In vitro drug susceptibility using a parasite-rescue and transformation assay of Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis amastigotes against antileishmanial drugs.

Leishmaniasis is an emerging infectious disease in Thailand, with Leishmania martiniquensis and Leishmania orientalis identified as the primary causative agents among immunocompetent and immunocompromised individuals. Variations in drug susceptibility among different Leishmania species have been reported in different regions. Therefore, drug susceptibility assays are essential to assess the effectiveness of antileishmanial drugs used or potentially used in the affected areas. This study investigated the in vitro drug sensitivity of L. martiniquensis and L. orientalis, along with two reference species causing VL, namely L. donovani and L. infantum, against six antileishmanial drugs. Using a parasite-rescue and transformation assay, the results demonstrated that the IC50 values of amphotericin B (AmB), miltefosine (MIL), and sodium stibogluconate (Sb(III)) against all Leishmania species tested were within the sensitive range of each drug. On the contrary, the IC50 values of artemisinin (ART) and dihydroartemisinin (DHA), drugs primarily used for malaria treatment, were outside the sensitive range of the Leishmania species tested except L. infantum. This in vitro study highlights that AmB could effectively exhibit good sensitivity against the intracellular amastigotes of L. martiniquensis and L. orientalis. Also, MIL and Sb(III) could be considered alternative drugs for antileishmanial treatment in Thailand.

Risk Factors of Leishmania Infection among HIV-Infected Patients in Trang Province, Southern Thailand: A Study on Three Prevalent Species.

There are two main species of Leishmania reported in Thailand, that is, Leishmania siamensis and Leishmania martiniquensis. Moreover, leishmaniasis cases caused by Leishmania donovani complex were also reported. There is still a lack of information concerning risk factors of Leishmania infection in Thailand. This study aimed to identify the risk factors of Leishmania infection caused by these three species among HIV-infected patients. A cross-sectional study was conducted in HIV clinic at Trang Hospital, Thailand. Nested PCR and sequencing were performed to detect Leishmania DNA in blood and saliva samples and identify Leishmania species. A standardized questionnaire was used to interview individuals. A total of 526 patients were recruited in this study. Sixty-three (12.0%) were positive for L. siamensis, 24 (4.6%) were positive for L. martiniquensis, and 23 (4.4%) were positive for L. donovani complex. Risk factors of L. siamensis infection included using intravenous drug (adjusted odds ratio [AOR] 2.01, 95% CI: 1.01-4.02). Risk factors of L. martiniquensis infection included female gender (AOR 4.23, 95% CI: 1.52-11.75), using recreational drug (AOR 3.43, 95% CI: 1.00-11.74), and having comorbidities (AOR 4.94, 95% CI: 2.00-12.21). Risk factors of L. donovani complex infection included having opportunistic infection (AOR 4.22, 95% CI: 1.00-17.79), CD4 count 200-500 cells/mm3 (AOR 3.64, 95% CI: 1.14-6.86), and not using insect repellent (AOR 3.04, 95% CI: 1.08-8.58). This study identified the risk factors of Leishmania infection caused by three Leishmania species in Thailand. The data could be useful for disease prevention and control. Further studies on trends of Leishmania infection and preventive measures are recommended.

An Epidemiological Survey of Opisthorchis viverrini Infection in a Lightly Infected Community, Eastern Thailand.

