A pilot study using hospital surveillance and a birth cohort to investigate enteric pathogens and malnutrition in children, Dili, Timor-Leste.

In low-to-middle-income countries (LMICs), enteric pathogens contribute to child malnutrition, affecting nutrient absorption, inducing inflammation, and causing diarrhoea. This is a substantial problem in LMICs due to high disease burden, poor sanitation and nutritional status, and the cyclical nature of pathogen infection and malnutrition. This relationship remains understudied in Timor-Leste. In our pilot study of enteric pathogens and malnutrition in Dili, Timor-Leste (July 2019-October 2020), we recruited 60 infants in a birth cohort from Hospital Nacional Guido Valadares (HNGV) with up to four home visits. We collected faecal samples and details of demographics, anthropometrics, diet and food practices, and animal husbandry. Additionally, we collected faecal samples, diagnostics, and anthropometrics from 160 children admitted to HNGV with a clinical diagnosis of severe diarrhoea or severe acute malnutrition (SAM). We tested faeces using the BioFire® FilmArray® Gastrointestinal Panel. We detected high prevalence of enteric pathogens in 68.8% (95%CI 60.4-76.2%) of infants at home, 88.6% of SAM cases (95%CI 81.7-93.3%) and 93.8% of severe diarrhoea cases (95%CI 67.7-99.7%). Diarrhoeagenic Escherichia coli and Campylobacter spp. were most frequently detected. Pathogen presence did not significantly differ in birth cohort diarrhoeal stool, but hospital data indicated associations between Salmonella and Shigella and diarrhoea. We observed wasting in 18.4% (95%CI 9.2-32.5%) to 30.8% (95%CI 17.5-47.7%) of infants across home visits, 57.9% (95%CI 34.0-78.9%) of severe diarrhoea cases, and 92.5% (95%CI 86.4-96.2%) of SAM cases. We associated bottle feeding with increased odds of pathogen detection when compared with exclusive breastfeeding at home (OR 8.3, 95%CI 1.1-62.7). We detected high prevalence of enteric pathogens and signs of malnutrition in children in Dili. Our pilot is proof of concept for a study to fully explore the risk factors and associations between enteric pathogens and malnutrition in Timor-Leste.

Seroprevalence and prevention of hepatitis B, measles and rubella among healthcare workers in Dili, Timor-Leste.

Introduction: The World Health Organisation recommends that healthcare workers (HCWs) are immune to measles and rubella, and those at risk of exposure are offered the hepatitis B vaccine. No formal programme for occupational assessment and provision of vaccinations to HCWs currently exists in Timor-Leste. Methods: A cross-sectional study was undertaken to determine the seroprevalence of hepatitis B, measles and rubella among HCWs in Dili, Timor-Leste. All patient-facing employees at three healthcare institutions during April-June 2021 were invited to participate. Epidemiological data were collected by interview-questionnaire and a serum sample was collected by phlebotomy and analysed at the National Health Laboratory. Participants were contacted to discuss their results. Relevant vaccines were offered to seronegative individuals and those with active hepatitis B infection were referred for further assessment and management in a hepatology clinic as per national guidelines. Results: Three-hundred-and-twenty-four HCWs were included (representing 51.3% of all eligible HCWs working at the three participating institutions). Sixteen (4.9%; 95% CI: 2.8-7.9%) had active hepatitis B infection, 121 (37.3%; 95% CI: 32.1-42.9%) had evidence of previous (cleared) hepatitis B infection, 134 (41.4%; 95% CI: 35.9-46.9%) were hepatitis B seronegative, and 53 (16.4%; 95% CI: 12.5-20.8%) had been vaccinated. Two-hundred-and-sixty-seven (82.4%; 95% CI: 77.8-86.4%) and 306 (94.4%; 95% CI: 91.4-96.7%) individuals exhibited antibodies to measles and rubella, respectively. Interpretation: There are significant immunity gaps and a high prevalence of hepatitis B infection among HCWs in Dili Municipality, Timor-Leste. Routine occupational assessment and targeted vaccination of this group would be beneficial and should include all types of HCWs. This study provided an opportunity to develop a programme for the occupational assessment and vaccination of HCWs and forms the template for a national guideline. Funding: This work was supported by the Department of Foreign Affairs and Trade, Australian Government [Complex Grant Agreement Number 75889].

Vaccine Preventable Disease Seroprevalence in a Nationwide Assessment of Timor-Leste (VASINA-TL): study protocol for a population-representative cross-sectional serosurvey.

