Strain-based HLA association analysis identified HLA-DRB1*09:01 associated with modern strain tuberculosis.

Tuberculosis (TB) occurs as a result of complex interactions between the host immune system and pathogen virulence factors. Human leukocyte antigen (HLA) class II molecules play an important role in the host immune system. However, no study has assessed the association between HLA class II genes and susceptibility to TB caused by specific strains. This study investigated the possible association of HLA class II genes with TB caused by modern and ancient Mycobacterium tuberculosis (MTB). The study included 682 patients with TB and 836 control subjects who were typed for HLA-DRB1 and HLA-DQB1 alleles. MTB strains were classified using a large sequence polymorphism typing method. Association analysis was performed using common HLA alleles and haplotypes in different MTB strains. HLA association analysis of patients infected with modern MTB strains showed significant association for HLA-DRB1*09:01 (odds ratio [OR] = 1.82; P-value = 9.88 × 10-4 ) and HLA-DQB1*03:03 alleles (OR = 1.76; P-value = 1.31 × 10-3 ) with susceptibility to TB. Haplotype analysis confirmed that these alleles were in strong linkage disequilibrium and did not exert an interactive effect. Thus, the results of this study showed an association between HLA class II genes and susceptibility to TB caused by modern MTB strains, suggesting the importance of strain-specific analysis to determine susceptibility genes associated with TB.

Development of a 5-minute rapid test for detecting Vibrio cholerae O139.

Vibrio cholerae O139 is an etiology of cholera in Thailand. We determined to prepare a rapid test to detect V cholerae O139 using an immunochromatographic method to be used for surveillance and use in community laboratories. We conjugated murine monoclonal antibodies specific to the lipopolysaccharide of V. cholerae O139 with colloidal gold particles. The sensitivity of the test was determined using 10-fold dilutions of V. cholerae O139. The lowest number of bacterial cells detected by the test was 10(6) cfu/ml. The specificity was determined using 51 isolates of pure cultures, including V. cholerae serogroups O1, O139, and non-O1/non-O139 and other enteric bacteria; 27 rectal swab specimens and 100 specimens of enriched alkaline peptone water, 50 of which contained V. cholerae O1 and the other did not contain V. cholerae O1. The rapid test showed specificity to V cholerae O139 and no cross reaction with V cholerae O1, V. cholerae non-O1/ non-O139, and other enteric bacteria. The test is rapid, simple and easy to use.

Chronic hepatitis B and C co-infection increased all-cause mortality in HAART-naive HIV patients in Northern Thailand.

A total of 755 highly active antiretroviral therapy (HAART)-naive HIV-infected patients were enrolled at a government hospital in Thailand from 1 June 2000 to 15 October 2002. Census dateo f survival was on 31 October 2004 or the date of HAART initiation. Of 700 (92.6%) patients with complete data, the prevalence of hepatitis B virus (HBV) surface antigen and anti-hepatitis C virus (HCV) antibody positivity was 11.9% and 3.3%, respectively. Eight (9.6%) HBV co-infected patients did not have anti-HBV core antibody (anti-HBcAb). During 1166.7 person-years of observation (pyo), 258 (36.9%) patients died [22.1/100 pyo, 95% confidence interval (CI) 16.7­27.8]. HBV and probably HCV co-infection was associated with a higher mortality with adjusted hazard ratios (aHRs) of 1.81 (95% CI 1.30­2.53) and 1.90 (95% CI0.98­3.69), respectively. Interestingly, HBV co-infection without anti-HBc Ab was strongly associated with death (aHR 6.34, 95% CI 3.99­10.3). The influence of hepatitis co-infection on the natural history of HAART-naive HIV patients requires greater attention.

MRSA carriage in a tertiary governmental hospital in Thailand: emphasis on prevalence and molecular epidemiology.

