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BackgroundThe 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.MethodsWe 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.ResultsThe 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).ConclusionsPCR 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.

Objective: To identify microorganisms in sputum samples of patients with stable non-cystic fibrosis bronchiectasis and to determine risk factors related to the isolation of Pseudomonas aeruginosa (PA) in those patients. Methods: Consecutive patients were recruited from a tertiary hospital outpatient clinic in the city of Fortaleza, Brazil. The patients were submitted to spirometry, six-minute walk test, HRCT, and sputum collection. Data on serum fibrinogen levels, disease severity, sputum color, and history of azithromycin treatment were collected. Results: The study included 112 patients, and females predominated (68%). The mean age was 51.6 ± 17.4 years. Most patients presented with mild-to-moderate disease (83%). The mean six-minute walk distance was 468.8 ± 87.9 m. Mean FEV1 and FVC, in % of predicted values, were 60.4 ± 21.8% and 69.9 ± 18.5%, respectively. The mean serum fibrinogen level was 396.1 ± 76.3 mg/dL. PA was isolated in 47 patients, other potentially pathogenic microorganisms (PPMs) were isolated in 31 patients, and non-PPMs were isolated in 34 patients. Purulent sputum was identified in 77 patients (68%). The patients with PA, when compared with those without it, presented with more severe disease, higher serum fibrinogen levels, and lower FVC%. In addition, purulent sputum and long-term azithromycin treatment were more common in those with PA. The multivariate regression analysis showed that the independent factors associated with PA were serum fibrinogen level > 400 mg/dL (OR = 3.0; 95% CI: 1.1-7.7) and purulent sputum (OR = 4.3; 95% CI: 1.6-11.3). Conclusions: In our sample, the prevalence of PA in sputum was 42%. Sputum color and inflammatory markers were able to predict the isolation of PA, emphasizing the importance of routine sputum monitoring. RESUMO Objetivo: Identificar microrganismos em amostras de escarro de pacientes com bronquiectasia não fibrocística estável e determinar os fatores de risco relacionados com o isolamento de Pseudomonas aeruginosa (PA) nesses pacientes. Métodos: Pacientes consecutivos foram recrutados em um ambulatório de um hospital terciário em Fortaleza (CE). Os pacientes foram submetidos a espirometria, teste de caminhada de seis minutos, TCAR e coleta de escarro. Foram coletados dados referentes ao fibrinogênio sérico, gravidade da doença, cor do escarro e histórico de tratamento com azitromicina. Resultados: O estudo incluiu 112 pacientes, com predomínio do sexo feminino (68%). A média de idade foi de 51,6 ± 17,4 anos. A maioria dos pacientes apresentou doença leve a moderada (83%). A média da distância percorrida no teste de caminhada de seis minutos foi de 468,8 ± 87,9 m. A média do VEF1 em % do previsto foi de 60,4 ± 21,8%, e a da CVF em % do previsto foi de 69,9 ± 18,5%. A média do fibrinogênio sérico foi de 396,1 ± 76,3 mg/dL. PA foi isolada em 47 pacientes; outros microrganismos potencialmente patogênicos (MPP) foram isolados em 31; não MPP foram isolados em 34. Escarro purulento foi identificado em 77 pacientes (68%). Os pacientes com PA, em comparação com aqueles sem, apresentaram doença mais grave, fibrinogênio sérico mais elevado e menor CVF%. Além disso, escarro purulento e tratamento prolongado com azitromicina foram mais comuns naqueles com PA. A análise de regressão multivariada mostrou que os fatores independentes relacionados com PA foram fibrinogênio sérico > 400 mg/dL (OR = 3,0; IC95%: 1,1-7,7) e escarro purulento (OR = 4,3; IC95%: 1,6-11,3). Conclusões: Em nossa amostra, a prevalência de PA no escarro foi de 42%. A cor do escarro e os marcadores inflamatórios foram capazes de prever o isolamento de PA, o que enfatiza a importância do monitoramento rotineiro do escarro.