INTRODUCTION: Historic disruption in health infrastructure combined with data from a recent vaccine coverage survey suggests there are likely significant immunity gaps to vaccine preventable diseases and high risk of outbreaks in Timor-Leste. Community-based serological surveillance is an important tool to augment understanding of population-level immunity achieved through vaccine coverage and/or derived from prior infection. METHODS AND ANALYSIS: This national population-representative serosurvey will take a three-stage cluster sample and aims to include 5600 individuals above 1 year of age. Serum samples will be collected by phlebotomy and analysed for measles IgG, rubella IgG, SARS-CoV-2 antispike protein IgG, hepatitis B surface antibody and hepatitis B core antigen using commercially available chemiluminescent immunoassays or ELISA. In addition to crude prevalence estimates and to account for differences in Timor-Leste's age structure, stratified age-standardised prevalence estimates will be calculated, using Asia in 2013 as the standard population. Additionally, this survey will derive a national asset of serum and dried blood spot samples which can be used for further investigation of infectious disease seroepidemiology and/or validation of existing and novel serological assays for infectious diseases. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Research Ethics and Technical Committee of the Instituto Nacional da Saúde, Timor-Leste and the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research, Australia. Co-designing this study with Timor-Leste's Ministry-of-Health and other relevant partner organisations will allow immediate translation of findings into public health policy, which may include changes to routine immunisation service delivery and/or plans for supplementary immunisation activities.

The COVID-19 laboratory response in Timor-Leste; a story of collaboration.

Timor-Leste is a small nation of 1.3 million people which shares a land border with Indonesia and is 550 km from Darwin, Australia. It is one of the poorest nations in Asia. The National Health Laboratory (NHL) and its network of smaller laboratories in Timor-Leste had limited capacity to perform molecular diagnostic testing before the coronavirus disease 2019 (COVID-19) pandemic began. With the support of international development partners, the NHL rapidly expanded its molecular testing service. From March 2020 to February 2022, over 200,000 molecular tests were performed; COVID-19 testing sites were established in hospital and community health center laboratories and all 13 municipalities, and the number of scientists and technicians at the molecular diagnostic laboratory at the NHL increased from five to 28 between 2019 and 2022. Molecular diagnostic testing for COVID-19 was successfully established at the NHL and in the municipalities. The molecular diagnostic laboratory at NHL is now equipped to respond to not only large-scale COVID-19 testing but also laboratory detection of other infectious diseases, preparing Timor-Leste for future outbreaks or pandemics.

The use of residual serum samples to perform serological surveillance of severe acute respiratory syndrome coronavirus 2 in Dili and regional areas of Timor-Leste.

BACKGROUND: Lack of access to diagnostic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can limit disease surveillance in remote areas. Serological surveillance can indicate the true extent and distribution of infections in such settings. METHODS: This study monitored SARS-CoV-2 seroprevalence in residual serum samples salvaged from laboratories at five healthcare facilities across Timor-Leste from March to October 2021. RESULTS: Seroprevalence increased from 8.3% to 87.0% during the study period. Potential immunity gaps were identified among children aged 0-15 y (who had not been eligible for vaccination) and individuals aged >60 y. CONCLUSIONS: Efforts to vaccinate vulnerable individuals including older people should be maintained. Residual serum samples can be analysed to give local, contemporary information about the extent and distribution of antibodies to infections, especially SARS-CoV-2, in areas where epidemiological information is limited.

Field evaluation of rapid diagnostic tests to determine dengue serostatus in Timor-Leste.

The live attenuated tetravalent CYD-TDV vaccine (Dengvaxia) is effective but has scarcely been used due to safety concerns among seronegative recipients. Rapid diagnostic tests (RDTs) which can accurately determine individual dengue serostatus are needed for use in pre-vaccination screening. This study aimed to determine the performance of existing RDTs (which have been designed to detect levels of immunoglobulin G, IgG, associated with acute post-primary dengue) when repurposed for detection of previous dengue infection (where concentrations of IgG are typically lower). A convenience sample of four-hundred-and-six participants including 217 children were recruited during a community serosurvey. Whole blood was collected by phlebotomy and tested using Bioline Dengue IgG/IgM (Abbott) and Standard Q Dengue IgM/IgG (SD Biosensor) RDTs in the field. Serum samples from the same individuals were also tested at National Health Laboratory. The Panbio indirect IgG ELISA was used as a reference test. Reference testing determined that 370 (91.1%) participants were dengue IgG seropositive. Both assays were highly specific (100.0%) but had low sensitivity (Bioline = 21.1% and Standard Q = 4.6%) when used in the field. Sensitivity was improved when RDTs were used under laboratory conditions, and when assays were allowed to run beyond manufacturer recommendations and read at a delayed time-point, but specificity was reduced. Efforts to develop RDTs with high sensitivity and specificity for prior dengue infection which can be operationalised for pre-vaccination screening are ongoing. Performance of forthcoming candidate assays should be tested under field conditions with blood samples, as well as in the laboratory.