We investigated prevalence and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) in a case-control study performed in a 900-bed tertiary governmental healthcare facility in Bangkok, Thailand. Multivariate unconditional logistic regression was used to identify risk profiles for MRSA carriage. Phage typing, pulsed-field gel electrophoresis (PFGE), polymorphisms of the coa and spa genes, hypervariable region (HVR) of SCCmec, multi-locus sequence typing (MLST), and identification of ST30/ST8 mosaic chromosome by heteroduplex-polymerase chain reaction (heteroduplex-PCR) were used to demonstrate a clonal relationship. Fifty-seven of 619 in-patients (9.2%) were positive for MRSA. Risk factors were being male, long admission, low modified McCabe score, history of MRSA infection, and use of broad spectrum cephalosporin. Molecular typing results indicated close relatedness among MRSA isolates. Successful epidemic subtypes were recovered from many different wards. However, all subtypes with different multi-locus sequence types were single locus variants (SLVs) of ST239. Heteroduplex-PCR gave two positive bands from ST8/ST30 mosaic chromosomal structures in all SLVs indicating all isolates were of the ST239 origin. The burden of MRSA nosocomial infections is high in the governmental tertiary hospital. The sole ST239 and its SLVs identified in this hospital is striking and calls for better policy for infection control and prevention.

Association analysis of susceptibility candidate region on chromosome 5q31 for tuberculosis.

Chromosome 5q31 spans the T helper (Th) 2-related cytokine gene cluster, which is potentially important in Th1/Th2 immune responses. The chromosome 5q23.2-31.3 has been recently identified as a region with suggestive evidence of linkage to tuberculosis in the Asian population. With the aim of fine-mapping a putative tuberculosis susceptibility locus, we investigated a family-based association test between the dense single nucleotide polymorphism (SNP) markers within chromosome 5q31 and tuberculosis in 205 Thai trio families. Of these, 75 SNPs located within candidate genes covering SLC22A4, SLC22A5, IRF1, IL5, RAD50, IL13, IL4, KIF3A and SEPT8 were genotyped using the DigiTag2 assay. Association analysis revealed the most significant association with tuberculosis in haplotypes comprising SNPs rs274559, rs274554 and rs274553 of SLC22A5 gene (P(Global)=2.02 x 10(-6)), which remained significant after multiple testing correction. In addition, two haplotypes within the SLC22A4 and KIF3A region were associated with tuberculosis. Haplotypes of SLC22A5 were significantly associated with the expression levels of RAD50 and IL13. The results show that the variants carried by the haplotypes of SLC22A4, SLC22A5 and KIF3A region potentially contribute to tuberculosis susceptibility among the Thai population.

Incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in Thailand.

Although pneumonia is a leading cause of death from infectious disease worldwide, comprehensive information about its causes and incidence in low- and middle-income countries is lacking. Active surveillance of hospitalized patients with pneumonia is ongoing in Thailand. Consenting patients are tested for seven bacterial and 14 viral respiratory pathogens by PCR and viral culture on nasopharyngeal swab specimens, serology on acute/convalescent sera, sputum smears and antigen detection tests on urine. Between September 2003 and December 2005, there were 1730 episodes of radiographically confirmed pneumonia (34·6% in children aged <5 years); 66 patients (3·8%) died. A recognized pathogen was identified in 42·5% of episodes. Respiratory syncytial virus (RSV) infection was associated with 16·7% of all pneumonias, 41·2% in children. The viral pathogen with the highest incidence in children aged <5 years was RSV (417·1/100,000 per year) and in persons aged ≥50 years, influenza virus A (38·8/100,000 per year). These data can help guide health policy towards effective prevention strategies.

Identification of colonization factors of enterotoxigenic Escherichia coli with PCR-based technique.

Colonization factors (CFs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhoea. We designed CF-specific polymerase chain reaction (PCR) primers, and developed a simple PCR-based genotypic CF identification method. ETEC strains (n=17) isolated from patients with diarrhoea in Thailand were examined for genotypical identification of CFs of ETEC strains. Coli surface antigen 6 (CS6) was the most common CF (29%), followed by CS13 (12%), colonization factor antigen I (CFA/I), CS2 and CS3, and CS17/CS19 (6% each), while 41% of the strains were negative. This simple PCR method for the detection of CF genes is useful for surveillance of ETEC infections in diagnostic laboratories.

Genome-wide SNP-based linkage analysis of tuberculosis in Thais.

