Performance of microbiological tests for tuberculosis diagnostic according to the type of respiratory specimen: A 10-year retrospective study.

Background: The microbial diagnosis of tuberculosis (TB) remains challenging and relies on multiple microbiological tests performed on different clinical specimens. Polymerase chain reactions (PCRs), introduced in the last decades has had a significant impact on the diagnosis of TB. However, questions remain about the use of PCRs in combination with conventional tests for TB, namely microscopy and culture. We aimed to determine the performance of microscopy, culture and PCR for the diagnosis of pulmonary tuberculosis according to the type of clinical specimen in order to improve the diagnostic yield and to avoid unnecessary, time and labor-intensive tests. Methods: We conducted a retrospective study (2008-2018) on analysis (34'429 specimens, 14'358 patients) performed in our diagnostic laboratory located in the Lausanne University Hospital to compare the performance of microbiological tests on sputum, induced sputum, bronchial aspirate and bronchoalveolar lavage (BAL). We analysed the performance using a classical "per specimen" approach and a "per patient" approach for paired specimens collected from the same patient. Results: The overall sensitivities of microscopy, PCR and culture were 0.523 (0.489, 0.557), 0.798 (0.755, 0.836) and 0.988 (0.978, 0.994) and the specificity were 0.994 (0.993, 0.995), 1 (0.999, 1) and 1 (1, 1). Microscopy displayed no significant differences in sensitivity according to the type of sample. The sensitivities of PCR for sputum, induced sputum, bronchial aspirate and BAL were, 0.821 (0.762, 0.871), 0.643 (0.480, 0.784), 0.837 (0.748, 0.904) and 0.759 (0.624, 0.865) respectively and the sensitivity of culture were, 0.993 (0.981, 0.998), 0.980 (0.931, 0.998), 0.965 (0.919, 0.988), and 1 (0.961, 1) respectively. Pairwise comparison of specimens collected from the same patient reported a significantly higher sensitivity of PCR on bronchial aspirate over BAL (p < 0.001) and sputum (p < 0.05) and a significantly higher sensitivity of culture on bronchial aspirate over BAL (p < 0.0001). Conclusions: PCR displayed a higher sensitivity and specificity than microscopy for all respiratory specimens, a rational for a smear-independent PCR-based approach to initiate tuberculosis microbial diagnostic. The diagnosis yield of bronchial aspirate was higher than BAL. Therefore, PCR should be systematically performed also on bronchial aspirates when available.

Successful pregnancy in a cystic fibrosis patient with a severe impairment of lung function receiving Elexacaftor-Tezacaftor-Ivacaftor.

Before the arrival of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulators women with CF and impaired lung function were experiencing a high risk of complications and mortality during and the years after pregnancy. The arrival of the highly efficient CFTR modulator, Elexacaftor-Tezacaftor-Ivacaftor (ETI) resulted in an improvement of lung function, quality of life and fertility. Here we report a case of successful pregnancy and uncomplicated delivery for a CF patient with severely impaired lung function receiving ETI prior to conception.

Genome sequencing of Mycobacterium tuberculosis clinical isolates revealed isoniazid resistance mechanisms undetected by conventional molecular methods.

A combination of targeted molecular methods and phenotypic drug-susceptibility testing is the most widely used approach to detect drug resistance in Mycobacterium tuberculosis isolates. We report the delay in the introduction of an efficient anti-tuberculous drug regimen because of a M. tuberculosis strain displaying a high level of resistance to isoniazid, in the absence of the common mutations associated with isoniazid-resistance, including katG mutations and inhA promoter mutations. Whole-genome sequencing (WGS) identified a large loss-of-function insertion (>1000 pb) at the end of katG in the isolate together with a -57C>T ahpC mutation, a resistance mechanism that would have remained undetected by a conventional molecular targeted approach. A retrospective search using publicly available WGS data of more than 1200 isoniazid-resistant isolates and a similar sized control dataset of isoniazid-susceptible isolates revealed that most (22/31) isoniazid-resistant, KatG loss-of-function mutants had an associated rare ahpC promoter mutation. In contrast, only 7 of 1411 isoniazid-susceptible strains carried a rare ahpC promoter mutation, including shared mutations with the 31 isoniazid-resistant KatG loss-of-function mutants. These results indicate that rare ahpC promoter mutations could be used as a proxy for investigating simultaneous KatG loss-of-function or missense mutations. In addition, WGS in routine diagnosis would improve drug susceptibility testing in M. tuberculosis clinical isolates and is an efficient tool for detecting resistance mechanisms undetected by conventional molecular methods.

Improvement in Tuberculosis Outcomes With a Combined Medical and Social Approach.

