BACKGROUND: Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described. FINDINGS: Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0) for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. CONCLUSIONS: We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results from blood RNA collected and processed by different methods with the intention of biomarker discovery. Results of this study should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of quantitative gene expression measurements.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

BACKGROUND: Dengue is a disease of public health interest, and Tanzania experienced major outbreaks in 2014 and 2019. Here, we report our findings on the molecular characterization of dengue viruses (DENV) that circulated during two smaller outbreaks (2017 and 2018) and one major epidemic (2019) in Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: We tested archived serum samples from 1,381 suspected dengue fever patients, with a median age of 29 (IQR:22-40) years, referred to the National Public Health Laboratory for confirmation of DENV infection. DENV serotypes were identified by reverse transcription polymerase chain reaction (RT-PCR), and specific genotypes were identified by sequencing the envelope glycoprotein gene and phylogenetic inference methods. DENV was confirmed in 823 (59.6%) cases. More than half (54.7%) of patients with dengue fever infection were males, and nearly three-quarters (73%) of the infected individuals were living in Kinondoni district, Dar es Salaam. DENV-3 Genotype III caused the two smaller outbreaks in 2017 and 2018, while DENV-1 Genotype V caused the 2019 epidemic. DENV-1 Genotype I was also detected in one patient in 2019. CONCLUSION/SIGNIFICANCE: This study has demonstrated the molecular diversity of dengue viruses circulating in Tanzania. We found that contemporary circulating serotypes did not cause the major epidemic of 2019 but rather due to a serotype shift from DENV-3 (2017/2018) to DENV-1 in 2019. Such a change increases the risk for patients previously infected with a particular serotype to develop severe symptoms upon potential re-infection with a heterologous serotype due to antibody-dependent enhancement of infection. Therefore, the circulation of serotypes emphasizes the need to strengthen the country's dengue surveillance system for better management of patients, early detection of outbreaks, and vaccine development.

In 2016, Tanzania expanded sentinel surveillance for influenza-like illness (ILI) and severe acute respiratory infection (SARI) to include testing for non-influenza respiratory viruses (NIRVs) and additional respiratory pathogens at 9 sentinel sites. During 2017-2019, respiratory specimens from 2730 cases underwent expanded testing: 2475 specimens (90.7%) were tested using a U.S. Centers for Disease Control and Prevention (CDC)-developed assay covering 7 NIRVs (respiratory syncytial virus [RSV], rhinovirus, adenovirus, human metapneumovirus, parainfluenza virus 1, 2, and 3) and influenza A and B viruses. Additionally, 255 specimens (9.3%) were tested using the Fast-Track Diagnostics Respiratory Pathogens 33 (FTD-33) kit which covered the mentioned viruses and additional viral, bacterial, and fungal pathogens. Influenza viruses were identified in 7.5% of all specimens; however, use of the CDC assay and FTD-33 kit increased the number of specimens with a pathogen identified to 61.8% and 91.5%, respectively. Among the 9 common viruses between the CDC assay and FTD-33 kit, the most identified pathogens were RSV (22.9%), rhinovirus (21.8%), and adenovirus (14.0%); multi-pathogen co-detections were common. Odds of hospitalization (SARI vs. ILI) varied by sex, age, geographic zone, year of diagnosis, and pathogen identified; hospitalized illnesses were most common among children under the age of 5 years. The greatest number of specimens were submitted for testing during December-April, coinciding with rainy seasons in Tanzania, and several viral pathogens demonstrated seasonal variation (RSV, human metapneumovirus, influenza A and B, and parainfluenza viruses). This study demonstrates that expanding an existing influenza platform to include additional respiratory pathogens can provide valuable insight into the etiology, incidence, severity, and geographic/temporal patterns of respiratory illness. Continued respiratory surveillance in Tanzania, and globally, can provide valuable data, particularly in the context of emerging respiratory pathogens such as SARS-CoV-2, and guide public health interventions to reduce the burden of respiratory illnesses.

