Repurposing rapid diagnostic tests to detect falsified vaccines in supply chains.

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose but have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global 'Prevent, Detect and Respond' strategy.

Estimating the cost of illness of acute Japanese encephalitis and sequelae care in Vietnam and Laos: A cross-sectional study.

BACKGROUND: Japanese encephalitis (JE) is a leading cause of acute encephalitis syndrome and resulting neurological disability in Asia and the Western Pacific. This study aims to estimate the cost of acute care, initial rehabilitation and sequelae care, in Vietnam and Laos. METHODOLOGY: We conducted a cross-sectional retrospective study using a micro-costing approach from the health system and household perspectives. Out-of-pocket direct medical and non-medical costs, indirect costs, and family impact were reported by patients and/or caregivers. Hospitalization costs were extracted from hospital charts. Acute costs covered expenditures from pre-hospital to follow-up visits while sequelae care costs were estimated from expenditures in the last 90 days. All costs are in 2021 US dollars. PRINCIPAL FINDINGS: 242 patients in two major sentinel sites in the North and South of Vietnam and 65 patients in a central hospital in Vientiane, Laos, with laboratory-confirmed JE were recruited regardless of age, sex, and ethnicity. In Vietnam, the mean total cost was $3,371 per acute JE episode (median $2,071, standard error [SE] $464) while annual costs were $404 for initial sequelae care (median $0, SE $220) and $320 for long-term sequelae care (median $0, SE $108). In Laos, the mean hospitalization costs in acute stage were $2,005 (median $1,698, SE $279) and the mean annual costs were $2,317 (median $0, SE $2,233) for initial sequelae care and $89 (median $0, SE $57) for long-term sequelae care. In both countries, most patients did not seek care for their sequelae. Families perceived extreme impact from JE and 20% to 30% of households still had sustained debts years after acute JE. CONCLUSIONS: JE patients and families in Vietnam and Laos suffer extreme medical, economic, and social hardship. This has policy implications for improving JE prevention in these two JE-endemic countries.

Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

Orthopoxvirus Seroprevalence and Infection Susceptibility in France, Bolivia, Laos, and Mali.

To determine a demographic overview of orthopoxvirus seroprevalence, we tested blood samples collected during 2003-2019 from France (n = 4,876), Bolivia (n = 601), Laos (n = 657), and Mali (n = 255) for neutralizing antibodies against vaccinia virus. In addition, we tested 4,448 of the 4,876 samples from France for neutralizing antibodies against cowpox virus. We confirmed extensive cross-immunity between the 2 viruses. Seroprevalence of antibodies was <1% in Bolivia, <5% in Laos, and 17.25% in Mali. In France, we found low prevalence of neutralizing antibodies in persons who were unvaccinated and vaccinated for smallpox, suggesting immunosenescence occurred in vaccinated persons, and smallpox vaccination compliance declined before the end of compulsory vaccination. Our results suggest that populations in Europe, Africa, Asia, and South America are susceptible to orthopoxvirus infections, which might have precipitated the emergence of orthopoxvirus infections such as the 2022 spread of monkeypox in Europe.

Childhood encephalitis in the Greater Mekong region (the SouthEast Asia Encephalitis Project): a multicentre prospective study.

