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The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection.This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot).Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

The

Mycobacterium abscessus can cause true infection or be present in the host as a harmless colonist. The ability of M. abscessus to cause disease and develop drug resistance is known to have a genetic basis. We aimed to differentiate between persistent infection and reinfection using multilocus sequence typing (MLST) and to study the genetic diversity of M. abscessus relative to multi-organ infection and drug resistance in Northeast Thailand. DNA was extracted from 62 M. abscessus isolates (24 cases). The following genes were sequenced: argH, cya, glpK, gnd, murC, pta, purH and rpoB. Drug susceptibility tests were performed using broth microdilution. Subspecies classification and phylogeny were determined. Among the 24 cases (62 isolates), 19 cases (49 isolates) were of true NTM infection and 5 cases (13 isolates) examples of colonization. Two subspecies, M. abscessus subsp. massiliense (12 cases, 32 isolates) and M. abscessus subsp. abscessus (12 cases, 30 isolates) were identified. The major sequence type (ST) was ST227. Two clonal groups among patients were found; clonal cluster I (5 cases, 8 isolates) and clonal cluster II (2 cases, 4 isolates) but no epidemiological link was apparent. Reinfection (2 cases with different clones of M. abscessus strains; >9 SNPs different) and persistent infection (14 cases with the same clone

Our aim was to explore the microbial community composition (bacteria and fungi) of fermented fish (pla-ra) from Northeast Thailand. We also made functional predictions concerning these microbial communities. The association between the microbiota and odor intensity was also analyzed. Fourteen samples of 1-year fermented fish samples derived from seven local markets in Khon Kaen, Northeast Thailand were used. The microbial community composition of each was investigated by sequencing the V1-V9 regions of the 16S rRNA gene (bacteria) and the ITS gene (fungi) using an Illumina MiSeq platform. Functional prediction analysis was conducted through Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) based on the use of the bacterial 16S rRNA gene sequences. The bacterial communities were rich, comprising 402 genera from 28 phyla, including such genera as Tetragenococcus, Staphylococcus, Virgibacillus, Lactobacillus and Lentibacillus. The fungal communities comprised 7 phyla and 60 genera, such as Heterobasidion, Densospora, Exophiala and Monascus. The bacterial community functional analysis revealed an association with six biological metabolic pathway categories (e.g., metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems and human diseases) with 17 subfunctions, showing the richness of bacterial community functions. Odor-association analysis revealed that Brevibacterium, Brachybacterium and Chromohalobacter were more abundant in the weak-odor group, while Noviherbaspirillum was more abundant in the strong-odor group. This study provides a preliminary analysis of pla-ra microbial community structure and function in popular traditional Thai foods. Functional prediction analysis might be helpful to improve our knowledge of the microbiota in fermented fish.

The existence of latent tuberculosis infection (LTBI) is one of the main obstacles hindering eradication of tuberculosis (TB). To better understand molecular mechanisms and explore biomarkers for the pathogen during LTBI, we cultured strains of Mycobacterium tuberculosis (Mtb) under stress conditions, mimicking those in the host granuloma intracellular environment, to induce entry into the non-replicating persistence stage. The stresses included hypoxia, low pH (5.0), iron deprivation (100 μM of 2, 2’˗dipyridyl) and nutrient starvation (10% M7H9 medium). Three Mtb strains were studied: two clinical isolates (drug-susceptible Beijing (BJ) and multidrug-resistant Beijing (MDR-BJ) strains) and the reference laboratory strain, H37Rv. We investigated the proteomics profiles of these strains cultured in stressful conditions and then validated the findings by transcriptional analysis. NarJ (respiratory nitrate reductase delta chain) was significantly up-regulated at the protein level and the mRNA level in all three Mtb strains. The narJ gene is a member of the narGHJI operon encoding all nitrate reductase subunits, which play a role in nitrate metabolism during the adaptation of Mtb to stressful intracellular environments and the subsequent establishment of latent TB. The identification of up-regulated mRNAs and proteins of Mtb under stress conditions could assist development of biomarkers, drug targets and vaccine antigens., Highlights • The proteomics profiles between BJ and MDR-BJ strains of M. tuberculosis (Mtb) cultured in vitro in stressful conditions. • NarJ is a common protein and significantly up-regulated protein of BJ and MDR-BJ Mtb strains. • The unique proteins found on BJ and MDR-BJ Mtb strain were of “Rv3764c/tcrY” and “Rv1356c and Rv1420/uvrC”, respectively.

