Intestinal parasitic infections and environmental water contamination in a rural village of northern Lao PDR

A field survey studying intestinal parasites in humans and microbial pathogen contamination at environment was performed in a Laotian rural village to identify potential risks for disease outbreaks. A parasitological investigation was conducted in Ban Lak Sip village, Luang Prabang, Lao PDR involving fecal samples from 305 inhabitants as well as water samples taken from 3 sites of the local stream. Water analysis indicated the presence of several enteric pathogens, i.e., Aeromonas spp., Vibrio spp., E. coli H7, E. coli O157: H7, verocytotoxin-producing E. coli (VTEC), Shigella spp., and enteric adenovirus. The level of microbial pathogens contamination was associated with human activity, with greater levels of contamination found at the downstream site compared to the site at the village and upstream, respectively. Regarding intestinal parasites, the prevalence of helminth and protozoan infections were 68.9% and 27.2%, respectively. Eight helminth taxa were identified in fecal samples, i.e., 2 tapeworm species (Taenia sp. and Hymenolepis diminuta), 1 trematode (Opisthorchis sp.), and 5 nematodes (Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, trichostrongylids, and hookworms). Six species of intestinal protists were identified, i.e., Blastocystis hominis, Cyclospora spp., Endolimax nana, Entamoeba histolytica/E. dispar, Entamoeba coli, and Giardia lamblia. Questionnaires and interviews were also conducted to determine risk factors of infection. These analyses together with a prevailing infection level suggested that most of villagers were exposed to parasites in a similar degree due to limited socio-economic differences and sharing of similar practices. Limited access to effective public health facilities is also a significant contributing factor

Pooling sputum testing to diagnose tuberculosis using xpert MTB/RIF and xpert ultra: a cost-effectiveness analysis

Background: The World Health Organization (WHO) recommends the diagnosis of tuberculosis (TB) using molecular tests, such as Xpert MTB/RIF (MTB/RIF) or Xpert Ultra (Ultra). These tests are expensive and resource-consuming, and cost-effective approaches are needed for greater coverage. Methods: We evaluated the cost-effectiveness of pooling sputum samples for TB testing by using a fixed amount of 1,000 MTB/RIF or Ultra cartridges. We used the number of people with TB detected as the indicator for cost-effectiveness. Cost-minimization analysis was conducted from the healthcare system perspective and included the costs to the healthcare system using pooled and individual testing. Results: There was no significant difference in the overall performance of the pooled testing using MTB/RIF or Ultra (sensitivity, 93.9% vs. 97.6%, specificity 98% vs. 97%, p-value > 0.1 for both). The mean unit cost across all studies to test one person was 34.10 international dollars for the individual testing and 21.95 international dollars for the pooled testing, resulting in a savings of 12.15 international dollars per test performed (35.6% decrease). The mean unit cost per bacteriologically confirmed TB case was 249.64 international dollars for the individual testing and 162.44 international dollars for the pooled testing (34.9% decrease). Cost-minimization analysis indicates savings are directly associated with the proportion of samples that are positive. If the TB prevalence is ≥ 30%, pooled testing is not cost-effective. Conclusion: Pooled sputum testing can be a cost-effective strategy for diagnosis of TB, resulting in significant resource savings. This approach could increase testing capacity and affordability in resource-limited settings and support increased testing towards achievement of WHO End TB strategy

Pooled testing of sputum with Xpert MTB/RIF and Xpert Ultra during tuberculosis active case finding campaigns in Lao People’s Democratic Republic

Introduction: Active case finding (ACF) of individuals with tuberculosis (TB) is a key intervention to find the 30% of people missed every year. However, ACF requires screening large numbers of individuals who have a low probability of positive results, typically <5%, which makes using the recommended molecular tests expensive. Methods: We conducted two ACF surveys (in 2020 and 2021) in high TB burden areas of Lao PDR. Participants were screened for TB symptoms and received a chest X-ray. Sputum samples of four consecutive individuals were pooled and tested with Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) (Xpert-MTB/RIF) (2020) or Xpert-Ultra (2021). The agreement of the individual and pooled samples was compared and the reasons for discrepant results and potential cartridge savings were assessed. Results: Each survey included 436 participants, which were tested in 109 pools. In the Xpert-MTB/RIF survey, 25 (sensitivity 89%, 95% CI 72.8% to 96.3%) of 28 pools containing MTB-positive samples tested positive and 81 pools containing only MTB-negative samples tested negative (specificity 100%, 95% CI 95.5% to 100%). In the Xpert-Ultra survey, all 32 (sensitivity 100%, 95% CI 89.3% to 100%) pools containing MTB-positive samples tested positive and all 77 (specificity 100%, 95% CI 95.3% to 100%) containing only MTB-negative samples tested negative. Pooling with Xpert-MTB/RIF and Xpert-Ultra saved 52% and 46% (227/436 and 199/436, respectively) of cartridge costs alone. Conclusion: Testing single and pooled specimens had a high level of agreement, with complete concordance when using Xpert-Ultra. Pooling samples could generate significant cartridge savings during ACF campaigns

