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 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.

Management of Central Nervous System Infections, Vientiane, Laos, 2003-2011.

During 2003-2011, we recruited 1,065 patients of all ages admitted to Mahosot Hospital (Vientiane, Laos) with suspected central nervous system (CNS) infection. Etiologies were laboratory confirmed for 42.3% of patients, who mostly had infections with emerging pathogens: viruses in 16.2% (mainly Japanese encephalitis virus [8.8%]); bacteria in 16.4% (including Orientia tsutsugamushi [2.9%], Leptospira spp. [2.3%], and Rickettsia spp. [2.3%]); and Cryptococcus spp. fungi in 6.6%. We observed no significant differences in distribution of clinical encephalitis and meningitis by bacterial or viral etiology. However, patients with bacterial CNS infection were more likely to have a history of diabetes than others. Death (26.3%) was associated with low Glasgow Coma Scale score, and the mortality rate was higher for patients with bacterial than viral infections. No clinical or laboratory variables could guide antibiotic selection. We conclude that high-dependency units and first-line treatment with ceftriaxone and doxycycline for suspected CNS infections could improve patient survival in Laos.

Rotavirus diarrhea in hospitalized children under 5 years of age in Vientiane, Lao PDR, 2009-2015.

BACKGROUND: Diarrhea is a leading cause of death in children <5 years worldwide, causing an estimated 215,000 deaths in 2013. This evaluation tracks the epidemiologic patterns and most common rotavirus genotypes among hospitalized children in this age group with acute gastroenteritis in Lao PDR. METHODS: Children <5 years in a central tertiary hospital in the capital city were prospectively enrolled into the surveillance platform during January 2009-December 2015. We collected information regarding clinical characteristics of enrolled children. Stool samples were obtained within 24 h of hospital admission and tested for rotavirus using rotavirus antigen detection enzyme immunoassay. Samples were sent to the regional reference laboratories in Australia and South Korea for genotyping. Bivariate analyses compared demographic and clinical characteristics between rotavirus positive and negative children using Chi-square statistical testing. Seasonality of rotavirus and annual genotype distribution are also described. RESULTS: We enrolled 1853 children <5 years with acute gastroenteritis during the surveillance period and collected 1772 fecal specimens, 982 (55%) of which tested positive for rotavirus. A higher proportion of rotavirus acute gastroenteritis was observed among children 12-23 months of age as compared to rotavirus negative children in the same age group, 41% vs 36%. Eighty-six percent of rotavirus positive children experienced vomiting, as compared to 65% of rotavirus negative children. Eighty-five percent (n = 830/982) of rotavirus positive specimens occurred during the dry season (January-April). The most common genotypes identified were G1, G2, G3 and P8 and P4. The most prevalent combined genotype differed annually during the surveillance period. CONCLUSION: Surveillance continues to be important in documenting the burden of rotavirus in children <5 years in Lao PDR as well as providing a baseline for determining the impact of rotavirus vaccine once it is introduced into Lao PDR's national immunization schedule.

Molecular epidemiology of dengue viruses in three provinces of Lao PDR, 2006-2010.

Few data on dengue epidemiology are available for Lao PDR. Here, we provide information on the complexity of dengue epidemiology in the country, demonstrating dynamic circulation that varies over space and time, according to serotype. We recruited 1,912 consenting patients presenting with WHO dengue criteria at Mahosot Hospital, Vientiane (central Laos), between 2006 and 2010. Between 2008 and 2010, 1,413 patients with undifferentiated fever were also recruited at Luang Namtha (LNT) Provincial Hospital (northern Laos) and 555 at Salavan (SV) Provincial Hospital (southern Laos). We report significant variations in Dengue virus (DENV) circulation between the three sites. Peaks of DENV infection were observed in the rainy seasons, although 11% of confirmed cases in the provinces and 4.6% in the capital were detected during the dry and cool seasons (between December and February). Four DENV serotypes were detected among the 867 RT-PCR positive patients: 76.9% DENV-1, 9.6% DENV-2, 7.7% DENV-4 and 5.3% DENV-3. DENV-1 was the predominant serotype throughout the study except in LNT in 2008 and 2009 when it was DENV-2. Before July 2009, DENV-2 was not detected in SV and only rarely detected in Vientiane. DENV-3 and DENV-4 were commonly detected in Vientiane, before 2008 for DENV-4 and after 2009 for DENV-3. The phylogenetic analyses of DENV envelope sequences suggest concurrent multiple introductions of new strains as well as active DENV circulation throughout Laos and with neighboring countries. It is therefore of great importance to develop and strengthen a year-round nation-wide surveillance network in order to collect data that would allow anticipation of public health issues caused by the occurrence of large dengue outbreaks.