The exploration of vaginal microbiota by using next-generation sequencing (NGS) of 16S ribosomal RNA (rRNA) gene is widely used. Up to now, different hypervariable regions have been selected to study vaginal microbiota by NGS and there is no standard method for analysis. The study aimed to characterize vaginal microbiota from clinical samples using NGS targeting the 16S rRNA gene and to determine the performance of individual and concatenated hypervariable region sequences to generate the taxonomic profiles of the vaginal microbiota. Fifty-one vaginal DNA samples were subjected to 16S rRNA gene NGS based on the Ion Torrent PGM platform with the use of two primer sets spanning seven hypervariable regions of the 16S rRNA gene. Our analysis revealed that the predominant bacterial genera were Lactobacillus, Gardnerella and Atopobium, which accounted for 78%, 14% and 2%, respectively, of sequences from all vaginal bacterial genera. At the species level, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae accounted for 72%, 10% and 6%, respectively, of the bacterial cells present. Analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6–V7 and V4 regions, while the V9 region gave the lowest bacterial resolution. NGS based on the 16S rRNA gene can give comprehensive estimates of the diversity of vaginal bacterial communities. Selection of sequences from appropriate hypervariable regions is necessary to provide reliable information on bacterial community diversity.

© 2020 by the authors., Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances., This study was supported by projects from the Royal Golden Jubilee (RGJ)-PhD Program Grant of the Thailand Research Fund (TRF), Thailand (Ref. PHD/0133/2558), and Asturias Principality, Spain (Ref. IDI/2018/000114).

In this present study, we investigated the phenol-soluble modulin (psm-mec) mutations, the staphylococcal cassette chromosome mec (SCCmec) types, and toxin production in 102 methicillin-resistant Staphylococcus aureus (MRSA) isolates from the northeast and central regions of Thailand. The MRSA isolates carrying -7T>C psm-mec in Type II SCCmec (n = 18) and the MRSA isolates carrying no psm-mec in Type IV (n = 8) or Type IX SCCmec (n = 4) had higher hemolytic activity against sheep erythrocytes than MRSA isolates carrying intact psm-mec in Type III SCCmec (n = 34), but MRSA isolates carrying no psm-mec in Type I SCCmec (n = 27) did not.

We report the genome sequence of Lactobacillus fermentum 47-7, a good in vitro probiotic strain isolated from an infant. Its genome size is 1.83 Mb, it is assembled from 180 contigs, and it consists of 1,636 protein-coding genes, 15 rRNAs, 57 tRNAs, and 4 noncoding RNAs. This genome sequence will be useful for a variety of applications.

We report the genome sequence of Lactobacillus fermentum 47-7, a good in vitro probiotic strain isolated from an infant. Its genome size is 1.83 Mb, it is assembled from 180 contigs, and it consists of 1,636 protein-coding genes, 15 rRNAs, 57 tRNAs, and 4 noncoding RNAs. This genome sequence will be useful for a variety of applications.

Lymphatic filariasis is principally caused by Wuchereria bancrofti, and Brugia malayi. The other two filarial nematode species, Brugia pahangi and Dirofilaria immitis, possibly cause human zoonotic diseases.We propose the development of a PCR assay linked with DNA pyrosequencing as a rapid tool to identify W. bancrofti, B. malayi, B. pahangi, and D. immitis in blood samples and mosquitoes. Primers targeting the fragment of the 5S ribosomal RNA and spliced leader sequences were newly designed and developed to identify these four filarial nematodes. Analytical sensitivity and specificity were evaluated.Pyrosequencing determination of nucleotide variations within 36 nucleotides for B. malayi and B. pahangi, and 32 nucleotides for W. bancrofti and D. immitis is sufficient for differentiation of those filarial nematodes, and for detection of intraspecies genetic variation of B. malayi. This analysis could detect a single B. malayi, B. pahangi, W. bancrofti, and D. immitis microfilaria in blood samples.Overall, the PCR-linked pyrosequencing-based method was faster than direct sequencing and less expensive than real-time PCR or direct sequencing. This is the possibility of choice that can be applied in a high-throughput platform for identification and surveillance of reservoirs and vectors infected with lymphatic filaria in endemic areas.