Tuberculosis, a potentially fatal infectious disease, affects millions of individuals annually worldwide. Human protective immunity that contains tuberculosis after infection has not been clearly defined. To gain insight into host genetic factors, nonparametric linkage analysis was performed using high-throughput microarray-based single nucleotide polymorphism (SNP) genotyping platform, a GeneChip array comprised 59 860 bi-allelic markers, in 93 Thai families with multiple siblings, 195 individuals affected with tuberculosis. Genotyping revealed a region on chromosome 5q showing suggestive evidence of linkage with tuberculosis (Z(lr) statistics=3.01, logarithm of odds (LOD) score=2.29, empirical P-value=0.0005), and two candidate regions on chromosomes 17p and 20p by an ordered subset analysis using minimum age at onset of tuberculosis as the covariate (maximum LOD score=2.57 and 3.33, permutation P-value=0.0187 and 0.0183, respectively). These results imply a new evidence of genetic risk factors for tuberculosis in the Asian population. The significance of these ordered subset results supports a clinicopathological concept that immunological impairment in the disease differs between young and old tuberculosis patients. The linkage information from a specific ethnicity may provide unique candidate regions for the identification of the susceptibility genes and further help elucidate the immunopathogenesis of tuberculosis.

Genetic analysis of two influenza A (H1) swine viruses isolated from humans in Thailand and the Philippines.

Influenza viruses A/Philippines/341/2004 (H1N2) and A/Thailand/271/2005 (H1N1) were isolated from two males, with mild influenza providing evidence of sporadic human infection by contemporary swine influenza. Both viruses were antigenically and genetically distinct from influenza A (H1N1 and H1N2) viruses that have circulated in the human population. Genetic analysis of the haemagglutinin genes found these viruses to have the highest degree of similarity to the classical swine H1 viruses circulating in Asia and North America. The neuraminidase gene and the internal genes were found to be more closely related to viruses circulating in European swine, which appear to have undergone multiple reassorting events. Although transmission of swine influenza to humans appears to be a relatively rare event, swine have been proposed as the intermediate host in the generation of potential pandemic influenza virus that may have the capacity to cause human epidemics resulting in high morbidity and mortality.

Specific IgM and IgG responses in primary and secondary dengue virus infections determined by enzyme-linked immunosorbent assay.

IgM- and IgG-capture ELISAs are widely used as diagnostic tests for confirmation of dengue virus infection. The positive rate of anti-dengue IgM and IgG detection was examined in primary and secondary dengue virus infections in the setting of a provincial hospital using IgM- and IgG-capture ELISAs. Disease day 1 was defined as the day of onset of symptoms. In total, 232 plasma samples were collected from 106 confirmed dengue cases consisting of 12 primary and 94 secondary infections. In primary infection, anti-dengue IgM was detected in 4 out of 5 samples collected on disease day 5 and in all the 21 samples collected on disease day 6 or later. Specific IgG was detected in 2 out of 5 samples collected on day 12, and in 5 out of 6 samples collected on disease days 13-15, but was not detected in samples collected on disease day 10 or earlier. In secondary infection, IgM was not detected in the samples on disease days 2 and 3, but detected in 20 out of 79 samples collected on days 4-6, in 44 out of 65 on disease days 7-11 and in 40 out of 51 samples on disease days 12-14. In contrast, specific IgG was detected in 21 out of 60 samples on disease days 4 and 5, in 13 out of 19 on disease day 6, in 62 out of 65 on disease days 7-11 and in all the samples collected on disease day 12 or later. The result indicate that seroconversion rates of IgM and IgG are different between primary and secondary infections, and suggest that detection of specific IgM and IgG is necessary for determining dengue virus infection and for differentiating primary and secondary dengue infections.

Serological and virological features of dengue fever and dengue haemorrhagic fever in Thailand from 1999 to 2002.

Serological and virological features of dengue fever (DF) and dengue haemorrhagic fever (DHF) in Thailand were analysed in 2715 patients from 1999 to 2002. The illness was caused by DEN-1 in 45%, DEN-2 in 32%, DEN-3 in 18% and DEN-4 in 5% of patients. Almost all of the DHF cases caused by DEN-2 and DEN-4 were in secondary infection, while approximately 20% of the DHF cases caused by DEN-1 and DEN-3 were in primary infection. Male:female ratio and age distribution were not different among four serotypes in primary and secondary infections. These results indicate that DEN-1 and DEN-3 induce DHF in both primary and secondary infections, and suggest that DEN-2 and DEN-4 in Thailand are less likely to cause DHF in primary infections.

