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Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia− SEA deletion/α+-thalassemia− 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease− SEA/−3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia− 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia− SEA deletion was combined for Hb H disease− SEA/−3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia− 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease− SEA/−3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease− SEA/−3.7kb which is optimal for PGT-M. [ABSTRACT FROM AUTHOR]

Seafood mislabeling threatens customer rights and causes economic loss worldwide. The information on seafood misrepresentation in Thailand is still lacking, and the investigation and monitoring program must be well established. This study investigated the mislabeling status of imported seafood in Thailand using the DNA barcoding technique. A total of 45 imported seafood products from five distributors were included. Scientific, common, local, and market names of seafood samples were obtained from FAO and Fishbase databases. DNA was extracted, and PCR was performed using a universal primer targeting the COI gene. Species of each sample were identified with over 98% similarity based on COI sequence analysis. DNA sequence revealed 11 mislabeled samples. Among substituted species, Pangasianodon hypophthalmus and Thunnus maccoyii were found to be endangered species according to IUCN status. Products obtained from Brand-C showed the highest mislabeling rate (42.85%). The phylogenetic analysis adopted with the TIM2+F+I+G4 model showed the sequenced DNA similar to the NCBI database reference sequence. Overall, mislabeled products of imported seafood were found at the rate of 24.44%, suggesting that strict surveillance for seafood substitution should be implemented in Thailand. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) poses a serious global health threat, and wastewater treatment (WWT), including septic tanks, is a source of AMR. In Thailand, antibiotics are unregulated, and septic tanks are commonly used. Yet, their impact on the spread or mitigation of AMR is unknown. We monitored household and healthcare conventional septic tanks (CST) and household solar septic tanks (SST) in Thailand using the class 1 integron-integrase (intI1) gene abundance as a proxy for AMR. A systematic review of the literature found 65 intI1 primers. We evaluated the coverage and specificity of each, including a new MGB TaqMan primer-probe, against clinical and environmental intI1, intI1-like, and non-intI1 databases. The three best primers were selected, laboratory validated for DNA and mRNA quantification, and used to quantify septic tank intI1 gene abundance. No primer set could distinguish between intI1 and intI1-like sequences. While primer choice did not affect gene abundance of the same sample (P-value > 0.05), sometimes when comparing the same samples quantified by different primers, statistical differences were observed for one but not the other primer set. This may lead to different interpretations of AMR risk. Irrespective of primers or reactor type intI1 gene abundance was greatest in influent > effluent > sludge. intI1 gene abundance was lowest in the effluent of the SST-household < CST-household < CST-healthcare. 31% to 42% of intI1 was removed by the CST-household tank, indicating while septic tanks remove some intI1 they remain a source to the surrounding environment. Toward the goal of achieving standardization across studies, we recommend the F3-R3 primer for intI1 quantification. IMPORTANCE Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies. [ABSTRACT FROM AUTHOR]

An outbreak of citrus greening or Huanglongbing disease bacteria occurs in many areas. We sampled and identified an ongoing ~year 2020 orange tree endemic in northern Thailand as Candidatus Liberibacter asiaticus. We thereby developed a plant greening disease (C. Liberibacter asiaticus) detection assay using simple alkaline heat DNA lysis and loop-mediated isothermal amplification coupled hydroxynaphthol blue (AL-LAMP-HNB), and evaluated the developed assay for its feasibility as point-of-care detection on 65 plant leaf samples with 100–1×104 copies of C. Liberibacter asiaticus or mocked injection compared with commercial DNA lysis kit and PCR-GE. Our assay is sensitive to 5–8.9 copies of omp (equaling 0.0056–0.01 fg) compatible with PCR-GE limit of detection. This ultra sensitive limit of detection could allow the disease detection before clinical apparent state of disease when C. Liberibacter asiaticus infection number is few, i.e. fewer than 100 copies of C. Liberibacter asiaticus. The assay is also specific with 6 degenerate primers targeting every strain of C. Liberibacter asiaticus omp from GenBank database, rapid (40 min total assay time), inexpensive (~2–3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay accuracy (93.85–100% accuracy, 100% specificity, and 89.74–100% sensitivity) to bacterial DNA extraction by a commercial kit followed by PCR and gel electrophoresis (92.31% accuracy, 100% specificity, and 87.18% sensitivity) based on the real sample tests. Hence, the technique could be used in local or laboratory resource-restricted settings. The test result could be read by naked eyes through the color change from violet (negative) to sky blue (positive) for a C. Liberibacter asiaticus-infected specimen. Furthermore, this assay uses safe chemical reagents and, thus, is safe for the users. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is an important infectious disease pathogen that can cause melioidosis. Melioidosis is mainly prevalent in Thailand, northern Australia and southern China and has become a global public health problem. Early identification of B. pseudomallei is of great significance for the diagnosis and prognosis of melioidosis. In this study, a simple and visual device combined with lateral flow strip-based recombinase polymerase amplification (LF-RPA) was developed, and the utility of the LF-RPA assay for identifying B. pseudomallei was evaluated. In order to screen out the optimal primer probe, a total of 16 pairs of specific primers targeting the orf2 gene of B. pseudomallei type III secretion system (T3SS) cluster genes were designed for screening, and F1/R3 was selected as an optimal set of primers for the identification of B. pseudomallei , and parameters for LF-RPA were optimized. The LF-RPA can be amplified at 30-45°C and complete the entire reaction in 5-30 min. This reaction does not cross-amplify the DNA of other non- B. pseudomallei species. The limit of detection (LOD) of this assay for B. pseudomallei genomic DNA was as low as 30 femtograms (fg), which was comparable to the results of real-time PCR. Moreover, 21 clinical B. pseudomallei isolates identified by 16S rRNA gene sequencing were retrospectively confirmed by the newly developed LF-RPA system. Our results showed that the newly developed LF-RPA system has a simple and short time of operation and has good application prospect in the identification of B. pseudomallei. [ABSTRACT FROM AUTHOR]

Aim: Although at least five Tylototriton species were recorded in Thailand, only Tylototriton verrucosus is registered as a protected species under the Wildlife Preservation and Protection Act in Thailand. Populations of T. uyenoi are now severely declining, caused by anthropogenic activities. As intense human pressure is having profound effects on the diminishment in T. uyenoi populations, a conservation plan is needed. Information such as the abundance and distribution of a species is necessary. Yet, current established survey methods are either time‐consuming or labour‐intensive. Here, eDNA‐based detection was developed for tracking the presence of the T. uyenoi. Location: Thailand. Methods: We target the surveillance of T. uyenoi using eDNA. Primers and a probe specific to T. uyenoi were designed and tested for their specificity and sensitivity. Water samples were collected once a month from August to January at three sites in Doi Suthep and at three extra sites within the range of the species. Three hundred ml samples of water were collected and filtered. Environmental DNA was extracted and then subjected to qPCR assay in an attempt to detect T. uyenoi. Results: The qPCR assay was found to be species‐specific to T. uyenoi. Both PCR and qPCR did not result in any positive detection of three congeners or other non‐target species. The LOD and the LOQ of the assay were determined by an analysis of the standard curve, and it was found that the LOD and the LOQ were 7.99 and 9.0 copies per reaction, respectively. Environmental DNA was detected in water samples from all sites where T. uyenoi has been known to occur, but detection varied among sites and sampling times. In addition, low amounts of eDNA were detected in three sites with unknown occupancy of newts, but within the species' range. Conclusions: Our findings suggest that eDNA survey is a powerful tool for tracking T. uyenoi throughout the year regardless of the sampling site conditions. Similar to other amphibians, T. uyenoi is severely declining due to anthropogenic factors. In order to have an effective conservation plan, knowledge of a species' distribution is needed. To our knowledge, this was the first study that used eDNA to track the crocodile newt in Thailand. [ABSTRACT FROM AUTHOR]

