Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study.

Background: In Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. Methods: Between September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 µg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. Results: A total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 µg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 µg/ml, 12/15 were mcr-1. Conclusions: We found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Diagnostic accuracy of DPP Fever Panel II Asia tests for tropical fever diagnosis.

BACKGROUND: Fever is the most frequent symptom in patients seeking care in South and Southeast Asia. The introduction of rapid diagnostic tests (RDTs) for malaria continues to drive patient management and care. Malaria-negative cases are commonly treated with antibiotics without confirmation of bacteraemia. Conventional laboratory tests for differential diagnosis require skilled staff and appropriate access to healthcare facilities. In addition, introducing single-disease RDTs instead of conventional laboratory tests remains costly. To overcome some of the delivery challenges of multiple separate tests, a multiplexed RDT with the capacity to diagnose a diverse range of tropical fevers would be a cost-effective solution. In this study, a multiplex lateral flow immunoassay (DPP Fever Panel II Assay) that can detect serum immunoglobulin M (IgM) and specific microbial antigens of common fever agents in Asia (Orientia tsutsugamushi, Rickettsia typhi, Leptospira spp., Burkholderia pseudomallei, Dengue virus, Chikungunya virus, and Zika virus), was evaluated. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood (WB) and serum samples from 300 patients with undefined febrile illness (UFI) recruited in Vientiane, Laos PDR were tested using the DPP Fever Panel II, which consists of an Antibody panel and Antigen panel. To compare reader performance, results were recorded using two DPP readers, DPP Micro Reader (Micro Reader 1) and DPP Micro Reader Next Generation (Micro Reader 2). WB and serum samples were run on the same fever panel and read on both micro readers in order to compare results. ROC analysis and equal variance analysis were performed to inform the diagnostic validity of the test compared against the respective reference standards of each fever agent (S1 Table). Overall better AUC values were observed in whole blood results. No significant difference in AUC performance was observed when comparing whole blood and serum sample testing, except for when testing for R. typhi IgM (p = 0.04), Leptospira IgM (p = 0.02), and Dengue IgG (p = 0.03). Linear regression depicted R2 values had ~70% agreement across WB and serum samples, except when testing for leptospirosis and Zika, where the R2 values were 0.37 and 0.47, respectively. No significant difference was observed between the performance of Micro Reader 1 and Micro Reader 2, except when testing for the following pathogens: Zika IgM, Zika IgG, and B pseudomallei CPS Ag. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the diagnostic accuracy of the DPP Fever Panel II is comparable to that of commonly used RDTs. The optimal 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 multiplex RDT offers diagnostic benefits in areas with limited access to healthcare and has the potential to improve field testing capacities. This could improve tropical fever management and reduce the public health burden in endemic low-resource areas.

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis.IMPORTANCEBurkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

Evolutionary and functional history of the Escherichia coli K1 capsule.

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.

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.

Bartonella Species in Cambodia, Ghana, Laos, and Peru: Results from Vector and Serosurveys.

Background: Bartonella species are fastidious gram-negative vector-borne bacteria with a wide range of mammalian reservoirs. While it is understood that some species of Bartonella are human pathogens, the extent of human exposure to Bartonella species (both pathogenic and nonpathogenic) is yet to be fully understood. Materials and Methods: To this end, residual sera from participants enrolled in undifferentiated fever studies in Cambodia, Ghana, Laos, and Peru were screened for the presence of IgG antibodies against Bartonella quintana and Bartonella henselae, using the FOCUS diagnostics Dual Spot- Bartonella IgG Immunofluorescence assay. Forty-eight patients with suspected or confirmed Bartonella bacilliformis exposure or infection in Peru were screened to assess cross-reactivity of the FOCUS assay for IgG against other Bartonella species. Results: Ten of 13 patients with confirmed B. bacilliformis infection were Bartonella-specific IgG positive, and overall, 36/48 of the samples were positive. In addition, 79/206, 44/200, 101/180, and 57/100 of the samples from Peru, Laos, Cambodia, and Ghana, respectively, were Bartonella-specific IgG positive. Furthermore, ectoparasite pools from Cambodia, Laos, and Peru were tested using quantitative real-time PCR (qPCR) for the presence of Bartonella DNA. Of the sand fly pools collected in Peru, 0/196 were qPCR positive; 15/140 flea pools collected in Cambodia were qPCR positive; while 0/105 ticks, 0/22 fleas, and 0/3 louse pools collected in Laos tested positive for Bartonella DNA. Conclusion: Evidence of Bartonella in fleas from Cambodia supports the possibility that humans are exposed to Bartonella through this traditional vector. However, Bartonella species were not found in fleas, ticks, or lice from Laos, or sand flies from Peru. This could account for the lower positive serology among the population in Laos and the strictly localized nature of B. bacilliformis infections in Peru. Human exposure to the Bartonella species and Bartonella as a human pathogen warrants further investigation.