Methamphetamine users in northern Thailand: changing demographics and risks for HIV and STD among treatment-seeking substance abusers.

Our objective was to determine sociodemographic, sexual and drug-use-related risk factors among methamphetamine (MA) users presenting for drug treatment in northern Thailand. Patients admitted for drug detoxification for MA and other drugs were studied cross-sectionally for risk factors associated with substance abuse and blood-borne and sexually transmitted pathogens. In all, 1865 (200 women) patients treated for MA, opiate, and mixed substance abuse between 1 February 1999 and 31 January 2000 completed all study procedures. Among 1865 participants, 750 (40.2%) were admitted for MA detoxification and 1115 (59.8%) for opiate (heroin, opium, or both) treatment. MA users were significantly younger, better educated, more likely to be Thai than highland ethnic minorities, and had significantly different sexual risks and sexually transmitted disease rates, including lower syphilis seropositivity and higher chlamydial prevalence, than persons admitted for opiate or mixed drug treatment. For those who reported MA use only, use by injection was rare and HIV infections significantly less common than among all other drug users. Thailand is undergoing an epidemic of MA use. These young users are a strikingly different population from opiate/heroin users in northern Thailand. MA users had higher rates of chlamydia infection than opiate users, reflecting their young age, and HIV rates in this population were lower than injecting drug users, but still elevated. MA use is a serious public health problem in Thailand and both improved prevention and treatment methodologies are urgently needed.

Temporal trends of dengue fever/dengue hemorrhagic fever in Bangkok, Thailand from 1981 to 2000: an age-period-cohort analysis.

The aim of this study was to examine the effects of age, time period, and birth cohorts with dengue fever/dengue hemorrhagic fever (DF/DHF) in Bangkok, Thailand over the period 1981-2000. The age group at greatest risk for DF/DHF was 5-9 years old. The period effect shows a remittent pattern, with significant increases in 1986-1990 and 1996-2000. The birth cohort group showed a significant decreasing trend from the 1961-1965 group to the 1991-1995 group (R2 = 0.7620) with a decreasing rate of 0.1. We concluded that the temporal trend of DF/DHF is decreasing; especially for DHF.

Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients.

OBJECTIVES: To characterise genotypes of Clostridium difficile strains isolated from asymptomatic individuals and patients with diarrhea. METHODS: Fecal specimens from 235 asymptomatic infants <12 months, 76 asymptomatic children 1-11 years and 132 adult patients with antibiotic-associated and non-antibiotic-associated diarrhea obtained from Siriraj Hospital, Bangkok from October 1998 to April 1999 were examined for C. difficile by cycloserine-cefoxitin-fructose agar culture. The presence of the C. difficile toxin A gene was determined by specific PCR with the use of primers 5-(CCC AAT AGA AGA TTC AAT ATT AAG CTT)-3 and 5-(GGA AGA AAA GAA CTT CTG GCT CAC TCA GGT)-3. All C. difficile isolates were subsequently genotyped by pulsed-field gel electrophoresis (PFGE). RESULTS: The C. difficile strains were found in 28 (11.9%) asymptomatic infants, 16 (21.1%) asymptomatic children and 33 (25%) adult patients. In total, 14 PFGE types and eight subtypes designated as types A, B, C, D, E, F, G, H, I, J, K, L, M and N, and A1, A2, A3, A4, B1, B2, B3 and E1, respectively, were identified. Only two isolates from infants and 18 isolates from adult patients were toxin A gene positive by PCR. Both isolates of toxigenic C. difficile were from infants in the same ward and were PFGE type B. PFGE type A was the predominant type among all toxigenic isolates (12 of 18 isolates) from adult patients. The other PFGE types of toxigenic C. difficile found in adult patients were: type A1, one isolate; type B, four isolates; and type C, one isolate. Types B2 and D were identified in 38.5% and 46.2%, respectively, of the toxin A gene-negative isolates of C. difficile from infants. CONCLUSIONS: These results revealed the occurrence of three distinct clusters from different wards in Siriraj Hospital. The toxigenic C. difficile of PFGE type A and related subtypes was a predominant infective clone in adult patients, whereas non-toxigenic C. difficile types B2 and D were encountered in asymptomatic infants. This information can be useful in epidemiologic investigations.

