Development of an Antigen Capture Lateral Flow Immunoassay for the Detection of Burkholderia pseudomallei.

Early diagnosis is essential for the successful management of Burkholderia pseudomallei infection, but it cannot be achieved by the current gold standard culture technique. Therefore, this study aimed to develop a lateral flow immunoassay (LFIA) targeting B. pseudomallei capsular polysaccharide. The development was performed by varying nitrocellulose membrane reaction pads and chase buffers. The prototype LFIA is composed of Unisart CN95 and chase buffer containing tris-base, casein, and Surfactant 10G. The assay showed no cross-reactivity with E. coli, S. aureus, P. aeruginosa, and P. acne. The limit of detections (LODs) of the prototype LFIA was 107 and 106 CFU/mL B. pseudomallei in hemoculture medium and artificial urine, respectively. These LODs suggest that this prototype can detect melioidosis from positive hemoculture bottles but not straight from urine. Additionally, these LODs are still inferior compared to Active Melioidosis Detect (AMDTM). Overall, this prototype holds the potential to be used clinically with hemoculture bottles. However, further improvements should be considered, especially for use with urine samples.

Natural infection of leptospirosis and melioidosis in long-tailed macaques (Macaca fascicularis) in Thailand.

This study aimed to determine the incidence of leptospirosis and melioidosis in long-tailed macaques (Macaca fascicularis) in Thailand. Serum samples from 223 monkeys were subjected to the Lepto Latex Test and indirect hemagglutination (IHA) test to detect antibodies against Leptospira spp. and Burkholderia pseudomallei. The microagglutination test (MAT) was used to identify serovars of Leptospira spp. Conventional PCR for the LipL32 gene of L. interogans and the BPSS0120 and btfc-orf18 genes of B. pseudomallei was used for molecular detection. The overall seroprevalence of leptospirosis and melioidosis was 2.69% (95% confidence interval (CI): 0.99-5.76%) and 14.35% (95% CI: 10.03-19.65%), respectively. Six samples that showed positive MAT results were also positive for IHA. The serovars of Leptospira were Ranarum (5/6), Shermani (6/6), and both (5/6). Conventional PCR for the LipL32 gene of Leptospira spp. was positive in 10.31% of the samples (95% CI: 5.56-13.51%). However, there were no positive results for BPSS0120 and btfc-orf18 in B. pseudomallei. Active infection was detected only for leptospirosis; however, it can be assumed that pathogen exposure occurred in this group of animals because immunity could be detected. The routes of infection and elimination pathways of both bacteria remain unclear, and the mechanism of protection in non-human primates needs to be elucidated in further studies. Moreover, this health issue should be considered to prevent human infections in monkeys and their environment.

First Molecular Detection of Babesia gibsoni in Stray Dogs from Thailand.

In southern Thailand, the increasingly growing population of stray dogs is a concern to public health and environmental safety because of the lack of medical attention and control. More importantly, these animals are considered reservoirs for many zoonotic pathogens. The objective of this study was to molecularly detect canine vector-borne pathogens, and to perform genetic characterization of Babesia gibsoni present in stray dogs from southern Thailand. Blood samples were collected from 174 stray dogs in two provinces (Songkhla and Narathiwat) in southern Thailand. PCR analyses were executed using specific primers based on the Babesia spp. 18S rRNA gene, Babesia gibsoni Internal transcribed spacer 1 (ITS1) region, Ehrlichia canis citrate synthase (gltA) gene, Hepatozoon spp. 18S rRNA gene and Anaplasma platys heat shock protein (groEL) gene. The most common canine vector-borne pathogen found infecting stray dogs in this study was Hepatozoon canis (24.7%) followed by A. platys (14.9%), Babesia vogeli (8.0%), B. gibsoni (6.3%), and E. canis (1.72%). Concurrent infection with more than one pathogen occurred in 72 cases. Phylogenetic analysis based on the ITS1 region and 18S rRNA gene revealed that the B. gibsoni isolates from this study shared a large proportion of their identities with each other and with other reported B. gibsoni genotypes from Asia. This study highlights the molecular detection of B. gibsoni in dogs in Thailand for the first time and presents the genetic characterization by sequencing the ITS1 region and 18S rRNA gene of B. gibsoni from Thailand. Follow-up studies are needed to elucidate the origin, distribution, and vectors of B. gibsoni parasites circulating in dogs in Thailand, as well as to determine to what extent dogs are important reservoir hosts for zoonotic canine vector-borne disease infection in the studied area.