Edible bird's nest (EBN) is a popular delicacy in the Asian Pacific region originating from Indonesia, Malaysia, Thailand and Vietnam, which consist of various potential medicine value in Traditional Chinese Medicine (TCM). Thailand is one of the main exporters of EBN. However, the genetic information of EBN, a key part of molecular biology, has yet to be reported in Thailand. It is necessary to explore the genetic information of EBN in Thailand based on a quick and simple method to help protect the rights and interests of consumers. This research aimed to systematically evaluate different methods of extracting EBN DNA to improve the efficiency of the analysis of cytochrome b (Cytb) and NADH dehydrogenase subunit 2 (ND2) gene sequences, the establishment of phylogenetic trees, and the genetic information of EBN in Thailand. Additionally, we aimed to develop a quick and simple method for identifying EBN from different species based on the genetic information and amplification-refractory mutation system PCR (ARMS-PCR). By comparing the four methods [cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), kit and guanidinium isothiocyanate methods] for EBN extraction, we found that the guanidinium isothiocyanate method was the optimal extraction method. Phylogenetic trees generated on the basis of Cytb and ND2 gene analyses showed that 26 samples of house EBN and 4 samples of cave EBN came from Aerodramus fuciphagus and Aerodramus maximus , respectively. In addition, to distinguish different samples from different species of Apodiformes , we designed 4 polymerase chain reaction (PCR) amplification primers based on the ND2 gene sequences of A. fuciphagus and A. maximus. The ARMS-PCR results showed band lengths for A. fuciphagus EBN of 533, 402, and 201 bp, while those for A. maximus EBN were 463, 317, and 201 bp. Collectively, the results showed that ARMS-PCR is a fast and simple method for the genetic identification of EBN based on designing specific original identification primers. [ABSTRACT FROM AUTHOR]

A banded linsang (Prionodon linsang) presented at our hospital with clinical signs of acute diarrhea. Fecal samples were positive for canine parvovirus (CPV) as determined by polymerase chain reaction with primers specific for both CPV and feline panleukopenia virus (FPV). The full-length VP2 was cloned, sequenced, and compared with sequences of FPV and CPV strains reported in GenBank. The amino acids that determined the host range were similar to those of FPV. Moreover, amino acid analysis of VP2 revealed over 98% homology to FPV. The FPV isolate was closely related with FPV isolates from Japan, South Korea, and China. To the best of our knowledge, this is the first study to report that banded linsang can be infected with FPV. [ABSTRACT FROM AUTHOR]

In present study, Loop Mediated Isothermal Amplification (LAMP) assay was conducted for diagnosis Nosema bombycis, the pebrine disease pathogen in domestic silkworm, Bombyx mori. Nine isolates of N. bombycis were collected from infected silkworms in rearing areas in Khon Kaen province, Thailand. N. bombycis genomic DNAs were extracted by boiling method and used as templates in LAMP and PCR reactions. A LAMP primer set was designed specific to N. bombycis small subunit ribosomal RNA (SSU rRNA) gene. The results revealed that the optimal condition was constantly performed at 63°C for 1 hour. The product was directly visualized by naked eye and confirmed with agarose gel eletrophoresis. LAMP assay is more sensitive than traditional PCR, since LAMP was able to detect the least 10 spores/ml while PCR needs 100 spores/ml. In addition, the present novel LAMP primer set was specific only to N. bombycis proven by the negative results when other B. mori pathogen DNAs were tested. In conclusion, the LAMP assay demonstrated a great potential alternative method in diagnosis N. bombycis with high sensitivity, rapidity and accuracy which can apply for pebrine disease surveillance. [ABSTRACT FROM AUTHOR]

Morphologically, species of fanged frogs (Limnonectes) are exceedingly similar, making it difficult to distinguish them within the complex. In Thailand, it has been difficult to distinguish between the sympatric species L. bannaensis and L. taylori, particularly among tadpoles, adolescents, and adult females. A precise identification contributes to a greater understanding of biodiversity, particularly for assessing distributions and population dynamics. Therefore, a novel approach is required. The objective of this study was to develop a high resolution melting analysis (HRM) for the rapid and accurate identification of six species of Limnonectes of the L. kuhlii complex found in Thailand, particularly the two sympatric fanged frogs. Here, HRM assays using 16S rRNA mitochondrial primers were designed and developed. There was as much as a 25.3% variation in the nucleotide sequence of the fragment amplified by HRM16S primers among the six species of Limnonectes. Prior to conducting an in vitro HRM, the DNA sequences were used in a simulation HRM, uMELT Quartz, to predict the melting curve for each species of Limnonectes. There were discrepancies between the predicted melting curves of each species generated by the programme. Consequently, in vitro HRM tests were conducted. The obtained melting curve and T m values were consistent with those predicted, albeit with a slightly different T m value and a more distinct melting curve. All evaluated species of Limnonectes could be easily distinguished from one another by comparing the melting curve shapes. The HRM assay was then used to confirm the species of 18 Limnonectes samples in comparison to the reference samples (confidence interval > 90%). In addition, the results of HRM were consistent with those of experts who used morphological analysis to identify species. The HRM was found to be useful, and therefore the method would also contribute to future ecological and systematic studies on the target species., (© 2023. The Author(s).)

Sarcocystis species are heteroxenous cyst-forming coccidian protozoan parasites with a wide host range, including rodents. In this study, Sarcocystis spp. samples were isolated from Bandicota indica, Rattus argentiventer, R. tiomanicus and R. norvegicus across five provinces of Thailand. Two major groups of Sarcocystis cysts were determined in this study: large and small cysts. By sequence comparisons and phylogenetic analyses based on the partial sequences of 28S ribosomal DNA, the large cysts showed the highest identity value (99%) with the S. zamani in GenBank database. While the small cysts could be divided into 2 groups of Sarcocystis: S. singaporensis and presupposed S. zuoi. The further analysis on 18S rDNA supported that the 2 isolates (S2 and B6 no.2) were as identified as S. singaporensis shared a high sequence identity with the S. singaporensis in GenBank database and the unidentified Sarcocystis (4 isolates, i.e., B6 no.10, B6 no.12, B10 no.4 and B10 no.7) showed 96.3-99.5% identity to S. zuoi as well as high distinct identity from others Sarcocystis spp. (=93%). The result indicated that these four samples should be S. zuoi. In this study, we provided complete sequence of internal transcribed spacer 1 (ITS1), 5.8S rDNA and internal transcribed spacer 2 (ITS2) of these three Sarcocystis species and our new primer set could be useful to study the evolution of Sarcocystis. [ABSTRACT FROM AUTHOR]