Tuberculosis preventative therapy as part of a care package for people living with HIV in a district of Thailand.

We studied the risk factors for default in isoniazid preventative therapy (IPT) against tuberculosis in 412 people living with HIV (PLWH) in Mae Chan Hospital, Thailand. Default rates decreased from 57% in 1995 to 17% in 1999. Female sex, residence inside Mae Chan District, and registered in the PLWH group at enrollment of IPT were associated with lower default rates. The integration of IPT into the PLWH day care centre activities over the years might contribute to improvements.

High HIV-1 prevalence among metamphetamine users in central Thailand, 1999-2000.

HIV-1 prevalence was studied in 1,890 metamphetamine users from Thanyarak Hospital from 1999 to 2000. 64.8 per cent positive urine metamphetamine and 2.3 per cent positive urine opiate were observed. The most common route of the drug intake was 93.92 per cent inhalation. HIV-1 prevalence was 2.44 per cent (95% Confidence interval; 1.65-3.18%). 44 out of 46 HIV-1 infected cases were typeable as 32 (72.73%) subtype E and the rest of subtype B'. Active opiate users had a higher rate of HIV-1 infection, 15.91 per cent, compared to 2.11 per cent of the non-opiate users (Fisher's exact test p=0.0002). This group of metamphetamine users is important to public health and more attention on intervention efforts towards HIV infection is urgently needed.

HIV incidence rates among drug users in northern Thailand, 1993-7.

Drug use is a major mode of HIV transmission in Thailand. This study determined HIV incidence rates among drug users in a regional drug treatment centre in northern Thailand. A retrospective cohort of repeatedly-hospitalized drug users between 1993 and 1997 was formed and HIV incidence rates were calculated. The overall incidence was 11.44 per 100 person-years of observation. Gender, age, religion, ethnicity, education, employment, income, reasons for drug use, type of drugs, mode of use, spending on drugs, and referral for treatment are associated with HIV incidence. However, there are no associations between HIV incidence and history of treatment and mode of discharge from the centre. This implies that current treatment modality has no impact on HIV infection risk and other therapeutic approaches should be explored.

Trend of HIV incidence rates among drug users in an HIV epicenter in northern Thailand (1989-1997).

OBJECTIVES: To determine trends and associated risk factors of HIV incidence (1989-1997) in a drug abuse treatment clinic in northern Thailand where HIV is epidemic. DESIGN: Retrospective cohort study. METHODS: Nine-years (1989-1997) of data (excluding names) from the logbook of drug abusers seeking treatments in Mae Chan Hospital in Chiangrai Thailand, were transcribed and double-entered into separate computer files which were later validated against each other. For each patient, the dates of the first HIV negative, the last HIV negative, and the first HIV positive were determined. A retrospective cohort of drug users who were initially HIV-negative and treated for more than once was constructed. HIV seroconversion was assumed to follow a uniform distribution between the last negative and the first positive HIV tests. The incidence rates and their 95% confidence intervals (CI) were calculated. RESULTS: Of the 378 repeat patients, 16 (4.2%) HIV seroconverted. This is equivalent to 5.11 per 100 person-years of observation (PYO) (95% CI = 3.13-8.35). The incidence remained relatively stable over the study period while the prevalence was on the decline. The younger, Thai lowlanders, drug injectors had higher incidence rates than the older, ethnic minorities and drug smokers, respectively. CONCLUSION: Prevalence can give illusional results. It is necessary to know baseline HIV incidence to monitor and evaluate an HIV intervention program.

HIV-related risk factors of blood donors in northern Thailand before and after knowing HIV test results.