Human liver fluke infection caused by Opisthorchis viverrini increases the risk of cholangiocarcinoma (CCA) reported along the Mekong basin including Thailand, Lao People's Democratic Republic (PDR), Cambodia, and Vietnam. The highest incidence of CCA has been reported in northeastern Thailand where liver fluke infection is prevalent. This study aimed to investigate the prevalence of O. viverrini infection in a northeastern-descendent community in rural Sa Kaeo Province, eastern Thailand, using stool examination and molecular technique. The Kato-Katz method was performed to determine eggs per gram (EPG) for infection intensity. Phosphate-buffered saline-ethyl acetate concentration was used to prepare specimens for polymerase chain reaction (PCR) and restriction fragment length polymorphism of the internal transcribed spacer 2 (ITS2) region of the ribosomal RNA. From 1,245 specimens, 105 (8.4%) samples were identified as Opisthorchis-like eggs from stool examination, and all positive specimens indicated light infection (< 1,000 EPG). From positive Opisthorchis-like egg samples, 55.2% (58/105) were identified as O. viverrini eggs from ITS2-PCR assay for which low infection intensity might result in a negative PCR result (44.8%). Using multiple logistic regression analysis, males were at 3.1 times higher risk of acquiring O. viverrini infection than females. From phylogenetic analysis, in eastern Thailand, nucleotide sequences of O. viverrini were grouped as a monoclade as those isolated from Greater Mekong, Vietnam, Myanmar, and west Siberia. The results revealed that the surveyed community is a low-grade endemic area of O. viverrini infection. Thus, data from this study can be used to improve health-promoting programs and activities to control the infection and its subsequent CCA.

In vitro growth characteristics and morphological differentiation of Leishmania martiniquensis promastigotes in different culture media.

The protozoan hemoflagellate, Leishmania martiniquensis, is the causative agent of cutaneous and visceral leishmaniasis among humans. This parasite was first isolated from an autochthonous case of cutaneous leishmaniasis in Martinique Island (French West Indies) in 1995 and its taxonomical position was later established in 2002. At present, the emergence of this globally infectious disease caused by L.martiniquensis raises serious concerns and has gained attention from the national public health policy. Epidemiological studies indicated that Thailand is one of the endemic areas of L.martiniquensis with hundreds of cases, both symptomatic and asymptomatic, have been reported among patients positive for HIV/AIDS. Information on its basic biology including suitable conditions for parasite propagation is limited. To assess this, we used four established media, that is, Medium 199 (M199), RPMI 1640 medium (RPMI), Grace's insect medium (GIM), and Schneider's insect medium (SIM) to investigate the promastigote growth by evaluating the growth characteristics, viability, and kinetics of stage differentiation in each medium. The findings from this study showed that parasites growing in different media exhibited different biological characteristics, which would be suitable for very specific research purposes, i.e., RPMI; for long term parasite maintenance, M199; for mass culture of parasites, M199 and GIM; for initial isolation of the parasites in clinical specimens, and SIM; for metacyclogenesis study.

Detection of Leishmania DNA in saliva among patients with HIV/AIDS in Trang Province, southern Thailand.

Leishmaniasis is a neglected tropical disease causing opportunistic infection among patients with HIV/AIDS. The fatal form of this disease is visceral leishmaniasis (VL). DNA of Leishmania can be detected in saliva, for which the collection is noninvasive and requires little expertise. This study aimed to evaluate the sensitivity and specificity of a nested-PCR to amplify the Internal Transcribed Spacer 1 (ITS1) to detect Leishmania DNA in paired saliva and buffy coat samples of 305 Thai patients with HIV/AIDS in Trang Hospital, Trang Province, southern Thailand. For asymptomatic Leishmania infection among Thai patients with HIV/AIDS, the sensitivity and specificity of the nested-PCR-ITS1 in buffy coat were 73.9 and 100%, respectively. However, the sensitivity in saliva was 26.1% and specificity was 100%. Using the nested-PCR-ITS1, saliva and buffy coat samples showed positive agreement in only 52.0% of patients. Saliva tested results with the nested-PCR-ITS1 showed positive agreement with the Direct Agglutination Test (DAT) in 46.5% of patients. Only 12.1% of the samples showed positive agreement for Leishmania infection among all the three tests: saliva, buffy coat and DAT results. Using nucleotide sequencing, at least three species of Leishmania infection were identified in saliva, i.e., L. siamensis (n = 28), L. martiniquensis (n = 9), and L. donovani complex (n = 1). As a result, buffy coat still appears to be a better specimen to diagnose asymptomatic VL infection among individuals with HIV. However, the use of both buffy coat and saliva together as clinical specimens would increase the sensitivity of Leishmania detection.