A purported parvovirus producing circular, eosinophilic inclusions in the nuclei of tubule epithelial cells of the hepatopancreas (HP) of the giant freshwater prawn Macrobrachium rosenbergii (Mr) was first reported from Malaysia in 1990. In 2009, similar inclusions in Mr from Thailand were confirmed to contain single-stranded DNA (ssDNA). In 2021, Mr samples showing similar HP inclusions were reported to give positive PCR test results for decapod hepanhamaparvovirus (DHPV) using universal DHPV primers designed from many viral isolates of 3 penaeid shrimp species. However, the matching DNA probe revealed positive in situ hybridization (ISH) results with host HP nuclei only, and not with the intranuclear inclusions. At the time, this anomaly could not be explained. Here we describe metagenomic analysis and de novo assembly from 2016 samples of Mr with similar intranuclear inclusions that showed negative PCR test results using the DHPV universal primers. Genomes identified through metagenomics revealed a novel ssDNA genome sequence of 6723 bases that was confirmed by a single PCR amplicon. ISH using specific probes derived from this genome sequence revealed that the ssDNA was present in both grossly normal nuclei and in the intranuclear inclusions. Tests with archived DNA from the samples used in the 2021 publication yielded positive PCR results for both DHPV and the novel sequence, suggesting that the shrimp had dual infections. Phylogenetic analysis of the novel sequence revealed no relationship to any known DHPV type. Instead, the sequence closely matched the polymerase B gene in the V1 fragment from Bombyx mori bidensovirus 2 (BmBDV2) that is characterized by 2 ssDNA genome fragments and has been classified in the family Bidnaviridae. The 2 BmBDV2 fragments V1 and V2 each produce their own independent capsid protein genes and virions. In contrast, the new Mr virus is independent with a single 6723 base ssDNA genome, and we propose the name Macrobrachium hepatopancreatic bidnavirus (MHBV). • Metagenomics revealed a novel ssDNA virus genome in the hepatopancreas of M. rosenbergii • Positive In situ hybridization signals arose from normal nuclei and intranuclear inclusions • Highest DNA homology was to the PolB gene in V1 fragment of Bombyx mori bidensovirus2 • The virus was tentatively named Macrobrachium hepatopancreatic bidnavirus (MHBV) • Dual infections with decapod hepanhamaparvovirus (DHPV) were associated with mortality [ABSTRACT FROM AUTHOR]

The causal agent of leaf spot disease of oil palm ( Elaeis guineensis) seedling nurseries in Thailand was identified as Curvularia oryzae. The fungus was isolated from leaves with disease symptoms, characterized by morphological properties, and pathogenicity tested. The identity of the phytopathogenic fungus was confirmed through polymerase chain reaction analysis using internal transcribed spacer (ITS) primers, which amplified about a 1 kb product. Sequencing this DNA product confirmed this pathogen was C. oryzae. Furthermore, the pathogenicity test showed that C. oryzae could infect oil palm seedlings. [ABSTRACT FROM AUTHOR]

Kamlang Suea Khrong (KSK) crude drug, a traditional Thai medicine used for oral tonic and analgesic purposes, is obtained from three origins: the inner stem bark of Betula alnoides (BA) or the stems of Strychnos axillaris (SA) or Ziziphus attopensis (ZA). According to the previous reports, SA contains strychnine-type alkaloids that probably cause poisoning; however, only organoleptic approaches are insufficient to differentiate SA from the other plant materials. To ensure the botanical origin of KSK crude drug, powerful and reliable tools are desperately needed. Therefore, molecular and chemical identification methods, DNA barcoding and thin-layer chromatography (TLC), were investigated. Reference databases, i.e., the ITS region and phytochemical profile of the authentic plant species, were conducted. In case of molecular analysis, multiplex polymerase chain reaction (PCR) based on species-specific primers was applied. Regarding species-specific primers designation, the suitability of three candidate barcode regions (ITS, ITS1, and ITS2) was evaluated by genetic distance using K2P model. ITS2 presented the highest interspecific variability was verified its discrimination power by tree topology. Accordingly, ITS2 was used to create primers that successfully specified plant species of authentic samples. For chemical analysis, TLC with toluene:ethyl acetate:ammonia (1:9:0.025) and hierarchical clustering were operated to identify the authentic crude drugs. The developed multiplex PCR and TLC methods were then applied to identify five commercial KSK crude drugs (CK1-CK5). Both methods correspondingly indicated that CK1-CK2 and CK3-CK5 were originated from BA and ZA, respectively. Molecular and chemical approaches are convenient and effective identification methods that can be performed for the routine quality-control of the KSK crude drugs for consumer reliance. According to chemical analysis, the results indicated BA, SA, and ZA have distinct chemical profiles, leading to differences in pharmacological activities. Consequently, further scientific investigations are required to ensure the quality and safety of Thai ethnobotanical medicine known as KSK., Competing Interests: The authors have declared that no competing interests exist.

Bamboo is a non-timber forest product and one of the most important grass plants of industrial and domestic use. It is widely distributed in tropical countries including India, China and Southeast Asian countries with wide genetic diversity. The diversity in the available genotypes becomes an important resource for the selection and improvement of the plants for ecological and commercial use. This study investigates eight commercially and ecologically important bamboo species of six genera (Bambusa, Dendrocalamus, Thyrsostachys, Vietnamosasa, Cephalostachyum and Indocalamus) from India, Thailand and Laos. These were evaluated for genetic differences by molecular makers, chemo-morphological variation and ability of silicon accumulation. The genetic cluster analyses of eight RAPD primers revealed genetic similarities in the ranges of 24-55%. The total silica content varied from 18.34 to 40.08 ppm in leaves of different bamboo species. Chemical analysis of the silicon content by ICP-OES and secondary metabolite profiling on TLC depicted the prominent distinction among the species. The PCA analysis of quantitative morphological data grouped the species in two major clusters and found to correlate with chemical pattern and genetic similarity to some extent. This is the first report that summarizes species-specific variability of leaf silica content, secondary metabolites, and quantitative morphological data towards delineation of genetic phylogeny of bamboo species.

This study aimed to investigate the prevalence of cercarial infections in freshwater snails collected from the Chai Nat province of Central Thailand. Moreover, we aimed to develop a duplex PCR technique, using the ITS2 and candidate MT-ND1 gene, to determine the dissemination of H. taichui and H. pumilio, respectively. Six types of cercariae were discovered with an overall prevalence of 4.71% (59/1,252). The parapleurolophocercous cercariae were demonstrated to be the dominant type, infecting only Melanoides tuberculata snails. The duplex PCR was optimized for specific amplification of ITS2 for H. taichui (115 bp) and MT-ND1 for H. pumilio (335 bp). Both specific primers confirmed the specificity of the duplex PCR reaction, with no cross-reactivity with other heterophyids or related species. In addition, this duplex PCR could be used to detect co-infection at a concentration of 3.0 ng/µL. For the molecular identification, 9 of 22 parapleurolophocercous cercaria specimens in Chai Nat province generated the specific DNA fragment of H. pumilio. These results proved that the MT-ND1 gene is a species-specific method for heterophyid detection and provides a rapid method for identification based on larval and adult stages of H. taichui and H. pumilio in their intermediate and/or definitive hosts in the infected area., (Copyright © 2020. Published by Elsevier B.V.)