Epstein-Barr virus strains defined by the latent membrane protein 1 sequence characterize Thai ethnic groups.

Epstein-Barr virus (EBV) is ubiquitous in the human population and seroepidemiological studies have revealed that more than 90% of adults are infected with the virus in Thailand. It has been suggested that latent membrane protein 1 (LMP1) variants may differ in their tumorigenicity and geographical localization. The distribution of LMP1 variants of EBV in the Thai population was studied. A total of 259 LMP1 sequences from ten Thai ethnic groups (Lahu, Lisu, Shan, Red Karen, White Karen, Hmong, Akha, Mlabri, Moken and Urak Lawoi) were studied using direct PCR sequencing. Nucleotide sequences corresponding to the C terminus of the LMP1, including previously published sequences from central and southern Thais, were used in the phylogenetic analysis. Five strains--the B95-8 prototype, China 1, China 2, Mediterranean (Med) and SEA 2--were identified in ethnic groups in Thailand. The major strain and the distribution pattern differed by group and location. When the ethnic groups were classified by linguistic group, the prevalence of the SEA 2 strain was significantly different between Austro-Thais and other linguistic groups (P=0.0001), whereas, among Tibeto-Burman linguistic groups, the prevalence of the Med strain was different between matrilocal and patrilocal groups (P=0.0245). The distribution of LMP1 strains in ethnic minorities in Thailand is associated with ethnogeographical factors and the social/marriage system. This study thus provides evidence for the importance of interactions between populations in virus diversity.

Prenatal diagnosis of α- and ß-thalassemias in southern Thailand.

We report our work on prenatal diagnosis of α-thalassemia, ß-thalassemia and other hemoglobinopathies in southern Thailand. DNA-based diagnosis was offered to 1906 pregnancies at risk for thalassemia using a combination method of multiplex-PCR and reverse dot blot analysis to detect seven α-globin and 47 ß-globin mutations. The most commonly detected mutation of α0-thalassemia was a South-East Asian deletion (98%), followed by a Thai deletion (2%). Twenty-eight ß-globin mutations were identified. Fourteen common mutations, including cod 19 A-G (18.6%), cod 41/42 -TCTT (14.4%), IVS1#5 G-C (13.2%), 3.5 kb deletion (9.2%), cod 17 A-T (7.7%), -28 A-G (7.3%), IVSI#1 G-T (7.1%), 12.5 kb deletion (δß)0 (5.7%), HPFH6 (4.2%), IVS2#654 C-T (2.7%), 45 kb deletion (1.9%), Asian Indian-inversion-deletion Gγ(Aγδß)0 (1.6%), cod 41 -C (1.5%) and cod 71/72 +A (1.3%) were detected, accounting for 96.5% of all mutations detected. The most common hemoglobin variant was Hb E, accounting for 97.86%. Prenatal diagnosis of 1906 couples at risk showed 22.0% normal, 51.2% carrier and 26.8% affected. The present study provides important information for diagnosis and control of severe thalassemia diseases.

Investigation of Leptospira Infection in Stray Animals in Songkhla, Thailand: Leptospirosis Risk Reduction in Human.

