Search

Background: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated., Methods: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis., Results: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species., Conclusions: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite., (© 2022. The Author(s).)

Background: Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA., Methods: LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification., Results: The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica., Conclusions: The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

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.

Background: Opisthorchis 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., Methods: A 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., Results: The 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., Conclusion: Although 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.

Background: Blastocystis sp. is one of the most common intestinal protozoa in humans. Unlike other intestinal parasitic infections such as giardiasis and cryptosporidiosis, the epidemiology of blastocystosis in children who live in crowded settings such as day-care centers and orphanages has been rarely explored., Methods: A retrospective cohort study was conducted to evaluate incidence and risk factors of Blastocystis infection in an orphanage every two consecutive months during April 2003 to April 2004, in Bangkok, Thailand. Blastocystis sp. was identified using direct simple smear, and in vitro cultivation in Jones' medium., Results: The incidence rate was 1.8/100 person-months and the independent risk factors associated with Blastocystis infection were age, nutritional status and orphans living in the room where their childcare workers were infected., Conclusions: Person-to-person transmission was most likely to occur either from orphans to childcare workers or from childcare workers to orphans living in the same room. Universal precautions such as regular hand washing and careful handling of fecally contaminated materials are indicated.

Blastocystis is a prevalent enteric protozoan that infects a variety of vertebrates. Infection with Blastocystis in humans has been associated with abdominal pain, diarrhea, constipation, fatigue, skin rash, and other symptoms. Researchers using different methods and examining different patient groups have reported asymptomatic infection, acute symptomatic infection, and chronic symptomatic infection. The variation in accounts has lead to disagreements concerning the role of Blastocystis in human disease, and the importance of treating it. A better understanding of the number of species of Blastocystis that can infect humans, along with realization of the limitations of the existing clinical laboratory diagnostic techniques may account for much of the disagreement. The possibility that disagreement was caused by the emergence of particular pathogenic variants of Blastocystis is discussed, along with the potential role of Blastocystis infection in irritable bowel syndrome (IBS). Findings are discussed concerning the role of protease-activated receptor-2 in enteric disease which may account for the presence of abdominal pain and diffuse symptoms in Blastocystis infection, even in the absence of fever and endoscopic findings. The availability of better diagnostic techniques and treatments for Blastocystis infection may be of value in understanding chronic gastrointestinal illness of unknown etiology.