BACKGROUND: Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and identifying clinical characteristics related to patients' conditions. METHODS: In this multicentre, observational, prospective study of childhood encephalitis, four referral hospitals in Cambodia, Vietnam, Laos, and Myanmar recruited children (aged 28 days to 16 years) who presented with altered mental status lasting more than 24 h and two of the following minor criteria: fever (within the 72 h before or after presentation), one or more generalised or partial seizures (excluding febrile seizures), a new-onset focal neurological deficit, cerebrospinal fluid (CSF) white blood cell count of 5 per mL or higher, or brain imaging (CT or MRI) suggestive of lesions of encephalitis. Comprehensive diagnostic procedures were harmonised across all centres, with first-line testing was done on samples taken at inclusion and results delivered within 24 h of inclusion for main treatable causes of disease and second-line testing was done thereafter for mostly non-treatable causes. An independent expert medical panel reviewed the charts and attribution of causes of all the included children. Using multivariate analyses, we assessed risk factors associated with unfavourable outcomes (ie, severe neurological sequelae and death) at discharge using data from baseline and day 2 after inclusion. This study is registered with ClinicalTrials.gov, NCT04089436, and is now complete. FINDINGS: Between July 28, 2014, and Dec 31, 2017, 664 children with encephalitis were enrolled. Median age was 4·3 years (1·8-8·8), 295 (44%) children were female, and 369 (56%) were male. A confirmed or probable cause of encephalitis was identified in 425 (64%) patients: 216 (33%) of 664 cases were due to Japanese encephalitis virus, 27 (4%) were due to dengue virus, 26 (4%) were due to influenza virus, 24 (4%) were due to herpes simplex virus 1, 18 (3%) were due to Mycobacterium tuberculosis, 17 (3%) were due to Streptococcus pneumoniae, 17 (3%) were due to enterovirus A71, 74 (9%) were due to other pathogens, and six (1%) were due to autoimmune encephalitis. Diagnosis was made within 24 h of admission to hospital for 83 (13%) of 664 children. 119 (18%) children had treatable conditions and 276 (42%) had conditions that could have been preventable by vaccination. At time of discharge, 153 (23%) of 664 children had severe neurological sequelae and 83 (13%) had died. In multivariate analyses, risk factors for unfavourable outcome were diagnosis of M tuberculosis infection upon admission (odds ratio 3·23 [95% CI 1·04-10·03]), coma on day 2 (2·90 [1·78-4·72]), supplementary oxygen requirement (1·89 [1·25-2·86]), and more than 1 week duration between symptom onset and admission to hospital (3·03 [1·68-5·48]). At 1 year after inclusion, of 432 children who were discharged alive from hospital with follow-up data, 24 (5%) had died, 129 (30%) had neurological sequelae, and 279 (65%) had completely recovered. INTERPRETATION: In southeast Asia, most causes of childhood encephalitis are either preventable or treatable, with Japanese encephalitis virus being the most common cause. We provide crucial information that could guide public health policy to improve diagnostic, vaccination, and early therapeutic guidelines on childhood encephalitis in the Greater Mekong region. FUNDING: Institut Pasteur, Institut Pasteur International Network, Fondation Merieux, Aviesan Sud, INSERM, Wellcome Trust, Institut de Recherche pour le Développement (IRD), and Fondation Total.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

Detection and significance of neuronal autoantibodies in patients with meningoencephalitis in Vientiane, Lao PDR.

BACKGROUND: The importance of autoimmune encephalitis and its overlap with infectious encephalitides are not well investigated in South-East Asia. METHODS: We report autoantibody testing, using antigen-specific live cell-based assays, in a series of 134 patients (cerebrospinal fluid and sera) and 55 blood donor controls (sera), undergoing lumbar puncture for suspected meningoencephalitis admitted in Vientiane, Lao People's Democratic Republic (PDR). RESULTS: Eight of 134 (6%) patients showed detectable serum neuronal autoantibodies, against the N-methyl-D-aspartate and gamma-aminobutyric acid A receptors (NMDAR and GABAAR), and contactin-associated protein-like 2 (CASPR2). Three of eight patients had accompanying autoantibodies in cerebrospinal fluid (two with NMDAR and one with GABAAR antibodies), and in two of these the clinical syndromes were typical of autoimmune encephalitis. Three of the other five patients had proven central nervous system infections, highlighting a complex overlap between diverse infectious and autoimmune causes of encephalitis. No patients in this cohort were treated with immunotherapy, and the outcomes were poor, with improvement observed in a single patient. CONCLUSIONS: In Lao PDR, autoimmune encephalitis is underdiagnosed and has a poor prognosis. Empiric immunotherapy should be considered after treatable infectious aetiologies are considered unlikely. Awareness and diagnostic testing resources for autoimmune encephalitis should be enhanced in South-East Asia.

Zoonotic Pathogens in Wildlife Traded in Markets for Human Consumption, Laos.

We tested animals from wildlife trade sites in Laos for the presence of zoonotic pathogens. Leptospira spp. were the most frequently detected infectious agents, found in 20.1% of animals. Rickettsia typhi and R. felis were also detected. These findings suggest a substantial risk for exposure through handling and consumption of wild animal meat.

A case-control study of the causes of acute respiratory infection among hospitalized patients in Northeastern Laos.