Leptospirosis is a zoonosis of public health concern in Thailand. Human leptospirosis presents severe illness and can be fatal due to pulmonary hemorrhage, kidney failure, or cardiac impairment. Infected animals show no clinical signs and play an important role in the Leptospira infection of humans and other hosts. The prevalence of leptospirosis in stray animals in Thailand is unknown. The aim of this study was to investigate the incidence of Leptospira infection in stray animals including dogs and cats in Songkhla province, Thailand. A total of 434 blood samples were collected from 370 stray dogs and 64 stray cats during a population control program from 2014 to 2018. Screening the serum samples using the latex agglutination test to detect antibodies against Leptospira interrogans showed that 29.26% (127/434) were positive. There were 120 positive samples for stray dogs and 7 positive samples for stray cats. The detection of positive samples by polymerase chain reaction specific to the LipL32 of L. interrogans showed 1.61% (7/434) were positive. Stray cats (5/64) showed a higher prevalence than stray dogs (2/370), which might be because they are more likely to come into contact with rodents in their habitat. Although the active infection detected was low, the seroprevalence was high. This result indicated that the stray animals might not have been infected at the time of sample collection, but that they had been infected in the past or were in a latent period of infection. Therefore, they might serve as a leptospirosis reservoir for domestic animals and humans present in the same environment. The results show that stray animals need health care, such as vaccination, surveillance, and treatment, when infected to prevent or reduce the risk of transmission to humans.

Investigation of Trypanosoma evansi infection in bullfighting cattle in Southern Thailand.

BACKGROUND AND AIM: Trypanosoma evansi infection has been reported in Thai livestock such as beef and dairy cattle. However, there is little information on T. evansi infection in bullfighting cattle in Southern Thailand. The aim of this study was to investigate the infection of T. evansi in bullfighting cattle presented for health checks at the Animal Hospital, Faculty of Veterinary Science, Prince of Songkla University, Thailand. MATERIALS AND METHODS: Blood and serum samples were collected from 177 bullfighting cattle from April 2016 to February 2017 after bullfighting matches. Animal inspected showed signs of fever, weight loss, or exercise intolerance. Investigation of T. evansi infection was tested using polymerase chain reaction (PCR) with TBR primers and using indirect enzyme-linked immunosorbent assay with T. evansi crude antigen. RESULTS: The seroprevalence of T. evansi in bullfighting cattle was 22.60% (40/177). The PCR results detected no parasite DNA in this study. However, bullfighting cattle may serve as T. evansi reservoirs. CONCLUSION: Health checking procedures for T. evansi should be promoted for bullfighting events so that infected animals can be quarantined in the preparatory stages of such events.

Hematological profile of blood parasitic infected dogs in Southern Thailand.

BACKGROUND AND AIM: Tick-borne pathogens such as Babesia canis, Hepatozoon canis, and Ehrlichia canis can cause serious disease in canines. Each blood parasite can be associated with different hematological characteristics in infected dogs. Identification of hematological alterations during routine laboratory screening of blood samples from dogs displaying clinical signs is essential for diagnosing blood parasitic infections. This study aimed to evaluate parasitic infections and hematological alterations in blood samples of infected dogs in Southern Thailand. MATERIALS AND METHODS: A total of 474 blood samples were collected from dogs presented at the Veterinary Teaching Hospital of the Prince of Songkla University between 2016 and 2019. An automatic hematology analyzer was used to establish hematological values; peripheral blood films were screened for blood parasites and their detection was associated with hematological alterations to determine the odds ratio (OR). RESULTS: This study found that E. canis (n=127) was the most common blood parasite infecting dogs in southern Thailand, followed by H. canis (n=100) and B. canis (n=24). Hematological alterations caused by Ehrlichia infections included anemia, thrombocytopenia, monocytosis, and eosinophilia (OR=14.64, 17.63, 20.34, and 13.43, respectively; p<0.01). The blood samples of Hepatozoon-infected dogs were characterized by anemia, thrombocytopenia, leukocytosis, neutrophilia, and monocytosis (OR=6.35, 3.16, 12.80, 11.11, and 17.37, respectively; p<0.01). Anemia, thrombocytopenia, eosinopenia, and lymphopenia (OR=10.09, 33.00, 20.02, and 66.47 respectively; p<0.01) were associated with B. canis-infected dogs. CONCLUSION: These data support the fact that hematological abnormalities are a hallmark for the identification of tick-borne infections. The hematological values, hereby reported, can be used as a guideline for the clinical diagnosis of canine blood parasitic infections in Southern Thailand.