Pooling samples to increase testing capacity with Xpert Xpress SARS-CoV-2 during the Covid-19 pandemic in Lao People's Democratic Republic.

The COVID-19 pandemic created the need for large-scale testing of populations. However, most laboratories do not have sufficient testing capacity for mass screening. We evaluated pooled testing of samples, as a strategy to increase testing capacity in Lao PDR. Samples of consecutive patients were tested in pools of four using the Xpert Xpress SARS CoV-2 assay. Positive pools were confirmed by individual testing, and we describe the performance of the test and savings achieved. We also diluted selected positive samples to describe its effect on the assays CT values. 1,568 patients were tested in 392 pools of four. 361 (92.1%) pools were negative and 31 (7.9%) positive. 29/31 (93.5% (95%CI 77-99%) positive pools were confirmed by individual testing of the samples but, in 2/31 (6.5%) the four individual samples were negative, suggesting contamination. Pools with only one positive sample had higher CT values (lower RNA concentrations) than the respective individual samples, indicating a dilution effect, which suggested an increased risk of false negative results with dilutions >1:10. However, this risk may be low if the prevalence of infection is high, when pools are more likely to contain more than one positive sample. Pooling saved 67% of cartridges and substantially increased testing capacity. Pooling samples increased SARS-CoV-2 testing capacity and resulted in considerable cartridge savings. Given the need for high-volume testing, countries may consider implementation of pooling for SARS-CoV-2 screening

Epidémiologie moléculaire de Mycobacterium tuberculosis et sa résistance aux antibiotiques en RDP Lao

