National cross-sectional cluster survey of tuberculosis prevalence in Timor-Leste: a study protocol.

INTRODUCTION: Timor-Leste has one of the world's highest estimated tuberculosis (TB) incidences, yet the data which informs this estimate is limited and the true burden of TB disease is not known. TB prevalence surveys offer the best means of determining robust estimates of disease burden. This study aims to provide an estimate of the prevalence of bacteriologically confirmed pulmonary TB in Timor-Leste and provide additional insights into diagnostic coverage and health-seeking behaviour of persons with symptoms suggestive of TB. METHODS AND ANALYSIS: A national population-based cross-sectional cluster survey will be conducted in which participants aged 15 years and older will be screened for pulmonary TB using an algorithm consisting of symptom screening and digital X-ray of the chest with computer-aided detection software for X-ray interpretation. Xpert Ultra and liquid culture methods will be used to confirm survey TB cases. Additional data will be collected from persons reporting symptoms suggestive of TB to assess health-seeking behaviour and access to TB diagnosis and care. The survey aims to screen a target sample population of 20 068 people, living within 50 clusters, representing every municipality of Timor-Leste. Bacteriologically confirmed pulmonary TB prevalence will be estimated using WHO-recommended methods. ETHICS AND DISSEMINATION: Research ethics approval has been granted by the human research ethics committee of the Northern Territory, Australia, and the Instituto Nacional da Saúde, Timor-Leste. The results will be published in a peer-reviewed scientific journal and disseminated with relevant stakeholders. TRIAL REGISTRATION NUMBER: ACTRN12623000718640.

A pilot study investigating severe community-acquired febrile illness through implementation of an innovative microbiological and nucleic acid amplification testing strategy in Timor-Leste (ISIN-MANAS-TL).

Objectives: Acute febrile illness (AFI) causes significant health-seeking, morbidity, and mortality in Southeast Asia. This pilot study aimed to describe presentation, etiology, treatment, and outcomes of patients with AFI at one hospital in Timor-Leste and assessing the feasibility of conducting larger studies in this setting. Methods: Patients attending Hospital Nacional Guido Valadares with tympanic or axillary temperature ≥37.5°C in whom a blood culture was taken as part of routine clinical care were eligible. Participants were followed up daily for 10 days and again after 30 days. Whole blood was analyzed using a real-time quantitative polymerase chain reaction assay detecting dengue virus serotypes 1-4 and other arthropod-borne infections. Results: A total of 82 participants were recruited. Polymerase chain reaction testing was positive for dengue in 14 of 82 (17.1%) participants and blood culture identified a bacterial pathogen in three of 82 (3.7%) participants. Follow-up was completed by 75 of 82 (91.5%) participants. High rates of hospital admission (58 of 82, 70.7%), broad-spectrum antimicrobial treatment (34 of 82, 41.5%), and mortality (9 of 82, 11.0%) were observed. Conclusions: Patients with AFI experience poor clinical outcomes. Prospective observational and interventional studies assessing interventions, such as enhanced diagnostic testing, clinical decision support tools, or antimicrobial stewardship interventions, are required and would be feasible to conduct in this setting.

Prevalence of Antimicrobial Resistance in Escherichia coli and Salmonella Species Isolates from Chickens in Live Bird Markets and Boot Swabs from Layer Farms in Timor-Leste.

The rapid emergence of antimicrobial resistance is a global concern, and high levels of resistance have been detected in chicken populations worldwide. The purpose of this study was to determine the prevalence of antimicrobial resistance in Escherichia coli and Salmonella spp. isolated from healthy chickens in Timor-Leste. Through a cross-sectional study, cloacal swabs and boot swabs were collected from 25 live bird markets and two layer farms respectively. E. coli and Salmonella spp. from these samples were tested for susceptibility to six antimicrobials using a disk diffusion test, and a subset was tested for susceptibility to 27 antimicrobials using broth-based microdilution. E. coli and Salmonella spp. isolates showed the highest resistance towards either tetracycline or ampicillin on the disk diffusion test. E. coli from layer farms (odds ratio:5.2; 95%CI 2.0-13.1) and broilers (odds ratio:18.1; 95%CI 5.3-61.2) were more likely to be multi-drug resistant than those from local chickens. Based on the broth-based microdilution test, resistance to antimicrobials in the Timor-Leste Antimicrobial Guidelines for humans were low, except for resistance to ciprofloxacin in Salmonella spp. (47.1%). Colistin resistance in E. coli was 6.6%. Although this study shows that antimicrobial resistance in chickens was generally low in Timor-Leste, there should be ongoing monitoring in commercial chickens as industry growth might be accompanied with increased antimicrobial use.

