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.

First insights into the genetic characteristics and drug resistance of Mycobacterium tuberculosis population collected during the first national tuberculosis prevalence survey of Lao PDR (2010-2011).

BACKGROUND: In Lao People's Democratic Republic (PDR), tuberculosis (TB) prevalence was estimated at 540/100,000 in 2011. Nevertheless, little is known about the genetic characteristics and anti-TB drug resistance of the Mycobacterium tuberculosis population. The main objective of this work was to study the genetic characteristics and drug resistance of M. tuberculosis population collected during the first National TB Prevalence Survey (TBPS) of Lao PDR (2010-2011). METHODS: Two hundred and twenty two isolates collected during TBPS (2010-2011) were analyzed with the GenoType MTBDRplus test for M. tuberculosis identification and drug resistance detection. Then, 206 of the 222 isolates were characterized by spoligotyping and MIRU-VNTR typing. RESULTS: Among the 222 M. tuberculosis isolates, 11 were mono-resistant to isoniazid and 2 were resistant to isoniazid and rifampicin (MDR-TB), using the GenoType MTBDRplus test. Among the 202 genetically characterized isolates, the East African-Indian (EAI) family was predominant (76.7%) followed by the Beijing (14.4%) and T (5.5%) families. EAI isolates came from all the country provinces, whereas Beijing isolates were found mainly in the northern and central provinces. A higher proportion of Beijing isolates was observed in people younger than 35 years compared to EAI. Moreover, the percentage of drug resistance was higher among Beijing (17.2%) than EAI (5.2%) isolates, and the two MDR-TB isolates belonged to the Beijing family. Combined analysis of the MIRU-VNTR and spoligotyping results (n = 202 isolates) revealed an estimated clustering rate of 11% and the occurrence of mini-outbreaks of drug-resistant TB caused by Beijing genotypes. CONCLUSIONS: The EAI family, the ancient and endemic family in Asia, is predominant in Lao PDR whereas the prevalence of Beijing, the most harmful M. tuberculosis family for humans, is still low, differently from neighboring countries. However, its association with drug resistance, its presence in young patients and its potential association with recent transmission suggest that the Beijing family could change TB epidemiological pattern in Lao PDR. Therefore, efficient TB control and surveillance systems must be maintained and reinforced to prevent the emergence of highly transmissible and drug-resistant strains in Lao PDR, as observed in neighboring countries.

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.

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

The 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.

The first national tuberculosis prevalence survey of Lao PDR (2010-2011).

OBJECTIVE: The objective of the study was to measure the prevalence of bacteriologically confirmed pulmonary tuberculosis (TB) in Lao PDR in 2010-2011. METHOD: A nationwide, multistage cluster-sampled cross-sectional survey was undertaken in 2010-2011. All consenting participants ≥15 years were screened for pulmonary TB with chest X-ray and symptom questionnaire. Two sputum specimens for bacteriological examination by microscopy and culture were collected from those who screened positive. Prevalence was estimated using multiple imputation and inverse probability weighting methods. RESULTS: Of 39 212 eligible participants from 50 clusters, 6290 participants provided at least one sputum sample for smear and culture. There were 237 bacteriologically confirmed pulmonary TB cases, 107 of which were smear-positive. Chest X-ray screening alone identified 230 (97.0%) cases compared with 118 (49.8%) by symptom screening alone. The estimated prevalence of smear-positive and bacteriologically confirmed TB in those ≥15 years was 278 per 100 000 (95%C.I. 199-356) and 595 per 100 000 (95%C.I. 457-733), respectively. Prevalence significantly increased with age and was higher in men than women. CONCLUSIONS: The prevalence of TB in Lao PDR is almost twice as high than previous estimates, with the greatest burden in the older population. Case detection efforts remain the primary goal of the national TB programme with case notifications being very low in comparison with the estimated number of prevalent cases. The survey observed major limitations with the diagnostic strategy of passive (symptom based) case finding that uses only direct smear microscopy for confirmation.