Integrated serological surveillance of acute febrile illness in the context of a lymphatic filariasis survey in Timor-Leste: a pilot study using dried blood spots.

BACKGROUND: Acute febrile illnesses (AFIs), including dengue, scrub typhus and leptospirosis, cause significant morbidity and mortality in Southeast Asia. Serological surveillance can be used to investigate the force and distribution of infections. Dried blood spot (DBS) samples are an attractive alternative to serum because they are easier to collect and transport and require less cold storage. We conducted a pilot study to determine the feasibility of integrating serological surveillance for dengue, scrub typhus and leptospirosis into a population-representative lymphatic filariasis seroprevalence survey in Timor-Leste using DBSs. METHODS: A total of 272 DBSs were collected from healthy community participants. DBSs were analysed at the National Health Laboratory using commercially available enzyme-linked immunosorbent assays. To validate assays for DBSs, 20 anonymised serum samples of unknown serostatus were used to create dried serum spots (DSSs). These were analysed with optical densities compared with those of serum. Where low variance was observed (dengue assay) the published kit cut-offs for serum were applied to the analysis of DBSs. For the other assays (scrub typhus and leptospirosis), index values (IVs) were calculated and cut-offs were determined to be at 2 standard deviations (SDs) above the mean. RESULTS: Of the 272 samples analysed, 19 (7.0% [95% confidence interval {CI} 4.3 to 10.7]) were positive for dengue immunoglobulin G (IgG), 11 (4.0% [95% CI 2.1 to 7.1]) were positive for scrub typhus IgG and 16 (5.9% [95% CI 3.4 to 9.4%]) were positive for leptospira IgG. CONCLUSIONS: While dengue seroprevalence was lower than in nearby countries, results represent the first evidence of scrub typhus and leptospirosis transmission in Timor-Leste. Integrated programmes of serological surveillance could greatly improve our understanding of infectious disease epidemiology in remote areas and would incur minimal additional fieldwork costs. However, when planning such studies, the choice of assays, their validation for DBSs and the laboratory infrastructure and technical expertise at the proposed location of analysis must be considered.

Epidemiology of the Rhinovirus (RV) in African and Southeast Asian Children: A Case-Control Pneumonia Etiology Study.

Rhinovirus (RV) is commonly detected in asymptomatic children; hence, its pathogenicity during childhood pneumonia remains controversial. We evaluated RV epidemiology in HIV-uninfected children hospitalized with clinical pneumonia and among community controls. PERCH was a case-control study that enrolled children (1-59 months) hospitalized with severe and very severe pneumonia per World Health Organization clinical criteria and age-frequency-matched community controls in seven countries. Nasopharyngeal/oropharyngeal swabs were collected for all participants, combined, and tested for RV and 18 other respiratory viruses using the Fast Track multiplex real-time PCR assay. RV detection was more common among cases (24%) than controls (21%) (aOR = 1.5, 95%CI:1.3-1.6). This association was driven by the children aged 12-59 months, where 28% of cases vs. 18% of controls were RV-positive (aOR = 2.1, 95%CI:1.8-2.5). Wheezing was 1.8-fold (aOR 95%CI:1.4-2.2) more prevalent among pneumonia cases who were RV-positive vs. RV-negative. Of the RV-positive cases, 13% had a higher probability (>75%) that RV was the cause of their pneumonia based on the PERCH integrated etiology analysis; 99% of these cases occurred in children over 12 months in Bangladesh. RV was commonly identified in both cases and controls and was significantly associated with severe pneumonia status among children over 12 months of age, particularly those in Bangladesh. RV-positive pneumonia was associated with wheezing.

Training physicians in India to interpret pediatric chest radiographs according to World Health Organization research methodology.

BACKGROUND: Chest radiography is the standard for diagnosing pediatric lower respiratory infections in low-income and middle-income countries. A method for interpreting pediatric chest radiographs for research endpoints was recently updated by the World Health Organization (WHO) Chest Radiography in Epidemiological Studies project. Research in India required training local physicians to interpret chest radiographs following the WHO method. OBJECTIVE: To describe the methodology for training Indian physicians and evaluate the training's effectiveness. MATERIALS AND METHODS: Twenty-nine physicians (15 radiologists and 14 pediatricians) from India were trained by two WHO Chest Radiography in Epidemiological Studies members over 3 days in May 2019. Training materials were adapted from WHO Chest Radiography in Epidemiological Studies resources. Participants followed WHO methodology to interpret 60 unique chest radiographs before and after the training. Participants needed to correctly classify ≥80% of radiographs for primary endpoint pneumonia on the post-training test to be certified to interpret research images. We analyzed participant performance on both examinations. RESULTS: Twenty-six of 29 participants (89.7%) completed both examinations. The average score increased by 9.6% (95% confidence interval [CI] 5.0-14.1%) between examinations (P<0.001). Participants correctly classifying ≥80% of images for primary endpoint pneumonia increased from 69.2% (18/26) on the pretraining to 92.3% (24/26) on the post-training examination (P=0.003). The mean scores of radiologists and pediatricians on the post-training examination were not statistically different (P=0.43). CONCLUSION: Our results demonstrate this training approach using revised WHO definitions and tools was successful, and that non-radiologists can learn to apply these methods as effectively as radiologists. Such capacity strengthening is important for enabling research to support national policy decision-making in these settings. We recommend future research incorporating WHO chest radiograph methodology to consider modelling trainings after this approach.