Phenotypic and genotypic characterization of Thai isolates of Plasmodium falciparum after an artemisinin resistance containment project.

BACKGROUND: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone®, a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before. RESULTS: One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC50s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. CONCLUSIONS: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified.

In Vitro Sensitivity of Pyronaridine in Thai Isolates of Plasmodium falciparum.

Pyronaridine, a Mannich base antimalarial agent with a high activity against chloroquine-resistant Plasmodium falciparum, has been combined with artesunate as a new artemisinin based combination therapy (ACT). Pyronaridine-artesunate combination could be one of the choices for the treatment of uncomplicated falciparum malaria in multidrug-resistant areas including Thailand. The aim of this study was to determine in vitro sensitivity and cross-resistance pattern of pyronaridine in Thai isolates of P. falciparum. In addition, the influence of resistant genes concerning in vitro pyronaridine sensitivity was determined. The mean pyronaridine 50% inhibitory concentration (IC50) of 118 parasite isolates was 5.6 ± 3.1 nM (range = 0.2-15.4 nM) with a significant positive correlation with artesunate IC50 (r = 0.246, P = 0.008) and amodiaquine IC50 (r = 0.220, P = 0.042) and a significant negative correlation with quinine IC50 (r = -0.185, P = 0.047). Parasites containing the pfmdr1 86Y allele exhibited significantly reduced pyronaridine sensitivity compared with those with the pfmdr1 N86 allele (7.6 ± 3.3 nM and 5.4 ± 3.0 nM, respectively, P = 0.032, independent t test); however, the difference may not be clinically relevant. Pyronaridine-artesunate could be the candidate ACT to treat multidrug-resistant falciparum malaria in Thailand with careful monitoring.

Molecular discrimination of Opisthorchis-like eggs from residents in a rural community of central Thailand.

BACKGROUND: Opisthorchis viverrini infection is a major public health problem in northern and northeastern Thailand. The chronic infection of O. viverrini is related to cholangiocarcinoma which causes high mortality in endemic areas. Therefore, the diagnosis, treatment, control and prevention of O. viverrini infection are necessary. The morphology of the egg is very similar to that of other species of human liver flukes (Opisthorchis felineus and Clonorchis sinensis) as well as that of small intestinal flukes in the family Heterophyidae. Thus, molecular characterization is crucially required to discriminate species of Opisthorchis-like eggs in fecal examination. METHODOLOGY/PRINCIPAL FINDINGS: We aimed to determine the prevalence of O. viverrini infection among villagers living in Sanamchaikate District, Chachoengsao Province, in central Thailand, where O. viverrini infection has previously been reported. A total of 2,609 fecal samples were examined for Opisthorchis-like eggs using microscopic examination. PCR-RFLP analysis of the ITS2 region was used to discriminate Opisthorchis-like eggs. The genetic structure of O. viverrini infection was demonstrated using nucleotide sequencing of cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 1 (nad1). Testing of evolutionary neutrality of the cox1 and nad1 sequences of O. viverrini was performed using Tajima's D tests and Fu's Fs tests. Moreover, the haplotype networks and phylogenetic trees were constructed to study the relationships of O. viverrini isolated from different endemic areas. A high prevalence of O. viverrini infection is still observed in a rural community of Chachoengsao Province, central Thailand. The overall prevalence of Opisthorchis-like eggs using microscopic examination was 16.8%. PCR-RFLP profiles showed the predominant infection of O. viverrini (9.6%) including very low infections of other small intestinal flukes, Haplorchis taichui (0.08%) and Euparyphium albuferensis (0.08%). The genetic structure of O. viverrini populations in central Thailand was also described and revealed a non-significant difference in genetic diversity. In addition, the genetic background of the O. viverrini populations was closely related to the isolate from Lao PDR. CONCLUSIONS/SIGNIFICANCE: Our study highlighted the prevalence of O. viverrini infection in central Thailand indicating that control programs and health education regarding opisthorchiasis is still required in this endemic area. Additionally, the study demonstrated the genetic structure of O. viverrini, in central Thailand which could provide information on the molecular epidemiology of this parasite.