Resistance of Mycobacterium tuberculosis to antibiotics in Lao PDR: first multicentric study conducted in 3 hospitals.

BACKGROUND: It is estimated that Lao People's Democratic Republic (Lao PDR) ranks fifth among the seven countries most affected by TB in the WHO Western Pacific Region. However, because of late implementation of mycobacterial culture, no study on resistance to anti-TB drugs had been performed yet. The objective of this study was to document drug resistance rate among patients hospitalized for pulmonary TB in threeprovinces of Lao PDR. METHODS: A cross-sectional study was conducted in three sites, one central and two regional hospitals, from April to November 2010. For each TB suspected patient sputum smear microscopy and culture on Lowenstein-Jensen media were performed. GenoType® MTBDRplus assay was used to test the susceptibility to isoniazid (INH) and rifampicin (RMP), GenoType® MTBDRsl for second-line drugs and GenoType® Mycobacterium CMAS for non-tuberculous mycobacteria (NTM). RESULTS: Out of 104 positive culture on Lowenstein-Jensen, 87 (83.6%) were M. tuberculosis and 17 (16.4%) were NTM. Of 73 new TB cases, 5 isolates (6.8%) were resistant to INH. Of 14 previously treated cases, 2 isolates (14.3%) were resistant to INH and one isolate was XDR. CONCLUSION: Despite an overall rate of resistance still moderate, the frequency of mutations conferring INH monoresistance and identification of the first strain of XDR require strengthening surveillance of drug resistant tuberculosis in Lao PDR.

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 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.

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.

Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study.

The emergence of colistin-resistant Klebsiella pneumoniae (CRKP) is a major public health concern worldwide. In this study, the prevalence and molecular basis of colistin resistance in CRKP isolated from healthy individuals and patients in Lao PDR, Thailand, Nigeria and France were investigated. Stool samples were screened by culture for the presence of colistin-resistant Klebsiella spp. Whole-genome sequence analysis was used to decipher the molecular mechanism of colistin resistance in a blaNDM-1-positive in vitro-selected CRKP mutant. PCR amplification and sequencing of the mgrB genetic environment was performed for all CRKP isolates as well as control colistin-susceptible K. pneumoniae (CSKP) isolates recovered from the same stools. A total of 869 stool samples were screened for colistin-resistant Klebsiella spp., yielding 32 CRKP and 2 colistin-resistant Klebsiella oxytoca. Comparative whole-genome sequence analysis revealed that an in vitro-selected CRKP mutant had an insertion sequence in its mgrB gene, as well as missense mutations in other selected clones. Of the 34 colistin-resistant Klebsiella spp. isolates, 14 (41.2%; 13 CRKP and 1 K. oxytoca) from the four countries also had various defects in their mgrB genes, but no such defects were found in the CSKP controls (P<10(-4)). Few mutations were observed in pmrAB compared with mgrB among the CRKP isolates. The worldwide emergence of CRKP is a major public health concern. Detection and surveillance of such strains are warranted to prevent an uncontrollable pandemic. Inactivation of the PhoP/PhoQ regulator gene mgrB is associated with ≥40% of colistin resistance among the CRKP isolates observed in this study.

Children's access to treatment for epilepsy: experience from the Lao People's Democratic Republic.

Epilepsy affects an estimated 10.5 million children worldwide, of whom 80% live in developing countries. The treatment gap is estimated at around 90% in the Lao People's Democratic Republic (Lao PDR). The present work analyses some of the reasons that could explain the low access to antiepileptic drugs (AED) for children with epilepsy in the Lao PDR. Epilepsy, the 'mad pig disease', is highly neglected. Traditional beliefs, fear and stigma are common among the general population and shared by 40% of health staff. Poor knowledge of the disease and its treatment, low trust in modern treatment, restricted access to AED and ignorance of long-term treatment explain the low access to AED. Improving understanding of epilepsy and its treatment, will improve the treatment gap for epileptic children in the Lao PDR.