A robust host-response-based signature distinguishes bacterial and viral infections across diverse global populations.

Limited sensitivity and specificity of current diagnostics lead to the erroneous prescription of antibiotics. Host-response-based diagnostics could address these challenges. However, using 4,200 samples across 69 blood transcriptome datasets from 20 countries from patients with bacterial or viral infections representing a broad spectrum of biological, clinical, and technical heterogeneity, we show current host-response-based gene signatures have lower accuracy to distinguish intracellular bacterial infections from viral infections than extracellular bacterial infections. Using these 69 datasets, we identify an 8-gene signature to distinguish intracellular or extracellular bacterial infections from viral infections with an area under the receiver operating characteristic curve (AUROC) > 0.91 (85.9% specificity and 90.2% sensitivity). In prospective cohorts from Nepal and Laos, the 8-gene classifier distinguished bacterial infections from viral infections with an AUROC of 0.94 (87.9% specificity and 91% sensitivity). The 8-gene signature meets the target product profile proposed by the World Health Organization and others for distinguishing bacterial and viral infections.

Antibiotic knowledge, attitudes and reported practice during pregnancy and six months after birth: a follow- up study in Lao PDR.

BACKGROUND: Antibiotics are important medicines to prevent maternal and child morbidity and mortality. Women's knowledge and attitudes towards antibiotic use influence their practice. When they become mothers, this may be mirrored in the use of antibiotics for their newborn children. The current study aimed to assess knowledge, attitudes, and reported practice of pregnant women regarding antibiotic use and antibiotic resistance as well as their approach towards antibiotic use for their newborn babies. METHODS: This was a follow-up study with data collected via structured interviews between September 2019 and August 2020 in Feuang (rural) and Vangvieng (urban) districts in Vientiane province, Lao PDR. We identified and invited all women attending antenatal care in their third trimester of pregnancy in the selected areas. Using a structured questionnaire at third trimester of pregnancy we captured data on knowledge regarding antibiotic use and resistance. We collected information on attitudes and reported practice at two time points: (i) at third trimester of pregnancy and (ii) 6 months after birth. Univariate analysis and frequency distributions were used to study pattern of responses. Chi-square and Mann-Whitney tests were used to compare categorical and continuous variables respectively. P value < 0.05 was considered statistically significant. RESULTS: We surveyed 539 women with a mean age of 25 years. Two oral antibiotics, i) ampicillin and ii) amoxicillin were correctly identified by 68 and 47% of participants respectively. Only 24% of women (19% in Feuang and 29% in Vangvieng) answered correctly that antibiotics are effective against bacterial infections. The most prevalent response was "I don't know" suggesting the questions were challenging. Significantly less women would use antibiotics from a previous illness for their child than for themselves (16% vs 29%), however they would be more willing to use antibiotics for their baby even in case of mild symptoms (29% vs 17% while pregnant). The majority of antibiotics were prescribed by healthcare providers and 46% of children with the common cold received antibiotics. CONCLUSIONS: Women's knowledge was sub-optimal, still, they manifested appropriate attitudes towards antibiotic use during pregnancy and for their child. Nearly half of children received antibiotics for the common cold. There is a need for context adapted programs aiming at improving women's knowledge, as well as healthcare providers, emphasising rational antibiotic prescribing during pregnancy and for children.

Evaluation of trends in hospital antimicrobial use in the Lao PDR using repeated point-prevalence surveys-evidence to improve treatment guideline use.

Background: Antimicrobial use (AMU) is a key driver of antimicrobial resistance (AMR). There are few data on AMU, to inform optimizing antibiotic stewardship, in the Lao PDR (Laos). Methods: Point prevalence surveys (PPS) of AMU were conducted at four-month intervals in six general hospitals across Laos from 2017 to 2020, using modified Global-PPS data collection tools. The surveys focused on AMU amongst hospitalized inpatients. Findings: The overall prevalence of inpatient AMU was 71% (4,377/6,188), varying by hospital and survey round from 50·4% (135/268) to 88·4% (61/69). Of 4,377 patients, 44% received >one antimicrobial. The total number of prescriptions assessed was 6,555. Ceftriaxone was the most commonly used (39·6%) antimicrobial, followed by metronidazole (17%) and gentamicin (10%). Pneumonia was the most common diagnosis among those prescribed antimicrobials in both children aged ≤5 years (29% among aged ≤1 year and 27% among aged >1 to ≤5years) and adults aged ≥15 years at 9%. The percentage of antimicrobial use compliant with local treatment guidelines was 26%; inappropriate use was mainly found for surgical prophylaxis (99%). Adult patients received ACCESS group antimicrobials less commonly than children (47% vs 63%, p-value<0·0001). Most WATCH group prescriptions (99%) were without a microbiological indication. Interpretation: AMU among hospitalized patients in Laos is high with frequent inappropriate use of antimicrobials, especially as surgical prophylaxis. Continued monitoring and enhanced antimicrobial stewardship interventions are needed in Lao hospitals. Funding: The Wellcome Trust [Grant numbers 220211/Z/20/Z and 214207/Z/18/Z] and bioMérieux.