Lymphatic filariasis, a mosquito-borne disease, is still a major public health problem in tropical and sub-tropical countries. Effective diagnostic tools are required for identification of infected individuals, for epidemiological assessment, and for monitoring of control programs. A duplex droplet digital polymerase chain reaction (ddPCR) was conducted to differentiate and quantify Wuchereria bancrofti DNA by targeting the long DNA repeat (LDR) element and Brugia malayi DNA by targeting the HhaI element in blood samples and mosquito vectors. The analytical sensitivity and specificity were evaluated. Our results indicated that the duplex ddPCR assay could differentiate and quantify W. bancrofti and B. malayi DNA from blood samples and mosquitoes. DNA from a single larva in 50 μl of a blood sample, or in one mosquito vector, could be detected. The analytical sensitivity and specificity for W. bancrofti are both 100 %. Corresponding values for B. malayi are 100 and 98.3 %, respectively. Therefore, duplex ddPCR is a potential tool for simultaneous diagnosis and monitoring of bancroftian and brugian filariasis in endemic areas.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p

This study investigated cloning and expression of enterovirus 71 viral capsid protein 1 (EV71‐VP1) in Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) M115. To achieve this, a codon‐optimized gene coding for EV71‐VP1 was analysed, designed, synthesized and cloned into a plasmid vector flanked by a transcriptional promoter and terminator sequences. The promoter was based on that of P919, a constitutive promoter of the gene encoding the large ribosomal protein of B. bifidum BGN4, while the terminator was based on that of the peptidase N gene of Lactococcus lactis. The construct was amplified in Escherichia coli XL1‐blue and then transferred into B. pseudocatenulatum M115 by electrotransformation. Western blot analysis revealed that the EV71‐VP1 was intracellularly expressed in B. pseudocatenulatum M115 under the control of the selected heterologous promoter. In addition, plasmid stability analysis showed the construct was maintained stably for more than 160 generations, enough for most future applications. The results derived from this study open the possibility to utilize the bacterium carrying a specific expression plasmid as cell factory for the production of proteins with high commercial and health‐promoting value., This study was supported by the Office of the HigherEducation Commission, the College of Medicine andPublic Health, and Ubon Ratchathani University, and theThailand Research Fund (TRF Grant No. MRG5680081 toMP), the Higher Education Research Promotion andNational Research University Project of Thailand to VL,and the Spanish Ministry of Economy and Competitive-ness (Project No. AGL-2014-57820-R to BM).

BackgroundThe vaginal microbiota (VMB) plays a key role in women’s reproductive health. VMB composition varies with ethnicity, making it necessary to characterize the VMB of the target population before interventions to maintain and/or improve the vaginal health are undertaken. Information on the VMB of Thai women is currently unavailable. We therefore characterized the VMB in normal Thai women.MethodsVaginal samples derived from 25 Thai women were subjected to 16S rRNA gene next-generation sequencing (NGS) on the Ion Torrent PGM platform.ResultsTwo groups of VMB were detected, lactobacilli-dominated (LD) and non-lactobacilli dominated (NLD) groups.Lactobacillus inerswas the most common species found in the LD group whileGardnerella vaginalisfollowed byAtopobium vaginaeandPseudumonas stutzeriwere commonly found in the NLD group.ConclusionsThe VMB patterns present in normal Thai women is essential information to further determine the factors associated with VMB patterns in vaginal health and disease and to develop proper management of reproductive health of Thai women.

