Genomic Characterization of Recrudescent Plasmodium malariae after Treatment with Artemether/Lumefantrine.

Plasmodium malariae is the only human malaria parasite species with a 72-hour intraerythrocytic cycle and the ability to persist in the host for life. We present a case of a P. malariae infection with clinical recrudescence after directly observed administration of artemether/lumefantrine. By using whole-genome sequencing, we show that the initial infection was polyclonal and the recrudescent isolate was a single clone present at low density in the initial infection. Haplotypic analysis of the clones in the initial infection revealed that they were all closely related and were presumably recombinant progeny originating from the same infective mosquito bite. We review possible explanations for the P. malariae treatment failure and conclude that a 3-day artemether/lumefantrine regimen is suboptimal for this species because of its long asexual life cycle.

Staphylococcus aureus Prostatic abscess: a clinical case report and a review of the literature.

BACKGROUND: Prostatic abscess is a rare complication of acute bacterial prostatitis and is most commonly caused by Enterobacteriaceae. We report on a case of prostatic abscess caused by Staphylococcus aureus and conduct a review of the literature. CASE PRESENTATIVE: We present a case of S. aureus prostatic abscess that was successfully treated with a combination of antibiotic and surgical therapy. The isolate was non­multidrug-resistant, methicillin-resistant Staphylococcus aureus and was genotyped as clonal complex 5, an emerging regional clone that is trimethoprim resistant and Panton-Valentine leukocidin positive. This current case report is the first to describe the use of clindamycin step-down therapy. A literature review identified a further 39 cases of S. aureus prostatic abscesses, of which 26 were methicillin resistant. CONCLUSION: S. aureus is an uncommon cause of prostatic abscess. Optimal management includes both antibiotic therapy and surgical drainage. Our use of clindamycin as step-down therapy was guided by its excellent prostatic penetration.

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.

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.

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.

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.

Development, deployment and in-field demonstration of mobile coronavirus SARS-CoV-2 Nucleic acid amplification test.

Introduction. The evolving SARS-CoV-2 coronavirus pandemic presents a series of challenges to clinical diagnostic services. Many proprietary PCR platforms deployed outside centralised laboratories have limited capacity to upscale when public health demands increase. We set out to develop and validate an open-platform mobile laboratory for remote area COVID-19 diagnosis, with a subsequent field trial.Gap Statement. In regional Western Australia, molecular diagnostic support is limited to near point-of-care devices. We therefore aimed to demonstrate open-platform capability in a rapidly deployable format within the context of the COVID-19 pandemic.Methodology. We compared, selected and validated components of a SARS-CoV-2 RT-PCR assay in order to establish a portable molecular diagnostics laboratory. The optimal combination of PCR assay equipment, reagents and consumables required for operation to national standards in regional laboratories was identified. This comprised RNA extraction and purification (QuickGene-480, Kurabo, Japan), a duplex RT-PCR assay (Logix Smart COVID-19, Co-Diagnostics, USA), a Myra liquid handling robot (Biomolecular Systems, Australia) and a magnetic induction thermal cycler (MIC, Biomolecular Systems).Results The 95 and 99% limits of detection were 1.01 copies µl-1 (5.05 copies per reaction) and 2.80 copies µl-1 (14.00 copies per reaction) respectively. The Co-Diagnostics assay amplified both SARS-CoV-1 and -2 RNA but showed no other cross reactivity. Qualitative results aligned with the reference laboratory SARS-CoV-2 assay (sensitivity 100% [95 % CI 96.48-100%], specificity 100% [95% CI 96.52-100%]). In field trials, the laboratory was operational within an hour of arrival on-site, can process up to 36 samples simultaneously, produces results in two and a half hours from specimen reception, and performed well during six consecutive runs during a 1 week deployment.Conclusion. Our mobile laboratory enables an adaptive response to increased test demand, and unlike many proprietary point-of-care PCR systems, rapid substitution with an alternative assay if gene targets change or reagent supply chains fail. We envisage operation of this RT-PCR assay as a standby capability to meet varying regional test demands under public health emergency operations guidance.

