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We report the coding-complete genome sequences of 25 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineage B.1.1.529 Omicron strains obtained from Bangladeshi individuals in samples collected between December 2021 and January 2022. Genomic data were generated by Nanopore sequencing using the amplicon sequencing approach developed by the ARTIC Network.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex, is still the number one deadly contagious disease. Mtb infection results in a wide spectrum of clinical presentations and severity symptoms, but without proven Mtb genetic determinants. Thanks to a collection of 355 clinical isolates with associated patient’s clinical data, we showed that Mtb micro-diversity within patient isolates is strongly correlated with TB-associated severity scores. Interestingly, this diversity is driven by a selection pressure to adapt to different lifestyles related to the infection site. Taken together, these results provide a new insight to better understand TB pathophysiology. Furthermore, Mtb micro-diversity could be envisioned as a new prognostic tool to improve the management of TB patients.

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management., Author summary Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), bacteria that are able to persist inside the patient for many months or years, thus requiring long antibiotic treatments. Here we focused on TB patients with delayed response to treatment and we performed genetic characterization of Mtb isolates to search for sub-populations that may tolerate anti-TB drugs. We found that Mtb cultured from 9/15 patients contained different sub-populations, and in vitro drug exposure revealed Mtb sub-populations in 6/15 isolates, none related to known drug-resistance mechanisms. By contrast, drug exposure revealed Mtb sup-populations in 2/20 isolates in the control cohort of patients with fast culture conversion. Furthermore, we characterized a Mtb variant isolated from a sub-population growing in the presence of rifampicin (RIF), a major anti-TB drug. We found that this variant featured a modified lipidic envelope, and that it was able to develop in the presence of RIF and inside human macrophage cells. We performed pharmacological modelling and found that this kind of variant may be related to a poor response to treatment. In conclusion, searching for particular Mtb sub-populations may help to detect patients at risk of treatment failure and provide additional guidance for TB management.

International audience; Objectives: Tuberculosis (TB) is the leading infectious cause of death in the world. Cheaper and more accessible TB treatment monitoring methods are needed. Here, we evaluated white blood cell (WBC) absolute counts, lymphocyte, and monocyte proportions during TB treatment, and characterized their association with treatment failure.Methods: This multicentered prospective cohort study was based in Bangladesh, Georgia, Lebanon, Madagascar, and Paraguay. Adult, non-immunocompromised patients with culture-confirmed pulmonary TB were included and followed up after two months of treatment and at the end of therapy. Blood counts were compared to treatment outcome using descriptive statistics, logistic regression, and Receiver Operating Characteristic (ROC) analyses.Results: Between December 2017 and August 2020, 198 participants were enrolled, and 152 completed treatment, including 28 (18.5%) drug-resistant patients. The rate of cure at the end of treatment was 90.8% (138/152). WBC absolute counts decreased, and lymphocyte proportions increased throughout treatment. In multivariate analyses, baseline high WBC counts and low lymphocyte proportions were associated with positive sputum culture results at the end of treatment (WBC > 11,450 cells/mm3: p = 0.048; lymphocytes 11,450 cells/mm3 and lymphocytes

Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.

14 páginas, 3 figuras, 1 tabla, Advanced epidemiological surveillance supported on genomic epidemiology were necessary to fully describe a Mycobacterium tuberculosis Beijing strain (ARA, Auvergne-Rhône-Alpes region) outbreak in France. The index case was a migrant from Cape Verde with a two-year history of tuberculosis, high bacillary load, and frequent trips within France and to Portugal. We designed an ARA-specific PCR to complete the tracking of this outbreak strain. The ARA-specific PCR was applied on 160 Beijing isolates from independent cases from the Auvergne-Rhône-Alpes region and on 25 cases from Cape Verde migrants in the Île-de-France region. No more cases were found, indicating that the previous surveillance had been exhaustive enough to capture the whole dimension of the outbreak in France. Next, we performed a cross-border surveillance, applying the PCR on 38 Beijing isolates in Portuthe gal. In all four cases (all migrants from Cape Verde) the ARA strain was identified. Whole genome sequences analysis of all ARA isolates representatives indicated a diversification of the strain into two variants present one in France and one in Portugal, This work was supported by ERANet-LAC [ELAC2015/T08-0664] and Instituto de Salud Carlos III [AC16/00057, FIS15/01554, FIS13/ 01207, CP15/00075, PI16/01449, 1PI19/00331] and cofounded by European Regional Development Funds from the European Commission: “A way of making Europe”. Miguel Servet Contract CP15/00075 and CPII20/00001) to LPL. JP [CEECIND/00394/2017] is supported by FCT through Estímulo Individual ao Emprego Cientifico.