High prevalence of Tropheryma whipplei in Lao kindergarten children.

BACKGROUND: Tropheryma whipplei is a bacterium commonly found in feces of young children in Africa, but with no data from Asia. We estimated the prevalence of T. whipplei carriage in feces of children in Lao PDR (Laos). METHODS/PRINCIPAL FINDINGS: Using specific quantitative real-time PCR, followed by genotyping for each positive specimen, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Those aged ≤ 4 years were significantly less frequently positive (17/52, 33%) than older children (34/54, 63%; p< 0.001). Positive samples were genotyped. Eight genotypes were detected including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively) whereas genotype 139 was specific to Akad (10.55%). CONCLUSIONS/SIGNIFICANCE: T. whipplei is a widely distributed bacterium, highly prevalent in feces of healthy children in Laos. Further research is needed to identify the public health significance of this finding.

SYBR green real-time PCR for the detection of all enterovirus-A71 genogroups.

Enterovirus A71 (EV-A71) has recently become an important public health threat, especially in South-East Asia, where it has caused massive outbreaks of Hand, Foot and Mouth disease every year, resulting in significant mortality. Rapid detection of EV-A71 early in outbreaks would facilitate implementation of prevention and control measures to limit spread. Real-time RT-PCR is the technique of choice for the rapid diagnosis of EV-A71 infection and several systems have been developed to detect circulating strains. Although eight genogroups have been described globally, none of these PCR techniques detect all eight. We describe, for the first time, a SYBR Green real-time RT-PCR system validated to detect all 8 EV-A71 genogroups. This tool could permit the early detection and shift in genogroup circulation and the standardization of HFMD virological diagnosis, facilitating networking of laboratories working on EV-A71 in different regions.

An epidemic of dengue-1 in a remote village in rural Laos.

In the Lao PDR (Laos), urban dengue is an increasingly recognised public health problem. We describe a dengue-1 virus outbreak in a rural northwestern Lao forest village during the cool season of 2008. The isolated strain was genotypically "endemic" and not "sylvatic," belonging to the genotype 1, Asia 3 clade. Phylogenetic analyses of 37 other dengue-1 sequences from diverse areas of Laos between 2007 and 2010 showed that the geographic distribution of some strains remained focal overtime while others were dispersed throughout the country. Evidence that dengue viruses have broad circulation in the region, crossing country borders, was also obtained. Whether the outbreak arose from dengue importation from an urban centre into a dengue-naïve community or crossed into the village from a forest cycle is unknown. More epidemiological and entomological investigations are required to understand dengue epidemiology and the importance of rural and forest dengue dynamics in Laos.

Inter- and intra-operator variability in the reading of indirect immunofluorescence assays for the serological diagnosis of scrub typhus and murine typhus.

Inter- and intra-observer variation was examined among six microscopists who read 50 scrub typhus (ST) and murine typhus (MT) indirect immunofluorescence assay (IFA) immunoglobulin M (IgM) slides. Inter-observer agreement was moderate (κ = 0.45) for MT and fair (κ = 0.32) for ST, and was significantly correlated with experience (P = 0.03 and P = 0.004, respectively); κ-scores for intra-observer agreement between morning and afternoon readings (range = 0.35-0.86) were not correlated between years of experience for ST and MT IFAs (Spearman's ρ = 0.31, P = 0.54 and P = 0.14, respectively; P = 0.78). Storage at 4°C for 2 days showed a change from positive to negative in 20-32% of slides. Although the titers did not dramatically change after 14 days of storage, the final interpretation (positive to negative) did change in 36-50% of samples, and it, therefore, recommended that slides should be read as soon as possible after processing.

Complete Genome of a Genotype I Japanese Encephalitis Virus Isolated from a Patient with Encephalitis in Vientiane, Lao PDR.

Japanese encephalitis virus (JEV) (Flaviviridae, Flavivirus) is an arthropod-borne flavivirus transmitted by Culex species mosquitoes. We report here the complete genome of the JEV genotype I strain JEV_CNS769_Laos_2009 isolated from an infected patient in Vientiane, Lao People's Democratic Republic (PDR) (Laos).

Causes of non-malarial fever in Laos: a prospective study.

BACKGROUND: Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. METHODS: For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5-49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. FINDINGS: With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. INTERPRETATION: Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. FUNDING: Wellcome Trust, WHO-Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention