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BackgroundNational Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures.MethodsSamples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs.ResultsGenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs.ConclusionsGenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community.FundingThe GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

Abstract Background New anti-malarial drugs are needed urgently to address the increasing challenges of drug-resistant falciparum malaria. Two rhinacanthin analogues containing a naphthoquinone moiety resembling atovaquone showed promising in-vitro activity against a P. falciparum laboratory reference strain (K1). The anti-malarial activity of these 2 compounds was further evaluated for P. falciparum field isolates from an area of multi-drug resistance in Northeast Thailand. Methods Using a pLDH enzyme-linked immunosorbent assay, four P. falciparum isolates from Northeast Thailand in 2018 were tested for in vitro sensitivity to the two synthetic rhinacanthin analogues 1 and 2 as well as established anti-malarials. Mutations in the P. falciparum cytochrome b gene, a marker for atovaquone (ATQ) resistance, were genotyped in all four field isolates as well as 100 other clinical isolates from the same area using PCR-artificial Restriction Fragment Length Polymorphisms. Pfkelch13 mutations, a marker for artemisinin (ART) resistance, were also examined in all isolates. Results The 50% inhibitory concentrations (IC50) of P. falciparum field isolates for rhinacanthin analogue 1 was 321.9–791.1 nM (median = 403.1 nM). Parasites were more sensitive to analogue 2: IC50 48.6–63.3 nM (median = 52.2 nM). Similar results were obtained against P. falciparum reference laboratory strains 3D7 and W2. The ART-resistant IPC-5202 laboratory strain was more sensitive to these compounds with a median IC50 45.9 and 3.3 nM for rhinacanthin analogues 1 and 2, respectively. The ATQ-resistant C2B laboratory strain showed high-grade resistance towards both compounds (IC50 > 15,000 nM), and there was a strong positive correlation between the IC50 values for these compounds and ATQ (r = 0.83–0.97, P

Abstract Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.

Abstract Background Thailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand. Methods Under Thailand’s integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed. Results The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance. Conclusions This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.

Abstract Background Resistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine–pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples. Methods Plasmodium falciparum DNA was collected from the Thailand–Myanmar, Thailand–Malaysia and Thailand–Cambodia borders during 2008–2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated. Results Mutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes. In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016. Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification. Conclusions Despite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

BACKGROUND:In the HIV-1 vaccine trial RV144, ALVAC-HIV prime with an AIDSVAX® B/E boost reduced HIV-1 acquisition by 31% at 42 months post first vaccination. The bivalent AIDSVAX® B/E vaccine contains two gp120 envelope glycoproteins, one from the subtype B HIV-1 MN isolate and one from the subtype CRF01_AE A244 isolate. Each envelope glycoprotein harbors a highly conserved 27-amino acid HSV-1 glycoprotein D (gD) tag sequence that shares 93% sequence identity with the HSV-2 gD sequence. We assessed whether vaccine-induced anti-gD antibodies protected females against HSV-2 acquisition in RV144. METHODS:Of the women enrolled in RV144, 777 vaccine and 807 placebo recipients were eligible and randomly selected according to their pre-vaccination HSV-1 and HSV-2 serostatus for analysis. Immunoglobulin G (IgG) and IgA responses to gD were determined by a binding antibody multiplex assay and HSV-2 serostatus was determined by Western blot analysis. Ninety-three percent and 75% of the vaccine recipients had anti-gD IgG and IgA responses two weeks post last vaccination, respectively. There was no evidence of reduction in HSV-2 infection by vaccination compared to placebo recipients over 78 weeks of follow-up. The annual incidence of HSV-2 infection in individuals who were HSV-2 negative at baseline or HSV-1 positive and HSV-2 indeterminate at baseline were 4.38/100 person-years (py) and 3.28/100 py in the vaccine and placebo groups, respectively. Baseline HSV-1 status did not affect subsequent HSV-2 acquisition. Specifically, the estimated odds ratio of HSV-2 infection by Week 78 for female placebo recipients who were baseline HSV-1 positive (n = 422) vs. negative (n = 1120) was 1.14 [95% confidence interval 0.66 to 1.94, p = 0.64)]. No evidence of reduction in the incidence of HSV-2 infection by vaccination was detected. CONCLUSIONS:AIDSVAX® B/E containing gD did not confer protection from HSV-2 acquisition in HSV-2 seronegative women, despite eliciting anti-gD serum antibodies.