Objectives: The contribution of intracellular and fastidious bacteria in Cystic fibrosis (CF) pulmonary exacerbations, and progressive lung function decline remains unknown. This project aimed to explore their impact on bacterial microbiota diversity over time in CF children. Methods: Sixty-one children enrolled in the MUCOVIB multicentre prospective cohort provided 746 samples, mostly nasopharyngeal swabs, throat swabs and sputa which were analysed using culture, specific real-time qPCRs and 16S rRNA amplicon metagenomics. Results: Chlamydia pneumoniae (n = 3) and Mycoplasma pneumoniae (n = 1) were prospectively documented in 6.6% of CF children. Microbiota alpha-diversity in children with a documented C. pneumoniae was highly variable, similarly to children infected with Staphylococcus aureus or Pseudomonas aeruginosa. The transition from routine follow-up visits to pulmonary exacerbation (n = 17) yielded variable changes in diversity indexes with some extreme loss of diversity. Conclusions: The high rate of C. pneumoniae detection supports the need for regular screenings in CF patients. A minor impact of C. pneumoniae on the microbial community structure was documented. Although detected in a single patient, M. pneumoniae should also be considered as a possible aetiology of lung infection in CF subjects., This study documented Chlamydia pneumoniae in 4.9% of children with cystic fibrosis, stressing the need for routine screening, and investigated its impact on airway microbiota by 16S amplicon-based metagenomics.

To assess the risk of tuberculosis (infection and disease) in children less than 15 years' old who are household contacts of pulmonary tuberculosis patients in three Colombian cities (Medellín, Cali, and Popayán).A cohort of 1,040 children household contacts of 380 adults with smear-positive pulmonary tuberculosis was followed up for 24 months. Study period 2005-2009.Tuberculin skin test was positive (≥10 mm) in 43.7% (95% CI: 39.2-48.2). Tuberculin skin test positivity was associated with age 10-14 years (Prevalence Ratio -PR= 1.43, 95% CI: 1.1-1.9), having a BCG vaccine scar (PR= 1.52, 95% CI: 1.1-2.1), underweight, closer proximity to the index case and exposure time3 months. The annual risk of infection (tuberculin skin test induration increase of 6 mm or more per year) was 17% (95% CI: 11.8-22.2) and was associated with a bacillary load of the adult index case (Relative Risk -RR= 2.12, 95% CI: 1.0-4.3). The incidence rate of active tuberculosis was 12.4 cases per 1,000 persons-year. Children5 years without BCG vaccine scar had a greater risk of developing active disease (Hazard Ratio -HR= 6.00, 95% CI: 1.3-28.3) than those with scar (HR= 1.33, 95% CI: 0.5-3.4). The risk of developing active tuberculosis augmented along with the increase from initial tuberculin skin test (tuberculin skin test 5-9 mm HR= 8.55, 95% CI: 2.5-29.2; tuberculin skin test ≥10 mm HR= 8.16, 95% CI: 2.0-32.9).There is a need for prompt interruption of adult-to-children tuberculosis transmission within households. Conducting proper contact investigation and offering chemoprophylaxis to infected children could reduce tuberculosis transmission.Evaluar el riesgo de tuberculosis (infección y enfermedad) en niños menores de 15 años de edad convivientes de pacientes con tuberculosis pulmonar en tres ciudades colombianas (Medellín, Cali y Popayán).Se siguió durante 24 meses una cohorte de 1,040 niños convivientes de 380 adultos con tuberculosis pulmonar bacilífera. Periodo de estudio 2005-2009.Es necesario interrumpir rápidamente la transmisión de tuberculosis de adultos a niños en los hogares. Realizar investigaciones de contacto apropiadas y ofrecer quimioprofilaxis a los niños infectados podría reducir la transmisión de la tuberculosis.