With the advent of highly sensitive real-time PCR, multiple pathogens have been identified from nasopharyngeal swabs of patients with acute respiratory infections (ARIs). However, the detection of microorganisms in the upper respiratory tract does not necessarily indicate disease causation. We conducted a matched case-control study, nested within a broader fever aetiology project, to facilitate determination of the aetiology of ARIs in hospitalised patients in Northeastern Laos. Consenting febrile patients of any age admitted to Xiengkhuang Provincial Hospital were included if they met the inclusion criteria for ARI presentation (at least one of the following: cough, rhinorrhoea, nasal congestion, sore throat, difficulty breathing, and/or abnormal chest auscultation). One healthy control for each patient, matched by sex, age, and village of residence, was recruited for the study. Nasopharyngeal swabs were collected from participants and tested for 33 pathogens by probe-based multiplex real-time RT-PCR (FastTrack Diagnostics Respiratory pathogen 33 kit). Attributable fraction of illness for a given microorganism was calculated by comparing results between patients and controls (= 100 * [OR - 1]/OR) (OR = odds ratio). Between 24th June 2019 and 24th June 2020, 205 consenting ARI patients and 205 matching controls were recruited. After excluding eight pairs due to age mismatch, 197 pairs were included in the analysis. Males were predominant with sex ratio 1.2:1 and children < 5 years old accounted for 59% of participants. At least one potential pathogen was detected in 173 (88%) patients and 175 (89%) controls. ARI in admitted patients were attributed to influenza B virus, influenza A virus, human metapneumovirus (HMPV), and respiratory syncytial virus (RSV) in 17.8%, 17.2%, 7.5%, and 6.5% of participants, respectively. SARS-CoV-2 was not detected in any cases or controls. Determining ARI aetiology in individual patients remains challenging. Among hospitalised patients with ARI symptoms presenting to a provincial hospital in Northeastern Laos, half were determined to be caused by one of several respiratory viruses, in particular influenza A virus, influenza B virus, HMPV, and RSV.

Nasopharyngeal Pneumococcal Colonization Density Is Associated With Severe Pneumonia in Young Children in the Lao People's Democratic Republic.

BACKGROUND: No studies have explored the association between pneumococcal nasopharyngeal density and severe pneumonia using the World Health Organization (WHO) 2013 definition. In Lao People's Democratic Republic (Lao PDR), we determine the association between nasopharyngeal pneumococcal density and severe pneumonia in children. METHODS: A prospective observational study was undertaken at Mahosot Hospital, Vientiane, from 2014 to mid-2018. Children <5 years admitted with acute respiratory infections (ARIs) were included. Clinical and demographic data were collected alongside nasopharyngeal swabs for pneumococcal quantification by lytA real-time quantitative polymerase chain reaction. Severe pneumonia was defined using the 2013 WHO definition. For pneumococcal carriers, a logistic regression model examined the association between pneumococcal density and severe pneumonia, after adjusting for potential confounders including demographic and household factors, 13-valent pneumococcal conjugate vaccine status, respiratory syncytial virus co-detection, and preadmission antibiotics. RESULTS: Of 1268 participants with ARI, 32.3% (n = 410) had severe pneumonia and 36.9% (n = 468) had pneumococcal carriage. For pneumococcal carriers, pneumococcal density was positively associated with severe pneumonia (adjusted odds ratio, 1.4 [95% confidence interval, 1.1-1.8]; P = .020). CONCLUSIONS: Among children with ARIs and pneumococcal carriage, pneumococcal carriage density was positively associated with severe pneumonia in Lao PDR. Further studies may determine if pneumococcal density is a useful marker for pneumococcal conjugate vaccine impact on childhood pneumonia.

A clinical, aetiological, and public health perspective on central nervous system infections in Bolivia, 2017-2018.

Central nervous system (CNS) infections are important causes of morbidity and mortality worldwide. In Bolivia, aetiologies, case fatality, and determinants of outcome are poorly characterised. We attempted to investigate such parameters to guide diagnosis, treatment, prevention, and health policy. From Nov-2017 to Oct-2018, we prospectively enrolled 257 inpatients (20.2% HIV-positive patients) of all ages from healthcare centers of Cochabamba and Santa Cruz, Bolivia with a suspected CNS infection and a lumbar puncture performed. Biological diagnosis included classical microbiology, molecular, serological and immunohistochemical tests. An infectious aetiology was confirmed in 128/257 (49.8%) inpatients, including, notably among confirmed single and co-infections, Cryptococcus spp. (41.7%) and Mycobacterium tuberculosis (27.8%) in HIV-positive patients, and Mycobacterium tuberculosis (26.1%) and Streptococcus pneumoniae (18.5%) in HIV-negative patients. The total mortality rate was high (94/223, 42.1%), including six rabies cases. In multivariate logistic regression analysis, mortality was associated with thrombocytopenia (Odds ratio (OR) 5.40, 95%-CI 2.40-11.83) and hydrocephalus (OR 4.07, 95%-CI 1.35-12.23). The proportion of untreated HIV patients, late presentations of neurotuberculosis, the rate of pneumococcal cases, and rabies patients who did not benefit from a post-exposure prophylaxis, suggest that decreasing the burden of CNS infections requires reinforcing health policy regarding tuberculosis, rabies, S. pneumoniae vaccination, and HIV-infections.