BACKGROUND: The introduction of human immunodeficiency virus (HIV) antibody rapid testing (RT) in resource-limited settings has proven to be a successful intervention to increase access to prevention measures and improve timely linkage to care. However, the quality of testing has not always kept pace with the scale-up of this testing strategy. To monitor the accuracy of HIV RT test results, a national proficiency testing (PT) program was rolled out at selected testing sites in Ghana using the dried tube specimen (DTS) approach. METHODS: Between 2015 and 2018, 635 HIV testing sites, located in five regions and supported by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), were enrolled in the HIV PT program of the Ghana Health Service National AIDS/STI Control Programme. These sites offered various services: HIV Testing and Counselling (HTC), prevention of mother-to-child transmission (PMTCT) and Antiretroviral Treatment (ART). The PT panels, composed of six DTS, were prepared by two regional laboratories, using fully characterized plasma obtained from the regional blood banks and distributed to the testing sites. The results were scored by the PT providers according to the predefined acceptable performance criteria which was set at ≥ 95%. RESULTS: Seven rounds of PT panels were completed successfully over three years. The number of sites enrolled increased from 205 in round 1 (June 2015) to 635 in round 7 (December 2018), with a noticeable increase in Greater Accra and Eastern regions. The average participation rates of enrolled sites ranged from 88.0% to 98.0% across the PT rounds. By round 7, HTC (257/635 (40.5%)) and PMTCT (237/635 (37.3%)) had a larger number of sites that participated in the PT program than laboratory (106/635 (16.7%)) and ART (12/635 (1.9%)) sites. The average testing performance rate improved significantly from 27% in round 1 to 80% in round 7 (p < 0.001). The highest performance rate was observed for ART (100%), HTC (92%), ANC/PMTCT (90%) and Laboratory (89%) in round 5. CONCLUSION: The DTS PT program showed a significant increase in the participation and performance rates during this period. Sub-optimal performances observed was attributed to non-compliance to the national testing algorithm and testing technique. However, the implementation of review meetings, peer-initiated corrective action, supportive supervisory training, and mentorship proved impactful. The decentralized approach to preparing the PT panels ensured ownership by the region and districts.

Encephalitis and meningitis (EM) are severe infections of the central nervous system associated with high morbidity and mortality. The etiology of EM in Kazakhstan is not clearly defined, so from February 1, 2017 to January 31, 2018 we conducted hospital-based syndromic surveillance for EM at the Shymkent City Hospital, in the South Kazakhstan region. All consenting inpatients meeting a standard case definition were enrolled. Blood and cerebrospinal fluid (CSF) samples were collected for bacterial culture, and CSF samples were additionally tested by PCR for four bacterial species and three viruses using a cascading algorithm. We enrolled 556 patients. Of these, 494 were of viral etiology (including 4 probable rabies cases), 37 were of bacterial etiology, 19 were of unknown etiology and 6 were not tested. The most commonly identified pathogens included enterovirus (73%, n = 406 cases), herpes simplex virus (12.8%, n = 71), and Neisseria meningitidis (3.8%, n = 21). The incidence rates (IRs) for enteroviral and meningococcal EM were found to be 14.5 and 0.7 per 100,000 persons, respectively. The IR for bacterial EM using both PCR and culture results was 3-5 times higher compared to culture-only results. Antibacterial medicines were used to treat 97.2% (480/494) of virus-associated EM. Incorporation of PCR into routine laboratory diagnostics of EM improves diagnosis, pathogen identification, ensures IRs are not underestimated, and can help avoid unnecessary antibacterial treatment.

INTRODUCTION: Etiology studies of severe acute respiratory infections (SARI) in adults are limited. We studied potential etiologies of SARI among adults in six countries using multi-pathogen diagnostics. METHODS: We enrolled both adults with SARI (acute respiratory illness onset with fever and cough requiring hospitalization) and asymptomatic adults (adults hospitalized with non-infectious illnesses, non-household members accompanying SARI patients, adults enrolled from outpatient departments, and community members) in each country. Demographics, clinical data, and nasopharyngeal and oropharyngeal specimens were collected from both SARI patients and asymptomatic adults. Specimens were tested for presence of 29 pathogens utilizing the Taqman® Array Card platform. We applied a non-parametric Bayesian regression extension of a partially latent class model approach to estimate proportions of SARI caused by specific pathogens. RESULTS: We enrolled 2,388 SARI patients and 1,135 asymptomatic adults from October 2013 through October 2015. We detected ≥1 pathogen in 76% of SARI patients and 67% of asymptomatic adults. Haemophilus influenzae and Streptococcus pneumoniae were most commonly detected (≥23% of SARI patients and asymptomatic adults). Through modeling, etiology was attributed to a pathogen in most SARI patients (range among countries: 57.3-93.2%); pathogens commonly attributed to SARI etiology included influenza A (14.4-54.4%), influenza B (1.9-19.1%), rhino/enterovirus (1.8-42.6%), and RSV (3.6-14.6%). CONCLUSIONS: Use of multi-pathogen diagnostics and modeling enabled attribution of etiology in most adult SARI patients, despite frequent detection of multiple pathogens in the upper respiratory tract. Seasonal flu vaccination and development of RSV vaccine would likely reduce the burden of SARI in these populations.