BACKGROUND: The epidemic of Human Immunodeficiency Virus (HIV) infection led blood banks to initiate donation deferral criteria based on self-reported risk factors. However little information is available on the differences in reporting risk factors before and after HIV status is known. METHODS: Between April and July 1994, blood donors in a provincial hospital in northern. Thailand were interviewed at the time of donation, about their demographic characteristics and risk factors. All donors had agreed to learn their test results and were called back for post-test counselling and reinterview. RESULTS: HIV-positive blood donors were more likely to change from 'denying' to 'acknowledging' risk factors while HIV-negatives were more likely to change from 'acknowledging' to 'denying'. The differences between risk factors obtained before and after test results were known resulted in stronger, weaker or even opposite risk measures. CONCLUSION: The study results raise questions about the impact of the differences in reporting HIV-related risk factors by the donors on how effective donation deferral criteria can be developed.

PIP: The widespread prevalence of HIV infection in countries and regions around the world has led blood banks to refuse blood donations from donors who are at high risk of being infected with HIV. Low-risk donors are instead recruited and all donors are tested for infection with HIV. The criteria for donor deferral are based mainly upon the epidemiology of HIV infection among the donors. The authors investigated the differences in reporting risk factors before and after HIV status is known. During April-July 1994, blood donors at a hospital in Chiangrai province were interviewed at the time of donation about their demographic characteristics and risk factors. 134 of the 175 HIV-positive donors returned for post-HIV-test counseling and reinterview. 423 of the 1062 HIV-negative donors returned. The donors were of median age 32 years. HIV-positive donors were more likely to change from denying to acknowledging risk factors, while HIV-seronegative donors were more likely to change from acknowledging to denying. The differences between risk factors observed before and after test results were known resulted in stronger, weaker, or even opposite risk measures.

Adherence to tuberculosis preventive therapy among HIV-infected persons in Chiang Rai, Thailand.

OBJECTIVE: To determine the level of and reasons associated with adherence to tuberculosis preventive therapy among asymptomatic HIV-infected individuals in northern Thailand. DESIGN: A prospective cohort study with a 9-month follow-up. METHODS: A total of 412 HIV-infected persons were enrolled in a tuberculosis preventive therapy programme in a hospital. A 9-month isoniazid regimen was prescribed. Adherence was determined by pill count. Participants who missed a scheduled appointment for more than a month were interviewed. Five focus group discussion sessions were held among those who successfully completed the therapy. RESULTS: Of the 412 participants, 69.4% (286) completed the 9-month regimen. The adherence rate, defined as the proportion of those who took more than 80% of pills, was 67.5% (n = 278). Sex, source of participants and history of physical symptoms were associated with adherence. A significant portion of defaults took place at the beginning of the therapy. Out-migration, denial of HIV status, and perceived side effects of isoniazid were frequently cited as reasons for non-adherence. For those adhering participants, the acceptance of personal HIV status, concern about children and family, and a good health provider relationship were important reasons motivating adherence. Several reminder systems were developed by the participants. CONCLUSIONS: Although a isoniazid preventive therapy programme was shown to be feasible, further adjustments on the selection of participants, enrollment process, and follow-up system based on these findings are necessary to increase the adherence.

PIP: A prospective study of 412 men and women infected with human immunodeficiency virus (HIV) who were enrolled in a hospital-based tuberculosis prevention program in Chiang Rai, Thailand, identified factors that contribute to non-adherence. Program participants were provided a 9-month supply of isoniazid, which has been shown to be effective in reducing the risk of developing clinical tuberculosis among asymptomatic HIV-infected individuals. Of the 412 individuals enrolled in the program, 286 (69.4%) completed the treatment regimen, 109 (26.5%) defaulted by failing to take the medicine for more than 60 consecutive days, and 17 (4.1%) developed acquired immunodeficiency syndrome (AIDS) or died. Among defaulters, 20 restarted the regimen and eventually complied with treatment, raising the completion rate to 74.3%. Married people, women, outpatients, surviving spouses, the self-employed, and those with no history of physical symptoms were more likely to be adherent. The primary reasons for default included outmigration for job search, denial of HIV status, perceived drug side effects, and confusion about the duration of treatment. Although this study suggests that good adherence to a tuberculosis prevention regime is possible among HIV patients, it highlights factors that should be considered (especially migration potential) in the selection of participants.