Four species of echinostomes, Echinostoma revolutum (Froelich, 1802), Echinostoma ilocanum (Garrison, 1908), Hypoderaeum conoideum (Bloch, 1872) Dietz, 1909, and Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 commonly infect humans in Thailand, but their eggs present similar morphologies resulting in difficult differentiation for diagnosis. Present molecular methods have a great potential to provide superior detection/diagnosis. DNA sequences, especially the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene, have already been used to differentiate among echinostomes; thus, we aimed to develop species-specific primers for the differential detection of four medically important echinostomes by multiplex PCR. The species-specific reverse primers and a forward primer were based on variable regions and conserved regions of the ND1 gene, respectively. Four reverse primers and a forward primer were combined in a multiplex PCR reaction to amplify the ND1 fragment. Different ND1 fragment sizes were amplified: 108, 209, 384 and 419 bp of E. revolutum H. conoideum, E. ilocanum and A. malayanum, respectively. Specificity was tested with other medically important parasite DNA; no cross-reaction occurred. Sensitivity ranged between 0.1 and 0.05 ng. The species-specific primers developed in this study could be of further use in differential diagnosis for these medically important echinostomes infection in human and animal hosts., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The Barbirostris Complex comprises six formally described species that cannot be differentiated based on morphology alone. Out of these six species, two have been reported as putative malaria vectors, An. campestris and An. wejchoochotei. Five species are present in Thailand, An. barbirostris, An. campestris, An. dissidens, An. saeungae and An. wejchoochotei, while An. vanderwulpi occurs in Indonesia. As these species cannot be accurately differentiated by morphological characters, there is a crucial lack of information on their bionomics and role in the transmission of malaria and filariasis agents., Results: For differentiating the six species, an allele-specific amplification (AS-PCR) based on the second internal transcribed spacer (ITS2) sequence was developed. From 862 mosquitoes in the Barbirostris Complex collected in 23 provinces throughout Thailand, the AS-PCR was able to identify five species and its validation was undertaken on 185 specimens., Conclusions: This multiplex-PCR assay is potentially able to definitely identify all six species of the Barbirostris Complex and was validated on five species present in Thailand.

Echinostomiasis caused by the Echinostoma group, in particular E. revolutum are a significant problem for both humans and other animals. This group has a large number of morphological similarities that are difficult and time-consuming to identify. The present study aimed to develop high-performance tools for the detection of the prevalence of E. revolutum and to reveal the prevalence of E. revolutum infections in intermediate snail hosts in Lopburi province, Thailand. The snail specimens were collected by stratified sampling method and examined to collect trematodes in the larval stage. The specific primer was manually designed and based on 18 s rDNA and verified the specificity and sensitivity for use as an identification tool to compare with classical method, constructed by epidemic mapping. The overall prevalence value of E. revolutum was found to be 16.26%. Tha Luang district had the highest prevalence (70.14%), followed by Chai Badan, Phatthana Nikhom, Tha Wung, Ban Mi, Khok Samrong, Nong Muang and Sa Bot at 42%, 25.14%, 2.52%, 1.73%, 2%, 1.33% and 0.40%, respectively. With regard to the specific primer, it can amplify both cercarial and metacercarial DNA (90 pg/μl.) and discriminated E. revolutum from its hosts, other trematodes and other echinostome larvae with no cross-reactions. Therefore, the developed specific primer can be used as a species-specific identification tool with a high degree of sensitivity and specificity. Consequently, this data is important for monitoring the outbreak of E. revolutum. It can be applied for initiating surveillance programs of snail-borne diseases in both medical and veterinary studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ammonia-oxidizing archaea (AOA) have recently been proposed as potential players for ammonia removal in wastewater treatment plants (WWTPs). However, there is little evidence directly showing the contribution of AOA to ammonia oxidation in these engineered systems. In this study, DNA-stable isotope probing (DNA-SIP) with labeled 13 C-HCO 3 - was introduced to sludge from a municipal WWTP. Quantitative PCR demonstrated that AOA amoA genes outnumbered AOB amoA genes in this WWTP sludge. AOA amoA gene sequence analysis revealed that AOA present in this WWTP were specific to one subcluster within the group 1.1b Thaumarchaeota. When ammonia was supplied to DNA-SIP incubation, the DNA-SIP profiles demonstrated the incorporation of the 13 C into AOA and AOB. However, the 13 C was not found to be assimilated into both microorganisms in the incubation without ammonia. Specific primers were designed to target amoA genes of AOA belonging to the subcluster found in this WWTP. Applying the primers to DNA-SIP experiment revealed that AOA of this subcluter most likely utilized inorganic carbon during ammonia oxidation under the studied conditions.

Obviously, suitable sex chromosome markers have not been investigated for Thai population. Our research determined standard set of four primer regions, namely amelogenin and three centromeric alphoid repeats. Hair shafts with roots from 73 female and 72 male individuals were collected from southern, northern, central and northeastern Thailand, and the DNA was extracted and tested with these primers. Amelogenin and centromeric alphoid repeat 3 regions showed an error rate of 10.96 and 5.56%, with successful amplification rate of 89.04 and 94.44%, respectively. The most effective regions, which showed 100% accuracy on sex identification were the centromeric alphoid repeats 1 and 2. Therefore, these two regions should be the standard specific sex markers used for Thais applicable in forensic DNA analysis. [ABSTRACT FROM AUTHOR]

What Is Known and Objective: Abacavir (ABC) is a commonly used nucleoside reverse-transcriptase inhibitor with potent antiviral activity against HIV-1. The US Food and Drug Administration and international HIV treatment guidelines recommend HLA-B*57:01 screening before initiating treatment with ABC. The current standard method for HLA-B*57:01 screening is limited by its high-cost, time-consuming and labour-intensive procedure with the requirement of a specialized laboratory. Our study aims to develop a more reliable screening test by selecting rs3093726 as an additional single nucleotide polymorphism (SNP) to combine with rs2395029 for multiplex pyrosequencing development. It offers high-accuracy, cost-effective and rapid detection., Methods: Multiplex pyrosequencing was developed for HLA-B*57:01 screening using rs2395029 and rs3093726 as a surrogate marker and tested in 130 Thai subjects in parallel with singleplex pyrosequencing of each SNP and the standard sequence-based method., Results and Discussion: Multiplex pyrosequencing showed 100% concordance when compared with both singleplex pyrosequencing and standard sequence-based method. This method showed 100% of negative predictive value (NPV), positive predictive value (PPV), specificity and sensitivity., What Is New and Conclusion: Multiplex pyrosequencing is a powerful tool for HLA-B*57:01 screening using the rs2395029 and rs3093726 haplotype genotyping as surrogate marker for this HLA-B. The assay provides accurate, cost-effective and rapid detection of this haplotype. It can be applied for ABC hypersensitivity screening of the Thai population before initiating treatment with ABC., (© 2014 John Wiley & Sons Ltd.)

Shells of certain freshwater mussel (Unionoida) species are highly demanded and serve as raw material for a range of decorative and pharmaceutical products. In Thailand, most animals for this purpose are currently harvested from wild populations, with unionoid culture still being in its infancy. Whilst reliable species identification is a prerequisite for developing a large-scale industry, identification by morphological means is hampered by extensive phenotypic plasticity and poor knowledge of species delimitations. To facilitate alternative molecular identification, we developed species-specific markers for the three Thai unionoids with considerable economic potential (CEP): that is, Chamberlainia hainesiana, Hyriopsis desowitzi and Hyriopsis myersiana. For this purpose, amplified fragment length polymorphism (AFLP) fingerprints using 24 specific primer pairs were generated for eight samples of each CEP species and four samples of the closely related, non-CEP species Contradens contradens. Cloning and sequencing of 13 CEP species-specific AFLP bands revealed fragment collision at three occasions. In total, 16 species-specific primer pairs were designed and tested on 92 Thai specimens spanning seven species and four genera. Thereby, specificity of (1) three primers to C. hainesiana, (2) one primer to H. desowitzi + Hyriopsis bialata, (3) one primer to H. myersiana + H. bialata and (4) four primers to all three Hyriopsis species tested was confirmed. Respective multiplex PCR protocols are provided. The developed primers enable cheap, quick and reliable identification of the Thai CEP species by one to three PCRs and offer a tool for a range of additional applications within mussel culture and ecological and evolutionary research on these important organisms., (© 2013 Stichting International Foundation for Animal Genetics.)