To assess qualities and outcomes of women participating in a large, community-based HIV vaccine trial, the present study was conducted among female participants of the RV 144 prime-boost trial in Thailand from 2003 to 2009. Qualities of participation refer to complete vaccination, retention, and status change. Outcomes of participation refer to incident rate, adverse event, and participation impact event. A total of 6,334 (38.6%) women participated in the trial, of whom about 50% were classified as low risk and 11% as high risk. About 85% of participants completed four vaccinations and 76% were included in the per-protocol analysis of the on-time vaccination schedule. More women (88%) completed 42 months follow-up compared with men (85%). Women aged 21 and above had more adverse events compared to younger age groups. More women (5%) compared with men (3%) reported participation impact events (PIEs). High-risk women had more PIEs and a higher infection rate compared to the low-risk group. Complete vaccination and retention on last follow-up were more common in married women aged above 21, and being a housewife. Female volunteers showed the same qualities and outcomes of participation as males in the HIV vaccine trial. There was no statistically significant difference in vaccine efficacy between men and women, especially among the high-risk and married women. The study highlighted the important behavioral, social, and cultural issues that could be considered for future HIV vaccine trial designs. [ABSTRACT FROM AUTHOR]

Background: In the HIV-1 vaccine trial RV144, ALVAC-HIV prime with an AIDSVAX® B/E boost reduced HIV-1 acquisition by 31% at 42 months post first vaccination. The bivalent AIDSVAX® B/E vaccine contains two gp120 envelope glycoproteins, one from the subtype B HIV-1 MN isolate and one from the subtype CRF01_AE A244 isolate. Each envelope glycoprotein harbors a highly conserved 27-amino acid HSV-1 glycoprotein D (gD) tag sequence that shares 93% sequence identity with the HSV-2 gD sequence. We assessed whether vaccine-induced anti-gD antibodies protected females against HSV-2 acquisition in RV144., Methods: Of the women enrolled in RV144, 777 vaccine and 807 placebo recipients were eligible and randomly selected according to their pre-vaccination HSV-1 and HSV-2 serostatus for analysis. Immunoglobulin G (IgG) and IgA responses to gD were determined by a binding antibody multiplex assay and HSV-2 serostatus was determined by Western blot analysis. Ninety-three percent and 75% of the vaccine recipients had anti-gD IgG and IgA responses two weeks post last vaccination, respectively. There was no evidence of reduction in HSV-2 infection by vaccination compared to placebo recipients over 78 weeks of follow-up. The annual incidence of HSV-2 infection in individuals who were HSV-2 negative at baseline or HSV-1 positive and HSV-2 indeterminate at baseline were 4.38/100 person-years (py) and 3.28/100 py in the vaccine and placebo groups, respectively. Baseline HSV-1 status did not affect subsequent HSV-2 acquisition. Specifically, the estimated odds ratio of HSV-2 infection by Week 78 for female placebo recipients who were baseline HSV-1 positive (n = 422) vs. negative (n = 1120) was 1.14 [95% confidence interval 0.66 to 1.94, p = 0.64)]. No evidence of reduction in the incidence of HSV-2 infection by vaccination was detected., Conclusions: AIDSVAX® B/E containing gD did not confer protection from HSV-2 acquisition in HSV-2 seronegative women, despite eliciting anti-gD serum antibodies.

Background: The RV144 ALVAC-HIV prime, AIDSVAX B/E boost afforded 60% efficacy against human immunodeficiency virus (HIV) acquisition at 1 year, waning to 31.2% after 3.5 years. We hypothesized that additional vaccinations might augment immune correlates of protection., Methods: In a randomized placebo-controlled double-blind study of 162 HIV-negative RV144 vaccine recipients, we evaluated 2 additional boosts, given 6-8 years since RV144 vaccination, for safety and immunogenicity, at weeks 0 and 24. Study groups 1-3 received ALVAC-HIV+AIDSVAX B/E, AIDSVAX B/E, and ALVAC-HIV, respectively, or placebo., Results: Vaccines were well tolerated. For groups 1 and 2, plasma immunoglobulin (Ig) G, IgA, and neutralizing antibody responses at week 2 were all significantly higher than 2 weeks after the last RV144 vaccination. IgG titers against glycoprotein (gp) 70V1V2 92TH023 increased 14-fold compared with 2 weeks after the last RV144 vaccination (14069 vs 999; P < .001). Groups 1 and 2 did not differ significantly from each other, whereas group 3 was similar to placebo recipients. Responses in groups 1 and 2 declined by week 24 but were boosted by the second vaccination, albeit at lower magnitude than for week 2., Conclusions: In RV144 vaccinees, AIDSVAX B/E with or without ALVAC-HIV 6-8 years after initial vaccination generated higher humoral responses than after RV144, but these responses were short-lived, and their magnitude did not increase with subsequent boost., Clinical Trials Registration: NCT01435135., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01&#95;AE, 3.5% subtype B, 4.3% B/CRF01&#95;AE recombinants, and 0.5% dual infections. CRF01&#95;AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01&#95;AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01&#95;AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01&#95;AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.