Perceptions and reported practices of pregnant women and mothers of children under two years of age regarding antibiotic use and resistance in Vientiane province, Lao PDR: a qualitative study.

BACKGROUND: Understanding pregnant women and mothers' perceptions towards antibiotic use and resistance is essential for appropriate antibiotic use and limiting antibiotic resistance. This study aimed to explore perceptions and reported practices of pregnant women and mothers with children under two years of age regarding correct antibiotic use and antibiotic resistance in Vientiane Province, Lao PDR. METHODS: The study employed an exploratory qualitative research design using focus groups discussions (FGDs). Participants were purposively selected based on: being pregnant at third trimester and attending antenatal care and mothers with children under two years of age, attending the health facility for postpartum visit /vaccinations. Six focus group discussions were conducted in September 2019 with a total of 55 women. The FGDs were transcribed verbatim, data were analyzed first by coding then categorizing the data as we looked for patterns and themes by using the qualitative content analysis. RESULTS: Most participants had some understanding of antibiotics but wrongly believed antibiotics can be used to treat viral disease. Over half of the participants had heard the term "antibiotic resistance", but often believed it was their bodies, not the bacteria that developed antibiotic resistance. During pregnancy and for their infants, women preferred to use antibiotics only when prescribed by a doctor. Outside of pregnancy however, consuming antibiotics without a prescription was commonly reported. Participants wanted more information about the indications for antibiotic use and antibiotic resistance. CONCLUSIONS: More effort is required to increase the level of understanding, and practice of mothers to promote optimal antibiotic use. Mothers' desire to learn more, and their fundamental concern for their children, can be used to promote appropriate antibiotic use. Awareness raising should be complemented by efforts to address other determinants of inappropriate antibiotic use, including educating healthcare workers, and pharmacists and addressing health service determinants that contribute to inappropriate antibiotic use.

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.

Antibiotic Prescribing in Connection to Childbirth: An Observational Study in Two Districts in Lao PDR.

Overuse and misuse of antibiotics has frequently been reported for obstetric conditions and procedures, which may impact both the mother and the unborn baby and increase antibiotic resistance. This study aimed to investigate the antibiotic prescribing pattern in connection to childbirth in two districts in Lao PDR. It is a cross-sectional observational study. Antibiotic prescription data related to childbirth was collected via reviews of medical records in two district hospitals and five health centers in Lao PDR from September 2019 to November 2020. In total, antibiotic prescription data for 1777 women were extracted from their medical records. It was found that all women received antibiotics during in-patient care irrespective of delivery mode. When in hospital, 85.5% of the women who underwent a caesarean section got antibiotic treatment for 5 days and women who had a vaginal delivery usually had antibiotic treatment for one day or less. All the women got oral antibiotics for an additional 4-5 days upon discharge. Antibiotic prescription rate in connection to childbirth was very high in comparison with the WHO guidelines, and antibiotics were used extensively in the participating health facilities. Interventions to guide appropriate prescribing behavior in relation to childbirth are urgently needed in Lao PDR.

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.

Utility of InTray COLOREX Screen agar and InTray COLOREX ESBL agar for urine culture in the Lao PDR.