Large sequence polymorphisms (LSPs) or regions of differences (RDs) are molecular epidemiological and evolutionary markers used to classify Mycobacterium tuberculosis (MTB) into East Asian (Beijing), Indo-Oceanic (IO), Euro-American (EuA) and East African Indian (EAI) lineages. The most used method is separate PCR and sequencing for each RD. We developed a single-tube multiplex PCR using four primer pairs specific to the four MTB lineages and a primer pair for species-specific RD9 with genomic DNA extracted from isolated colonies. The single-tube multiplex PCR produced lineage-specific amplicon patterns capable of differentiating the four MTB lineages. Sensitivity and specificity of the assay were 100% when differentiating MTB lineages from other species and strains of bacteria. The limit of detection of genomic MTB DNA was 12.5 ng. This single-tube multiplex PCR method offers a simple, rapid and reliable method for classification of MTB lineages based on LSPs.

Background: Gracilaria tenuistipitata is an agarophyte with substantial economic potential because of its high growth rate and tolerance to a wide range of environment factors. This red seaweed is intensively cultured in China for the production of agar and fodder for abalone. Microsatellite markers were developed from the chloroplast genome of G. tenuistipitata var. liui to differentiate G. tenuistipitata obtained from six different localities: four from Peninsular Malaysia, one from Thailand and one from Vietnam. Eighty G. tenuistipitata specimens were analyzed using eight simple sequence repeat (SSR) primer-pairs that we developed for polymerase chain reaction (PCR) amplification., Findings: Five mononucleotide primer-pairs and one trinucleotide primer-pair exhibited monomorphic alleles, whereas the other two primer-pairs separated the G. tenuistipitata specimens into two main clades. G. tenuistipitata from Thailand and Vietnam were grouped into one clade, and the populations from Batu Laut, Middle Banks and Kuah (Malaysia) were grouped into another clade. The combined dataset of these two primer-pairs separated G. tenuistipitata obtained from Kelantan, Malaysia from that obtained from other localities., Conclusions: Based on the variations in repeated nucleotides of microsatellite markers, our results suggested that the populations of G. tenuistipitata were distributed into two main geographical regions: (i) populations in the west coast of Peninsular Malaysia and (ii) populations facing the South China Sea. The correct identification of G. tenuistipitata strains with traits of high economic potential will be advantageous for the mass cultivation of seaweeds.

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

Summary Non-syndromic oral clefts comprising cleft lip with and without cleft palate (CL/P) and cleft palate only (CPO) are common birth defects worldwide. Their aetiology involves both environmental and genetic factors. FOXE1 has previously been reported to be associated with oral clefts in some populations. Here, we investigate whether mutations in FOXE1 play a part in the formation of oral cleft in a Thai population. We first performed PCR-RFLP to genotype a previously reported associated polymorphism, c.-1204C > G (rs111846096), in our cohort. No association was found. In addition, two unrelated unaffected controls were found to be homozygous GG, indicating that homozygous GG at this c.-1204 position was not sufficient for the development of oral clefts. We then sequenced the entire coding region of FOXE1 in 458 unrelated individuals (146 CPOs, 108 CL/Ps and 204 Thai controls). Five different non-synonymous variants, c.274G > T (p.D92Y), c.569C > G (p.P190R), c.569C > T (p.P190L), c.664C > T (p.R222C) and c.1090G > A (p.G364S), were identified in CPOs and one, c.572C > G (p.P191R), in CL/P. All these six variants were in heterozygous state, each identified in one patient, and absent in 204 controls. Except the p.P190R, which was previously reported, the other five variants were novel. Our study identifies probable susceptibility variants of FOXE1 for oral clefts in the Thai population.

The presence of Bartonella species in Xenopsylla cheopis fleas collected from Rattus spp. (R. exulans, R. norvegicus, and R. rattus) in Khon Kaen Province, Thailand was investigated. One hundred ninety-three fleas obtained from 62 rats, were screened by polymerase chain reaction using primers specific for the 16S-23S intergenic spacer region, and the presence of Bartonella DNA was confirmed by using the citrate synthase gene. Bartonella DNA was detected in 59.1% (114 of 193) of fleas examined. Sequencing demonstrated the presence of Bartonella spp. similar to B. elizabethae, B. rattimassiliensis, B. rochalimae, and B. tribocorum in the samples tested with a cutoff for sequence similarity ≥ 96% and 4 clustered together with the closest match with B. grahamii (95.5% identity). If X. cheopis proves to be a competent vector of these species, our results suggest that humans and animals residing in this area may be at risk for infection by several zoonotic Bartonella species.

Background: We developed an allele-specific polymerase chain reaction (AS-PCR) technique for Kidd blood group genotyping., Methods: Altogether, 340 blood samples from Thai blood donors at the National Blood Centre, Thai Red Cross Society, were tested with anti-Jk(a) and anti-Jk(b) using the gel technique and the direct urea lysis test was used for screening Jk(a-b-) phenotype. For AS-PCR technique, different types of primers were used for JK*01 and JK*02 allele detections in known DNA controls., Results: Regarding JK*02 allele detection, the pseudopositve amplification products were found when using correctly matched forward primer and a single mismatch forward primer. Interestingly, one type of two mismatch pairing at the 3' end of the forward primer can be used together with the newly designed reverse primer for Kidd blood group genotyping. It was found that the typing results in all samples obtained by serological techniques and newly developed AS-PCR technique were in agreement and this PCR technique also gave 100% concordance of results in 30 samples randomly tested twice and demonstrated reproducible results., Conclusion: This study shows that the in-house AS-PCR is simple, cost-effective, and convenient for Kidd blood group genotyping in routine laboratories, especially, in resolving serologic investigations., (© 2012 Wiley Periodicals, Inc.)

Purpose: The purpose of this study was to investigate the vertical transmission of mutans streptococci (MS) and lactobacilli in a group of Thai families., Methods: One hundred eighty-one mother-child pairs were included in this study. Unstimulated saliva was collected using the spatula method and counted for evaluating the number of MS and lactobacilli on a selective medium. Genotyping of MS and Lactobacillus species were performed in 37 and 22 child-mother pairs, respectively. Typically, 3 to 4 isolates of MS and/or Lactobacillus strains from each mother and child were collected for genotyping by an arbitrarily primed polymerase chain reaction (OPA-02 primer for MS and enterobacterial repetivive intergenic consensus primers for Lactobacillus species)., Results: Generally, MS and lactobacilli levels in children were associated with their mothers' levels. Genotyping of most isolates of MS and Lactobacillus strains in both mothers and children found diversity, and each individual showed a distinct genotypic pattern. The presence of matching genotypes of MS and Lactobacillus strains of mother-child was approximately 76% and 50%, respectively. The genotypes acquired from the maternal route show effective persistence in the children's oral cavities., Conclusions: In Thai families, mothers can be the source for transmission of mutans streptococci and Lactobacillus strains to their children.

Flea and tick specimens (5-10 fleas or ticks) on dogs and cats from various sites in Bangkok were tested by polymerase chain reaction and DNA sequencing to detect DNA of bacteria Rickettsia (gltA and 17 kDa genes), Anaplasmataceae (16S rRNA gene), and Bartonella (pap31 and its genes). We confirmed that Rickettsia sp. related to Rickettsia felis was detected in 66 of 98 (67.4%) flea specimens from dogs, whereas 8 Bartonella henselae and 2 Bartonella clarridgeiae were detected in 10 of 54 (18.5%) flea specimens from cats. Further, this work provides the first evidence of 10 Ehrlichia canis (3.3%), 7 Anaplasma platys (2.3%), and 2 Wolbachia spp. (0.66%) in 304 Rhipicephalus sanguineus tick specimens in Thailand.