The first confirmed outbreak of chikungunya reported in Timor-Leste, 2024.

Abstract: Timor-Leste is a mountainous, half-island nation with a population of 1.3 million, which shares a land border with Indonesia and is 550 km from Darwin, Australia. Since independence in 2002, Timor-Leste has achieved significant development; however, high levels of poverty remain. Chikungunya virus (CHIKV) is endemic in over 100 countries in Africa, Asia, Europe and in the Americas. It is transmitted by the bite of infected Aedes aegypti or Ae. albopictus mosquitoes, which are present in Timor-Leste and which contribute to annual rainy-season dengue virus (DENV) outbreaks. Symptomatic people typically suffer from acute onset of fever, usually accompanied by severe arthritis or arthralgia. Joint pain can be debilitating for several days, and may sometimes last for weeks, months or years. Unlike DENV infection which has significant mortality, most people recover completely. Between 2002 and 2023, there were 26 cases of CHIKV notified in Australia who acquired their infection in Timor-Leste; however, laboratory testing capability for CHIKV in Timor-Leste only became available in 2021 using polymerase chain reaction (PCR). The first locally diagnosed case was notified in November 2023. In January 2024, an outbreak of CHIKV was recognised in Timor-Leste for the first time, with 195 outbreak cases reported during 1-31 January 2024; all were PCR positive. There were no cases hospitalised, and no deaths. The median age of cases was 17 years (range 1-76 years); 51% were males. Cases were reported across the country; most (88/195) were from Dili, although the highest incidence was seen in the neighbouring municipality of Ermera (monthly incidence rate of 58.8 cases per 100,000 population). This first reported outbreak of CHIKV in Timor-Leste highlights the need for improved mosquito-borne illness control and response strategies, including minimising breeding sites and promoting early presentation for treatment and differential diagnosis from DENV, and consideration of the deployment of Wolbachia-infected mosquitoes, particularly as they have shown to reduce the transmission of CHIKV, DENV and Zika virus, all of which pose threats in Timor-Leste.

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.

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].

Melioidosis in Timor-Leste: First Case Description and Phylogenetic Analysis.

Burkholderia pseudomallei, the causative agent of melioidosis, has not yet been reported in Timor-Leste, a sovereign state northwest of Australia. In the context of improved access to diagnostic resources and expanding clinical networks in the Australasian region, we report the first 3 cases of culture-confirmed melioidosis in Timor-Leste. These cases describe a broad range of typical presentations, including sepsis, pneumonia, multifocal abscesses, and cutaneous infection. Phylogenetic analysis revealed that the Timor-Leste isolates belong to the Australasian clade of B. pseudomallei, rather than the Asian clade, consistent with the phylogeographic separation across the Wallace Line. This study underscores an urgent need to increase awareness of this pathogen in Timor-Leste and establish diagnostic laboratories with improved culture capacity in regional hospitals. Clinical suspicion should prompt appropriate sampling and communication with laboratory staff to target diagnostic testing. Local antimicrobial guidelines have recently been revised to include recommendations for empiric treatment of severe sepsis.

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 epidemiology of the COVID-19 pandemic in the small, low-resource country of Timor-Leste, January 2020 - June 2022.