Successful Treatments and Management of A Case of Canine Melioidosis.

This communication presents a successful story of an attempt to treat and manage a case of canine melioidosis, a severe tropical disease caused by Burkholderia pseudomallei. A 10-year-old dog was trapped with barbed wires, causing an infected wound around its neck and back, which was later diagnosed as severe melioidosis. The dog was treated based on a modified human protocol. Intravenous meropenem injections (20 mg/kg twice daily) were given for 14 days to prevent death from sepsis prior to treatment with oral sulfamethoxazole-trimethoprim (25 mg/kg twice daily) for 20 weeks to eliminate the bacteria. Canine melioidosis is an unusual infection in dogs, even in Thailand where melioidosis is highly endemic. This successful case management was solely based on proper diagnosis and appropriate treatments.

Analysis of LMP1 variants of EBV in Southern Thailand: evidence for strain-associated T-cell tropism and pathogenicity.

BACKGROUND: The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) has sequence heterogeneity. Some of the variants are associated with altered tumorigenic activity and show geographically specific localization. In Thailand, the EBV genome is frequently detected in circulating T cells of T-cell diseases. OBJECTIVE: To determine the role of EBV LMP1 variation in the genesis of T-cell diseases, we focused on virus factors and analyzed EBV strains in Thailand. STUDY DESIGN: EBV DNA was extracted from 18 healthy individuals and 45 patients with T-cell diseases in Southern Thailand and 30 healthy individuals in Central Thailand. By using PCR-direct sequencing method, nucleotide sequences corresponding to the carboxyl terminus of the LMP1 were determined. RESULTS: Four known strains, B95-8 prototype, China 1, China 2 and Mediterranean (Med) and two novel strains, Southeast Asia 1 (SEA 1) and Southeast Asia 2 (SEA 2) were identified. The prevalence of China 2 strain was significantly different (p=0.006) between Central and Southern Thailand. Higher prevalence (p=0.026) of 30-bp deletion type in the Southern Thais was observed. The LMP1 Med strain was associated with the worse prognosis (p=0.029). Among T-cell diseases patients, CD3(+)-cell oriented infection was recognized in SEA1 strain (p=0.025). CONCLUSION: The distribution of EBV strains may be associated with geographic/ethnic and clinical background in the Thai population. Certain EBV strains defined by their LMP1 sequence may influence cell tropism, disease association, or disease severity.

Molecular survey of canine vector-borne diseases in stray dogs in Thailand.

Despite the large population of stray dogs in Thailand, there is limited information on the prevalence of canine vector-borne diseases (CVBDs). In this study, a molecular survey was conducted to determine the prevalence of Babesia spp., Ehrlichia canis, Hepatozoon spp., Anaplasma platys and Mycoplasma spp. in dogs in Thailand. Of the 181 dog blood samples tested by PCR, 78/181 (43.1%) were found to be infected with one or more pathogens. The overall prevalence rates of Mycoplasma spp., Hepatozoon spp., Babesia spp., A. platys and E. canis infections were 19.9%, 18.8%, 9.4%, 4.4% and 3.9%, respectively. To the authors' knowledge, this is the first report of Mycoplasma infection in Thailand in dogs. The current findings are important for future surveillance of CVBDs and designing appropriate approaches for diagnosis and control for the diseases in Thailand.

Epstein-barr virus-associated non-Hodgkin's lymphoma of B-cell origin, Hodgkin's disease, acute leukemia, and systemic lupus erythematosus: a serologic and molecular analysis.