To substantiate reports of greater emergence of the K65R nucleoside reverse transcriptase inhibitor (NRTI) mutation in human immunodeficiency virus type 1 (HIV-1) subtype C, we examined natural low-level K65R expression in subtype C relative to subtypes B and AE. We used allele-specific polymerase chain reaction to screen HIV-1 amplified by reverse-transcription high-fidelity polymerase chain reaction from subtype C-infected South African women and infants and CRF01(subtype AE) from Thailand; all subjects were NRTI naive. We found low-level K65R of unknown clinical significance in NRTI-naive subtype C-infected women and infants at frequencies above the natural occurrence in subtypes B and AE. The frequent appearance of subtype C frameshift deletions at codon 65 supports a propensity for transcription error in this region.

Treatment failure of chloroquine for Plasmodium vivax infection has increased in endemic countries. However, the molecular mechanisms for resistance and in vitro susceptibility of P. vivax to chloroquine remain elusive. We investigated the prevalence of mutations in the pvmdr1 and pvcrt-o genes, and the copy number of the pvmdr1 gene in isolates from the Republic of Korea (ROK), Thailand, the Union of Myanmar (Myanmar), and Papua New Guinea (PNG). We also measured in vitro susceptibility of Korean isolates to antimalarial drugs. The pvmdr1 analysis showed that mutations at amino acid position Y976F of pvmdr1 were found in isolates from Thailand (17.9%), Myanmar (13.3%), and PNG (100%), but none from the ROK, and mutation at position F1076L was present in isolates from the ROK (100%), Thailand (60.7%), and Myanmar (46.7%). One copy of the pvmdr1 gene was observed in most isolates and double copy numbers of the gene were observed in two Thai isolates. In the exons of the pvcrt-o gene that were sequenced, a K10 insertion was present in isolates from Thailand (56.0%) and Myanmar (46.2%), and the wild type was found in all Korean isolates. The results suggest that gene polymorphisms and copy number variation was observed in isolates of P. vivax from Southeast Asian countries. In Korean isolates polymorphism as limited to the F1076L variant, and no isolates with high level of resistance were found by in vitro susceptibility determinations. Moreover, our results provide a baseline for future prospective drug studies in malaria-endemic areas., (2010 Elsevier B.V. All rights reserved.)

Background: The envelope glycoproteins (Env), gp120 and gp41, are the most variable proteins of human immunodeficiency virus type 1 (HIV-1), and are the major targets of humoral immune responses against HIV-1. A circulating recombinant form of HIV-1, CRF01&#95;AE, is prevalent throughout Southeast Asia; however, only limited information regarding the immunological characteristics of CRF01&#95;AE Env is currently available. In this study, we attempted to examine the evolutionary pattern of CRF01&#95;AE Env under the selection pressure of host immune responses., Methodology/principal Findings: Peripheral blood samples were collected periodically over 3 years from 15 HIV-1-infected individuals residing in northern Thailand, and amplified env genes from the samples were subjected to computational analysis. The V5 region of gp120 showed highest variability in several samples over 3 years, whereas the V1/V2 and/or V4 regions of gp120 also showed high variability in many samples. In addition, the N-terminal part of the C3 region of gp120 showed highest amino acid diversity among the conserved regions of gp120. Chronological changes in the numbers of amino acid residues in gp120 variable regions and potential N-linked glycosylation (PNLG) sites are involved in increasing the variability of Env gp120. Furthermore, the C3 region contained several amino acid residues potentially under positive selection, and APOBEC3 family protein-mediated G to A mutations were frequently detected in such residues., Conclusions/significance: Several factors, including amino acid substitutions particularly in gp120 C3 and V5 regions as well as changes in the number of PNLG sites and in the length of gp120 variable regions, were revealed to be involved in the molecular evolution of CRF01&#95;AE Env. In addition, a similar tendency was observed between CRF01&#95;AE and subtype C Env with regard to the amino acid variation of gp120 V3 and C3 regions. These results may provide important information for understanding the immunological characteristics of CRF01&#95;AE Env.

Oral cancer ranks as one of the top ten cancers in Thailand. Molecular carcinogenesis of this disease remains unknown. The purpose of this report was to identify the genetic alteration profile in Thai oral squamous cell carcinoma (OSCC) patients using arbitrarily primed PCR and to determine the association between genetic alterations and clinico-pathological characteristics. Band alteration profiles in the 32 OSCC tissues were compared with corresponding normal tissues amplified from 60 arbitrary primers using arbitrarily primed polymerase chain reaction (AP-PCR) were identified with 12 primers. Among these, 45 band patterns presented the alteration ranged from 36% to 88%. Primer AD15 at 750 base pairs (AD15-750 bp) was found to have both the highest band alteration (88%) and the highest band loss (37%). The highest DNA band amplification was found in primer AX11-1300 bp (56%). Primer AX-11 at 1300 base pairs at the altered frequency of 78% was significantly associated with smoking (p=0.007), and primer N20 at 800 base pairs showed association with low grade tumors (p=0.030). Our results indicate that AP-PCR is a useful technique for detect genetic alteration in oral squamous cell carcinoma and provide various genetic alternative data.

Genetic variation and species authentication of 71 Kaempferia accessions (representing 15 recognized, six new, and four unidentified species) found indigenously in Thailand were examined by determining chloroplast psbA-trnH and partial petA-psbJ spacer sequences. Ten closely related species (Boesenbergia rotunda, Gagnepainia godefroyi, G. thoreliana, Globba substrigosa, Smithatris myanmarensis, S. supraneanae, Scaphochlamys biloba, S. minutiflora, S. rubescens, and Stahlianthus sp) were also included. After sequence alignments, 1010 and 865 bp in length were obtained for the respective chloroplast DNA sequences. Intraspecific sequence variation was not observed in Kaempferia candida, K. angustifolia, K. laotica, K. galanga, K. pardi sp nov., K. bambusetorum sp nov., K. albomaculata sp nov., K. minuta sp nov., Kaempferia sp nov. 1, and G. thoreliana, for which more than one specimen was available. In contrast, intraspecific sequence polymorphisms were observed in various populations of K. fallax, K. filifolia, K. elegans, K. pulchra, K. rotunda, K. marginata, K. parviflora, K. larsenii, K. roscoeana, K. siamensis, and G. godefroyi. A strict consensus tree based on combined psbA-trnH and partial petA-psbJ sequences revealed four major groups of Kaempferia species. We suggest that the genus Kaempferia is a polyphyletic group, as K. candida was distantly related and did not group with other Kaempferia species. Polymorphic sites and indels of psbA-trnH and petA-psbJ can be used as DNA barcodes for species diagnosis of most Kaempferia and outgroup species. Nuclear DNA polymorphism should be examined to determine if there has been interspecific hybridization and chloroplast DNA introgression in these taxa.

To face the worldwide threat of Surra caused by Trypanosoma evansi, international organizations have stressed the need to evaluate and standardize diagnostic tools. PCR detection of T. evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared. In this work, we compared the performances of six primer pairs-TBR1/2 (Masiga et al., 1992), ESAG6/7 (Holland et al., 2001a, b), TEPAN1/2 (Panyim et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples. TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitaemia below one parasite per ml in rat blood. They presented the highest sensitivity in cattle samples as well as a high specificity, without non-specific products nor false positive reactions out of 84 negative cattle samples tested. ESAG6/7 showed equivalent results with purified DNA and rat samples but presented non-specific products with Thai dairy cattle samples, leading to non interpretable results. TEPAN1/2 was not able to detect less than 0.1 pg of purified DNA or 50 trypanosomes/ml in rat blood. In cattle, TEPAN1/2 primers detected only 36% of the positives detected by TBR1/2. Given the parasitemic level detected, pMUTEC F/R, TRYP1 R/S and TRYP4 R/S were not more sensitive than classical microscopic examination of the buffy coat. TBR1/2, TEPAN1/2, pMUTEC F/R and TRYP4 R/S did not cross-reacted with Babesia sp., Trypanosoma theileri and Anaplasma marginale. TBR1/2 was the most sensitive primer set to detect T. evansi in purified DNA, rodent blood and cattle blood, and did not show cross reaction with the other pathogens tested: it should be therefore preferred for epidemiological surveys. These results confirmed that TBR1/2 primers remain the reference for the detection of Trypanozoon DNA and should therefore be included in subsequent evaluations of new diagnosis tools based on DNA detection., (Copyright 2010 Elsevier B.V. All rights reserved.)

Unlabelled: Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma., Introduction: Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation 34. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis 56.GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost 7. Therefore, rapid and more cost effective technique are being sought.LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye 89. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product 1011. Therefore, current investigations using melting curve analysis are being developed 1213.In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.

A modified set of primers was developed to increase the sensitivity of nested PCR amplification of glutamate dehydrogenase (gdh) gene to detect and genotype Giardia duodenalis cysts in stool specimens. This modified set of primers had a significantly higher sensitivity (82%) than that of a previously published PCR primer set (53%).

The beta giardin locus of Giardia duodenalis encodes a structural component of ventral disc and exhibits sequence variation among isolates rendering it a useful marker for genotypic analysis. To determine the distribution of genotypes of G. duodenalis in Thailand and to explore the extent of sequence variation in this locus, we deployed the PCR-RFLP method and sequence analysis of recombinant subclones from 30 clinical isolates. In total, assemblage B was more prevalent than assemblage A. Sequence analysis revealed that 13 isolates had clonal mixtures, comprising three to five distinct sequences per isolate. Nucleotide diversity of assemblage B was greater than that of assemblage A. A striking transitional bias was noted at the first and the third positions of codons in both assemblages; however, they differed in the patterns of nucleotide diversity at 0-fold and 4-fold-degenerate sites. Most amino acid exchanges were conservative in terms of polarity, charge and volume. Both assemblages have evolved under purifying selection as evidenced by a significantly greater number of mean synonymous substitutions per synonymous site (d(S)) than that of nonsynonymous substitutions per nonsynonymous site (d(N)) as well as significant negative Tajima's D values and its related statistics. The significant negative Tajima's D test at nonsynonymous sites further suggests that elimination of slightly deleterious mutations at these sites by purifying selection is ongoing as predicted in the nearly neutral theory. Furthermore, a minimum number of seven recombination sites was detected by the four gamete test in assemblage B, consistent with previous reports on meiotic recombination in G. duodenalis. Therefore, accurate subassemblage assignment of clinical isolates that has practical consequence for disease control could be complicated by the presence of intra-isolate clonal diversity and interallelic recombination.

Lung cancer is a major cause of cancer-related death in developed countries, and its incidence in developing countries is increasing. In Thailand, cancer incidences differ greatly from region to region, and lung cancer is the most common cancer in the northern Thai population. The polymorphic frequency of 10 genetic susceptibility genes and their association with lung cancer were examined in a northern Thai population: CYP1A1 (MspI), CYP1A1 (Ile462Val), CYP2E1 (PstI), CYP2E1 (DraI), GSTM1, GSTT1, MPO (AciI), OGG1 (Ser326Cys), TP53 (Arg72Pro), and MMP1(AluI). The 173 subjects were 91 lung cancer patients and 82 healthy volunteers. Although no significant association between any single genetic variant and lung cancer risk was observed, when genetic variants were analyzed in combination, a significant effect on lung cancer risk was found for the variant allele in a combination of five genes involved in oxidative stress and inflammatory response: GSTM1 (null), MPO (-463A), OGG1 (326Cys), TP53 (72Pro) (alias p53), MMP1 (2G). With a reference group of individuals carrying at least two wild-type genotypes of these five genes, it was found that an individual carrying three or more variant genotypes is at significantly higher risk of developing lung cancer with the increasing of odds ratios (OR) in concurrence with the number of variant genes. The OR was 2.41 (95% CI = 0.76-7.64), 3.90 (95% CI = 1.23-12.34), and 5.20 (95% CI = 1.31-20.54) for individuals carrying three, four, and five variants, respectively. After stratifying by sex, the OR was higher for women: OR 4.05 (95% CI = 0.44-36.94), 9.00 (95% CI = 0.95-84.89) and 18.00 (95% CI = 1.49-216.62) for three, four, and five variant genotypes, respectively. This augmented effect on lung cancer risk of variant genes involved in oxidative stress and inflammatory response in women with a low prevalence of smoking indicates their modifying effect on other risk factors, such as environmental cigarette smoke, air pollution, radon radiation, or infection of the airway. Confirmation would require further investigations with larger sample sizes.

Multidrug resistant Acinetobacter baumannii has become the most common cause of health care-associated infections at Maharaj Nakhon Si Thammarat Hospital, Thailand. The objective of the study was to detect integrons using PCR-based method from 96 A. baumannii isolates from ventilator-associated pneumonia (VAP) patients and their environment. Antibiotic susceptibility was determined using a disk diffusion technique. Forty-six isolates exhibited integrase genes, with only class I and class II integron detected in 43 and 3 A. baumannii isolates, respectively. Twenty-seven of 52 clinical and 19 of 44 environmental isolates were integron-positive. Detection rate of integron-positive A. baumannii isolated from VAP patients increased from 25% to 83% over the 4 month study period. The majority (91%) of integron-positive A. baumannii showed resistance to 6 or more of 11 antibiotics tested and 72% of class I integron-positive isolates were imipenem-resistant. Thus, class I integron-positive A. baumannii had spread among the VAP patients and into hospital environment, the latter acting as reservoirs of potential pathogens possessing drug resistance genes.

Dengue virus serotype 3 (DENV-3) was associated with severe dengue epidemics in Thailand during 1973-1999. We studied Thai DENV-3 viruses isolated from hospitalized children in Bangkok with differing disease severity during that period. Viruses were sequenced at their 5' and 3' untranslated regions (UTRs), which are regions that play a pivotal role in viral replication. Our results indicated that the primary sequences as well as the secondary structures at both ends of Thai DENV-3 viruses were highly conserved over almost three decades. We found nucleotide insertions and deletions at the variable region (VR) that is located just downstream of the nonstructural protein 5 (NS5) stop codon among these viruses. The phylogenetic tree derived from the size heterogeneity of VR in the 3' UTR divided DENV-3 into four genotypes, and Thai DENV-3 viruses in this study belonged to genotype II. The replication efficiency of the candidate viruses with different lengths at the VR were assessed in the mosquito (C6/36) and human (HepG2) cell lines. Our results show that the viruses with nucleotide insertions at VR replicated better than the virus that contained deletions. Our findings indicate that Thai DENV-3 demonstrated a remarkable conservation of nucleotides over 28 years. Correlation with disease severity suggests that both primary sequences and secondary structures of the 3' UTR do not appear correlated with disease severity in humans.