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.

Mycobacterium tuberculosis (Mtb) whole genome sequencing (WGS) plays an increasingly important role in tuberculosis diagnosis and research. WGS is typically performed on biobanked isolates obtained by subculture during diagnosis. Genetic variation upon culturing is known to occur in other bacterial species. However, little is understood regarding the impact of different subculture media on genome-wide diversity and variant selection in Mtb. Here we compared WGS derived from direct sequencing of sputa samples to WGS sequences from isolates subcultured on 3 different media. Based on analysis of single nucleotide polymorphisms (SNPs), there was no evidence of variant selection caused by the different culture media used, indicating that subcultured clinical strains can be reliably used to explore genetic determinants of Mtb pathogenesis and epidemiological features.

Background: Molecular survey including whole-genome sequencing (WGS) of Mycobacterium tuberculosis isolates is a reliable tool to reveal clonal outbreaks. Here, both genomic and epidemiological investigations were coupled in a prospective approach to assess nosocomial tuberculosis (TB) transmission in a tertiary-care hospital. Methods: Since 2008, the Mycobacteria laboratory of the Lyon university hospital, has implemented a prospective population-based survey by systematic genotyping (first using spoligo- and MIRU typing, and WGS since 2017) of M. tuberculosis isolates in the Rhone-Alpes region. Conventional contact-tracing of TB cases was conducted in accordance with French guidelines. Findings: Since 2008, among 3230 cases of microbiologically-proven TB, 96 molecular clusters with confirmed transmissions were identified. Four clusters were health care-related, representing eight patients, including three independent transmissions of susceptible M. tuberculosis in lung transplant recipients, and one transmission of a multi-resistant strain. Epidemiological investigation validated nosocomial transmissions in these four health care-related clusters. One nosocomial transmission was imbedded in a much larger community transmission chain involving ten patients. Corrective measures implemented were to discard any admission of TB patients in the transplant surgery hospital, and to construct a building equipped with negative-pressure ventilation rooms dedicated to TB management. Interpretation: TB molecular survey is a valuable strategy to detect unsuspected transmission chains, particularly among health-care facilities. Prospective monitoring and tight partnership with public health agencies render survey-guided prevention control measures possible. Funding Statement: LABEX ECOFECT (ANR-11-LABX-0048) of the Lyon University as part of the research program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Lyon University Hospital Ethical Committee.

Rapid and correct determination of Mycobacterium tuberculosis (MTB) drug susceptibility is a challenge for tuberculosis (TB) management. Phenotypic drug susceptibility testing (DST) remains the reference method but is time consuming. In this study, genotypic prediction of the first-line drug susceptibility profile obtained by whole-genome sequencing (WGS) was compared with that obtained by phenotypic DST and the line probe assay (LPA). All MTB strains isolated from patients during routine practice at the mycobacteria laboratory of Lyon University Hospital, France, between November 2016 and July 2019 were included (n = 274). Isolates were tested for the first-line drugs using phenotypic DST (Mycobacteria Growth Indicator Tube) and for genotypic prediction of the susceptibility profile with LPA and WGS. Considering phenotypic DST as the reference, WGS predicted resistance to rifampicin, isoniazid, ethambutol and pyrazinamide with sensitivities of 100%, 100%, 100% and 93.8%, respectively, and susceptibility to these drugs with specificities of 99.6%, 100%, 98.5% and 100%, respectively. Performance of the LPA was poorer, with sensitivity of 83.3% for rifampicin and 85.7% for isoniazid resistance. Five isolates were classified as susceptible according to phenotypic DST (1 for rifampicin, 4 for ethambutol) while WGS detected resistance mutations in rpoB and embB genes. WGS, used under appropriate quality-control conditions, has good performance to predict the resistance profile for the four first-line drugs and can correct phenotypic DST results. This study highlights the need for future guidelines recommending WGS as the initial tool in routine practice in areas where the prevalences of TB and drug-resistant MTB are low.