Parallel studies of (a) patients with Epstein-Barr virus (EBV)-associated peripheral T-cell proliferative disease/lymphomas and (b) a group of patients with a prolonged fever from other causes were conducted at Songklanagarind University Hospital from 1997 through 2000. (Reports on EBV-associated peripheral T-cell and NK-cell proliferative disease/lymphomas have been published elsewhere) In this study, the authors identified 58 patients; 14 were non-Hodgkin's lymphoma of B-cell origin (NHL-B), 8 were Hodgkin's disease, 6 were acute leukemia, 9 were systemic lupus erythematosus (SLE), and 21 were patients with other diseases. Serologic tests for the EBV infection, the study of EBV genome in circulating non-T-cells (CD3-cells) and T-cells (CD3+ cells), and the EBV-RNA study in the tumor cells were performed. EBV internal repeat-1 region (IR-1) in peripheral blood CD3+ cells was detected in 10 of 14 patients (71.5%) with NHL-B, 3 of 8 patients (37.5%) with Hodgkin's disease, 1 of 6 patients (16.7%) with acute leukemia, 4 of 9 patients (44.5%) with SLE, and was not detected in any of the 21 patients with other diseases. Anti-viral capsid antigen-IgG was significantly elevated in hematologic malignancy patients with EBV IR-1 genome in the peripheral blood CD3+ cells when compared to hematologic malignancy patients with a negative result, whereas there was no significant difference in anti-EBV nuclear antigen among these two groups. EBV-RNA expression in tumor cells by in situ hybridization was detected in 4 of 13 patients (31%) with NHL-B (all showed EBV IR-1 genome in peripheral blood CD3+ cells), and 3 of 5 patients (60%) with Hodgkin's disease (only two showed EBV IR-1 genome in peripheral blood CD3+ cells). These data support the theory that chronic EBV infection is often found in association with cases of NHL-B, Hodgkin's disease, acute leukemia, and SLE.

Epstein-Barr virus-associated peripheral T-cell lymphoma with gastrointestinal tract involvement.

Peripheral T-cell lymphoma (PTCL) is a group of diseases which are common in Asia and areas of South and Central America. They are highly associated with the Epstein-Barr virus (EBV) infection. In the present study the authors evaluated patients with gastrointestinal involvement of PTCL with respect to clinical findings and outcome, pathologic features, and molecular analysis for EBV infection and the clonality of tumor cells. From January 1997 through December 2000, 7 patients with gastrointestinal tract involvement of PTCL were identified. The frequency of gastrointestinal tract involvement in the various types of PTCL was 5.4 per cent (7 of 129 cases). The pertinent clinical features were prolonged fever, weight loss, anemia, hepatosplenomegaly, lymphadenopathy, multiorgan involvement, and gastrointestinal bleeding. Laboratory results showed a significantly high serum level of alkaline phosphatase and lactate dehydrogenase, and abnormal coagulograms. Five patients died within 4 months after onset of illness, while two were in complete remission after chemotherapy. The tumor cell morphology was classified into three categories: small-sized cells, mixed medium- and large-sized cells, and large-sized cells. The antigenic phenotypes of the tumor cells were LCA+, CD3+, CD15-, CD16-, CD30-, CD45R0+, CD57-, CD68-, EMA-, betaF1-, granzyme B+, TIA-1+, and p53+. The expression of CD4, CD8, CD56 and CD20 was variable. EBV-RNA expression by in situ hybridization (EBER-ISH) study was positive and T-cell receptor (TCR) beta and/or gamma gene rearrangements were detected in all patients. DNA sequence analysis showed high identity to the human TCR germline gene. PTCL with gastrointestinal tract involvement was associated with EBV infection. The tumor cells were mature T cells with some NK-cell antigenic expression and all demonstrated TCR gene rearrangements.

Molecular basis and hematological features of hemoglobin variants in Southern Thailand.

Hemoglobinopathy (abnormal hemoglobin or hemoglobin variant) is an inherited disorder that results in the abnormal structure of globin chains of the hemoglobin (Hb) molecule. Many abnormal Hbs have been characterized worldwide, including more than 20 variants in Thailand. The Bio-Rad Variant II HPLC system is used for investigating hemoglobin variants at Songklanagarind Hospital. This system has been shown to be a sensitive, specific, and reproducible method, but some hemoglobin variants such as Hb Tak and Hb D-Punjab cannot, as yet, be clearly separated by this method. The aim of this study was to investigate the prevalence of hemoglobinopathy in southern Thailand using DNA sequencing and study the severity of each hemoglobin variant. A total of 58 hemoglobin variant samples were obtained from blood samples undergoing routine hemoglobin typing at Songklanagarind Hospital. Genomic DNAs were extracted from the samples, and the globin genes were analyzed by using PCR-direct sequencing. The molecular analysis revealed eight hemoglobin variants: 28 Hb C, 12 Hb D-Punjab, 7 Hb Tak, 4 Hb G-Makassar, 2 Hb Lepore-Hollandia, 2 Hb Q-Thailand, 2 Hb O-Indonesia, and 1 Hb Hope. The distribution of hemoglobin variants in southern Thailand is associated with geographic and/or ethnic backgrounds. This study may help hematologists understand better the prevalence of hemoglobin variants and their hematological features in this region.