Background: The Thai Phase III Trial of ALVAC-HIV and AIDSVAX B/E showed an estimated vaccine efficacy (VE) of 31% to prevent acquisition of human immunodeficiency virus (HIV). Here we evaluated the effect of vaccination on disease progression after infection., Methods: CD4(+) T-cell counts and HIV viral load (VL) were measured serially. The primary analysis evaluated vaccine efficacy (VEP) as the percent reduction (vaccine vs placebo) in cumulative probability of a primary composite endpoint of clinical and CD4(+) count components at prespecified time points after infection. Secondary analyses of biomarker-based endpoints were assessed using marginal mean and linear mixed models., Results: There were 61 endpoints in the modified intent-to-treat cohort (mITT; n = 114). There was no evidence for efficacy at 30, 42, 54, and 60 months in the mITT and per protocol (n = 90) cohorts. Estimated VEP (mITT) was15.8% (-21.9, 41.8) at 60 months postinfection. There was weak evidence of lower VL and higher CD4(+) count at 60 and 66 months in the vaccine group. Lower mucosal VL was observed among vaccine recipients, primarily in semen (P = .04)., Conclusions: Vaccination did not affect the clinical course of HIV disease after infection. A potential vaccine effect on the genital mucosa warrants further study.

Background: The Thai phase 3 HIV vaccine trial RV 144 showed modest efficacy of a vaccine against HIV acquisition. Baseline variables of age, sex, marital status, and risk did not modify vaccine efficacy. We did a post-hoc analysis of the trial's data to investigate behavioural risk and efficacy every 6 months after vaccination., Methods: RV 144 was a randomised, multicentre, double-blind, placebo-controlled efficacy trial testing the combination of the HIV vaccines ALVAC-HIV (vCP1521) and AIDSVAX B/E to prevent HIV infection or reduce setpoint viral load. Male and female volunteers aged 18-30 years were recruited from the community. In this post-hoc analysis of the modified intention-to-treat population (16,395 participants), HIV risk behaviour was assessed with a self-administered questionnaire at the time of initial vaccination in the trial and every 6 months thereafter for 3 years. We classified participants' behaviour as low, medium, or high risk. Both the acquisition endpoint and the early viral-load endpoint were examined for interactions with risk status over time and temporal effects after vaccination. Multiple proportional hazards regression models with treatment and time-varying risk covariates were analysed., Findings: Risk of acquisition of HIV was low in each risk group, but 9187 (58·2%) participants reported higher-risk behaviour at least once during the study. Participants classified as high or increasing risk at least once during follow-up were compared with those who maintained low-risk or medium-risk behaviour as a time-varying covariate, and the interaction of risk status and acquisition efficacy was significant (p=0·01), with greater benefit in low-risk individuals. Vaccine efficacy seemed to peak early--cumulative vaccine efficacy was estimated to be 60·5% (95% CI 22-80) through the 12 months after initial vaccination--and declined quickly. Vaccination did not seem to affect viral load in either early or late infections., Interpretation: Future HIV vaccine trials should recognise potential interactions between challenge intensity and risk heterogeneity in both population and treatment effects. The regimen tested in the RV 144 phase 3 trial might benefit from extended immunisation schedules., Funding: US Army Medical Research and Materiel Command and Division of AIDS, National Institute of Allergy and Infectious Disease, National Institutes of Health., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The development of a safe and effective vaccine against the human immunodeficiency virus type 1 (HIV-1) is critical to pandemic control., Methods: In a community-based, randomized, multicenter, double-blind, placebo-controlled efficacy trial, we evaluated four priming injections of a recombinant canarypox vector vaccine (ALVAC-HIV [vCP1521]) plus two booster injections of a recombinant glycoprotein 120 subunit vaccine (AIDSVAX B/E). The vaccine and placebo injections were administered to 16,402 healthy men and women between the ages of 18 and 30 years in Rayong and Chon Buri provinces in Thailand. The volunteers, primarily at heterosexual risk for HIV infection, were monitored for the coprimary end points: HIV-1 infection and early HIV-1 viremia, at the end of the 6-month vaccination series and every 6 months thereafter for 3 years., Results: In the intention-to-treat analysis involving 16,402 subjects, there was a trend toward the prevention of HIV-1 infection among the vaccine recipients, with a vaccine efficacy of 26.4% (95% confidence interval [CI], -4.0 to 47.9; P=0.08). In the per-protocol analysis involving 12,542 subjects, the vaccine efficacy was 26.2% (95% CI, -13.3 to 51.9; P=0.16). In the modified intention-to-treat analysis involving 16,395 subjects (with the exclusion of 7 subjects who were found to have had HIV-1 infection at baseline), the vaccine efficacy was 31.2% (95% CI, 1.1 to 52.1; P=0.04). Vaccination did not affect the degree of viremia or the CD4+ T-cell count in subjects in whom HIV-1 infection was subsequently diagnosed., Conclusions: This ALVAC-HIV and AIDSVAX B/E vaccine regimen may reduce the risk of HIV infection in a community-based population with largely heterosexual risk. Vaccination did not affect the viral load or CD4+ count in subjects with HIV infection. Although the results show only a modest benefit, they offer insight for future research. (ClinicalTrials.gov number, NCT00223080.), (2009 Massachusetts Medical Society)