Melioidosis is caused by Burkholderia pseudomallei and is predominantly seen in tropical regions. The clinical signs and symptoms of the disease are non-specific often resulting in misdiagnosis, failure of treatment, and poor clinical outcome. Septicemia with septic shock is the most common cause of death with mortality rates above 40%. Bacterial culture is the gold standard for diagnosis but it has low sensitivity and takes days to produce definitive results. Early laboratory diagnosis can help guide physicians to provide treatment specific to B. pseudomallei In our study, we adapted host gene expression signatures obtained from microarray data of B. pseudomallei infected cases to develop a real-time PCR diagnostic test using two differentially expressed genes, AIM2 (Absent in Melanoma 2) and FAM26F (Family with sequence similarity 26, member F). We tested blood from 33 patients with B. pseudomallei and 29 patients with other bacterial infections to validate the test and determine cutoff values for use in a cascading diagnostic algorithm. Differentiation of septicemic melioidosis from other sepsis cases had a sensitivity of 82%, specificity of 93%, and negative and positive predictive values (NPV and PPV) of 82% and 93% respectively. Separation of cases likely to be melioidosis from unlikely cases in non-bacteremic situations showed a sensitivity of 40%, specificity of 54%, and NPV and PPV of 44% and 50% respectively. We suggest that our AIM2 and FAM26F expression combination algorithm could be beneficial for early melioidosis diagnosis offering a result within 24 hours of admission.

INTRODUCTION: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. METHODS: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. RESULTS: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. CONCLUSION: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered.

BACKGROUND: In 2015, Singapore had the first and only reported foodborne outbreak of invasive disease caused by the group B Streptococcus (GBS; Streptococcus agalactiae). Disease, predominantly septic arthritis and meningitis, was associated with sequence type (ST)283, acquired from eating raw farmed freshwater fish. Although GBS sepsis is well-described in neonates and older adults with co-morbidities, this outbreak affected non-pregnant and younger adults with fewer co-morbidities, suggesting greater virulence. Before 2015 ST283 had only been reported from twenty humans in Hong Kong and two in France, and from one fish in Thailand. We hypothesised that ST283 was causing region-wide infection in Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: We performed a literature review, whole genome sequencing on 145 GBS isolates collected from six Southeast Asian countries, and phylogenetic analysis on 7,468 GBS sequences including 227 variants of ST283 from humans and animals. Although almost absent outside Asia, ST283 was found in all invasive Asian collections analysed, from 1995 to 2017. It accounted for 29/38 (76%) human isolates in Lao PDR, 102/139 (73%) in Thailand, 4/13 (31%) in Vietnam, and 167/739 (23%) in Singapore. ST283 and its variants were found in 62/62 (100%) tilapia from 14 outbreak sites in Malaysia and Vietnam, in seven fish species in Singapore markets, and a diseased frog in China. CONCLUSIONS: GBS ST283 is widespread in Southeast Asia, where it accounts for a large proportion of bacteraemic GBS, and causes disease and economic loss in aquaculture. If human ST283 is fishborne, as in the Singapore outbreak, then GBS sepsis in Thailand and Lao PDR is predominantly a foodborne disease. However, whether transmission is from aquaculture to humans, or vice versa, or involves an unidentified reservoir remains unknown. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture.