Estimates of Antibacterial Consumption in Timor-Leste Using Distribution Data and Variation in Municipality Usage Patterns.

The association between antimicrobial resistance and antimicrobial usage has become a growing global concern. Many lower-middle income countries including Timor-Leste (TL) have limited information on antimicrobial usage, although recent research suggests increasing resistance rates among human pathogens there. The aim of this study was to use distribution data to estimate antibiotic consumption at both the national and sub-national level in Timor-Leste, stratifying into resistance class and adherence to the national essential medicines list (EML) and WHO AWaRe guidelines. A retrospective review of distribution data from Timor-Leste central medical store (SAMES) was undertaken to give a defined daily dose (DDD)/1000 inhabitants/day using WHO methodology. National antibiotic distribution in the TL EML in 2019 was estimated at 11.1 DDD/1000 inhabitants/day, comparable to consumption rates observed in other lower-middle-income countries using similar methodology. Differences in distribution quantities were noted between municipalities, with 4 of the 13 municipalities notably above the national average and around 32% of listed restricted antimicrobials distributed incongruent with the EML. This study provides insights into estimated antimicrobial consumption in Timor-Leste that has previously been poorly defined. Estimates of consumption can be used to understand emerging resistance in this small island nation, add to the body of knowledge on antimicrobial use to advise policy and guideline development, and help with stewardship activities.

Development of a Mobile Laboratory for Sudden Onset Disasters.

OBJECTIVES: Clinical diagnostics in sudden onset disasters have historically been limited. We set out to design, implement, and evaluate a mobile diagnostic laboratory accompanying a type 2 emergency medical team (EMT) field hospital. METHODS: Available diagnostic platforms were reviewed and selected against in field need. Platforms included HemoCue301/WBC DIFF, i-STAT, BIOFIRE FILMARRAY multiplex rt-PCR, Olympus BX53 microscopy, ABO/Rh grouping, and specific rapid diagnostic tests. This equipment was trialed in Katherine, Australia, and Dili, Timor-Leste. RESULTS: During the initial deployment, an evaluation of FilmArray tests was successful using blood culture identification, gastrointestinal, and respiratory panels. HemoCue301 (n = 20) hemoglobin values were compared on Sysmex XN 550 (r = 0.94). HemoCue WBC DIFF had some variation, dependent on the cell, when compared with Sysmex XN 550 (r = 0.88-0.16). i-STAT showed nonsignificant differences against Vitros 250. Further evaluation of FilmArray in Dili, Timor-Leste, diagnosed 117 pathogens on 168 FilmArray pouches, including 25 separate organisms on blood culture and 4 separate cerebrospinal fluid pathogens. CONCLUSION: This mobile laboratory represents a major advance in sudden onset disaster. Setup of the service was quick (< 24 hr) and transport to site rapid. Future deployment in fragmented health systems after sudden onset disasters with EMT2 will now allow broader diagnostic capability.

Impact of Cerebrospinal Fluid Multiplex Assay on Diagnosis and Outcomes of Central Nervous System Infections in Children: A Before and After Cohort Study.

BACKGROUND: This study evaluated the performance of cerebrospinal fluid multiplex assay in the diagnosis of pediatric central nervous system (CNS) infection, and assessed for the effect on clinical management. METHODS: A 15-month prospective cohort of pediatric patients with confirmed CNS infection was compared with a 15-month retrospective cohort from the Top End region of the Northern Territory, Australia. The study characterized all the CNS infections over the 30-month period and compared the time to organism identification and antibiotic management before and after the introduction of the multiplex assay. RESULTS: Thirty-six cases of pediatric CNS infection were diagnosed before the introduction of the multiplex assay, and 29 afterwards. Multiplex assay was performed on 26/29 (90%) of the cerebrospinal fluid isolates from children with confirmed CNS infections in the prospective cohort. Enterovirus was the most common causative organism identified in 14 children, followed by human parechovirus in 4 children. The multiplex assay performed with 93.8% sensitivity and 90.0% specificity when compared with microbiologic culture or reference laboratory results. The median time to organism identification reduced from 6.0 to 2.0 days (P value <0.001), the median duration of antibiotic therapy from 3.0 to 2.0 days (P value <0.001) and median hospitalization reduced from 5.0 to 3.0 days (P value 0.016) after introduction of the multiplex assay. CONCLUSIONS: The multiplex assay is a useful adjunct diagnostic tool enabling prompt organism identification and reducing antibiotic treatment and hospitalization duration. The assay would be of most value to hospitals that do not have access to an onsite molecular laboratory.