Tuberculosis (TB) is still one of the top 10 leading causes of death worldwide; the emergence/re-emergence of drug resistant TB aggravates the situation globally and challenges the prospect of ending TB by 2035. Lao PDR is surrounded by TB and MDR-TB high burden countries and TB continues to be one of the priority infection diseases in this country. The prevalence of TB in 2010 was almost twice as high than previous estimates and little is known about drug resistance. Up to now, M. tuberculosis population data regarding drug resistance and genetic structure are totally absent. In this context, we aimed to study the diversity and the structure of M. tuberculosis population and the genetic determinants associated to drug resistance using clinical samples collected from the TB prevalence survey (TBPS), 2010-2011; from the Drug resistance survey (DRS), 2016-2017 and from presumptive MDR-TB cases in Lao PDR (2010-2014). Various methods and analyses were used, including drug susceptibility testing (phenotypic and genotypic), DNA sequencing and genotyping of M. tuberculosis using spoligotyping and MIRU-VNTR. The data were analyzed by statistical and phylogenetic analyses.Firstly, this work was focused on the diversity of M. tuberculosis families circulating in Lao PDR. According to the result form TBPS, EAI and Beijing family (76.7% and 14.4% respectively) were mainly observed, while Beijing family was more observed in DRS, and presumptive MDR-TB cases (35% and 41% respectively). The level of recent transmission in Lao PDR was non-negligible with a global clustering rate of 11.9% and in Beijing and EAI of 20% and 11%, respectively. Secondly, the results demonstrated the diversity of drug resistant patterns from mono-resistance to pre-extensively drug resistance (pre-XDR). A high diversity of mutations associated with drug resistance was also observed, however common mutations were mainly found (e.g: mutations in rpoB gene, katG and rpsL). The pattern was different for pncA gene, we observed a diversity of mutations without preeminent ones. Besides the number of known and unknown mutations associated with anti-TB drug resistance, deletion and insertion of bases were also observed. The sequencing showed its usefulness for drug resistance detection. Lastly, Beijing family, which is the more problematic family in the world in terms of resistance and transmissibility, was observed on a significant manner in young age group, mainly in the northern provinces, in recent transmission cases and among highly drug resistant isolates, suggesting an increasing risk of highly drug resistance TB due to highly transmissible Beijing strains in Lao PDR.In conclusion, this study provides the first genetic insights into the M. tuberculosis population in Lao PDR. The results underline the risk of increase of Beijing and drug resistant TB in the country. In order to prevent a more serious situation in the future regarding drug resistance as observed in neighboring countries, there is an urgent need of effective strategy improvement for drug resistance screening and the development of rapid molecular tests that cover a large number of drug resistance simultaneously with a feasible implementation in the limited resource countries. The results of genotyping from our study will be the baseline of families/subfamilies/genotype of M. tuberculosis population and of the mutations associated with drug resistance in Lao PDR. These data will be compared with further study/analysis to evaluate the trend of TB and drug resistant TB in the country and to determine if the drug resistance is under control after the set-up of new policies. The data of drug resistance associated mutations are used to build a regional database in collaboration with Vietnam and Cambodia in order to develop a diagnostic tool based on DNA chip technology to improve the drug resistance detection in the region.La tuberculose (TB) reste parmi les 10 premières causes de décès dans le monde ; l’émergence/réémergence de la TB résistante aux antituberculeux aggrave la situation et représente un défi majeur pour l’éradication de la TB. Le Laos est entouré par des pays fortement touchés par la TB et la TB multi-résistante (MDR) et cette maladie représente une priorité en termes de santé publique dans ce pays. Il n’existe encore aucune donnée sur la structure génétique et la résistance aux antibiotiques de la population de M. tuberculosis au Laos.Dans ce contexte, ce travail avait pour but d’analyser la diversité génétique et la structure des populations de M. tuberculosis ainsi que les déterminants génétiques associés à la résistance à partir d’échantillons collectés lors de l’enquête de prévalence nationale de la Tuberculose (TBPS) 2010-2011, l’enquête de résistance aux antituberculeux (DRS) 2016-2017 et chez les cas suspects de MDR-TB au Laos (2010-2014). Plusieurs techniques d’analyses ont été utilisées, comprenant les tests de sensibilité aux médicaments (phénotypique et génotypique), le séquençage et le génotypage par spoligotypage et MIRU-VNTR. Les données ont été analysées par des méthodes statistiques et phylogénétiques.Premièrement, ce travail s’est focalisé sur la diversité des familles de M. tuberculosis circulant au Laos. Les familles EAI et Beijing (76.7% et 14.4% respectivement) ont été principalement observées dans les échantillons de TBPS, alors que la famille Beijing était plus fréquente dans les échantillons de DRS et chez les patients suspectés de MDR-TB (35% et 41% respectivement). La transmission récente était non-négligeable avec un taux de « clustering » global de 11.9%, et des taux pour Beijing de 20 % et EAI de 11 %. Deuxièmement, les résultats ont révélé des profils de résistance très diverses allant de la mono-résistance jusqu’à la pré-XDR (ultrarésistance). Les mutations associées aux profils de résistance ont montré une grande diversité, avec cependant certaines mutations majeures dans les gènes rpoB, katG, et rpsL. Le gène pncA a montré un pattern différent avec de la diversité sans mutations prééminentes. En plus des mutations détectées, des délétions et insertions de bases ont été également observées. Le séquençage a montré son utilité pour la détection de la résistance aux antibiotiques dans les trois échantillons à l’étude. Enfin, la famille Beijing, famille la plus problématique au niveau mondial en termes de résistance et de transmissibilité, a été identifiée de manière significative dans le groupe de patients <35 ans, principalement dans les provinces du Nord, dans les cas de transmissions récentes et chez les isolats très résistants. Tous ces points suggèrent un risque d’émergence de la MDR-TB accrue au Laos dû à la famille Beijing.En conclusion, cette étude permet d’avoir pour la première fois un aperçu de la structure des populations de M. tuberculosis au Laos. Les résultats soulignent le risque d’augmentation du nombre de cas infectés par la famille Beijing et donc des cas de résistance. Pour empêcher une dégradation de la situation, il est essentiel d’améliorer les stratégies pour le dépistage des résistances et de développer des tests moléculaires capables de couvrir un large nombre de mutations qui soit simple à implémenter dans les pays à ressources limités. Les résultats de ce travail serviront de base en termes de famille/sous-famille/génotype et de mutations associées à la résistance au Laos. Ces données pourront être comparées avec de futures études/analyses pour étudier l’évolution de la TB et de la TB résistante et ainsi d’évaluer l’efficacité des politiques de contrôle mises en place. La description des mutations associées aux résistances est utilisée pour créer une base de données régionale en collaboration avec le Vietnam et le Cambodge pour développer un outil de diagnostic basé sur la technologie des puces à ADN pour améliorer la détection de la résistance dans la région