Enterobiasis, caused by the nematode Enterobius vermicularis, is a common health problem among schoolchildren in Thailand. We provide the first molecular identification of this nematode from Thai schoolchildren and document genetic variation among E. vermicularis eggs using sequence analyses of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). A cross-sectional parasitological survey was conducted in schoolchildren (n = 491) in five regions of Thailand between May 2015 and December 2016. The diagnosis of Enterobius infection was made using the adhesive tape perianal swab technique. Enterobius eggs were recovered from 43 participants (8.75%). DNA was extracted from these eggs and the cox1 gene and partial ITS2 region amplified using the polymerase chain reaction (PCR). Nineteen amplified PCR products of the cox1 gene (441 bp) and 18 of the ITS2 region (623 bp) were subsequently sequenced. All sequences were identified as belonging to E. vermicularis based on database searches. Phylogenetic analysis and a median-joining network of available E. vermicularis cox1 sequences showed 66 haplotypes. We found haploclusters (types A and B) represented among the Thai sequences. Six haplotypes from Thailand fell into type A (of Nakano et al., 2006) (along with sequences from Japan and Korea) and five haplotypes into type B (with sequences from Japan, Iran, Czech Republic, Greece, Denmark and Sudan). The overall haplotype diversity (Hd) was 0.9888. Transmission of worms with type B haplotypes from primates to humans in Asia or from humans in Europe possibly occurs in Thailand.

A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples ( n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate.

Canine babesiosis, hepatozoonosis, ehrlichiosis, and anaplasmosis are tick-borne diseases caused by different hemopathogens. These diseases are causes of morbidity and mortality in dogs. The classic method for parasite detection and differentiation is based on microscopic observation of blood smears. The limitations of the microscopic method are that its performance requires a specially qualified person with professional competence, and it is ineffective in differentiating closely related species. This study applied PCR amplification with high throughput pyrosequencing for molecular differential detection of the following 4 hemoparasites common to tropical areas in dog blood samples: Babesia vogeli, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. PCR was initially used to amplify specific target regions of the ribosomal RNA genes of each parasite using 2 primer pairs that included 18S rRNA for protozoa (B. vogeli and H. canis) and 16S rRNA for rickettsia (E. canis and A. platys). Babesia vogeli and H. canis were discriminated using 9 nucleotide positions out of 30 base pairs, whereas E. canis and A. platys were differentiated using 15 nucleotide positions out of 34 base pairs that were determined from regions adjacent to 3' ends of the sequencing primers. This method provides a challenging alternative for a rapid diagnosis and surveillance of these tick-borne diseases in canines.

Summary 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.

Natural killer (NK) cell plays an important role in an innate immune response against viral infection. The kinetics regulation and functional consequences of NK cells in the pathogeneses of diseases are uncertain. We analyzed NK cell distribution and function of successfully combination antiretroviral therapy (cART)-treated HIV-1 infected individuals in Khon Kaen Regional Hospital, Thailand. The results demonstrated that increased percentage and the total number of NK cell in cART-treated HIV-1 infected patients with preferential high levels of CD56dimCD16+ and CD56-CD16+ subsets when compared with a control group even in undetectable viral load (γ production implied the impairment of cytolytic activity was not recovered after cART treatment. Thus, altered NK cell frequency and function by HIV-1 infection are not completely recovered with cART, which may contribute to impaired cellular immune response and persistence of HIV-1.

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

Nematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Evidence has been presented that herpes simplex virus (HSV) immunoglobulin (IgG) Fc receptors are composed of a complex of two glycoproteins, gE and gI. In previous studies, cells infected with HSV-1 mutants lacking either gE or gI bound lower levels of soluble IgG than cells infected with wild-type viruses suggesting that both gE and gI were required for IgG binding. We have reevaluated the Fc receptor activity of these mutants using a more sensitive assay involving IgG-coated erythrocytes and have found that cells infected with a gE- mutant HSV-1 did not bind IgG-coated erythrocytes whereas cells infected with a gI- mutant retained some Fc binding activity. To further study HSV-induced Fc receptors recombinant adenovirus vectors expressing gE or gI were constructed. Cells expressing gE alone bound both soluble IgG and IgG-coated red cells, although the binding was consistently lower than that observed with HSV-infected cells or cells expressing both gE and gI. Cells expressing only gI were unable to bind either soluble IgG or IgG-coated erythrocytes. These results support the conclusion that both gE and gI are required for full Fc receptor activity, although gE alone can bind IgG to a lesser extent.

MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3′ UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.