Acute bacterial meningitis is an important cause of morbidity and mortality in children. To estimate the incidence of meningitis caused by all types of bacteria in Thai children under five years of age, data were collected using a rapid assessment tool (RAT) and analyzed. Clinical and laboratory data from suspected meningitis cases for a one-year period were retrospectively collected from 5 selected catchment areas located in the 4 regions of the country. Adjusted incidences of confirmed bacterial meningitis were calculated based on laboratory quality and lumbar puncture rates. Seventy-five suspected meningitis cases were identified among 305,023 children under age five in the catchment areas, with an unadjusted incidence of 24.6 per 100,000. Of these, 66.2, 55.9, and 33.8% were unconfirmed bacterial, purulent, and confirmed bacterial meningitis cases, respectively. Among the confirmed bacterial meningitis cases, 39.1, 26.1, 21.7 and 13.0% were caused by Haemophilus influenzae type B, gram-positive cocci, gram-negative bacilli, and Neisseria meningitidis, respectively. After adjusting based on the RAT application, the incidence of confirmed bacterial meningitis was about double that of the unadjusted incidence. This study gives an interval of possible incidences of bacterial meningitis in children under age five, which is between the unadjusted (low estimate) and adjusted (high estimate) incidences.

There are limited prospective data for Haemophilus influenzae type b (Hib) disease in Asia, where some countries are considering vaccine introduction. A prospective population-based study was conducted to measure the incidence of Hib meningitis in children in two northern provinces of Thailand. Children <5 years with symptoms consistent with bacterial meningitis were enrolled in the study if inclusion criteria were met. The study enrolled 598 children with clinical meningitis, 76% of whom received lumbar puncture. The rate of probable bacterial meningitis was 26.6/100,000 children <5 years per year. There were four cases of laboratory confirmed Hib meningitis (rate 3.8/100,000 children <5 years per year). These findings suggest a relatively low incidence of Hib meningitis. However, additional data from studies of pneumonia are needed to define the Hib disease burden in Thailand.

