was highly genetic diverse when compare among all isolates. The predominant variant type of PvCSP gene was VK210 type. CONCLUSIONS: The multiple genotypes are common found in Thailand and it might hide the real genotype. PvCSP does not have extensive genetic diversity in this study. However, PvMSP1 marker due to multiple genotypes is difficult to be analyzed. The multiple genotypes findings might stem from population migration and vector species findings.

Novel ellipsoid spatial analysis for determining malaria risk at the village level.

The distribution patterns of malaria incidence at a village level in Thailand were demonstrated with the use of a geographical information system (GIS), and provided the study of the malaria situation at a household level. Mosaic imageries from aerial photographs were used to create maps that contained X and Y coordinates. These digitized base maps were kept as computerized files. Standard Distance Ellipse (SDE) was used to measure the prevalence of dispersion around the mean center of malaria cases and points. Households in the SDE were at greater risk of malaria infection than those located outside the SDE. The spatial pattern of malaria incidence was investigated using spatial autocorrelation using Geary's ratio and Moran's index. Five of seven villages had a clustered spatial distribution of malaria incidence, the vector point of which had a 2-3km range from the patient's houses. Only one village had a significant clustered spatial distribution of malaria incidence (p<0.05). Control efforts should be focused on high-risk areas, especially those households with the heaviest caseloads. This approach would probably be more cost effective than the conventional malaria control methods. This SDE analytical technique would be a novel and useful epidemiological control method for use by public health administrators. The ellipsoidal areas required malaria control intervention.

Effective and cheap removal of leukocytes and platelets from Plasmodium vivax infected blood.

BACKGROUND: Investigations of Plasmodium vivax are restricted to samples collected from infected persons or primates, because this parasite cannot be maintained in in vitro cultures. Contamination of P. vivax isolates with host leukocytes and platelets is detrimental to a range of ex vivo and molecular investigations. Easy-to-produce CF11 cellulose filters have recently provided us with an inexpensive method for the removal of leukocytes and platelets. This contrasted with previous reports of unacceptably high levels of infected red blood cell (IRBC) retention by CF11. The aims of this study were to compare the ability of CF11 cellulose filters and the commercial filter Plasmodipur at removing leukocyte and platelet, and to investigate the retention of P. vivax IRBCs by CF11 cellulose filtration. METHODS AND RESULTS: Side-by-side comparison of six leukocyte removal methods using blood samples from five healthy donor showed that CF11 filtration reduced the mean initial leukocyte counts from 9.4 x 103 per microl [95%CI 5.2-13.5] to 0.01 x 103 [95%CI 0.01-0.03]. The CF11 was particularly effective at removing neutrophils. CF11 treatment also reduced initial platelet counts from 211.6 x 103 per microl [95%CI 107.5-315.7] to 0.8 x 103 per microl [95%CI -0.7-2.2]. Analysis of 30 P. vivax blood samples before and after CF11 filtration showed only a minor loss in parasitaemia (

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method.

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.

Morphological features of Acanthamoeba causing keratitis contaminated from contact lens cases.

OBJECTIVE: To study the morphological characteristics of genus Acanthamoeba which is an opportunistic organism associated with wearing contact lenses that the biofilm phenomenon in contact lens cases contained Acanthamoeba causing keratitis by conventional culture technique. MATERIAL AND METHOD: A total of 150 contact lens cases were biofilm scraped in March till September 2007, at an institution in Nakhornpathom Province, Thailand. The 'gold standard' culture technique was used for the excystation growth development observation. Cysts of Acanthamoeba spp. contained 50 microlitres of Escherichia coli and contact lens solution were incubated and observed for the presence of cysts and/or trophozoites for 12 days. An infected slide was stained with giemsa solution and other non-stained and non-fixed slides were carried out for morphological characteristics study by different microscopes. RESULTS: The prevalence of Acanthamoeba spp. in scraping of contact lens cases was 6.7% (10/150). These Acanthamoeba isolates at temperature around 37 degrees C were consisted of all three groups, which in summary; the average diameter of cysts in Astronyxids (group I) was relatively large. They were > or = 18 micrometers, while those of Polyphagids (group II) and Culbertsonids (group III) were < or = 18 micron. The typical morphology of Acanthamoeba trophozoites moving freely in water were recognized by the presence of lobopodium and acanthopodia within 12 observed days. The average size of Acanthamoeba trophozoites was in the range of 12-45 micron. Three different images of cyst were feature studied. CONCLUSION: Three Acanthamoeba groups by biofilm scraping from contact lens cases should be differentiated. Morphological characteristics cysts and trophozoites should be confirmed. In addition, to improve contact lens wearer education, compliance with contact lens cases, hygiene recommendations and regular disposal of contact lens cases might help to solve contact lens cases.

Plasmodium vivax trophozoites insensitive to chloroquine.

BACKGROUND: Plasmodium vivax is a major cause of malaria and is still primarily treated with chloroquine. Chloroquine inhibits the polymerization of haem to inert haemozoin. Free haem monomers are thought to catalyze oxidative damage to the Plasmodium spp. trophozoite, the stage when haemoglobin catabolism is maximal. However preliminary in vitro observations on P. vivax clinical isolates suggest that only ring stages (early trophozoites) are sensitive to chloroquine. In this study, the stage specific action of chloroquine was investigated in synchronous cryopreserved isolates of P. vivax. METHODS: The in vitro chloroquine sensitivity of paired ring and trophozoite stages from 11 cryopreserved P. vivax clinical isolates from Thailand and two Plasmodium falciparum clones (chloroquine resistant K1 and chloroquine sensitive FC27) was measured using a modified WHO microtest method and fluorometric SYBR Green I Assay. The time each stage was exposed to chloroquine treatment was controlled by washing the chloroquine off at 20 hours after the beginning of treatment. RESULTS: Plasmodium vivax isolates added to the assay at ring stage had significantly lower median IC50s to chloroquine than the same isolates added at trophozoite stage (median IC50 12 nM vs 415 nM p < 0.01). Although only 36% (4/11) of the SYBR Green I assays for P. vivax were successful, both microscopy and SYBR Green I assays indicated that only P. vivax trophozoites were able to develop to schizonts at chloroquine concentrations above 100 nM. CONCLUSION: Data from this study confirms the diminished sensitivity of P. vivax trophozoites to chloroquine, the stage thought to be the target of this drug. These results raise important questions about the pharmacodynamic action of chloroquine, and highlight a fundamental difference in the activity of chloroquine between P. vivax and P. falciparum.