Background: Bloodstream infection (BSI) surveillance is essential to characterize the public health threat of bacteremia. We summarize BSI epidemiology in rural Thailand over an eight year period. Methods: Population-based surveillance captured clinically indicated blood cultures and associated antimicrobial susceptibility results performed in all 20 hospitals in Nakhon Phanom (NP) and Sa Kaeo (SK) provinces. BSIs were classified as community-onset (CO) when positive cultures were obtained ≤2 days after hospital admission and hospital-onset (HO) thereafter. Hospitalization denominator data were available for incidence estimates for 2009-2014. Results: From 2007 to 2014 a total of 11,166 BSIs were identified from 134,441 blood cultures. Annual CO BSI incidence ranged between 89.2 and 123.5 cases per 100,000 persons in SK and NP until 2011. Afterwards, CO incidence remained stable in SK and increased in NP, reaching 155.7 in 2013. Increases in CO BSI incidence over time were limited to persons aged ≥50 years. Ten pathogens, in rank order, accounted for > 65% of CO BSIs in both provinces, all age-groups, and all years: Escherichia coli, Klebsiella pneumoniae, Burkholderia pseudomallei, Staphylococcus aureus, Salmonella non-typhi spp., Streptococcus pneumoniae, Acinetobacter spp., Streptococcus agalactiae, Streptococcus pyogenes, Pseudomonas aeruginosa. HO BSI incidence increased in NP from 0.58 cases per 1000 hospitalizations in 2009 to 0.91 in 2014, but were higher (ranging from 1.9 to 2.3) in SK throughout the study period. Extended-spectrum beta-lactamase production among E. coli isolates and multi-drug resistance among Acinetobacter spp. isolates was common (> 25% of isolates), especially among HO cases (> 50% of isolates), and became more common over time, while methicillin-resistance among S. aureus isolates (10%) showed no clear trend. Carbapenem-resistant Enterobacteriaceae were documented in 2011-2014. Conclusions: Population-based surveillance documented CO BSI incidence estimates higher than previously reported from Thailand and the region, with temporal increases seen in older populations. The most commonly observed pathogens including resistance profiles were similar to leading pathogens and resistance profiles worldwide, thus; prevention strategies with demonstrated success elsewhere may prove effective in Thailand.

BACKGROUND: Determining the etiology of pneumonia is essential to guide public health interventions. Diagnostic test results, including from polymerase chain reaction (PCR) assays of upper respiratory tract specimens, have been used to estimate prevalence of pneumococcal pneumonia. However limitations in test sensitivity and specificity and the specimen types available make establishing a definitive diagnosis challenging. Prevalence estimates for pneumococcal pneumonia could be biased in the absence of a true gold standard reference test for detecting Streptococcus pneumoniae. METHODS: We conducted a case control study to identify etiologies of community acquired pneumonia (CAP) from April 2014 through August 2015 in Thailand. We estimated the prevalence of pneumococcal pneumonia among adults hospitalized for CAP using Bayesian latent class models (BLCMs) incorporating results of real-time polymerase chain reaction (qPCR) testing of upper respiratory tract specimens and a urine antigen test (UAT) from cases and controls. We compared the prevalence estimate to conventional analyses using only UAT as a reference test. RESULTS: The estimated prevalence of pneumococcal pneumonia was 8% (95% CI: 5-11%) by conventional analyses. By BLCM, we estimated the prevalence to be 10% (95% CrI: 7-16%) using binary qPCR and UAT results, and 11% (95% CrI: 7-17%) using binary UAT results and qPCR cycle threshold (Ct) values. CONCLUSIONS: BLCM suggests a > 25% higher prevalence of pneumococcal pneumonia than estimated by a conventional approach assuming UAT as a gold standard reference test. Higher quantities of pneumococcal DNA in the upper respiratory tract were associated with pneumococcal pneumonia in adults but the addition of a second specific pneumococcal test was required to accurately estimate disease status and prevalence. By incorporating the inherent uncertainty of diagnostic tests, BLCM can obtain more reliable estimates of disease status and improve understanding of underlying etiology.

Bloodstream infection surveillance conducted from 2008 to 2014 in all 20 hospitals in Sa Kaeo and Nakhon Phanom provinces, Thailand, allowed us to look at disease burden, antibiotic susceptibilities, and recurrent infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. Of 97,832 blood specimens, 3,338 were positive for E. coli and 1,086 for K. pneumoniae. The proportion of E. coli isolates producing ESBL significantly increased from 19% to 22% in 2008-2010 to approximately 30% from 2011 to 2014 (P-value for trend = 0.02), whereas ESBL production among K. pneumoniae cases was 27.4% with no significant trend over time. Incidence of community-onset ESBL-producing E. coli increased from 5.4 per 100,000 population in 2008 to 12.8 in 2014, with the highest rates among persons aged >/= 70 years at 79 cases per 100,000 persons in 2014. From 2008 to 2014, community-onset ESBL-producing K. pneumoniae incidence was 2.7 per 100,000, with a rate of 12.9 among those aged >/= 70 years. Although most (93.6% of E. coli and 87.6% of K. pneumoniae) infections were community-onset, hospital-onset infections were twice as likely to be ESBL. Population-based surveillance, as described, is vital to accurately monitor emergence and trends in antimicrobial resistance, and in guiding the development of rational antimicrobial therapy recommendations.