Abstract: Timor-Leste, a small, mountainous half-island nation which shares a land border with Indonesia and which is 550 km from Australia, has a population of 1.3 million and achieved independence for the second time in 2002. It is one of the poorest nations in Asia. In response to the global coronavirus disease 2019 (COVID-19) pandemic, the Timor-Leste Ministry of Health undertook surveillance and contact tracing activities on all notified COVID-19 cases. Between 1 January 2020 and 30 June 2022, there were 22,957 cases of COVID-19 notified which occurred in three waves, the first which was delayed until April 2021 (community transmission of B.1.466.2 variant following major flooding), followed by waves in August 2021 (B.1.617.2 Delta variant transmission) and February 2022 (B.1.1.529 Omicron variant transmission). There were 753 people hospitalised due to COVID-19 and 133 deaths. Of the 133 deaths, 122 (92%) were considered not fully vaccinated (< 2 COVID-19 vaccines) and none had received boosters. Timor-Leste implemented measures to control COVID-19, including: rapid closure of international borders; isolation of cases; quarantining of international arrivals and close contacts; restrictions on internal travel; social and physical distancing; and, finally, a country-wide vaccination program. The health system's capacity was never exceeded.

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.

ACORN (A Clinically-Oriented Antimicrobial Resistance Surveillance Network) II: protocol for case based antimicrobial resistance surveillance.

Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs). Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies. Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest.

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.

Gastrointestinal Carriage of Antimicrobial Resistance in School-Aged Children in Three Municipalities of Timor-Leste.

Invasive bacterial infections are a leading cause of death in children, primarily in low- and middle-income countries (LMIC). Links between carriage of antimicrobial-resistant organisms and more resistant infections have been established; however, little has been reported regarding community carriage of antibiotic-resistant organisms such as extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in LMIC. The aim of this study was to determine colonic carriage of ESBL-producing fluoroquinolone- and aminoglycoside-resistant Enterobacterales in healthy children in three municipalities of Timor-Leste. In November 2020, 621 stool samples were collected from school-aged children and underwent screening for the presence of Enterobacterales species and antimicrobial resistance (AMR). Ciprofloxacin-resistant Gram-negative organisms were cultured from 16.5% (95% CI 6.2−26.9), and gentamicin resistance was identified in 6.8% (95% CI 2.8−10.7). Compared to the prevalence of ciprofloxacin resistance in Dili (36.1%), there was significantly lower prevalence in the rural municipalities of Ermera (12.9%; AOR 0.38, 95% CI 0.24−0.60, p < 0.001) and Manufahi (4.5%; AOR 0.07, 95% CI 0.01−0.51, p = 0.009). The overall cluster-adjusted prevalence of ESBL-producing bacteria was 8.3%, with no significant differences between municipalities. This study demonstrates high rates of carriage of AMR among school-aged children in Timor-Leste, with higher rates observed in Dili compared to rural municipalities. Empiric antibiotic guidelines should include recommendations for treating community-acquired infections that account for the possibility of antimicrobial resistance.

Serological surveillance of healthcare workers to evaluate natural infection- and vaccine-derived immunity to SARS-CoV-2 during an outbreak in Dili, Timor-Leste.

Background Serosurveillance can be used to investigate the extent and distribution of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a population. Characterisation of humoral immune responses gives insight into whether immunity is infection- or vaccine-derived. Methods A longitudinal study of health care workers (HCWs) in Dili, Timor-Leste, was conducted during vaccine rollout (ChAdOx1) and a concurrent SARS-CoV-2 outbreak. Results A total of 324 HCWs were included at baseline (April-May 2021). Out of those, 32 (9.9%) were seropositive for anti-nucleocapsid protein (anti-N) IgG antibodies, indicating a significant sub-clinical infection among HCWs early in the local outbreak. Follow-up was conducted in 157 (48.5%) participants (July-September 2021), by which time there had been high uptake of vaccination (91.7%), and 86.0% were seropositive for anti-spike protein antibodies. Acquisition of anti-N antibodies was observed in partially vaccinated HCWs (30/76, 39.5%), indicating some post-dose-1 infections. Discussion Serosurveillance of HCWs may provide early warning of SARS-CoV-2 outbreaks and should be considered in non-endemic settings, particularly where there is limited availability/uptake of testing for acute infection. Characterisation of humoral immune responses may be used to assess vaccine impact and coverage. Such studies should be considered in national and international efforts to investigate and mitigate against future emerging pathogens.