Prevalence and risk factors associated with Leishmania infection in Trang Province, southern Thailand.

BACKGROUND: Autochthonous cutaneous and visceral leishmaniasis (VL) caused by Leishmania martiniquensis and Leishmania siamensis have been considered emerging infectious diseases in Thailand. The disease burden is significantly underestimated, especially the prevalence of Leishmania infection among HIV-positive patients. METHODS: A cross-sectional study was conducted to determine the prevalence and risk factors associated with Leishmania infection among patients with HIV/AIDS living in Trang province, southern Thailand, between 2015 and 2016. Antibodies against Leishmania infection were assayed using the direct agglutination test (DAT). DNA of Leishmania was detected by ITS1-PCR using the buffy coat. Species of Leishmania were also identified. RESULTS: Of 724 participants, the prevalence of Leishmania infection was 25.1% (182/724) using either DAT or PCR assays. Seroprevalence of Leishmania infection was 18.5% (134/724), while Leishmania DNA detected by the PCR method was 8.4% (61/724). Of these, 24.9% (180/724) were asymptomatic, whereas 0.3% (2/724) were symptomatic VL and VL/CL (cutaneous leishmaniasis). At least five species were identified: L. siamensis, L. martiniquensis, L. donovani complex, L. lainsoni, and L. major. Multivariate analysis showed that CD4+ levels <500 cells/µL and living in stilt houses were independently associated with Leishmania infection. Those who were PCR positive for Leishmania DNA were significantly associated with a detectable viral load, whereas non-injection drug use (NIDU) and CD4+ levels <500 cells/µL were potential risk factors of Leishmania seropositivity. CONCLUSIONS: A magnitude of the prevalence of underreporting Leishmania infection among Thai patients with HIV was revealed in this study. Effective public health policy to prevent and control disease transmission is urgently needed.

Comparison of Recombinant Proteins of Kinesin 39, Heat Shock Protein 70, Heat Shock Protein 83, and Glycoprotein 63 for Antibody Detection of Leishmania martiniquensis Infection.

Leishmania martiniquensis, a zoonotic hemoflagellate, is a causative agent of cutaneous (CL) and visceral leishmaniasis (VL) among humans and animals. This organism, first reported in Martinique Island, now has become an emerging infectious agent in Thailand. Symptomatic cases of L. martiniquensis infection among humans have continuously increased. In the meantime, asymptomatic infection of this novel species has seriously created national public health awareness and concern to prevent and control disease transmission. The unsuccessful serological test using the commercial rK39 dipstick based on antigen from Leishmania donovani to detect the antibodies against VL among infected Thai patients has encouraged us to further explore a new sensitive and specific antigenic epitope. In this study, we determined the sequences and expressed recombinant proteins of kinesin 39 (k39), heat shock protein 70 (hsp70), heat shock protein 83 (hsp83), and glycoprotein 63 (gp63) of L. martiniquensis to evaluate the diagnostic efficiency to detect antibodies against L. martiniquensis in patient sera. The preliminary results from western blot analysis have suggested that K39 is the most sensitive recombinant protein to detect L. martiniquensis. Moreover, this recombinant protein reacts with antibodies against L. donovani and Leishmania infantum, making it a promising antigen for further development of a universal rapid diagnostic tool for VL.

Leishmaniasis in Thailand: A Review of Causative Agents and Situations.