Control of Mycobacterium tuberculosis (Mtb) transmission in high-income health care settings and in low tuberculosis (TB) prevalence countries remains a public health priority given the constant changes in Mtb epidemiology worldwide. Though Europe is a low prevalence area [1], TB burden among precarious and migrant populations contributes to this evolving landscape, as addressed by the action framework towards TB elimination [2]. At the core of the national health-care system, tertiary care hospitals manage both patients with greater susceptibility to TB, and patients with complex and/or advanced TB disease. Key measures for TB control rely on enabling linkage of cases and identification of transmission chains, often supported by molecular survey tools [3, 4]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Genestet reports grants from LABEX ECOFECT (ANR-11-LABX-0048) of Lyon University, within the program “Investissements d'Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), during the conduct of the study. Conflict of interest: Dr. Paret has nothing to disclose. Conflict of interest: Mrs. Pichat has nothing to disclose. Conflict of interest: Mr. Berland has nothing to disclose. Conflict of interest: Dr. Jacomo has nothing to disclose. Conflict of interest: Dr. Carret has nothing to disclose. Conflict of interest: Dr. Fredenucci has nothing to disclose. Conflict of interest: Dr. Hodille has nothing to disclose. Conflict of interest: Dr. Rasigade has nothing to disclose. Conflict of interest: Dr. Boisset has nothing to disclose. Conflict of interest: Dr. Carricajo has nothing to disclose. Conflict of interest: Pr. Lina has nothing to disclose. Conflict of interest: Dr. Ronnaux-Baron has nothing to disclose. Conflict of interest: Dr. Mornex has nothing to disclose. Conflict of interest: Dr. Grando has nothing to disclose. Conflict of interest: Dr. Senechal has nothing to disclose. Conflict of interest: Pr. Ader has nothing to disclose. Conflict of interest: Dr. Dumitrescu has nothing to disclose.