INTRODUCTION: Invasive salmonellosis is a common cause of bloodstream infection in Southeast Asia. Limited epidemiologic and antimicrobial resistance data are available from the region. METHODS: Blood cultures performed in all 20 hospitals in the northeastern province of Nakhon Phanom (NP) and eastern province of Sa Kaeo (SK), Thailand were captured in a bloodstream infection surveillance system. Cultures were performed as clinically indicated in hospitalized patients; patients with multiple positive cultures had only the first included. Bottles were incubated using the BacT/Alert system (bioMerieux, Thailand) and isolates were identified using standard microbiological techniques; all Salmonella isolates were classified to at least the serogroup level. Antimicrobial resistance was assessed using disk diffusion. RESULTS: Salmonella was the fifth most common pathogen identified in 147,535 cultures with 525 cases (211 in Nakhon Phanom (NP) and 314 in Sa Kaeo (SK)). The overall adjusted iNTS incidence rate in NP was 4.0 cases/100,000 person-years (95% CI 3.5-4.5) and in SK 6.4 cases/100,000 person-years (95% CI 5.7-7.1; p = 0.001). The most common serogroups were C (39.4%), D (35.0%) and B (9.9%). Serogroup D predominated in NP (103/211) with 59.2% of this serogroup being Salmonella serovar Enteritidis. Serogroup C predominated in SK (166/314) with 84.3% of this serogroup being Salmonella serovar Choleraesuis. Antibiotic resistance was 68.2% (343/503) for ampicillin, 1.2% (6/482) for ciprofloxacin (or 58.1% (280/482) if both intermediate and resistant phenotypes are considered), 17.0% (87/512) for trimethoprim-sulfamethoxazole, and 12.2% (59/484) for third-generation cephalosporins (cefotaxime or ceftazidime). Multidrug resistance was seen in 99/516 isolates (19.2%). CONCLUSIONS: The NTS isolates causing bloodstream infections in rural Thailand are commonly resistant to ampicillin, cefotaxime, and TMP-SMX. Observed differences between NP and SK indicate that serogroup distribution and antibiotic resistance may substantially differ throughout Thailand and the region.

Staphylococcus aureus is a common cause of bloodstream infection and methicillin-resistant S. aureus (MRSA) is a growing threat worldwide. We evaluated the incidence rate of S. aureus bacteremia (SAB) and MRSA from population-based surveillance in all hospitals from two Thai provinces. Infections were classified as community-onset (CO) when blood cultures were obtained </= 2 days after hospital admission and as hospital-onset (HO) thereafter. The incidence rate of HO-SAB could only be calculated for 2009-2014 when hospitalization denominator data were available. Among 147,524 blood cultures, 919 SAB cases were identified. Community-onset S. aureus bacteremia incidence rate doubled from 4.4 (95% confidence interval [CI]: 3.3-5.8) in 2006 to 9.3 per 100,000 persons per year (95% CI: 7.6-11.2) in 2014. The highest CO-SAB incidence rate was among adults aged 50 years and older. Children less than 5 years old had the next highest incidence rate, with most cases occurring among neonates. During 2009-2014, there were 89 HO-SAB cases at a rate of 0.13 per 1,000 hospitalizations per year (95% CI: 0.10-0.16). Overall, MRSA prevalence among SAB cases was 10% (90/911) and constituted 7% (55/736) of CO-SAB and 20% (22/111) of HO-SAB without a clear temporal trend in incidence rate. In conclusion, CO-SAB incidence rate has increased, whereas MRSA incidence rate remained stable. The increasing CO-SAB incidence rate, especially the burden on older adults and neonates, underscores the importance of strong SAB surveillance to identify and respond to changes in bacteremia trends and antimicrobial resistance.

Artemisinin-based combination therapy (ACT) is the most effective and widely used treatment for uncomplicated Plasmodium falciparum (Pf) malaria and is a cornerstone for malaria control and prevention globally. Resistance to artemisinin derivatives has been confirmed in the Greater Mekong Subregion (GMS), which manifest as slow parasite clearance in patients and reduced ring-stage susceptibility to artemisinins in survival assays. The Pf kelch 13 gene mutations associated with artemisinin resistant parasites are now wide-spread in the GMS. We genotyped 277 samples collected during an observational study from 2012-2016 from eight provinces in Thailand to identify Pf kelch 13 mutations. The results were combined with previously reported genotyping results from Thailand to construct a map illustrating the evolution of Pf kelch 13 mutations from 2007 - 2016 in the country. Different mutant alleles were found in strains with different geographical origins. The artemisinin resistant Y493H and R539T mutations were detected mainly in eastern Thailand (bordering Cambodia), while P574L was only found in western Thailand and R561H in northwestern Thailand. The C580Y mutation was found across the entire country and was nearing fixation along the Thai-Cambodia border. Overall the prevalence of artemisinin resistant mutations increased over the last ten years across Thailand, especially along the Thai-Cambodia border. Molecular surveillance and therapeutic efficacy monitoring should be intensified in the region to further assess the extent and spread of artemisinin resistance.