BACKGROUND: There is a need for simple microbiology diagnostics to enable antimicrobial resistance surveillance in low- and middle-income countries. OBJECTIVES: To investigate the field utility of InTray COLOREX plates for urine culture and ESBL detection. METHODS: Clinical urine samples from Mahosot Hospital, Vientiane, Lao PDR were inoculated onto chromogenic media and InTray COLOREX Screen plates between June and August 2020. Urine and isolates from other clinical specimens were inoculated onto COLOREX ESBL plates. A simulated field study investigating the field utility of the InTray COLOREX plates was also completed. RESULTS: In total, 355 urine samples were inoculated onto standard chromogenic agar and InTray COLOREX Screen plates, and 154 urine samples and 54 isolates from other clinical specimens on the COLOREX ESBL plates. Growth was similar for the two methods (COLOREX Screen 41%, standard method 38%) with 20% discordant results, mainly due to differences in colony counts or colonial appearance. Contamination occurred in 13% of samples, with the COLOREX Screen plates showing increased contamination rates, potentially due to condensation. ESBL producers were confirmed from 80% of isolates from the COLOREX ESBL plates, and direct plating provided rapid detection of presumptive ESBL producers. Burkholderia pseudomallei also grew well on the ESBL plates, a relevant finding in this melioidosis-endemic area. CONCLUSIONS: The InTray COLOREX Screen and ESBL plates were simple to use and interpret, permitting rapid detection of uropathogens and ESBLs, and have the potential for easy transport and storage from field sites and use in laboratories with low capacity.

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.

A Clinically Oriented antimicrobial Resistance surveillance Network (ACORN): pilot implementation in three countries in Southeast Asia, 2019-2020.

Background: Case-based surveillance of antimicrobial resistance (AMR) provides more actionable data than isolate- or sample-based surveillance. We developed A Clinically Oriented antimicrobial Resistance surveillance Network (ACORN) as a lightweight but comprehensive platform, in which we combine clinical data collection with diagnostic stewardship, microbiological data collection and visualisation of the linked clinical-microbiology dataset. Data are compatible with WHO GLASS surveillance and can be stratified by syndrome and other metadata. Summary metrics can be visualised and fed back directly for clinical decision-making and to inform local treatment guidelines and national policy. Methods: An ACORN pilot was implemented in three hospitals in Southeast Asia (1 paediatric, 2 general) to collect clinical and microbiological data from patients with community- or hospital-acquired pneumonia, sepsis, or meningitis. The implementation package included tools to capture site and laboratory capacity information, guidelines on diagnostic stewardship, and a web-based data visualisation and analysis platform. Results: Between December 2019 and October 2020, 2294 patients were enrolled with 2464 discrete infection episodes (1786 community-acquired, 518 healthcare-associated and 160 hospital-acquired). Overall, 28-day mortality was 8.7%. Third generation cephalosporin resistance was identified in 54.2% (39/72) of E. coli and 38.7% (12/31) of K. pneumoniae isolates . Almost a quarter of S. aureus isolates were methicillin resistant (23.0%, 14/61). 290/2464 episodes could be linked to a pathogen, highlighting the level of enrolment required to achieve an acceptable volume of isolate data. However, the combination with clinical metadata allowed for more nuanced interpretation and immediate feedback of results. Conclusions: ACORN was technically feasible to implement and acceptable at site level. With minor changes from lessons learned during the pilot ACORN is now being scaled up and implemented in 15 hospitals in 9 low- and middle-income countries to generate sufficient case-based data to determine incidence, outcomes, and susceptibility of target pathogens among patients with infectious syndromes.

Knowledge, Attitudes, Perception and Reported Practices of Healthcare Providers on Antibiotic Use and Resistance in Pregnancy, Childbirth and Children under Two in Lao PDR: A Mixed Methods Study.

BACKGROUND: Overuse and misuse of antibiotics contribute unnecessarily to antibiotic resistance (ABR), and are thereby global health threats. Inappropriate prescriptions of antibiotics during pregnancy, delivery and early childhood are widespread across the world. This study aimed to assess knowledge, attitudes, and reported practices of healthcare providers (HCPs) and to explore their perceptions regarding antibiotic use and ABR related to pregnancy, childbirth, and children under two in Lao PDR. METHODS: This is a mixed methods study with data collection in 2019 via structured interviews among 217 HCPs (medical doctors/assistant doctors, midwives/nurses, pharmacists/assistant pharmacists and drug sellers), who prescribed/dispensed antibiotics in one rural and one urban district in Vientiane province and individual qualitative interviews with 30 HCPs and stakeholders. RESULTS: Of the HCPs, 36% had below average knowledge regarding antibiotic use and ABR, and 67% reported prescribing antibiotics for uncomplicated vaginal delivery. Half of the HCPs did not believe that their prescribing contributed to ABR, and only 9% had participated in antibiotic education. CONCLUSION: A substantial number of HCPs had suboptimal knowledge and prescribed antibiotics unnecessarily, thereby contributing to ABR. Continuous education and regular supervision of HCPs is recommended to improve the use of antibiotics related to pregnancy, childbirth, and young children.

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain.

BACKGROUND: Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. METHODOLOGY/PRINCIPAL FINDINGS: We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. CONCLUSIONS/SIGNIFICANCE: The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.