Clonal transmission of a colistin-resistant Escherichia coli from a domesticated pig to a human in Laos

International audienc

Pooling sputum for Xpert MTB/RIF and Xpert Ultra testing during the Covid-19 pandemic in Lao People’s Democratic Republic

The global Covid-19 pandemic has limited access to molecular TB diagnostics and National Programmes are struggling to maintain essential services. The pooling method (testing several samples together) could reduce the number of cartridges and staff time needed for TB diagnosis but has not been tested within the pandemic. We conducted two independent cross-sectional surveys. Pools composed of four sputum samples were tested using either Xpert-MTB/RIF or Xpert-Ultra. Pooled and individual results were compared to determine the level of agreement. Each survey included 840 participants and 210 pools. In the Xpert MTB/RIF survey, 77/81 (sensitivity 95.1%, 95%CI 87.8%-98.6%) pools containing ≥1 positive sample tested MTB-positive and 4/81 (4.9%, 95%CI 1.4%-12.2%) tested MTB-negative. All 129/129 pools containing MTB-negative samples tested MTB-negative (specificity 100%, 95%CI 97.2%-100%), with 98.1% agreement (Kappa: 0.959). In the Xpert-Ultra survey, 70/70 (sensitivity 100%, 95%CI 94.9%-100%) pools containing ≥ 1 MTB-positive sample tested MTB-positive and 140/140 (specificity 100%, 95%CI 97.4%-100%) pools containing only MTB-negative samples tested MTB-negative, with 100% agreement (Kappa: 1). Pooled testing with Xpert-MTB/RIF and Xpert-Ultra saved 38.3% and 41.7% (322/840 and 350/840, respectively) in cartridge costs alone. The pooling method with Xpert-MTB/RIF and Xpert-Ultra has similar performance to individual testing and can reduce the number of cartridges needed. These efficiencies can facilitate maintenance of stocks and sustain essential services as countries face difficulties for laboratory procurement during the pandemic and will provide cost and time savings post-pandemic.</jats:p

High-throughput mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping for Mycobacterium tuberculosis epidemiological studies

International audienceThe emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner

Intestinal parasitic infections and environmental water contamination in a rural village of northern Lao PDR

A field survey studying intestinal parasites in humans and microbial pathogen contamination at environment was performed in a Laotian rural village to identify potential risks for disease outbreaks. A parasitological investigation was conducted in Ban Lak Sip village, Luang Prabang, Lao PDR involving fecal samples from 305 inhabitants as well as water samples taken from 3 sites of the local stream. Water analysis indicated the presence of several enteric pathogens, i.e., Aeromonas spp., Vibrio spp., E. coli H7, E. coli O157: H7, verocytotoxin-producing E. coli (VTEC), Shigella spp., and enteric adenovirus. The level of microbial pathogens contamination was associated with human activity, with greater levels of contamination found at the downstream site compared to the site at the village and upstream, respectively. Regarding intestinal parasites, the prevalence of helminth and protozoan infections were 68.9% and 27.2%, respectively. Eight helminth taxa were identified in fecal samples, i.e., 2 tapeworm species (Taenia sp. and Hymenolepis diminuta), 1 trematode (Opisthorchis sp.), and 5 nematodes (Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, trichostrongylids, and hookworms). Six species of intestinal protists were identified, i.e., Blastocystis hominis, Cyclospora spp., Endolimax nana, Entamoeba histolytica/E. dispar, Entamoeba coli, and Giardia lamblia. Questionnaires and interviews were also conducted to determine risk factors of infection. These analyses together with a prevailing infection level suggested that most of villagers were exposed to parasites in a similar degree due to limited socio-economic differences and sharing of similar practices. Limited access to effective public health facilities is also a significant contributing factor