Before 1999, leishmaniasis was considered an imported disease in Thailand. Since then, autochthonous leishmaniasis was reported in both immmunocompetent and immmunocompromised patients especially in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). A new species was identified and named as Leishmania siamensis consisting of two lineages, that is, lineages TR and PG. Analysis of isoenzymes has clarified the more commonly detected L. siamensis lineage PG as Leishmania martiniquensis (MON-229), a species originally reported from the Martinique Island, whereas the L. siamensis lineage TR has been identified as the true novel species, L. siamensis (MON-324). Both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been found among Thai patients. Disseminated CL and VL could be presented in some reported patients who had HIV/AIDS coinfection. So far, only sporadic cases have been reported; thus, the true prevalence of leishmaniasis should be determined in Thailand among the high-risk populations such as people with HIV/AIDS. A recent survey among animals identified L. martiniquensis DNA in black rats (Rattus rattus) suggesting a potential animal reservoir. In addition, L. martiniquensis DNA was identified in Sergentomyia gemmea and Sergentomyia barraudi, the predominant sandfly species in the affected areas. However, further studies are needed to prove that these sandflies could serve as the vector of leishmaniasis in Thailand.

PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC50. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

Development and evaluation of PCR methods based on cytochrome c oxidase subunit one (cox1) and NADH dehydrogenase subunit one gene (nad1) to detect Opisthorchis viverrini in human fecal samples.

Opisthorchis viverrini is highly prevalent throughout Southeast Asia. Chronic infection of this parasite leads to cholangiocarcinoma (CCA), a fatal bile duct cancer. The early and accurate detection of this parasite is very important; therefore, new PCR methods targeting cytochrome c oxidase subunit one and NADH dehydrogenase subunit one gene to detect O. viverrini in fecal specimens have been developed. Ninety O. viverrini-positive human fecal samples were used in this study. The PCR sensitivity of both genes was compared with internal transcribe spacer 2 (ITS2)-PCR. The sensitivity of cox1-PCR and nad1-PCR was 66.7 and 50 %, respectively. The sensitivity of cox1-PCR and nad1-PCR achieved 89.1 and 71.7 % in specimens containing O. viverrini eggs of >100 eggs per gram (EPG). Additionally, these primers can be used to provide the information on genetic diversity from mitochondrial genes of O. viverrini.

Incidence and risk factors of blastocystis infection in orphans at the Babies' Home, Nonthaburi Province, Thailand.

BACKGROUND: Blastocystis infection is one of the most common intestinal protozoan infections reported in Thai population of all age groups for which epidemiological information is important to understand patterns of transmission for developing methods ofprevention and control for each specific group. The authors aimed to study prevalence, incidence and riskfactors associated with Blastocystis infection in orphans and childcare workers. Additionally, subtypes ofBlastocystis were identified MATERIAL AND METHOD: A retrospective cohort study of Blastocystis sp. was conducted in orphans aged less than 5 years and their childcare workers at Babies' Home, Nonthaburi Province, Thailand. A base line survey was conducted in December 2009 and afollow-up survey was conducted in April 2010. A total of 336 and 331 stool samples were collected. Blastocystis infection was examined using short-term in vitro cultivation in Jones's medium supplemented with 10% fetal calf serum. To analyze subtypes ofBlastocystis sp., PCR-RFLP of the small subunit ribosomal RNA gene was performed. RESULTS: Theprevalence ofBlastocystis infection in December 2009 and April 2010 were 8.1% and 13.3%, respectively The inlcidence rate ofBlastocystis infection was 1.6/100 person-months. Subtype analysis ofBlastocystis sp. in December 2009 and in April 2010 showed that subtype 3 was the most predominant (76% and 76%), followed by subtype 1 (16% and 20%), and unidentified subtype (8% and 4%), respectively. Subtype 3 is of human origin, thus person-to-person transmission is considered a major route ofBlastocystis infection in this population. CONCLUSION: Person-to-person transmission of Blastocystis infection in orphans living in the same house had been proposed, thus the prevalence and incidence of Blastocystis infection could be used to reflect the hygienic condition in the orphanage. Infection prevention and control practice can be effectively implemented.