Meningitis due to an invasive Haemophilus influenzae type b (Hib) infection, has been previously perceived to be relatively uncommon in Asia. However, the incidence of disease and its impact may have been underestimated. In addition to a lack of microbiological facilities in some hospitals, difficulties in culturing the organism and the widespread use of antibiotics may have hidden the true incidence of the disease in some countries. Furthermore, the reported disease burden probably underestimates the incidence of Hib pneumonia. The epidemiology of invasive Hib disease for various Asian nations is reviewed in this paper. Hospital-based studies show that Hib is a major cause of bacterial meningitis and/or pneumonia in the Philippines, India, Thailand, Malaysia, Indonesia and Vietnam. Singapore and Hong Kong have a low incidence of infection compared with Western and other Asian nations. This low incidence is not due to a higher level of natural protective antibodies, but may be related to an interaction between environmental and genetic factors. Therefore the widespread belief that Hib infection is unimportant in Asia does not refer to Asia as a whole and possibly to Chinese patients only, and failure to recognize this has serious implications. The inclusion of Hib vaccine in the routine infant immunization schedule in many industrialized nations has significantly reduced the incidence of invasive disease. Recent studies have shown Hib vaccination is also effective in preventing invasive disease in children in developing countries. While population-based data may be required to confirm the need for public-funded infant Hib immunization in Asia, its introduction in countries with a high incidence of Hib meningitis and/or pneumonia has the potential to significantly improve pediatric health and survival.

The prevalence of hepatitis A virus (HAV) in a country largely reflects its standards of hygiene and socioeconomic conditions. Countries which undergo socioeconomic development show major change in HAV prevalence from high to low endemicity, and this is largely reflected in patterns of age-related seroprevalence. This paper presents age-related HAV seroprevalence patterns of SE Asian countries, and highlights how these patterns have changed over recent decades. Singapore, Thailand and Malaysia have experienced a decline in childhood and adolescent HAV seroprevalence, typical of countries which undergo socioeconomic development. By contrast, India has remained a country of high endemicity, with almost universal seroconversion in childhood. The Philippines and Vietnam show age-related seroprevalence patterns typical of high to moderate endemicity, while Indonesia shows significant regional variation in HAV seroprevalence. Populations within countries which exhibit major improvements in endemicity and age related HAV seroprevalence patterns are at risk of HAV epidemics, and a paradoxical increase in incidence tends to occur as seroconversion shifts from children to adults. The residents of these countries, a significant number of whom are at-risk, would benefit from a program of vaccination, as would non-infected individuals visiting high-risk areas.

Hepatitis B (HB) immunization was introduced as part of the expanded programme on immunization (EPI) in two provinces in Thailand and evaluated over a four year period. Three doses of HB vaccine were offered to 60,980 newborns at birth, 2 and 6 months of age. The overall coverage for complete HB immunization was 90.4%. Serosurveys of randomly selected children under the age of 5 years were undertaken before and at the end of the project. Levels of HBsAg positivity were reduced by 85%, and there was a corresponding 70% increase in protective immunity. These findings demonstrate that HB immunization can be successfully integrated into EPI without adverse effect on coverage rates of other antigens, and results in a marked reduction in the rate of chronic carriage of HB virus in preschool age children.

From November 1993 to December 1994, the seroprevalence of anti-HCV, HBsAg was studied among 346 HIV-infected persons (asymptomatic HIV-infected persons and AIDS patients) and 1,023 subjects from the general population (including 119 cord blood samples). The prevalence of anti-HCV, HBsAg among HIV-infected patients aged 15-45+ years was 11.0 and 11.6 per cent respectively which is significantly higher than the comparable levels for the general population (1.9% and 4.7%) in the age group 15-44 years. There was no statistically significant association of anti-HCV and HBsAg prevalence among 200 asymptomatic HIV-infected carriers and 146 AIDS patients. Assays for anti-HCV among blood donors are highly recommended to reduce the development of liver disease or cirrhosis in the immediate future.

In a village near Chiangmai, Thailand, during October 1987, there was an outbreak of cholera following a funeral in which 264 attendants were served food. The present article is a report of an epidemiological study performed to identify the source of infection and the mode of its transmission. All the attendants were screened for infection by bacteriological examination of their rectal swabs and were kept under diarrhoeal surveillance. Of them, 20 patients and 40 matched controls were interviewed about the details of their eating foods served at the funeral. Vibrio cholerae 01, Inaba, El Tor was detected from 24 persons (9.1%), 15 of whom suffered from mild diarrhoea and the rest 9 had inapparent infections. There was no death. Except one butcher whose rectal swab was positive for the same strain of V. cholerae, 3 other butchers and 4 women who had prepared food were free from the infection. Food remnants were not available for culture. The water used for cooking and the water from the cement well used for slaughter were negative for the organism. The only significant association (p less than .01, odds ratio = 15) was found between an attack of cholera and eating laebmoo--an uncooked pork preparation with Thai spices and chili. The transmission of cholera appeared to have occurred through eating the uncooked pork presumably due to its contamination with V. cholerae shed by the infected butcher. He was known to have earlier visits to Chiangmai where cholera epidemic was going on.