Метод GenoType MTBDRplus, v.2 позволяет исследовать клинический материал с положительных и отрицательных мазков, обследовать больных с новыми и повторными случаями туберкулёза лёгких и внелёгочной локализации. Но в настоящее время количество исследований касательно использования данного метода в диагностике туберкулёза невелико.Цель работы – оценить эффективность использования метода исследования клинического материала с применением метода GenoType MTBDRplus, v.2 в диагностике туберкулёза в Запорожской области в сравнении со стандартными методами исследования.Материалы и методы. Проведён анализ 52 результатов исследований клинического материала с использованием тест-системы GenoType MTBDRplus, v.2 от пациентов, которые обследовались и лечились в диспансерах Запорожской области в 2016 году. Среди пациентов – 67,3 % мужчин и 32,7 % женщин. Средний возраст больных составлял 46,0 ± 1,9 года. Исследование клинического материала с использованием теста GenoType MTBDRplus, v.2 проводили по стандарту, согласно инструкции.Результаты. При отрицательных результатах микроскопии клинического материала в 12,5 % случаев результаты оказались положительными при исследовании методом GenoType MTBDRplus, v.2 (р ˂ 0,05). Доля совпадений между культуральным и молекулярно-генетическим методами составила 90 % для мокроты и 100 % для другого клинического материала. При наличии мультирезистентности оба метода исследования совпадали по количеству выявленных случаев (50 % для метода GenoType MTBDRplus, v.2 и 42,9 % для культурального метода соответственно, р ˂ 0,05). В сравнении с результатами культурального метода, тест GenoType MTBDRplus, v.2 позволил установить устойчивость к рифампицину у 35,7 % (р ˂ 0,05) пациентов, у которых устойчивость не определялась. Ложно-положительные и ложно-отрицательные результаты обусловлены, возможно, гетерогенностью штаммов микобактерий туберкулёза в клинических образцах, отсутствием жизнеспособных микобактерий, отсутствием фенотипических проявлений генетических мутаций микобактерий туберкулёза, ошибками при проведении исследования. Чувствительность теста GenoType MTBDRplus, v.2 составляет около 78 %, но специфичность достигает 97 %. В то же время чувствительность теста по определению мультирезистентности достигает 100 %, специфичность – около 89 %. Большая прогностическая значимость положительного результата для случаев, в которых определяются ДНК микобактерий, а прогностическая значимость отрицательного результата – для случаев, в которых определяется мультирезистентность.Выводы. Это свидетельствует о том, что использование теста GenoType MTBDRplus, v.2 вместе с культуральным методом исследования имеет высокую диагностическую ценность, высокую чувствительность (100 %) и специфичность (89 %) по определению мультирезистентности и даёт возможность получить результат в более короткий срок сравнительно с культуральным исследованием., The method GenoType MTBDRplus, v.2 allows to study clinical material from positive and negative smears, to examine patients with new and previously treated cases of tuberculosis pulmonary and extrapulmonary localization. But now there is a small amount of researches concerning the use of this method in the diagnosis of tuberculosis.Aim. To evaluate the effectiveness of GenoType MTBDRplus, v.2 method using in clinical material research due to the diagnosis of tuberculosis in the Zaporizhzhia region in comparison with standard methods of investigation.Materials and methods. The analysis of 52 results of studies of clinical material using the GenoType MTBDRplus test system, v.2 from patients who were examined and treated in dispensaries of the Zaporizhzhia region in 2016 was carried out. There were 67.3 % of men and 32.7 % of women among the patients. The mean age of the patients was 46.0 ± 1.9 years. The study of the clinical material using the GenoType MTBDRplus test, v.2 was performed according to the standard instructions.Results. The results were positive in 12.5 % of the cases when tested by GenoType MTBDRplus, v.2 (p ˂ 0.05) among negative results of the microscopy of the clinical material. The proportion of coincidence between the cultural and molecular genetic methods was 90 % for sputum and 100 % for other clinical material. In the presence of multiresistance, both methods of investigation coincided in the number of detected cases (50 % for the GenoType MTBDRplus method, v.2 and 42.9 % for the cultural method, respectively, p ˂ 0.05). In comparison with the results of the cultural method, the GenoType MTBDRplus test, v.2 made it possible to establish resistance to rifampicin in 35.7 % (p < 0.05) of patients whose resistance was not determined. False-positive and false-negative results are due, perhaps, to the heterogeneity of strains of mycobacterium tuberculosis in clinical specimens, the absence of viable mycobacteria, the absence of phenotypic manifestations of mycobacterium tuberculosis genetic mutations, and errors in the investigations. The sensitivity of the GenoType MTBDRplus test, v.2 is about 78 %, but the specificity reaches 97 %. At the same time, the sensitivity of the test for multidrug resistance determining reaches 100 %, the specificity is about 89 %. The greater positive predictive value for cases in which the DNA of mycobacteria is determined and the negative predictive value is for cases in which multidrug resistance is determined.Conclusions. This indicates that the using of the GenoType MTBDRplus test, v.2, together with the cultural method of investigation, has a high diagnostic value, high sensitivity (100 %) and specificity (89 %) for the definition of multiresistance, and makes it possible to obtain a result in a short time compared to culture., Метод GenoType MTBDRplus 2 версії (GenoType MTBDRplus, v.2) дає можливість досліджувати клінічний матеріал із позитивних і негативних мазків, обстежувати хворих із новими та повторними випадками туберкульозу легень і позалегеневої локалізації. Але нині кількість досліджень щодо використання цього методу в діагностиці туберкульозу невелика.Мета роботи – оцінити ефективність застосування методу дослідження клінічного матеріалу з використанням методу GenoType MTBDRplus, v.2 у діагностиці туберкульозу в Запорізькій області порівняно зі стандартними методами дослідження.Матеріали та методи. Здійснили аналіз 52 результатів досліджень клінічного матеріалу за допомогою тест-системи GenoType MTBDRplus, v.2 від пацієнтів, які обстежувалися та лікувалися в диспансерах Запорізької області у 2016 році. Серед пацієнтів було 67,3 % чоловіків і 32,7 % жінок. Середній вік хворих становив 46,0 ± 1,9 року. Дослідження клінічного матеріалу з використанням тесту GenoType MTBDRplus, v.2 здійснили за стандартом, відповідно до інструкції виробника.Результати. При негативних результатах мікроскопії клінічного матеріалу у 12,5 % випадках результати виявилися позитивними під час дослідження за допомогою методу GenoType MTBDRplus, v.2 (р ˂ 0,05). Частка збігів між культуральним і молекулярно-генетичним методами становила 90 % для харкотиння та 100 % для іншого клінічного матеріалу. За наявності мультирезистентності обидва методи дослідження майже збігалися за кількістю виявлених випадків (50,0 % для методу GenoType MTBDRplus, v.2 та 42,9 % для культурального методу відповідно, р ˃ 0,05). Порівняно з результатами культурального методу, тест GenoType MTBDRplus, v.2 дав можливість встановити стійкість до рифампіцину у 35,7 % (р ˂ 0,05) пацієнтів, у яких не визначалася стійкість. Хибно-позитивні та хибно-негативні результати зумовлені, можливо, гетерогенністю штамів мікобактерій туберкульозу у клінічних зразках, відсутністю життєздатних мікобактерій, відсутністю фенотипічних проявів генетичних мутацій мікобактерій туберкульозу, помилками під час дослідження тощо. Чутливість тесту GenoType MTBDRplus, v.2 становить майже 78 %, але специфічність сягає 97 %. Натомість чутливість тесту щодо встановлення мультирезистентності сягає 100 %, специфічність – майже 89 %. Більша прогностична цінність позитивного результату для випадків, у яких визначаються ДНК МБТ, а прогностична цінність негативного результату – для випадків, у яких визначається мультирезистентність.Висновки. Застосування тесту GenoType MTBDRplus, v.2. поряд із культуральним методом дослідження має високу діагностичну цінність, високу чутливість (100 %) і специфічність (89 %) щодо визначення мультирезистентності й дає змогу отримати результат у коротший термін порівняно з культуральним дослідженням.