Purpose: Since May 2016, WHO recommended a 9–12 month short-treatment regimen for multidrug-resistant tuberculosis (MDR-TB) treatment known as the ‘Bangladesh Regimen’. However, limited data exist on the appropriateness thereof, and its implementation in low- and middle-income countries (LMIC). We report here on the pilot phase of the evaluation of the Bangladesh regimen in Gabon, prior to its endorsement by the WHO. Methods: This ongoing observational study started in September 2015. Intensive training of hospital health workers as well as community information and education were conducted. GeneXpert-confirmed MDR-TB patients received the second-line anti-tuberculosis drugs (4KmMfxPtoHCfzEZ/5MfxCfzEZ). Sputum smears and cultures were done monthly. Adverse events were monitored daily. Results: Eleven patients have been treated for MDR-TB piloting the short regimen. All were HIV-negative and presented in poor health with extensive pulmonary lesions. The overall sputum culture conversion rate was 64% after 4 months of treatment. Three patients developed marked hearing loss; one a transient cutaneous rash. Of 11 patients in our continuous care, 7 (63.6%) significantly improved clinically and bacteriologically. One (9.1%) patient experienced a treatment failure, two (18.2%) died, and one (9.1%) was lost to follow up. Conclusions: Our pioneering data on systematic MDR-TB treatment in Gabon, with currently almost total absence of resistance against the second-line drugs, demonstrate that a 9-month regimen has the capacity to facilitate early culture negativity and sustained clinical improvement. Close adverse events monitoring and continuous care are vital to success.

Establishing a correct diagnosis is challenging. We aimed to investigate the sensitivity and specificity of routine tuberculosis (TB) diagnostic work-up in lung clinics in Indonesia, a country with the third highest TB burden and the second highest gap between notifications of TB cases and the best estimate of incident cases in the world. In the lung clinics of the Province of Yogyakarta, Indonesia, we recruited all consecutive patients with symptoms suggesting TB, aged ≥18 years. Routine TB examination consisted of clinical evaluation, sputum smear microscopy, and chest radiography. For research purposes, we added sputum culture, Human Immunodeficiency Virus (HIV) testing, and follow-up for 1.5 years or 2.5 years if culture results disagreed with the initial clinical diagnosis. The initial diagnosis was considered incorrect if patients did not respond to treatment. We calculated sensitivity and specificity of the TB routine examination using culture and a composite reference standard (CRS – a combination of routine examination, culture, and follow-up) as the reference standards. All analyses were conducted with IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA). Between 2013 and 2015, we included 360 participants, and 21 were excluded due to incomplete data. Among those analyzed, 115 were initially diagnosed with smear-positive TB, 12 with smear-negative TB, and 212 non-TB. In 15 study participants, the diagnosis was changed after median 45 (range: 14–870) days; 14 participants initially not diagnosed with TB were later diagnosed with TB, while one subject initially diagnosed with TB actually did not have TB. Compared with culture and CRS, TB routine examination had sensitivity of 85% (95%CI: 77–91) and 90% (95%CI: 84–94), and specificity of 86.3% (95%CI: 81–91) and 99.5% (95%CI: 97–100), respectively. A combination of clinical evaluation with sputum microscopy and chest radiography provided high sensitivity and specificity in diagnosing TB in lung clinics; in only 4.4% the diagnosis was incorrect. There is a need to improve routine TB diagnostic work by using clinical evaluation, sputum smear microscopy, and chest radiography all together in other settings, such as in primary health centers. NCT02219945 , clinicaltrials.gov . Registered 19 August 2014 (retrospectively registered).