Setting: Studies performed locally in Switzerland in the late eighties reported unsatisfactory treatment outcomes. Better outcomes were observed since the introduction of directly observed therapy (DOT) in the late nineties and improvement in social support in recent years. Design: retrospective study of treatment outcomes for all tuberculosis (TB) patients notified in Vaud County (VD), Switzerland, between, 1st of January 2010 and 31st of December of 2014. Results: 375 patients were notified in VD during the study period. The global outcome was successful in 90.1% of patients (338/375). In 183 culture and PCR positive pulmonary TB, the documented cure rate was 57.9% (106/183), and the treatment completion was 59/183 (32.2%), i.e., a treatment success of 90.2%. DOT was applied globally in 234/375 (62.4%) and in 64/67 of the asylum seekers (AS) (95.5%) followed at the dispensary. Treatment outcomes were successful in 60/67 (89.6%) AS. Discussion: Improvements in tuberculosis outcomes resulted not only from the introduction of DOT in VD in the nineties but also from a change in the management, with increased attention to the social problems faced by the migrants. Conclusion: A combined medical and social approach of TB care in VD improved treatment outcomes.

Added diagnostic value of 16S rRNA gene pan-mycobacterial PCR for nontuberculous mycobacterial infections: a 10-year retrospective study.

The diagnosis of mycobacterial infections has been dramatically improved by the introduction of molecular methods aimed to reduce the time to diagnosis as compared with culture. The broad range pan-mycobacterial PCR can detect all the mycobacterial species directly from clinical specimens. We aimed to evaluate its usefulness and its clinical added value for the diagnosis of nontuberculous mycobacterial (NTM) infections. We performed a retrospective study (2003-2013) including 952 samples taken from 639 patients with clinical suspicion of NTM infection. The performance of smear microscopy, PCR and culture was established using clinical data to investigate discrepant results. We also compared the time to microbial diagnosis between the direct PCR and culture. The sensitivity, specificity, positive and negative predictive values of the PCR were 61.6% (53.5-69.1), 99.1% (98.2-99.6), 92.8% (85.8-96.5) and 93.4% (91.6-94.9), respectively, when considering all specimens. When considering smear-positive specimens and smear-negative specimens, the sensitivity was 81.6% and 40%, respectively. The sensitivity for pulmonary and extra-pulmonary smear-positive specimens was 85.2% versus 72.7%. The median time to identification at species level was 35 days (SD, 17.67) for culture and 6 days (SD, 2.67) for the PCR (when positive), which represents a 29-day shorter time to results (p < 0.0001). The 16S rRNA gene pan-mycobacterial PCR displays a substantial benefit in terms of time to diagnose NTM infections when compared with culture. Despite an excellent specificity, its sensitivity is yet limited in particular for smear-negative specimens, which might be improved by relying onto real-time PCRs.

[Consensus for the management of infectious diseases among asylum seekers and detainees in the canton of Vaud]. / Consensus vaudois de prise en charge de maladies infectieuses auprès des requérants d'asile et détenus.

Our work develops procedures and useful tools for the screening, assessment and management of prevalent infectious or parasitic diseases (tuberculosis, measles, chickenpox, scabies, bed bugs) among asylum seekers and detainees in the canton of Vaud, populations living in similar closed settings. Its aim is to support health professionals in their work, to maintain the health of the target population and to protect the health of the community. Through a literature review and a focus group with experts, it is proposed to harmonize the existing procedures in asylum seekers centres end prisons of the canton of Vaud. The proposed decision algorithms are coherent with the recommendations of the literature and relevant in terms of public health and ethics, as well as feasible logistically and acceptable by the field health professionals.

Ce travail développe des procédures dans le cadre du dépistage et de la prise en charge de maladies infectieuses ou parasitaires prévalentes (tuberculose, rougeole, varicelle, gale, punaises de lit) chez les requérants d'asile et détenus du canton de Vaud, populations vivant dans des environnements similaires, afin de soutenir le personnel soignant dans son travail de terrain, veiller à la santé des personnes concernées et protéger la population locale. Il s'agit donc d'harmoniser, grâce à une revue de littérature et un focus group d'experts, les procédures existant dans les centres de requérants et de détenus vaudois en s'assurant que celles-ci correspondent aux recommandations de la littérature et soient pertinentes en termes de santé publique et d'éthique, ainsi que réalisables d'un point de vue logistique et acceptables par les équipes de terrain.

Added Value of Xpert MTB/RIF Ultra for Diagnosis of Pulmonary Tuberculosis in a Low-Prevalence Setting.