From September through October 1987, a cholera outbreak involving 59 cases of biotype El Tor, serotype Inaba occurred in Sunpathong district, Chiang Mai. No cases died. Twenty-seven cases were males and 32 were females. The age ranged between 4 months and 85 years, with a median of 36 years. The outbreak affected 7 small communities, and showed different vehicles of infection. Six housewives and one girl were infected with cholera in the first localized outbreak. The transmission of infection appeared due to the consumption of packed food contaminated by an infected food handler. In the second localized outbreak, 6 young males acquired cholera after eating uncooked fish harvested from a canal contaminated with cholera organisms. Another outbreak of cholera with 24 culture-confirmed cases occurred among guests at a funeral held in one rural village. The source of infection was traced to uncooked pork contaminated from an infected butcher: Early detection of infected persons, rapid identification of possible vehicles of transmission, and prompt implementation of control measures effectively curtailed the extension of these outbreaks.

A study was conducted to determine the current situation of chemical foodborne outbreaks in Thailand for the period 1981-1987. Seventy-three outbreaks of chemical poisoning involving 1236 persons of whom 54 died were reported. Twenty outbreaks affecting 722 cases were caused by insecticide poisoning and methomyl was the most commonly recognized insecticide involved. Poisonous plants were responsible for 43 outbreaks with 420 cases. Mushroom poisoning was the most common entity (21 outbreaks, 211 cases), with plant seed poisoning next (9 outbreaks, 179 cases). There were 8 outbreaks following consumption of poisonous seafoods. Mussels were identified to be the vector in the outbreak of PSP. Horseshoe crabs which served as the vehicles for 4 outbreaks were also suspected to be associated with PSP. Puffer fish accounted for the remaining 3 outbreaks involving 6 cases of tetradotoxin poisoning. More complete reporting and more effort in outbreak investigations are needed for appropriate preventive and control measures.

National surveillance of foodborne diseases in Thailand has revealed an increasing incidence of from 29/100,000 population in 1979 to 74/100,000 in 1984. A total of 158,482 cases and 241 deaths were reported during this 6 year period. In each year, foodborne diseases occurred regularly in April-June. The highest mean annual reported rates were from the North-eastern region, followed by the Central region and the Northern region. Because of inadequate investigations, only 7% (10,567) of the total cases had known specific aetiology. Of these 10,567 cases, the majority (7788, 73%) were caused by micro-organisms which included bacteria (46%), parasites (12%), and viruses (15%), the remaining (2779, 27%) were caused by poisonous plants (19%), animals (1%), and chemicals (7%). In recent years surveillance has increasingly served as a means to control the outbreak. However, due to many limitations, the surveillance data presented here probably do not represent an accurate picture of the whole problem. Two very important factors identified are under-reporting and inadequate case and outbreak investigations.

A study was conducted to determine the incidence of neurological complications among a cohort of 6,980 recipients of Semple vaccine administered in Bangkok and 5 nearby provinces in 1984. A review of all patients admitted to public hospitals in these 6 provinces discovered a total of 32 cases, with neurological complications following Semple vaccine. Twenty-two cases (68.8%) were encephalitis or myelitis. The complication rate was 3.6 times higher for males than females and the rate was lowest in the 0-14 year age group. Vaccinees receiving large daily dose of vaccine had a higher rate of complications than those with low dose regimen. One patient died, giving the case-fatality rate of 3.13 per cent. Since the search was limited, the rate of neurological complications to Semple vaccine was a minimum of 4.6 cases per 1,000 vaccinees [1:220]. This complication rate was much higher than most rates reported previously. It is imperative to find economically feasible alternatives to Semple vaccine.