Emergence of dengue virus serotype 2 in Mauritania and molecular characterization of its circulation in West Africa.

The number of sporadic and epidemic dengue fever cases have reportedly been increasing in recent years in some West African countries, such as Senegal and Mali. The first epidemic of laboratory-confirmed dengue occurred in Nouakchott, the capital city of Mauritania situated in the Saharan desert, in 2014. On-site diagnosis of dengue fever was established using a rapid diagnostic test for dengue. In parallel, the presence of Aedes aegypti mosquitoes in the city was confirmed. The initial diagnosis was confirmed by RT-PCR, which showed that all samples from the 2014 dengue epidemic in Nouakchott were dengue virus serotype 2 (DENV-2). The whole genome or envelope protein gene of these strains, together with other DENV-2 strains obtained from travelers returning from West African countries to France between 2016 and 2019 (including two Mauritanian strains in 2017 and 2018), were sequenced. Phylogenetic analysis suggested a recent emergence of an epidemic strain from the cosmopolitan genotype belonging to West African cosmopolitan lineage II, which is genetically distinct from African sylvatic genotype. The origin of this DENV-2 lineage is still unknown, but our data seem to suggest a recent and rapid dispersion of the epidemic strain throughout the region. More complete genome sequences of West African DENV-2 are required for a better understanding of the dynamics of its circulation. Arboviral surveillance and outbreak forecasting are urgently needed in West Africa.

Indirect effects of 13-valent pneumococcal conjugate vaccine on pneumococcal carriage in children hospitalised with acute respiratory infection despite heterogeneous vaccine coverage: an observational study in Lao People's Democratic Republic.

INTRODUCTION: Empiric data on indirect (herd) effects of pneumococcal conjugate vaccines (PCVs) in settings with low or heterogeneous PCV coverage are limited. The indirect effects of PCV, which benefits both vaccinated and non-vaccinated individuals, are mediated by reductions in vaccine-type (VT) carriage (a prerequisite for disease). The aim of this study among hospitalised children in Lao People's Democratic Republic (Lao PDR) is to determine the effectiveness of a 13-valent PCV (PCV13) against VT pneumococcal nasopharyngeal carriage (direct effects) and the association between village-level PCV13 coverage and VT carriage (indirect effects). METHODS: Pneumococcal nasopharyngeal carriage surveillance commenced in December 2013, shortly after PCV13 introduction (October 2013). We recruited and swabbed children aged 2-59 months admitted to hospital with acute respiratory infection. Pneumococci were detected using lytA quantitative real-time PCR and serotyped using microarray. PCV13 status and village-level PCV13 coverage were determined using written immunisation records. Associations between both PCV13 status and village-level PCV13 coverage and VT carriage were calculated using generalised estimating equations, controlling for potential confounders. RESULTS: We enrolled 1423 participants and determined PCV13 coverage for 368 villages (269 863 children aged under 5 years). By 2017, median village-level vaccine coverage reached 37.5%, however, the IQR indicated wide variation among villages (24.1-56.4). Both receipt of PCV13 and the level of PCV13 coverage were independently associated with a reduced odds of VT carriage: adjusted PCV13 effectiveness was 38.1% (95% CI 4.1% to 60.0%; p=0.032); and for each per cent increase in PCV13 coverage, the estimated odds of VT carriage decreased by 1.1% (95% CI 0.0% to 2.2%; p=0.056). After adjustment, VT carriage decreased from 20.0% to 12.8% as PCV13 coverage increased from zero to 60% among under 5. CONCLUSIONS: Despite marked heterogeneity in PCV13 coverage, we found evidence of indirect effects in Lao PDR. Individual vaccination with PCV13 was effective against VT carriage.

A pilot study to investigate the risk of SARS-CoV-2 household transmission following hospital discharge of confirmed cases in Vientiane Capital, Lao PDR.