Tuberculosis (TB) control efforts are hampered by a mismatch in diagnostic technology. Lack of adequate early diagnostics and Multi-drug resistant (MDR) detection is a critical problem in control efforts. Alternate and novel diagnostic approaches are required, especially in low-resources settings where they are needed most. The Microscopic Observation Drug Susceptibility (MODS) assay is a cost-effective, highly sensitive, and specific method based on the detection of characteristic cording growth patterns of Mycobacterium tuberculosis (MTB), in microscopic examination of a liquid culture under an inverted microscope. By adding antimicrobials to the wells, MODS also determines antimicrobial susceptibility in both MDR and Extreme Drug Resistant (XDR) tuberculosis. The interpretation of a MODS culture performed in a 24 well plate, requires an extensive inspection over the entire surface to detect TB cords. This process requires significant time and effort from a trained microscopist. We evaluated a lens-free imager system, able to render microscopic images of live specimens, for the proof of principle to be used for MODS culture interpretation. The lens-free imager system is able to digitalize a 24-mm(2) surface with approximately 40X magnification in a single capture. The evaluation of the lens-free imager found that it produced microscopic images that were adequate for MODS interpretation by a human expert. Compared to the average time that takes a microscopist to completely examine a MODS culture sample, the lens free imager notably reduced the time of inspection. Therefore, lens-free imager variants may constitute promising systems to aid in the diagnostics of tuberculosis, by simplifying and reducing the time of inspection and permitting automatization of MODS interpretation.

Author(s): Walter, Nicholas D; Dolganov, Gregory M; Garcia, Benjamin J; Worodria, William; Andama, Alfred; Musisi, Emmanuel; Ayakaka, Irene; Van, Tran T; Voskuil, Martin I; de Jong, Bouke C; Davidson, Rebecca M; Fingerlin, Tasha E; Kechris, Katerina; Palmer, Claire; Nahid, Payam; Daley, Charles L; Geraci, Mark; Huang, Laurence; Cattamanchi, Adithya; Strong, Michael; Schoolnik, Gary K; Davis, John Lucian | Abstract: BackgroundTreatment initiation rapidly kills most drug-susceptible Mycobacterium tuberculosis, but a bacterial subpopulation tolerates prolonged drug exposure. We evaluated drug-tolerant bacilli in human sputum by comparing messenger RNA (mRNA) expression of drug-tolerant bacilli that survive the early bactericidal phase with treatment-naive bacilli.MethodsM. tuberculosis gene expression was quantified via reverse-transcription polymerase chain reaction in serial sputa from 17 Ugandans treated for drug-susceptible pulmonary tuberculosis.ResultsWithin 4 days, bacterial mRNA abundance declined g98%, indicating rapid killing. Thereafter, the rate of decline slowed g94%, indicating drug tolerance. After 14 days, 16S ribosomal RNA transcripts/genome declined 96%, indicating slow growth. Drug-tolerant bacilli displayed marked downregulation of genes associated with growth, metabolism, and lipid synthesis and upregulation in stress responses and key regulatory categories-including stress-associated sigma factors, transcription factors, and toxin-antitoxin genes. Drug efflux pumps were upregulated. The isoniazid stress signature was induced by initial drug exposure, then disappeared after 4 days.ConclusionsTranscriptional patterns suggest that drug-tolerant bacilli in sputum are in a slow-growing, metabolically and synthetically downregulated state. Absence of the isoniazid stress signature in drug-tolerant bacilli indicates that physiological state influences drug responsiveness in vivo. These results identify novel drug targets that should aid in development of novel shorter tuberculosis treatment regimens.

Summary Fluorescein diacetate microscopy was used to assess Mycobacterium tuberculosis metabolic activity in pretreatment sputa. Lower results predicted greater infectiousness in transmitting tuberculosis to household contacts. Fluorescein diacetate microscopy–negative bacteria may be better adapted to airborne tuberculosis transmission and consequently more contagious., Background Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness. Methods We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients’ 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years. Results FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47–386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%–11%) the concentration of acid-fast microscopy–positive bacteria (2069 [IQR, 1358–3734] bacteria/μL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy–positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02). Conclusions Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy–negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission.