Background.: We investigated the performance of polymerase chain reaction (PCR) on blood in the diagnosis of pneumococcal pneumonia among children from 7 low- and middle-income countries. Methods.: We tested blood by PCR for the pneumococcal autolysin gene in children aged 1-59 months in the Pneumonia Etiology Research for Child Health (PERCH) study. Children had World Health Organization-defined severe or very severe pneumonia or were age-frequency-matched community controls. Additionally, we tested blood from general pediatric admissions in Kilifi, Kenya, a PERCH site. The proportion PCR-positive was compared among cases with microbiologically confirmed pneumococcal pneumonia (MCPP), cases without a confirmed bacterial infection (nonconfirmed), cases confirmed for nonpneumococcal bacteria, and controls. Results.: In PERCH, 7.3% (n = 291/3995) of cases and 5.5% (n = 273/4987) of controls were blood pneumococcal PCR-positive (P < .001), compared with 64.3% (n = 36/56) of MCPP cases and 6.3% (n = 243/3832) of nonconfirmed cases (P < .001). Blood pneumococcal PCR positivity was higher in children from the 5 African countries (5.5%-11.5% among cases and 5.3%-10.2% among controls) than from the 2 Asian countries (1.3% and 1.0% among cases and 0.8% and 0.8% among controls). Among Kilifi general pediatric admissions, 3.9% (n = 274/6968) were PCR-positive, including 61.7% (n = 37/60) of those with positive blood cultures for pneumococcus. Discussion.: The utility of pneumococcal PCR on blood for diagnosing childhood pneumococcal pneumonia in the 7 low- and middle-income countries studied is limited by poor specificity and by poor sensitivity among MCPP cases.

The Pneumonia Etiology Research for Child Health study was conducted across 7 diverse research sites and relied on standardized clinical and laboratory methods for the accurate and meaningful interpretation of pneumonia etiology data. Blood, respiratory specimens, and urine were collected from children aged 1-59 months hospitalized with severe or very severe pneumonia and community controls of the same age without severe pneumonia and were tested with an extensive array of laboratory diagnostic tests. A standardized testing algorithm and standard operating procedures were applied across all study sites. Site laboratories received uniform training, equipment, and reagents for core testing methods. Standardization was further assured by routine teleconferences, in-person meetings, site monitoring visits, and internal and external quality assurance testing. Targeted confirmatory testing and testing by specialized assays were done at a central reference laboratory.

Background.: The etiologic inference of identifying a pathogen in the upper respiratory tract (URT) of children with pneumonia is unclear. To determine if viral load could provide evidence of causality of pneumonia, we compared viral load in the URT of children with World Health Organization-defined severe and very severe pneumonia and age-matched community controls. Methods.: In the 9 developing country sites, nasopharyngeal/oropharyngeal swabs from children with and without pneumonia were tested using quantitative real-time polymerase chain reaction for 17 viruses. The association of viral load with case status was evaluated using logistic regression. Receiver operating characteristic (ROC) curves were constructed to determine optimal discriminatory viral load cutoffs. Viral load density distributions were plotted. Results.: The mean viral load was higher in cases than controls for 7 viruses. However, there was substantial overlap in viral load distribution of cases and controls for all viruses. ROC curves to determine the optimal viral load cutoff produced an area under the curve of <0.80 for all viruses, suggesting poor to fair discrimination between cases and controls. Fatal and very severe pneumonia cases did not have higher viral load than less severe cases for most viruses. Conclusions.: Although we found higher viral loads among pneumonia cases than controls for some viruses, the utility in using viral load of URT specimens to define viral pneumonia was equivocal. Our analysis was limited by lack of a gold standard for viral pneumonia.

Studies on Mycoplasma pneumoniae in Thailand have focused on urban centers and have not included the molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3000 nasopharyngeal swab specimens, collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces. M. pneumoniae was detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality to M. pneumoniae infections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed all 141 tested to possess the genotype associated with macrolide susceptibility.