Background: Global guidelines from the World Health Organization on discharging patients diagnosed with COVID-19 changed in 2021 to a symptom-based rather than negative PCR-based approach. Studies have shown that shedding of viable virus continues for approximately eight days after symptom onset in most patients. In Vientiane, Laos, until now, patients diagnosed with asymptomatic or mild COVID-19 are hospitalised for 2 weeks and then, if they still test PCR positive for SARS-CoV-2, stay for a further week in a designated quarantine hotel before being discharged home. Objective: The aim of this pilot study was to investigate the risk of transmission of SARS-CoV-2 to household contacts of discharged patients who are still PCR-positive following 2-3 weeks quarantine in Vientiane, Lao PDR. Methods: Adult participants, who were resident in Vientiane Capital and who were about to be discharged from hospital (after 2 weeks hospitalisation), or from a quarantine hotel, following a further one-week quarantine, were screened to assess eligibility for the study. The household of each case was visited a maximum of 48 hours before or up to 24 hours after the participant was discharged and a nasopharyngeal swab was taken from all household members. Repeat nasopharyngeal swabs from cases and contacts were taken on day 7 and day 14 after discharge home of each case. Results: Between 20th May 2021 and 27th August 2021, 55 cases and 84 contacts in 27 households were enrolled in the study. The median [range] age of all 139 included participants was 26.5 years [3 months to 83 years] and 83 (60%) were female. By household, the median [range] number of cases and contacts were 1 [1-6] and 3 [1-13] respectively. At discharge home 32/48 (67%) cases tested positive for SARS-CoV-2. By day 7 11 of 47 cases (23%) still tested positive for SARS-CoV-2 by PCR and by day 14 this number was 2/24 (8%). No contacts tested positive during follow up and the numbers tested at the time of discharge of the case, 7 days later and 2 weeks later were 56, 57 and 37 respectively. Loss to follow up at day 7 and day 14 ranged from 15-50% (participants not at home at the time of visits). Conclusion: In this pilot study we found no evidence of onward transmission of SARS-CoV-2 to contacts of cases discharged home with a positive PCR result. This suggests the current discharge policy for mild to moderate COVID-19 case following 2 weeks in hospital in the Lao PDR is safe.

Outcome of Japanese Encephalitis Virus (JEV) Infection in Pediatric and Adult Patients at Mahosot Hospital, Vientiane, Lao PDR.

Although Japanese encephalitis virus (JEV) infection is an important cause of acute febrile illness in Lao PDR (Laos), patient outcome has not been evaluated. We prospectively followed up 123 JEV-infected patients (70 children < 15 years and 53 adults ≥ 15 years) admitted at Mahosot Hospital, Vientiane, from 2003 to 2013. Japanese encephalitis virus infection was diagnosed by the detection of anti-JEV IgM in cerebrospinal fluid and/or IgM seroconversion. Neurological sequelae were assessed using the Liverpool Outcome Score (LOS), total (maximum score = 75), and final (maximum score = 5). The median (interquartile range [IQR]) age of the patients was 12.0 (7.5-18.8) years, and 57% were male. The median (IQR) duration of patients' follow-up was 4.5 (3.2-7.3) years. Of all patients, 10/123 (8.1%) died during hospitalization, and 13/123 (10.6%) died at home after discharge, giving a mortality of 18.7% (23/123) (33 [26.8%] patients were lost to follow-up). The frequency of neurological sequelae at the last follow-up was 61.2% (48.4% in adults and 69.4% in children, P = 0.135). The proportion of patients with severe and moderate functional impairment at the last follow-up was significantly higher in children (25%) than in adults (6.5%), P = 0.042. Half of the patients who were still alive at the last follow-up (67) and for whom LOS data were available (22) had improvements in their total and final LOS between discharge and the last follow-up. The total and final LOS at discharge were not significantly different between children and adults, but total LOS at the last follow-up was significantly higher in adults than in children (median [IQR]: 74.5 [73-75] versus 73.0 [73-75], P = 0.019).

Diagnostic accuracy of an in-house Scrub Typhus enzyme linked immunoassay for the detection of IgM and IgG antibodies in Laos.