Severe dengue virus (DENV) infection is characterized by a cascade of cytokine production, including the production of tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-alpha (LT-alpha). We have analyzed a variety of polymorphisms in the TNF and LTA genes of 435 ethnic Thais who had subclinical DENV infection, primary or secondary dengue fever (DF), or primary or secondary dengue hemorrhagic fever (DHF). The TNF -238A polymorphism marking the TNF-4,LTA-3 haplotype occurred in a significantly greater number of patients with secondary DHF (20 [15.2%] of 132) than patients with secondary DF (7 [4.1%] of 169) (P < .001; P corrected by use of Bonferroni adjustment, .022; odds ratio, 4.13 [95% confidence interval, 1.59-11.17]). In a subset of patients, the LTA-3 haplotype was associated with in vivo intracellular production of LT-alpha and TNF-alpha during the acute viremic phase of infection. Two extended human major histocompatibility complex (MHC) haplotypes containing TNF-4 and LTA-3, together with HLA-B48, HLA-B57, and HLA-DPB1*0501, were detected only in patients with secondary DHF. These observations indicate that polymorphism in functionally distinct MHC-encoded proteins contributes to the risk of developing severe secondary DENV infection and warrants further investigation.

The tropical cattle tick, Rhipicephalus (Boophilus) microplus, is an important ectoparasite of livestock in Thailand that causes economic losses due to the direct effects of tick feeding and by the pathogens they transmit. Intensive acaricide use has several drawbacks, which spurred efforts to develop anti-tick vaccines. Vaccines targeting concealed antigens localized in the tick midgut result in reduced tick fecundity, but molecules localized in the tick salivary glands, which could play a role in pathogen transmission, remain largely unexplored for R. microplus. Calreticulin (CRT) is a protein found in tick salivary glands and saliva, and CRT might facilitate tick feeding and pathogen transmission through anti-thrombotic and complement-inhibition activities. This then suggests that CRT should be evaluated as a vaccine candidate antigen to control cattle ticks in Thailand. The objective of this work was to clone, sequence, and analyze cDNA encoding CRT from salivary glands of R. microplus indigenous to Thailand. Nucleotide sequence analysis showed an open reading frame of 1233 bp. Comparison of the amino acid sequence showed 65-99% identities to other known CRTs from Oryctolagus cuniculus, Rattus norvegicus, Homo sapiens, Bos taurus, R. sanguineus, and R. microplus. The N- and P-domains of CRT were the most conserved, whereas the C-domain was high acid and more variable. CRT primary sequences were most conserved among mammals. Further investigations are warranted to determine whether immunization with Thai B. microplus CRT can affect tick performances and experimental pathogen transmission.

We studied the occurrence of swine influenza virus (SIV) infection in piglets with respiratory symptoms resembling porcine respiratory disease complex (PRDC). A total of 106 samples including nasal swab and lung suspension from sick piglets were collected from 30 farms of medium size in the central and eastern parts of Thailand from August 2006 to February 2007. Samples were inoculated onto Mardin-Darby Canine Kidney (MDCK) cells and SIV infection was confirmed by immunofluorescent assay (IFA) and reverse transcriptase polymerase chain reaction (RT-PCR) specific for M gene. Of 106 samples, 3 pigs from 3 different farms were found to be SIV positive on all assays. The positive samples were further identified by RT-PCR as H3N2 subtype using specific primers for hemagglutinin (HA) and neuraminidase (NA) genes. SIV infection was found in 2.8% of swine suffering from respiratory distress suggesting SIV may not be the major pathogen for PRDC in the central and eastern Thailand. SIV was present in 3 of 30 farms (10%) indicating the prevalence of SIV in these regions is considerable. Since pigs are vulnerable to infection from both human and avian influenza viruses and interspecies transmission between humans and swine occurs sporadically, it is essential to continue surveillance and monitoring of SIV infection in the swine population.

Objectives: To develop and apply the polymerase chain reaction with confronting two-pair primers (PCR-CTPP) for detection and identification of hemoglobin E (Hb E)., Material and Method: Fifty unrelated northern Thais were included in the present study. DNA was extracted from peripheral blood mononuclear cells and targeted to amplify by PCR-CTPP. The amplified product was analyzed and compared with the reference hemoglobin electrophoresis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis., Results: The results validated a completely concordant among these three methods consisting of 74%, 24%, and 2% identified as normal, heterozygous, and homozygous Hb E type, respectively., Conclusion: Successful Hb E genotyping by PCR-CTPP was introduced. It allows for confirming and simultaneously detection with other thalassemia mutations.

A total of 134 isolates of Bacillus thuringiensis obtained from different geographical and ecological origins in Thailand were analyzed to determine the distribution and diversity of cry1, cry2 and cry9 genes encoding for Cry proteins toxic to lepidopteran insects. Strains containing cry1-type genes (109/134 or 81.3%) were found at the same frequency as strains harboring cry2 gene (108/134 or 80.6%) whereas only 50 strains contained cry9 gene (50/134 or 37.3%). Seventeen percent (23/134) of the B. thuringiensis isolates did not harbor any cry1, cry2 or cry9 genes. Among cry1 containing isolates, cry1A (49.3%), cry1B (50.0%), cry1G (48.5%), cry1I (49.3%), cry1J (35.1%) and cry1L (47.0%) were considered abundant. The cry2 gene was distributed with high frequency (>70%) in every region of the country. The study of cry gene combinations revealed 14 cry gene profiles.

Four mosquito densovirus strains were assayed for mortality and infectivity against Aedes aegypti larvae from different geographic regions. The viral titers were quantified by real-time PCR using TaqMan technology. Firstinstar larvae were exposed to the same titer of each densovirus strain for 48 hours. All strains of densoviruses exhibited larvicidal activity and caused more than 80% mortality and infectivity in the three mosquito strains. AalDNV-exposed larvae had the highest mortality rate. The mean time to death of AalDNV-exposed larvae was shorter than other DNVs-exposed larvae. We can conclude that different densovirus strains exhibit some variations in their pathogenicity to different populations of Ae. aegypti mosquitoes. A few mosquitoes from Chachoengsao and Bangkok exposed to AeDNV and AThDNV survived to the adult stage to lay eggs and showed 22% to 50% vertical transmission in the F1 generation. Phylogenetic analysis of four densovirus strains indicated that mosquito densoviruses are separated into two distinct clades.

Objectives: The aims of this study were to determine the prevalence of HPV infection and distribution of HPV genotypes in Northern Thai women and thereby estimate the benefit of administering the HPV vaccine in the population., Methods: Formaldehyde-fixed, paraffin-embedded samples of invasive squamous cell carcinoma from 99 patients were tested for HPV genotypes using the Linear Array HPV Genotyping Test., Results: HPV was detected in 96/99 (96.9%) cases. Seventy-five (78.1%) cases were single infections and 21 (21.9%) multiple. HPV16 and HPV18 were the most common subtypes, detected in 62/96 (64.4%) cases. HPV52 and HPV58 infections were found in 17/96 (17.7%) cases. Co-infection always involved HPV16. The most common co-infection was HPV16 and 52 (7 cases) but never HPV16 and 18., Conclusions: Although the prevalence of HPV infection in cervical cancer of Northern Thai women is comparable to the other regions worldwide, the distribution of HPV subtypes differs with lower frequencies of HPV16 and 18, and higher frequencies of HPV52 and 58. Moreover, multiple infections are common. The vaccine against HPV16 and HPV18 can be estimated to prevent approximately two thirds of the cervical cancer cases in Northern Thailand. Although designed for use on unfixed tissue, this study shows that the Linear Array HPV Genotyping Test can be successfully used for HPV genotyping on paraffin-embedded archival tissue. This methodology also provides a means for retrospective studies on serial samples for a greater understanding of HPV genotypes, co-infections, and relationship to cervical cancer.