Biological safety cabinets (BSCs) are the primary means of containment used in laboratories worldwide for the safe handling of infectious microorganisms. They provide protection to the laboratory worker and the surrounding environment from pathogens. To ensure the correct functioning of BSCs, they need to be properly maintained beyond the daily care routines of the laboratory. This involves annual maintenance and certification by a qualified technician in accordance to the NSF/American National Standards Institute 49-2014 Biosafety Cabinetry: Design, Construction, Performance, and Field Certification. Service programs can be direct from the manufacturer or through third-party service companies, but in many instances, technicians are not accredited by international bodies, and these services are expensive. This means that a large number of BSCs may not be operating in a safe manner. In this article, we discuss our approach to addressing the lack of trained and qualified personnel in Thailand who can install, maintain, and certify BSCs in a cost-effective and practical manner. We initiated a program to create both local and regional capacity for repair, maintenance, and certification of BSCs and share our experiences with the reader.

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

Community-acquired bloodstream infections cause substantial morbidity and mortality worldwide but microbiology capacity and surveillance limitations have challenged good descriptions of pathogen distribution in many regions, including Southeast Asia. Active surveillance for bloodstream infections has been conducted in two rural Thailand provinces for >7 years. Blood specimens were divided into two culture bottles: one optimized for aerobic growth (F bottle) and a second for enhanced growth of mycobacteria (MB bottle), and processed used the BactT/ALERT 3D(R) system. Because routine use of MB culture bottles is resource intensive (expensive and prolonged incubation), we assessed the added yield of MB bottles by comparing the proportion of pathogens detected by MB vs. F bottles from 2005 - 2012. Of 63,066 blood cultures, 7,296(12%) were positive for at least one pathogen, the most common pathogens were Escherichia coli (28%), Burkholderia pseudomallei (11%), Klebsiella pneumoniae (9%) and Staphylococcus aureus (6%). Two bottles improved yield overall, but added yield attributable to the MB bottles was limited to a few pathogens. In addition to mycobacteria and some fungi, MB bottles improved B. pseudomallei detection (MB 27% vs. F 8%; p-value < 0.0001) with added benefit if therapy was initiated prior to the blood draw culture. Targeted use of MB bottles is warranted for patients at risk for mycobacterial and fungal infections, as well as B. pseudomallei, a common cause of septicemia in Thailand.

Despite rigorous diagnostic testing, the cause of infective endocarditis was identified for just 60 (45.5%) of 132 patients admitted to hospitals in Khon Kaen, Thailand, during January 2010-July 2012. Most pathogens identified were Viridans streptococci and zoonotic bacteria species, as found in other resource-limited countries where underlying rheumatic heart disease is common.

Since 2001, the Centers for Disease Control and Prevention (CDC) has established 10 GDD Regional Centers, serving primarily resource-constrained locations in Thailand, Kenya, Guatemala, Egypt, China, Bangladesh, Kazakhstan, India, South Africa, and Georgia [1]. GDD laboratories support the following GDD Center programs that require diagnostic testing for emerging infectious diseases: the International Emerging Infections Program (IEIP), the One Health Program, the Field Epidemiology Training Program, the Influenza Program, and the Refugee Health Program (unique to the GDD Regional Center in Kenya). The laboratory leaders at each GDD Center also serve as the center's advisor for the Strengthening Laboratory Capacity Program, through which they advise the host country on means of improving laboratory capacity to support the International Health Regulations [2]. In the 6 GDD Centers whose IEIP programs conduct population-based surveillance for acute respiratory illness and other syndromes, laboratory support is provided through GDD laboratories (in Thailand, Kenya, and Guatemala), through laboratories run by the GDD partner institution (in China and Bangladesh), or a combination of both (in Egypt).