The national immunization coverage in Thailand for all types of vaccine has been steadily increasing since 1978, when the EPI was formally launched. The coverage in 1987 was 96% for BCG, 75% for DPT, 74% for OPV, and 60% for TT. Measles vaccine, which started only in late 1984, had the lowest coverage, 51%, in 1987. During the period 1982-1987, the drop-out rates between the first and third dose of DPT and OPV decreased dramatically from 69% to 13% and from 42% to 13% respectively. Sampling surveys of immunization coverage showed higher coverage for DPT and OPV than those from reporting in all regions, especially in the capital city which has a high concentration of the private health sector. Only the northeastern region had less coverage from surveys than from reporting. Following the launch of EPI, the disease incidence demonstrated a clearly downward trend for diphtheria, poliomyelitis, and measles, while in the case of pertussis and neonatal tetanus, slower of still fluctuating declines were observed. The reported age-specific incidences per 100,000 population in 1986 for children 0-4 years were as follows: 4 for diphtheria, 0.9 for poliomyelities, 180 for measles, 14 for pertussis, and 10 for tetanus.

Information on the morbidity and mortality of neonatal tetanus was reviewed to evaluate the impact of the immunization programme among pregnant women in Thailand from 1977. We also analysed the epidemiological characteristics of investigated neonatal tetanus cases during the period 1984-1986. The neonatal tetanus case rate declined from 72.1 per 100,000 livebirths in 1977 to 53.7 per 100,000 livebirths in 1986. Reduction in the incidence rate was inversely associated with increasing vaccination coverage of pregnant women with tetanus toxoid. The ratio of male to female neonatal tetanus was 1.5 to 1. Approximately 95 per cent of the cases occurred within the first 14 days of life, with the highest number recorded at 6-8 days of life. The majority of investigated cases were infants who became ill following delivery assisted by traditional birth attendants or relatives of mothers. About 88 per cent of these cases were infants whose mothers had no tetanus immunization. Razor blades and bamboo splits were the most frequently used instruments for cutting the umbilical cord. Strategies to control neonatal tetanus in Thailand should include immunization of pregnant women with tetanus toxoid, and more comprehensive training and supervision of untrained birth attendants.

Antibody to the human T-lymphotropic virus, type III/lymphadenopathy-associated virus (HTLV-III/LAV) by ELISA test was detected in one (1%) of 101 male homosexual prostitutes (confidence limit 95%:0.03-5.4%, in two (2%) of 100 thalassemia patients, and in none (C.L. 95%:0-3.6%) of 100 female prostitutes, 99 parenteral drug abusers, 100 male VD patients, 100 consecutive blood donors in serum collected from February through June 1985. Serum from the positive homosexual subject was strongly positive on repeated ELISA testing, and was also positive by Western Blot test. The two thalassemia patients, who were repeatedly weakly-positive by ELISA, were negative by Western Blot test and presumed to be false positive reactors. Prevalence of HTLV-III/LAV virus in sexually-active homosexuals in Thailand in 1985 appears to be similar to the 1% rate among homosexuals in San Francisco in 1978 at the start of the AIDS epidemic there.

A cost-effectiveness study of the Thai expanded programme on immunization was carried out in district hospitals and health centres in Thailand during early 1987. The total annual spending on immunization was US $3852 in hospitals and US $813 in health centres. The percentage distribution of annual costs was similar in both facilities. Salaries were the largest component, followed by building and vaccine costs. The frequency of immunization sessions was the most important factor in determining total costs--immunization costs increasing with the frequency of sessions. In hospitals the average number of fully immunized children was 184, compared with 49 in health centres. The cost per fully immunized child varied widely from US $5.30 to US $33.20, and the most cost-effective facilities were those that immunized the greatest number of children. With the present number of health facilities in all areas of the country, which correspond to saturation levels, the most likely way for the Thai programme to reduce costs would be to make better use of staff time by decreasing the frequency of the services offered, thereby increasing the efficiency of each session. Hospitals should adjust the frequency of their immunization sessions according to the number of children being served, but health centres should offer sessions only monthly or once every two months.

From 30 October to 7 December 1984, an outbreak of nosocomial cholera involving 11 cases of biotype El Tor, serotype Inaba, took place in a 755-bed hospital in southern Thailand. The outbreak occurred primarily among patients admitted with severe illness. Of the 11 cases, 7 were children and 4 were adults. Most cases had mild symptoms of cholera and no case died in this outbreak. The first 2 cases occurred sporadically with a subsequent cluster of cases showing an explosive pattern. A case-control study found that a history of receiving liquid tube-fed diet was significantly more common among cholera cases than their matched controls, but it could not be determined how the diet was contaminated with cholera. Cases were also significantly more likely than controls to be on oral antacid medication which could increase risk of infection by neutralizing gastric acidity. No additional cases occurred after extensive implementation of control measures.