BACKGROUND: The performance of laboratory tests to diagnose pulmonary tuberculosis is dependent on the quality of the sputum sample tested. The relative merits of sputum collection methods to improve tuberculosis diagnosis are poorly characterised. We therefore aimed to investigate the effects of sputum collection methods on tuberculosis diagnosis. METHODS: We did a systematic review and meta-analysis to investigate whether non-invasive sputum collection methods in people aged at least 12 years improve the diagnostic performance of laboratory testing for pulmonary tuberculosis. We searched PubMed, Google Scholar, ProQuest, Web of Science, CINAHL, and Embase up to April 14, 2017, to identify relevant experimental, case-control, or cohort studies. We analysed data by pairwise meta-analyses with a random-effects model and by network meta-analysis. All diagnostic performance data were calculated at the sputum-sample level, except where authors only reported data at the individual patient-level. Heterogeneity was assessed, with potential causes identified by logistic meta-regression. FINDINGS: We identified 23 eligible studies published between 1959 and 2017, involving 8967 participants who provided 19 252 sputum samples. Brief, on-demand spot sputum collection was the main reference standard. Pooled sputum collection increased tuberculosis diagnosis by microscopy (odds ratio [OR] 1·6, 95% CI 1·3-1·9, p

SummaryPathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value

Airborne precautions are required at hospital admission for patients with suspected pulmonary tuberculosis. The isolation is maintained until 3 serially collected sputum smears are acid-fast bacilli negative, a time- and labor-intensive method with limited sensitivity and specificity, which has a great impact on patient flow management. We evaluated the possibility of replacing the result of microscopy by the semiquantitative result of the molecular point-of-care test Xpert MTB/RIF to assess patients' transmission risk to quickly guide airborne isolation decisions in low-endemic countries. The performance of the Xpert MTB/RIF, used as a first-line test, was compared to the results of microscopy for specimens (n=242) collected from May 2010 to December 2014 in Lausanne, Switzerland. The sensitivity and specificity of Xpert MTB/RIF were 91.5% (65/71) and 99.6% (170/171), respectively, vs. 64.8% (46/71) and 94.2% (161/171) for microscopy. Samples with negative Xpert MTB/RIF were all smear negative for Mycobacterium tuberculosis (negative predictive value, 100%). The semiquantitative results of Xpert MTB/RIF—high, medium, low or very low—were found to correlate with acid-fast bacilli detection: positive predictive value of 100% (6/6), 96.5% (27/28), 52.2% (12/23) and 11.1% (1/9) respectively. Finally, when including clinical criteria, we identified 11 smear-negative but Xpert MTB/RIF–positive patients with a significant transmission potential. In conclusion, our data support the introduction of an Xpert MTB/RIF–based strategy as a replacement of smear microscopy for a faster and more accurate management of tuberculosis patients' transmission risk in a low-prevalence country.

OBJECTIVE: To evaluate the diagnostic performance of manual MGIT (MMGIT) compared to the gold standard, Lowenstein-Jensen (LJ), in the diagnosis of pulmonary tuberculosis (TB) in a high-burden setting. METHODS: Individuals with suspected TB enrolled in parallel diagnostic trials during 2007-2011 were included. Two samples were obtained from each patient and inoculated into MMGIT and LJ medium. Diagnostic tests were performed, and the incremental yield of a second test and time to detection (TTD) were calculated. Analyses were performed per patient and per sample. Gold standard was based on LJ culture. RESULTS: In the per patient and per sample analysis, we evaluated 1436 patients and 4142 samples. The sensitivity and specificity for smear and MMGIT per sample were respectively 89.9%/92.2% and 97.1%/98.9%. Contamination was observed in 1.4% of samples on MMGIT. The mean TTD (days) was 11.8 for MMGIT and 22.9 for LJ. The sensitivity and specificity for smear and MMGIT per patient were respectively 89.9% and 92.2% and 97.1% and 98.3%. A second MMGIT culture had an incremental yield of 1.6%. CONCLUSIONS: MMGIT has high sensitivity and specificity, regardless of smear result, with a 50% reduction in TTD compared to LJ. These features make MMGIT an acceptable TB diagnostic method for use in resource-limited settings.