Xpert MTB/RIF (Xpert) for direct molecular detection of Mycobacterium tuberculosis and rifampin resistance from clinical specimens has dramatically improved the diagnosis of tuberculosis (TB). Xpert MTB/RIF Ultra (Ultra) is proposed as a substitute of Xpert with increased sensitivity and improved rifampin resistance detection. We evaluated the diagnostic performance of Ultra and Xpert for pulmonary TB diagnosis in a low-TB-burden setting. Performance of Ultra and Xpert were compared to culture on respiratory specimens from patients with suspected pulmonary TB (November 2016 to August 2018; n = 196) in Lausanne (Switzerland). Clinical data were used to investigate discrepant results. Correlation between semiquantitative result of Ultra and smear microscopy status for the detection of acid-fast bacilli (AFB) was established. The sensitivities of Xpert and Ultra were 82.9% (39/47) and 95.8% (45/47), respectively, when considering all culture-positive specimens, 100% (23/23) for both assays on smear-positive specimens, and 66.7% (16/24) and 91.7% (22/24) on smear-negative specimens. Using culture as gold standard, the specificities of Xpert and Ultra were 97.3% (145/149) and 96.64% (144/149), respectively. All the patients with Ultra-positive results with the new category "trace" were diagnosed with active TB based on clinical findings and microbiological culture. The semiquantitative results of both Xpert and of Ultra positively correlated with the semiquantitative result of AFB detection. Our data support an increased sensitivity of Ultra compared to Xpert in a low-prevalence setting. Correlation between the Ultra semiquantitative result and AFB burden can help in evaluating a patient's transmission potential.

Health-seeking behaviour and treatment delay in patients with pulmonary tuberculosis in Switzerland: some slip through the net.

AIMS OF THE STUDY: To assess the health-seeking behaviour, the patient delay (onset of symptoms to first consultation) and the health system delay (first consultation to start of tuberculosis treatment) among patients with pulmonary tuberculosis (TB) diagnosed in Switzerland, and to assess the predictors of the various types of delay. METHODS: A survey among pulmonary TB patients was carried out in six cantons, covering 42% of all pulmonary adult TB cases notified in Switzerland. Data were collected by collaborators of the cantonal lung associations in charge of the follow-up of TB patients to investigate treatment seeking behaviour and to establish various delays and its predictors. Predictors of percentiles of delay (median and 75th percentile) were assessed using quantile regression. RESULTS: Among 252 eligible patients, 162 patients could be interviewed. Of these, 20.4% were born in Switzerland. Cough as a symptom was mentioned by 76% of the interviewed patients. Almost half of the 162 patients (46%) consulted first a general practitioner in an ambulatory care setting and 26% approached a hospital first. The median delay between symptom onset and first healthcare contact (patient delay) was 5.2 weeks, which is slightly longer than findings in other low prevalence countries. The interquartile range was 1.6 to 14.2 weeks. The median delay between first consultation in Switzerland and the start of TB treatment (health system delay) was 2 weeks. The interquartile range was 0.6 to 7.1 weeks. There were no clear predictors of patient delay. The main predictors of a longer median health system delay were the presence of fever (1.6 weeks, 95% confidence interval [CI] 0.5 to 2.6 weeks), having visited first a general practitioner or a paediatrician (1 week, 95% CI 0.1 to 1.9 weeks) and having seen three or four doctors before beginning TB treatment (2.9 weeks, 95% CI 0.7 to 5.1 weeks). A clear predictor of a shorter median health system delay was having undergone an X-ray at the first consultation (-2.9 weeks, 95% CI -4.8 to -0.9 weeks). Marginally significant for shorter delay was male sex (-2.6 weeks, 95% CI -5.4 to 0.1 weeks). CONCLUSIONS: No predictor of patient delay was found among the variables collected. For one fourth of the patients, the health system delay was longer than 7 weeks. General practitioners are commonly approached first, and they have to consider TB, also for patients not considered at high-risk for TB.

Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

Combined use of Mycobacterium tuberculosis-specific CD4 and CD8 T-cell responses is a powerful diagnostic tool of active tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

Mycobacterium tuberculosis-specific CD8+ T cells are functionally and phenotypically different between latent infection and active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8(+) T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb-specific CD8(+) T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb-specific CD8(+) T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb-specific CD8(+) T cells in LTBI subjects were mostly T EMRA cells (CD45RA(+) CCR7(-)), coexpressing 2B4 and CD160, and in TB patients were mostly TEM cells (CD45RA(-) CCR7(-)), expressing 2B4 but lacking PD-1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb-specific CD8(+) T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro-expanded Mtb-specific CD8(+) T cells expressed perforin and granulysin. Finally, Mtb-specific CD8(+) T-cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb-specific CD8(+) T-cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb-specific CD8(+) T-cell responses. These results provide new insights in the interaction between Mtb and the host immune response.