Antimicrobial resistance in urine and skin isolates in Timor-Leste.

OBJECTIVES: High rates of antimicrobial resistance (AMR) are seen throughout Southeast Asia. However, limited AMR data exist for Timor-Leste, which is situated on the south-eastern portion of the Malay Archipelago. The purpose of this study was to identify AMR in bacteria isolated from urine and skin swabs from patients in Dili, the capital of Timor-Leste. METHODS: Urine and skin swabs were collected from symptomatic patients in Timor-Leste and were processed for bacterial culture. Isolates were processed in Australia using a VITEK®2 system for bacterial identification and to determine antimicrobial susceptibility according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: A total of 154 urine isolates and 57 skin isolates were analysed. Of the Enterobacteriaceae, 35% were resistant to ceftriaxone with an extended-spectrum ß-lactamase (ESBL)-producing phenotype. Carbapenem resistance was not observed in any of the Gram-negative isolates. Of the Staphylococcus aureus isolates, 11% were of the community-associated methicillin-resistant S. aureus (CA-MRSA) phenotype. CONCLUSIONS: A moderately high proportion of Gram-negative urine isolates in Timor-Leste demonstrate phenotypic ESBL production, and a relatively low proportion of S. aureus isolates were methicillin-resistant. Improved understanding of AMR rates in Timor-Leste can help guide antimicrobial prescribing and inform antimicrobial stewardship strategies.

Hepatitis B virus and human T-cell lymphotropic virus type 1 co-infection in the Northern Territory, Australia.

OBJECTIVE: To establish the relationship between hepatitis B virus (HBV) and human T-cell lymphotropic virus type 1 (HTLV-1) serological markers in the Northern Territory, Australia. METHODS: A retrospective serological study of patients presenting to public healthcare facilities in the Northern Territory between 2008 and 2015 was performed in order to determine the presence and relationships of serological markers of HBV and HTLV-1. RESULTS: Seven hundred and forty individual patients were found to be serologically positive for HTLV-1 in the Northern Territory over the 8-year period. Hepatitis B results were available for 521 of these patients. Hepatitis B surface antigen (HBsAg) positivity was demonstrated in 15.9% (83/521) of this cohort, which was significantly different to the HTLV-1-negative group (3.7%, 125/3354) (p<0.001). Excluding individuals with isolated hepatitis B surface antibody (anti-HBs), those in the HTLV-1-positive group had a higher HBV exposure history (67.5%, 352/521) when compared to HTLV-1-negative individuals (37.8%, 1259/3354) (p<0.001). HTLV-1-positive individuals had a lower prevalence of HBV combined anti-HBs and hepatitis B core antibody (anti-HBc) positive markers compared to those who were HTLV-1-negative (56.3% (198/352) versus 73.8% (937/1269), respectively; p<0.001). CONCLUSIONS: A significantly higher prevalence rate of HBV was found in HTLV-1-positive individuals from the Northern Territory. When considering the higher exposure to HBV in HTLV-1-positive individuals, the clearance of HBV appears lower than in those individuals testing HTLV-1-negative. A lower prevalence of clearance in HTVL-1-positive individuals than in HTLV-1-negative individuals, as signified by formation of HBVcAb and HBVsAb in HTVL-1 positive individual's may equate to higher prevalence of ongoing coinfection.