Background Household contacts (HHCs) of TB cases are at increased risk for TB disease compared to the general population but the risk may be modified by individual or household factors. We conducted a study to determine incident TB among HHCs over two years after exposure and to identify individual and household level risk factors. Methods Adults newly diagnosed with a first episode of smear-positive pulmonary TB (index cases) between March 2010 and December 2011 in eastern Lima, were interviewed to identify their HHC and household characteristics. TB registers were reviewed for up to two years after the index case diagnosis and house visits were made to ascertain TB cases among HHC. The TB incidence rate ratio among HHCs as a function of risk factors was determined using generalized linear mixed models. Results The 1178 index cases reported 5466 HHCs. In 402/1178 (34.1 %) households, at least one HHC had experienced a TB episode ever. The TB incidence among HHCs was 1918 (95%CI 1669–2194) per 100,000 person-years overall, and was 2392 (95%CI 2005–2833) and 1435 (95%CI 1139–1787) per 100,000 person-years in the first and second year, respectively. Incident TB occurred more than six months following the index case’s TB diagnosis in 121/205 (59.0 %) HHCs. In HHCs, bacillary load and time between symptoms and treatment initiation in the index case, as well as the relationship to the index case and the sex of the HHC all had a significant association with TB incidence in HHCs. Conclusions Incidence of TB among HHCs was more than ten times higher than in the general population. Certain HHC and households were at higher risk of TB, we recommend studies to compare HHC investigation to households at highest risk versus current practice, in terms of efficiency. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1616-x) contains supplementary material, which is available to authorized users.

Background: Early identification of patients with drug-resistant tuberculosis (DR-TB) increases the likelihood of treatment success and interrupts transmission. Resource-constrained settings use risk profiling to ration the use of drug susceptibility testing (DST). Nevertheless, no studies have yet quantified how many patients with DR-TB this strategy will miss. Methods: A total of 1,545 subjects, who presented to Lima health centres with possible TB symptoms, completed a clinic-epidemiological questionnaire and provided sputum samples for TB culture and DST. The proportion of drug resistance in this population was calculated and the data was analysed to demonstrate the effect of rationing tests to patients with multidrug-resistant TB (MDR-TB) risk factors on the number of tests needed and corresponding proportion of missed patients with DR-TB. Results: Overall, 147/1,545 (9.5 %) subjects had culture-positive TB, of which 32 (21.8 %) had DR-TB (MDR, 13.6 %; isoniazid mono-resistant, 7.5 %; rifampicin mono-resistant, 0.7 %). A total of 553 subjects (35.8 %) reported one or more MDR-TB risk factors; of these, 506 (91.5 %; 95 % CI, 88.9–93.7 %) did not have TB, 32/553 (5.8 %; 95 % CI, 3.4–8.1 %) had drug-susceptible TB, and only 15/553 (2.7 %; 95 % CI, 1.5–4.4 %) had DR-TB. Rationing DST to those with an MDR-TB risk factor would have missed more than half of the DR-TB population (17/32, 53.2 %; 95 % CI, 34.7–70.9). Conclusions: Rationing DST based on known MDR-TB risk factors misses an unacceptable proportion of patients with drug-resistance in settings with ongoing DR-TB transmission. Investment in diagnostic services to allow universal DST for people with presumptive TB should be a high priority.

Summary Background Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. Methods We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 μg/ml and 800 μg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. Results The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 μg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 μg/ml PZA were more accurate than 800 μg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. Conclusions The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

© 2015 Shah et al. Background: We designed a pragmatic stepped-wedge cluster randomized controlled trial in order to evaluate provider-initiated evaluation of household contacts (HCs) of smear positive tuberculosis (TB) cases within a routine TB program in Lima, Peru. Methods/Design: National TB program (NTP) officers of San Juan de Lurigancho District (Lima, Peru) and university-based researchers jointly designed a pragmatic stepped-wedge cluster randomized trial design in order to evaluate a planned active case finding (ACF) program for all HCs of smear-positive TB cases in 34 district healthcare centres. Randomization of time to intervention initiation was stratified by health centre TB case rate. The ACF intervention included provider-initiated home visits of all new sputum smear positive TB patients in order to evaluate household contacts for active TB. Active TB was diagnosed using symptom screening, sputum screening, chest x-ray and clinical evaluation. Once initiated, ACF was provided by NTP staff and integrated into the routine DOTS TB program activities. Discussion: This study protocol describes the pragmatic stepped-wedge cluster randomized trial of active household contact evaluations within an NTP. The stepped-wedge design met overlapping needs of local TB programmers and researchers to adequately evaluate the large-scale roll out of a new control program in a TB endemic setting. Multiple planning meetings were required to develop the necessary networks and in order to understand the operations, needs and goals of the NTP staff and researchers collaborating on this project. The advantages and challenges of using this study design in practice and within existing routine TB programs in a middle-income country context are discussed. Trial registration: ClinicalTrials.gov NCT02174380. Registered 24 Jun 2014