Prevalence and gB genotype distribution of human cytomegalovirus among HIV sero-negative and HIV sero-positive orphans in Thailand.

BACKGROUND: Human Cytomegalovirus (HCMIV infects humans in all geographic areas. Polymorphisms ofglycoprotein B (gB) have been usedforgenotypic characterization of HCMV However information of gB genotyping of HCMV in Thailand is not clearly known especially in children. MATERIAL AND METHOD: A cross-sectional study was conducted to assess HCMV infection in 236 HIV seronegative and HIV seropositive children who attended an orphanage in Nonthaburi, Thailand by nested-PCR technique using urine specimens. HCMV gB genotypes were determined by restrictionfragment length polymorphism (RFLP), andDNA sequencing technique. RESULTS: Sixty-one percent (144/236) of the samples were HCMV positive, which consisted of 66.1% (37/56) of the HIV seropositive children and 59.4% (107/180) of the HIVsero-negative children. Multivariate analysis showed that children who living in one particular room were independently associated with HCMVinfection. Genotypic analysis revealed that the most prevalent genotype in these children was gB1; 85.4% (111/130) followed by gB3; 4.6% (6/130), gB2 and gB4 each at 2.3% (3/130). Mixed gB genotypes were identified in 5.4% (7/130) of the samples. CONCLUSION: HCMV infection, in particular gB1 genotype was commonly ident fled among these Thai orphans. Living in one particular room was associated with getting the infection. To prevent the transmission of HCMV infection in this setting, improvement in hygienic behavior ofchildcare workers should be focused.

Comparison of PCR methods for detection of Leishmania siamensis infection.

BACKGROUND: Leishmania siamensis, a newly identified species, has been reported as a causative agent of leishmaniasis in Thailand. This organism has been identified and genetically characterized using PCR techniques based on several target genes. However, the sensitivities and specificities of these methods for the diagnosis of L. siamensis infection have never been evaluated. METHODS: To evaluate the sensitivities and specificities of PCR methods to detect L. siamensis infection, PCR for different genetic markers, i.e., the small subunit ribosomal RNA region (SSU-rRNA), the internal transcribed spacer 1 region (ITS1), cysteine protease B (cpb), cytochrome b (cyt b), heat shock protein 70 (hsp70), the spliced leader mini-exon, and the triose-phosphate isomerase (tim) genes were compared. RESULTS: Both the ITS1-PCR and the SSU rRNA-PCR could detect promastigote of L. siamensis at concentrations as low as 0.05 parasites/µl or the DNA concentration at 2.3 pg/µl. Though the ITS1-PCR method only recognized 8 samples as positive, all of these could be assessed as true positive according to microscopic diagnosis and/or amplifying the results of the PCR and their sequencing, while other methods also produced false positive results. Compared with the ITS1-PCR method, the PCR amplified SSU-rRNA and cpb gene showed 100% sensitivity for the detection of L. siamensis in clinical specimens. The PCR amplified mini-exon and hsp70 gene also gave a high sensitivity of 87.5%. In contrast, the PCR methods for cyt b and tim gene showed low sensitivity. The PCR methods for cyt b, mini-exon and tim gene showed 100% specificity compared with the ITS1-PCR. CONCLUSION: As a result, the ITS1-PCR method is a suitable target for PCR-based detection of L. siamensis infection in clinical specimens due to its high sensitivity and specificity. The results of this study can be used for molecular diagnosis as well as in epidemiological studies of L. siamensis in affected areas.

Human cytomegalovirus gB1 genotypes among children who live at the Phayathai Babies' home in Nonthaburi, Thailand.

We conducted a survey of human cytomegalovirus (HCMV) genotypes among 176 children aged 1 month to 5 years living at Phayathai Babies' Home in Nonthaburi Province, Thailand to determine the prevalence of HCMV glycoprotein B (gB) genotype. The study was conducted on urine samples using nested polymerase chain reaction and restriction fragment length polymorphism; the HCMV gB1 genotype was found in 89% of subjects, much higher than previous reports. Our results show a high proportion of HCMV gB1 infected children in this population.