Scrub typhus is a major cause of morbidity and mortality in Southeast Asia. Diagnosis of scrub typhus is difficult due to a lack of accessible validated diagnostic tools. Despite its objectivity, the diagnostic accuracy of ELISA tests is influenced by methodological and patient factors. This study aims to evaluate the performance of a novel in-house ELISA developed in the Mahidol Oxford Tropical Medicine Research Unit (MORU) for anti-scrub typhus group IgM and IgG compared to the "gold standard" reference IFA and PCR, and to determine whether the in-house ELISA can be used as a seroepidemiological screening tool and/or stand-alone test for scrub typhus. A total of 1,976 admission and 1,438 participant follow-up sera collected in the Lao PDR (Laos) were tested with ELISA for IgM and IgG. Samples with an ELISA OD≥0.50 were tested with IFA for IgM and/or IgG. A strong positive relationship was present between ELISA ODs and IFA titers for admission IgM (r2: 0.70, p <0.005) and IgG (r2: 0.76, p<0.005), and for follow-up IgM and IgG (both r2: 0.76, p<0.005) samples. The best compromise between sensitivity and specificity for the ELISA OD cut-off is likely to be between 0.8-1.0 for IgM antibodies and 1.2-1.8 for IgG antibodies. These results demonstrate that the diagnostic accuracy of the MORU in-house scrub typhus group ELISA is comparable to that of IFA, with similar results as reported for the commonly used InBios Scrub Typhus Detect ELISA, validating the use of the in-house ELISA. The optimal ELISA cut-off would depend on the use of the test, and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This in-house ELISA has the potential to replace the imperfect IFA, which could ultimately reduce the burden of scrub typhus by improving the rate of scrub typhus diagnoses in endemic low-resource areas.

Spatial epidemiology of Japanese encephalitis virus and other infections of the central nervous system infections in Lao PDR (2003-2011): A retrospective analysis.

BACKGROUND: Central nervous system (CNS) infections are important contributors to morbidity and mortality and the causative agents for ~50% patients are never identified. The causative agents of some CNS infections have distinct spatial and temporal patterns. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the results of a spatial epidemiological and ecological analysis of CNS infections in Lao PDR (2003-2011). The data came from hospitalizations for suspected CNS infection at Mahosot Hospital in Vientiane. Out of 1,065 patients, 450 were assigned a confirmed diagnosis. While many communities in Lao PDR are in rural and remote locations, most patients in these data came from villages along major roads. Japanese encephalitis virus ((JEV); n = 94) and Cryptococcus spp. (n = 70) were the most common infections. JEV infections peaked in the rainy season and JEV patients came from villages with higher surface flooding during the same month as admission. JEV infections were spatially dispersed throughout rural areas and were most common in children. Cryptococcus spp. infections clustered near Vientiane (an urban area) and among adults. CONCLUSIONS/SIGNIFICANCE: The spatial and temporal patterns identified in this analysis are related to complex environmental, social, and geographic factors. For example, JEV infected patients came from locations with environmental conditions (surface water) that are suitable to support larger mosquito vector populations. Most patients in these data came from villages that are near major roads; likely the result of geographic and financial access to healthcare and also indicating that CNS diseases are underestimated in the region (especially from more remote areas). As Lao PDR is undergoing major developmental and environmental changes, the space-time distributions of the causative agents of CNS infection will also likely change. There is a major need for increased diagnostic abilities; increased access to healthcare, especially for rural populations; and for increased surveillance throughout the nation.

Harnessing Dengue Rapid Diagnostic Tests for the Combined Surveillance of Dengue, Zika, and Chikungunya Viruses in Laos.

Recent expansions of vector-borne diseases highlight the need for improved surveillance, especially in resource-poor settings. Dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) share the same vectors as well as similar clinical presentations, suggesting that combined surveillance would be useful. We hypothesized that blood spotted on dengue rapid diagnostic tests (RDTs) could be harnessed for sample collection in remote areas for subsequent detection of DENV, CHIKV, and ZIKV by reverse transcription real-time polymerase chain reaction (RT-qPCR). CHIKV and ZIKV dilutions were spotted on dengue RDTs (SD BIOLINE Dengue DUO, Standard Diagnostics, Gyeonggi-do, Republic of Korea), dried, and extracted. As reference, aliquots of each viral dilution were directly extracted. Using specific RT-qPCR tests, both viruses were successfully detected from RDT extracts. However, the limit of detection was slightly lower in comparison to direct extracts, two logfold for CHIKV and one logfold for ZIKV. For analysis of temperature stability, DENV dilutions were spotted on RDTs and stored for up to 2 months at -80°C, 4°C, or 35°C before testing. Storage of RDTs for 2 months at 35°C did not compromise detection of RNA by RT-qPCR; only minimal degradation was observed. This proof-of-principle study demonstrates the potential of using dengue RDTs for DENV/CHIKV/ZIKV combined surveillance in areas without access to laboratory facilities. Further investigations are needed for evaluation of tri-viral surveillance under field conditions using patient samples. Large-scale implementation of surveillance for these viruses is of crucial public health importance for the early detection of epidemics. This method also has important implications for improving understanding of the molecular epidemiology of the three viruses.