Autoantibodies against interferon-γ are associated with severe disseminated opportunistic infection, but their importance and prevalence are unknown.We enrolled 203 persons from sites in Thailand and Taiwan in five groups: 52 patients with disseminated, rapidly or slowly growing, nontuberculous mycobacterial infection (group 1); 45 patients with another opportunistic infection, with or without nontuberculous mycobacterial infection (group 2); 9 patients with disseminated tuberculosis (group 3); 49 patients with pulmonary tuberculosis (group 4); and 48 healthy controls (group 5). Clinical histories were recorded, and blood specimens were obtained.Patients in groups 1 and 2 had CD4+ T-lymphocyte counts that were similar to those in patients in groups 4 and 5, and they were not infected with the human immunodeficiency virus (HIV). Washed cells obtained from patients in groups 1 and 2 had intact cytokine production and a response to cytokine stimulation. In contrast, plasma obtained from these patients inhibited the activity of interferon-γ in normal cells. High-titer anti-interferon-γ autoantibodies were detected in 81% of patients in group 1, 96% of patients in group 2, 11% of patients in group 3, 2% of patients in group 4, and 2% of controls (group 5). Forty other anticytokine autoantibodies were assayed. One patient with cryptococcal meningitis had autoantibodies only against granulocyte-macrophage colony-stimulating factor. No other anticytokine autoantibodies or genetic defects correlated with infections. There was no familial clustering.Neutralizing anti-interferon-γ autoantibodies were detected in 88% of Asian adults with multiple opportunistic infections and were associated with an adult-onset immunodeficiency akin to that of advanced HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research; ClinicalTrials.gov number, NCT00814827.).

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (Tm), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean Tm ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals.

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

Please cite this paper as: Phungoen et al. (2011) Clinical factors predictive of PCR positive in pandemic H1N1 2009 influenza virus infection. Influenza and Other Respiratory Viruses 5(6), e558–e562. Objective Pandemic H1N1 2009 influenza virus (H1N1) has been spreading globally. Clinical features might be predictive and may be different among countries. Even though the PCR test is a confirmatory test for this viral infection, it is expensive and limited in most Thai health care facilities. We studied predictive factors of PCR positive in H1N1 suspected patients. Methods Consecutive patients who had influenza-like illness less than seven days and had been tested for H1N1 by the real-time PCR method between May and July 2009 were enrolled. Clinical data was collected and compared between those who had positive and negative PCR tests. Results There were 6494 patients had flu-like symptoms. Of those, 166 patients were done PCR test and 75 patients (45·18%) had positive PCR test. There were four predictors for positive PCR test including history of contact with confirmed H1N1 patients, headache, body temperature, and coryza with the adjusted odds ratio (95% confidence interval) of 2·84 (1·09–7·40), 6·25 (1·42–27·49), 1·69 (1·08–2·66), and 0·31 (0·12–0·79), respectively. Conclusions Clinical factors can be both suggestive and protective factors for H1N1 infection. These factors may be helpful in clinical practice to assess the possibility of the H1N1 infection in people who are at risk; particularly in resource-limited health care facilities.

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%–3.67% and 0.79%–4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample.

A real-time fluorescence resonance energy transfer (FRET) PCR supplemented with melting curve analysis for the rapid molecular detection of Dirofilaria immitis in mosquito vectors and dog blood samples was developed. This real-time FRET PCR was based on the fluorescence melting curve analysis of a hybrid between an amplicon generated from the D. immitis ribosomal RNA gene sequence and specific fluorophore-labeled probes. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method were all 100%. Besides being highly sensitive and specific, this PCR is fast and offers a high throughput. Therefore it is a suitable and powerful tool for the diagnosis and for epidemiological surveys of canine dirofilariasis as well as for molecular xenomonitoring of D. immitis in mosquito vectors.

Real-time fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) supplemented with melting curve analysis is a highly sensitive and fast method offering a high throughput. We report the development of a real-time FRET PCR for molecular detection of Opisthorchis viverrini in human fecal samples. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of this method were 97.5%, 100%, 98.9%, 100%, and 98.2%, respectively. The sensitivity was not significantly different from that of the quantified formalin-ethyl acetate concentration technique, the gold standard (P > 0.05). The procedure has potential for diagnosis of human opisthorchiasis in disease-endemic areas, for large epidemiologic investigations involving at risk populations, and monitoring eradication programs of the liver fluke, which causes hepatobiliary diseases and induces cholangiocarcinoma.

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.