Sergentomyia (Neophlebotomus) gemmea, a potential vector of Leishmania siamensis in southern Thailand.

BACKGROUND: Leishmaniasis, caused by Leishmania siamensis, is an emerging disease in Thailand. Although reported cases have been increasing, epidemiological information of the disease including host and vector aspects is not clearly known. This study was a preliminary survey of the potential vector of L. siamensis in an affected area of leishmaniasis, Trang Province, southern Thailand. METHODS: The collection of sandflies was performed around the area where a case of leishmaniasis was reported using CDC light traps. Species of sandfly were identified based on morphological characteristics according to Lewis's key. PCR amplification and sequencing of the heat shock protein 70 gene (hsp70) was used to identify L. siamensis DNA in sandflies. RESULTS: A total of 146 male and female sandflies were collected in the affected areas. Of 71 female sandflies, four species were identified, i.e., Sergentomyia (Neophlebotomus) gemmea, S. (Neophlebotomus) iyengari, S. (Parrotomyia) barraudi and Phlebotomus (Anaphlebotomus) stantoni. Among these species, S. (Neophlebotomus) gemmea was the most predominant species in all areas. DNA of L. siamensis was identified in S. (Neophlebotomus) gemmea. Nucleotide sequences of PCR products using DNA extracted from S. (Neophlebotomus) gemmea showed 99.8% identity to L. siamensis. CONCLUSION: S. (Neophlebotomus) gemmea might be a potential vector of L. siamensis in an affected area, Trang Province, southern Thailand. However further studies are needed to prove whether these sandflies can be natural vectors of leishmaniasis.

Multilocus characterization and phylogenetic analysis of Leishmania siamensis isolated from autochthonous visceral leishmaniasis cases, southern Thailand.

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania siamensis is an emerging disease continuously reported in six southern provinces of Thailand. To date, the phylogenetic relationships among Leishmania isolates from Thai patients and other Leishmania species are still unclear and the taxonomic diversity needs to be established. In this study, the phylogenetic inference trees were constructed based on four genetic loci (i.e., SSU-rRNA, ITS1, hsp70, and cyt b), using DNA sequences obtained from autochthonous VL patients from southern Thailand and reference sequences of reported Leishmania isolates from other studies deposited in GenBank. RESULTS: Phylogenetic analyses of hsp70 and cyt b loci supported a clade comprised of L. siamensis isolates, which is independent to the other members in the genus Leishmania. In combination with genetic distance analysis, sequence polymorphisms were observed among L. siamensis isolates and two different lineages could be differentiated, lineages PG and TR. Phylogenetic analysis of the cyt b gene further showed that L. siamensis lineage TR is closely related to L. enrietti, a parasite of guinea pigs. CONCLUSION: The finding of this study sheds further light on the relationships of L. siamensis, both in intra- and inter-species aspects. This information would be useful for further in-depth studies on the biological properties of this important parasite.

Identification of Blastocystis subtype 1 variants in the Home for Girls, Bangkok, Thailand.

A cross-sectional study of Blastocystis infection was conducted to evaluate the prevalence, risk factors, and subtypes of Blastocystis at the Home for Girls, Bangkok, Thailand in November 2008. Of 370 stool samples, 118 (31.9%) were infected with Blastocystis. Genotypic characterization of Blastocystis was performed by polymerase chain reaction and sequence analysis of the partial small subunit ribosomal RNA (SSU rRNA) gene. Subtype 1 was the most predominant (94.8%), followed by subtype 6 (3.5%) and subtype 2 (1.7%). Sequence analyses revealed nucleotide polymorphisms for Blastocystis subtype 1, which were described as subtype 1/variant 1, subtype 1/variant 2. Blastocystis subtype 1/variant 1 was the most predominant infection occurring in almost every house. The results showed that subtype analysis of Blastocystis was useful for molecular epidemiological study.