Prevalence of scabies and impetigo in school-age children in Timor-Leste.

BACKGROUND: Scabies and impetigo are endemic in many tropical, low- and middle-income countries. Mass drug administration (MDA) with ivermectin has emerged as a control strategy for these conditions. In 2019, Timor-Leste Ministry of Health planned to implement MDA including ivermectin for the control of lymphatic filariasis, so we undertook a baseline assessment of scabies and impetigo to better understand local epidemiology and contribute to future surveys assessing the impact of MDA. METHODS: A cross-sectional school survey was conducted in April-May 2019 at six primary schools in a semi-urban (Dili) and two rural (Ermera and Manufahi) settings. Children under 19 years of age present at school on survey days were eligible to participate, of whom we enrolled 1183. Trained health workers interviewed and examined 1043 participants to clinically diagnose scabies using the 2020 International Alliance for the Control of Scabies (IACS) diagnostic criteria, as well as impetigo. Prevalence was adjusted for age and sex. Mixed-effects logistic regression models were used to analyse odds of scabies and impetigo infection. All models accounted for clustering at the school level through the use of random effect terms. Population attributable risk of scabies as a cause of impetigo was also estimated. RESULTS: The overall weighted prevalence of scabies was 30.6%. Children in rural Manufahi were more likely to have scabies than those in semi-urban Dili (53.6% vs 28.2%, adjusted odds ratio [AOR] 3.5). Most cases of scabies were mild (3 to 10 lesions), and lesions were usually distributed on more than one body region. Scabies prevalence was lower among 10 to 14 year olds compared to 5 to 9 year olds. Overall weighted prevalence of impetigo was 11.3%. Relative to Dili, children in rural Ermera and Manufahi were twice as likely to have impetigo. Impetigo was twice as common in children with scabies than in those without, corresponding to an attributable risk of scabies as a cause of impetigo of 22.7%. CONCLUSIONS: Scabies and impetigo prevalence in Timor-Leste is among the highest reported globally, particularly in rural areas. Scabies infestation was strongly associated with impetigo. Comprehensive control strategies are urgently needed in Timor-Leste.

Digital auscultation in PERCH: Associations with chest radiography and pneumonia mortality in children.

BACKGROUND: Whether digitally recorded lung sounds are associated with radiographic pneumonia or clinical outcomes among children in low-income and middle-income countries is unknown. We sought to address these knowledge gaps. METHODS: We enrolled 1 to 59monthold children hospitalized with pneumonia at eight African and Asian Pneumonia Etiology Research for Child Health sites in six countries, recorded digital stethoscope lung sounds, obtained chest radiographs, and collected clinical outcomes. Recordings were processed and classified into binary categories positive or negative for adventitial lung sounds. Listening and reading panels classified recordings and radiographs. Recording classification associations with chest radiographs with World Health Organization (WHO)-defined primary endpoint pneumonia (radiographic pneumonia) or mortality were evaluated. We also examined case fatality among risk strata. RESULTS: Among children without WHO danger signs, wheezing (without crackles) had a lower adjusted odds ratio (aOR) for radiographic pneumonia (0.35, 95% confidence interval (CI): 0.15, 0.82), compared to children with normal recordings. Neither crackle only (no wheeze) (aOR: 2.13, 95% CI: 0.91, 4.96) or any wheeze (with or without crackle) (aOR: 0.63, 95% CI: 0.34, 1.15) were associated with radiographic pneumonia. Among children with WHO danger signs no lung recording classification was independently associated with radiographic pneumonia, although trends toward greater odds of radiographic pneumonia were observed among children classified with crackle only (no wheeze) or any wheeze (with or without crackle). Among children without WHO danger signs, those with recorded wheezing had a lower case fatality than those without wheezing (3.8% vs. 9.1%, p = .03). CONCLUSIONS: Among lower risk children without WHO danger signs digitally recorded wheezing is associated with a lower odds for radiographic pneumonia and with lower mortality. Although further research is needed, these data indicate that with further development digital auscultation may eventually contribute to child pneumonia care.