Hepatic cytotoxic T-cell infiltrates in patients with peripheral T-cell proliferative diseases/lymphomas: clinicopathological and molecular analysis.

Seventy patients with various types of peripheral T-cell proliferative disease/lymphoma who manifested with prolonged fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly and elevated serum levels of alkaline phosphatase and/or lactate dehydrogenase were evaluated. Histopathological examination of the livers revealed T-cell infiltration into the hepatic sinusoids and portal tracts. The morphology of the infiltrated T cells varied from mature small lymphocytes to malignant lymphoid cells. The liver pathology was classified into four groups on the basis of cellular atypia. Group A and group B showed mature lymphoid cell infiltration; however, only group B had multiple large areas of hepatocellular necrosis. Group C showed atypical lymphoid cell infiltration and in group D malignant lymphoid cell infiltrates were demonstrated. The majority of the antigenic phenotypes of these T-cell infiltrates were CD3+, CD4-, CD8+, CD20-, CD45RO+, CD56-, CD57-, TIA-1+ and betaF1-. Epstein-Barr virus RNA in the nuclei of the infiltrated T cells was recorded in 38.6% of the patients and was more common in groups C and D. Patients in groups B, C and D had a very poor prognosis, median survival was only 1 month, whereas median survival in group A patients was 36 months. Chemotherapy was not effective in improving survival. Monoclonal band/s of T-cell receptors (TCR) beta and/or gamma gene rearrangements were detected in 88.6% of patients, and DNA-sequence analysis showed high identity to the human TCR germline gene.

Epstein-Barr virus-associated peripheral T-cell and NK-cell proliferative disease/lymphoma: clinicopathologic, serologic, and molecular analysis.

Peripheral T-cell proliferative disease/lymphoma is a group of diseases which exhibits heterogeneity in clinical manifestations, pathological findings and outcomes. They are highly associated with the Epstein-Barr virus (EBV) infection. It is likely that EBV plays an important role in the tumorigenesis. From January 1997 through April 2000, we identified 100 patients. One hundred healthy age- and sex- matched controls were selected. Serologic tests for the EBV infection and the study of EBV genomes in circulating non-T cells (CD3- cells), T cells (CD3+ cells), and T-cell subsets (CD4+ and CD8+ cells) were performed. The main features were prolonged fever, weight loss, hepatosplenomegaly, lymphadenopathy, multiorgan involvement, anemia, and high serum alkaline phosphatase and lactate dehydrogenase. Fifty-one patients had an aggressive course and died; median survival was 21 months. Chemotherapy was not effective in improving survival. Anti-viral capsid antigen-IgG and anti-early antigen-IgG were significantly elevated, whereas there was no significant difference in anti-EBV nuclear antigen. EBV internal repeat-1 region (IR-1) in the peripheral blood CD3+ cells was detected in 65% of the patients but in none of the controls. For the CD3- cells, EBV IR-1 was detected in 88% of the patients and 50% of the controls. Among twenty-five patients whose CD3+ cells were positive for EBV IR-1, 6 (24%) showed EBV IR-1 in only CD4+ cells, 6 (24%) in only CD8+ cells, and 13 (52%) in both CD4+ and CD8+ cells. The 30-bp deletion variant of the EBV latent membrane protein-1 gene was significantly higher in the patients than in the controls. These data support the chronic infective process. The EBV which is dormant in non-T cells may infect T cells and contribute to the pathogenesis of disease in a select group of patients.