Lack of Mycobacterium tuberculosis-specific interleukin-17A-producing CD4+ T cells in active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb-specific CD4(+) T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL-17A, IFN-γ, TNF-α, and IL-2 by Mtb-specific CD4(+) T cells was assessed both directly ex vivo and following in vitro antigen-specific T-cell expansion. Unlike for extracellular bacteria, Mtb-specific CD4(+) T-cell responses lacked immediate ex vivo IL-17A effector function in both LTBI and TB individuals. Furthermore, Mtb-specific Th17 cells were absent in BALs, while extracellular bacteria-specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb-specific CD4(+) T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL-17A following Mtb-specific T-cell expansion. Finally, IL-17A acquisition by Mtb-specific CD4(+) T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb-specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.

[Latent M. tuberculosis infection, update 2011]. / Infection latente a M. tuberculosis, mise a jour 2011.

It is estimated that one third of the world population is latently infected by Mycobacterium tuberculosis and thus at risk of reactivation. Latent tuberculosis (TB) impact in Switzerland is often overlooked. Diagnosis and prophylaxis are insufficiently undertaken, especially for people at higher risk of reactivation due to immunosuppression. Interferon-gamma release assays replace tuberculosis skin tests for diagnosis of latent infection in adults. It is still recommended to treat prophylactically a case of latent TB infection with 9 months of isoniazid; however therapy with rifampicin for 4 months, currently an alternative option, is linked to improved adherence and favorable cost-benefit ratio.

Low incidence of severe respiratory syncytial virus infections in lung transplant recipients despite the absence of specific therapy.

BACKGROUND: Respiratory syncytial virus (RSV) infections in lung transplant recipients (LTRs) have been associated with significant morbidity and mortality. Immunoglobulins, ribavirin, and palivizumab are suggested treatments for both pre-emptive and therapeutic purposes. However, in the absence of randomized, placebo-controlled trials, efficacy is controversial and there is toxicity as well as cost concerns. METHODS: We retrospectively reviewed cases of lower respiratory tract RSV infections in adult LTRs. Diagnosis was based on clinical history, combined with a positive polymerase chain reaction (PCR) and/or viral cultures of bronchoalveolar lavage (BAL) specimens. RESULTS: Ten symptomatic patients were identified (7 men and 3 women, age range 28 to 64 years). All were hospitalized for community-acquired respiratory tract infections. Two patients had a concomitant acute Grade A3 graft rejection, and 1 patient had a concomitant bacterial pneumonia. Eight patients did not receive a specific anti-RSV treatment because of clinical stability and/or improvement at the time of RSV diagnosis. Only 2 patients (1 with Grade A3 allograft rejection and 1 requiring mechanical ventilation) received ribavirin and palivizumab. All patients recovered without complications and with no persistent RSV infection. However, bronchiolitis obliterans (BOS) staging worsened in 6 patients during the mean follow-up of 45 months. CONCLUSIONS: Our data suggest that mild RSV infections in LTRs might evolve favorably in the absence of specific anti-viral therapy. However, this observation needs confirmation in a large clinical trial specifically investigating the development of BOS in untreated vs treated patients.

[Non tuberculous mycobacteria pulmonary disease: what's new?]. / Mycobactéries non tuberculeuses maladie pulmonaire: quoi de neuf?

The incidence of NTM (non tuberculous mycobacteria) pulmonary disease is increasing. The diagnosis must be established in the presence of clinical, radiological and microbiological findings. Groups at risk to contract pulmonary disease due to NTM are patients with underlying structural lung disease. Treatment of NTM is long and requires multiple drugs combinations. Relapses and re-infection are not rare. Our understanding in many matters of NTM pulmonary disease is incomplete. Further research is necessary in order to understand the host's defense mechanisms against NTM, and the factors that influence the evolution to lung disease.

[Immunization and immuno-modulation for prevention of respiratory tract infections]. / Immunisation, immunomodulation et prévention des infections respiratoires.

Respiratory tract infections are a major public health issue. Prevention in high risk populations relies mainly on vaccination against Influenza and S. pneumoniae. Vaccination of health-care workers is highly recommended, to decrease absenteism, but above all to protect high risk patients. New conjugate vaccines have shown their effectiveness in the paediatric population. In patients with chronic bronchitis or COPD, immunomodulatory agents (OM-85 BV) and anti-oxidants (NAC) are probably contributive in decreasing exacerbation rates. Inhaled corticosteroids decrease exacerbations in a well defined group of severe COPD. In patients with diffuse bronchiectasis, the immunomodulatory effect of macrolides, and the use of inhaled corticosteroids should be confirmed by larger clinical investigations.