Background Laos has a high prevalence of tuberculosis (TB) and a slowly increasing prevalence of human immunodeficiency virus/acquired immunedeficiency syndrome (HIV/AIDS). Sputum smear microscopy is the only method currently available for routine screening of pulmonary TB, although it only detects one in three cases among persons living with HIV (PLWH). Bleach treatment of sputum samples (bleach method) has been shown to significantly improve the sensitivity of the test; however, its effectiveness in PLWH remains to be determined in Laos. Objectives To determine the performance of the bleach method as a diagnostic tool for pulmonary TB in PLWH and to assess its cost-effectiveness in Laos. Results Of 174 sputum samples collected from 92 patients, 29 were culture-positive for Mycobacterium tuberculosis in 17 patients. The sensitivity of the direct method and the bleach method was respectively 59% and 93%, and specificity was 100% for both methods. The incremental cost-effectiveness ratio for screening an additional case was US$17.40. Conclusion The bleach method is simple, cheap, easy to perform and cost-effective in PLWH. Its implementation in laboratories involved in routine screening of pulmonary TB among PLWH would allow practitioners to start the treatment of this life-threatening co-infection earlier.

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.

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.

We have evaluated in C57/Bl6 and HLA-A2.1 transgenic mice the immunogenicity of three MVA vectors expressing either native HCV E1E2 polyprotein, truncated and secreted E1 (E'1(311)) and E2 (E'2(661)) proteins, or a chimeric E1E2 heterodimer presented at the plasma membrane. Immunization induced mainly a Th1 response in HLA-A2.1 transgenic mice while a Th2-type response was detected in C57/Bl6 mice. Comparison of the three vectors shows an increase in the humoral response when antigens are secreted or membrane bound, and slightly in the cellular response when antigens are exposed on the cell surface.

Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia.

Setting The uptake of tests endorsed by the World Health Organization to detect and appropriately confirm multidrug-resistant tuberculosis (MDR-TB) in low-income countries remains insufficient. Objective To validate the implementation of line-probe assays (LPA) and liquid culture to develop an algorithm to detect MDR-TB in the challenging setting of Haiti. Methods Through an EXPAND-TB (Expanding Access to New Diagnostics for TB) partnership, proficiency testing and validation of 221 acid-fast bacilli positive specimens were performed. Sensitivity, cost and processing time were analysed. Results Using liquid vs. solid culture shortened the turnaround time from 54 to 19 days, with a sensitivity of 100% vs. 98.6% and a total cost reduction of 13%. LPA detected all TB and MDR-TB cases at a lower cost than culture, in a mean time of 7.5 days. Conclusion The combined use of molecular and liquid culture techniques accelerates the accurate diagnosis of TB and susceptibility testing against first-line drugs in a significantly shorter time, and is less expensive. The implementation of this new algorithm could significantly and accurately improve the screening and treatment follow-up of patients affected with TB and MDR-TB.

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.

Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.

UNLABELLED: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

BACKGROUND: Preventive and therapeutic vaccine strategies aimed at controlling hepatitis C virus (HCV) infection should mimic the immune responses observed in patients who control or clear HCV, specifically T helper (Th) type 1 and CD8+ cell responses to multiple antigens, including nonstructural protein (NS) 3. Given the experience with human immunodeficiency virus, the best candidates for this are based on DNA prime, pox, or adenovirus boost regimens. METHODS: In rhesus macaques, we compared NS3-expressing DNA prime and adenovirus boost strategy with 2 alternative priming approaches aimed at modifying Th1 and CD8+ responses: DNA adjuvanted with interleukin (IL)-2- and -12-encoding plasmids or Semliki Forest virus (SFV). RESULTS: All prime-boost regimens elicited NS3-specific B and T cell responses in rhesus macaques, including CD8+ responses. SFV priming induced higher lymphoproliferation and longer Th1 memory responses. The use of IL-2- and IL-12-expressing vectors resulted in reduced Th2 and antibody responses, which led to increased Th1 skewing but not to an increase in the magnitude of the IFN- gamma and CD8+ responses. CONCLUSIONS: All strategies induced Th1 cellular responses to HCV NS3, with fine modulations depending on the different priming approaches. When they are developed for more HCV antigens, these strategies could be beneficial in therapeutic vaccine approaches.

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.