BACKGROUND: There is no consistent evidence of specific gene(s) or molecular pathways that contribute to the pathogenesis, therapeutic intervention or diagnosis of chronic fatigue syndrome (CFS). While multiple studies support a role for genetic variation in CFS, genome-wide efforts to identify associated loci remain unexplored. We employed a novel convergent functional genomics approach that incorporates the findings from single-nucleotide polymorphism (SNP) and mRNA expression studies to identify associations between CFS and novel candidate genes for further investigation. METHODS: We evaluated 116,204 SNPs in 40 CFS and 40 nonfatigued control subjects along with mRNA expression of 20,160 genes in a subset of these subjects (35 CFS subjects and 27 controls) derived from a population-based study. RESULTS: Sixty-five SNPs were nominally associated with CFS (p < 0.001), and 165 genes were differentially expressed (≥4-fold; p ≤ 0.05) in peripheral blood mononuclear cells of CFS subjects. Two genes, glutamate receptor, ionotropic, kinase 2 (GRIK2) and neuronal PAS domain protein 2 (NPAS2), were identified by both SNP and gene expression analyses. Subjects with the G allele of rs2247215 (GRIK2) were more likely to have CFS (p = 0.0005), and CFS subjects showed decreased GRIK2 expression (10-fold; p = 0.015). Subjects with the T allele of rs356653 (NPAS2) were more likely to have CFS (p = 0.0007), and NPAS2 expression was increased (10-fold; p = 0.027) in those with CFS. CONCLUSION: Using an integrated genomic strategy, this study suggests a possible role for genes involved in glutamatergic neurotransmission and circadian rhythm in CFS and supports further study of novel candidate genes in independent populations of CFS subjects.

BACKGROUND: The contribution of alternative splicing and isoform expression to cellular response is emerging as an area of considerable interest, and the newly developed exon arrays allow for systematic study of these processes. We use this pilot study to report on the feasibility of exon array implementation looking to replace the 3' in vitro transcription expression arrays in our laboratory. One of the most widely studied models of cellular response is T-cell activation from exogenous stimulation. Microarray studies have contributed to our understanding of key pathways activated during T-cell stimulation. We use this system to examine whole genome transcription and alternate exon usage events that are regulated during lymphocyte proliferation in an attempt to evaluate the exon arrays. RESULTS: Peripheral blood mononuclear cells form healthy donors were activated using phytohemagglutinin, IL2 and ionomycin and harvested at 5 points over a 7 day period. Flow cytometry measured cell cycle events and the Affymetrix exon array platform was used to identify the gene expression and alternate exon usage changes. Gene expression changes were noted in a total of 2105 transcripts, and alternate exon usage identified in 472 transcript clusters. There was an overlap of 263 transcripts which showed both differential expression and alternate exon usage over time. Gene ontology enrichment analysis showed a broader range of biological changes in biological processes for the differentially expressed genes, which include cell cycle, cell division, cell proliferation, chromosome segregation, cell death, component organization and biogenesis and metabolic process ontologies. The alternate exon usage ontological enrichments are in metabolism and component organization and biogenesis. We focus on alternate exon usage changes in the transcripts of the spliceosome complex. The real-time PCR validation rates were 86% for transcript expression and 71% for alternate exon usage. CONCLUSIONS: This study illustrates that the Exon array technology has the potential to provide information on both transcript expression and isoform usage, with very little increase in expense.

Understanding the biologic significance of alternative splicing has been impeded by the difficulty in systematically identifying and validating transcript isoforms. Current exon array workflows suggest several different filtration steps to reduce the number of tests and increase the detection of alternative splicing events. In this study, we examine the effects of the suggested pre-analysis filtration by detection above background P value or signal intensity. This is followed post-analytically by restriction of exon expression to a fivefold change between groups, limiting the analysis to known alternative splicing events, or using the intersection of the results from different algorithms. Combinations of the filters are also examined. We find that none of the filtering methods reduces the number of technical false-positive calls identified by visual inspection. These include edge effects, nonresponsive probe sets, and inclusion of intronic and untranslated region probe sets into transcript annotations. Modules for filtering the exon microarray data on the basis of annotation features are needed. We propose new approaches to data filtration that would reduce the number of technical false-positives and therefore, impact the time spent performing visual inspection of the exon arrays.

We investigated peripheral blood mononuclear cell gene expression responses to acute psychosocial stress to identify molecular pathways relevant to the stress response. Blood samples were obtained from 10 healthy male subjects before, during and after (at 0, 30, and 60 min) a standardized psychosocial laboratory stressor. Ribonucleic acid (RNA) was extracted and gene expression measured by hybridization to a 20,000-gene microarray. Gene Set Expression Comparisons (GSEC) using defined pathways were used for the analysis. Forty-nine pathways were significantly changed from baseline to immediately after the stressor (p<0.05), implicating cell cycle, cell signaling, adhesion and immune responses. The comparison between stress and recovery (measured 30 min later) identified 36 pathways, several involving stress-responsive signaling cascades and cellular defense mechanisms. These results have relevance for understanding molecular mechanisms of the physiological stress response, and might be used to further study adverse health outcomes of psychosocial stress.