Tawee Naaglor

Evaluation of DNA Extraction and PCR Methods for Detection of Enterocytozoon bienuesi in Stool Specimens

ABSTRACT An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.

Incidence and Risk Factors of Hookworm Infection in a Rural Community of Central Thailand

A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2-14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9-11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.

Evaluation of the Sensitivities of DNA Extraction and PCR Methods for Detection of Giardia duodenalis in Stool Specimens

ABSTRACT Sensitivities of DNA extraction methods and PCR methods for Giardia duodenalis were evaluated. A combination of the most sensitive methods, i.e., FTA filter paper and a PCR protocol using RH11/RH4 and GiarF/GiarR primers, showed no significant differences compared to immunofluorescence assay in terms of their sensitivities and specificities.

Autochthonous disseminated dermal and visceral leishmaniasis in an AIDS patient, southern Thailand, caused by Leishmania siamensis

We report the first establishment of in vitro cultivation and genotypic characterization of Leishmania siamensis isolated from an autochthonous disseminated dermal and visceral leishmaniasis in a Thai acquired immunodeficiency syndrome (AIDS) patient. The molecular identification has shown that the parasite was identical to L. siamensis, a recently described Leishmania species reported in the southern provinces of Thailand. The phylogenetic analysis has confirmed L. siamensis as closely related to the zoonotic Leishmania species L. enrietti.

Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

ABSTRACT We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.

A follow-up study of Opisthorchis viverrini infection after the implementation of control program in a rural community, central Thailand

BackgroundOpisthorchis viverrini infection is still one of the public health problems in Thailand. Our recent cohort study conducted in a rural community in central Thailand showed that the incidence rate of O. viverrini infection in 2002–2004 was 21.6/100 person-years. Conventional control activities including case diagnosis and treatment, hygienic defecation promotion and health education focusing on avoiding raw fish consumption was implemented. This study aimed to re-assess the status of infection after implementation of intervention programs, using both quantitative and qualitative methods in 2007–2009.MethodsA prospective cohort study was conducted to evaluate the incidence and risk factors of O. viverrini infection. Stool examination methods including wet preparation, Kato and formalin-ethyl acetate concentration technique were performed for the detection of O. viverrini eggs. A standardized questionnaire was used to assess risk behavior. In addition, qualitative information was collected from both O. viverrini negative and positive villagers using focus group discussions.ResultsThe incidence of O. viverrini infection was 21.4/100 person-years. Consumption of chopped raw fish salad, Koi pla and age 60 years and older were independently associated with O. viverrini infection, similar to our previous study. Findings from the qualitative study, indicated that inadequate knowledge, misbeliefs, and social and cultural mores were important factors leading to the maintenance of risk behaviors. Moreover, unhygienic defecation and insufficient diagnosis and treatment were found to facilitate O. viverrini transmission.ConclusionAlthough the conventional control program had been used in the study population, the incidence of O. viverrini infection remained the same. Precise and regular health education and promotion targeting the main risk factor, Koi pla consumption, improving diagnosis and treatment, and promoting hygienic defecation should be used in the prevention and control program.

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.

Comparison of PCR methods for detection of Leishmania siamensis infection

BackgroundLeishmania 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.MethodsTo 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 (hsp 70), the spliced leader mini-exon, and the triose-phosphate isomerase (tim) genes were compared.ResultsBoth 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 hsp 70 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.ConclusionAs 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.

Recurrences of visceral leishmaniasis caused by Leishmania siamensis after treatment with amphotericin B in a seronegative child.

We report the first case of visceral leishmaniasis caused by Leishmania siamensis in a seronegative child. She was treated with amphotericin B at 1 mg/kg/day for 3 weeks; however, recurrences occurred twice. The patient was cured after the administration of amphotericin B for 5 weeks and monthly prophylaxis for 6 months.

Comparative Diagnosis of Strongyloidiasis in Immunocompromised Patients.

Strongyloides hyperinfection syndrome and disseminated strongyloidiasis frequently occur in immunocompromised persons and can lead to high complication and mortality rates. Thus, detection of Strongyloides stercolaris in those patients is crucial. The present study aimed to determine the prevalence of strongyloidiasis and compare the detection rates of different strongyloidiasis detection methods. We conducted a cross-sectional study of 135 adults with various immunocompromising conditions (corticosteroid usage, chemotherapy, hematologic malignancies, organ transplants, use of immunosuppressive agents, and symptomatic human immunodeficiency virus infection) in Phramongkutklao Hospital, Bangkok, Thailand. All patients were asked to undergo serology testing for Strongyloides IgG by indirect enzyme-linked immunosorbent assay (ELISA), and 3 days of stool collection for use in a simple smear along with formalin-ether concentration and agar plate techniques. Prevalence rates of strongyloidiasis were 5% by stool concentration technique, 5.4% by IgG-ELISA, and 6.7% by agar plate culture. Three of the eight strongyloidiasis cases in this study had hyperinfection syndrome. The tested risk factors of age, sex, occupation, and immunocompromising condition were not associated with Strongyloides infestation. Serology was only 42.9% sensitive (positive predictive value), but it was 96.3% specific (negative predictive value). In conclusion, prevalence rates of strongyloidiasis in this study were 5-7%. Although agar plate culture was the most sensitive technique, the other diagnostic methods might be alternatively used.