Background: Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in HIV-positive people worldwide. Diagnosing TB is difficult, and is more challenging in resource-scarce settings where culture-based diagnostic methods rely on poorly sensitive smear microscopy by Ziehl-Neelsen stain (ZN). Methods: We performed a cross-sectional study examining the diagnostic utility of Microscopic Observation Drug Susceptibility liquid culture (MODS) versus traditional Ziehl-Neelsen staining (ZN) and Lowenstein Jensen culture (LJ) of pulmonary tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB) in HIV-infected patients in Bolivia. For sputum scarce individuals we assessed the value of the string test and induced sputum for TB diagnosis. The presence of Mycobacterium tuberculosis (Mtb) in the sputum of 107 HIV-positive patients was evaluated by ZN, LJ, and MODS. Gastric secretion samples obtained by the string test were evaluated by MODS in 102 patients. Results: The TB-HIV co-infection rate of HIV patients with respiratory symptoms by sputum sample was 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, and 30/48 (63 %) by ZN. The rate of MDRTB was 9 % (4/48). Median time to positive culture was 10 days by MODS versus 34 days by LJ (p

Improved tuberculosis (TB) diagnostics are required. Induced sputum sampling is superior to spontaneous sputum analysis for diagnosis of pulmonary TB. Therefore, we examined the applicability of induced sputum in primary health centers of the Peruvian TB program and studied the safety and tolerability of this procedure. We show that induced sputum is safe, inexpensive, and well-tolerated in a resource-limited environment. Widespread use of induced sputum at primary health centers can be implemented and may improve TB diagnosis.

Tuberculosis viability microscopy predicted, within 1 hour, quantitative culture results that became available weeks later. Viability microscopy provides promising results for informing decisions concerning drug susceptibility testing, treatment changes, and infection control measures in resource-constrained settings where most tuberculosis occurs., Background. It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis. Methods. To assess treatment response, sputum samples (n = 124) from unselected patients (n = 35) with sputum microscopy–positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate. Results. Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (rS = 0.85; P < .0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P < .001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a >10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P = .4) or quantitative culture (P = .6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P < .001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P = .6). Conclusions. Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.

Real-time polymerase chain reaction (qPCR) was optimized for detecting Mycobacterium tuberculosis in sputum. Sputum was collected from patients (N = 112) with suspected pulmonary tuberculosis, tested by smear microscopy, decontaminated, and split into equal aliquots that were cultured in Lowenstein-Jensen medium and tested by qPCR for the small mobile genetic element 1S6110. The human ERV3 sequence was used as an internal control. 3 of 112 (3%) qPCR failed. For the remaining 109 samples, qPCR diagnosed tuberculosis in 79 of 84 patients with culture-proven tuberculosis, and sensitivity was greater than microscopy (94% versus 76%, respectively, P < 0.05). The qPCR sensitivity was similar (P = 0.9) for smear-positive (94%, 60 of 64) and smear-negative (95%, 19 of 20) samples. The qPCR was negative for 24 of 25 of the sputa with negative microscopy and culture (diagnostic specificity 96%). The qPCR had 99.5% sensitivity and specificity for 211 quality control samples including 84 non-tuberculosis mycobacteria. The qPCR cost similar to 5US$ per sample and provided same-day results compared with 2-6 weeks for culture.

Thirty-five patients with clinical features and histologically or microbiologically proven infection met predetermined stringent criteria for invasive aspergillosis over a 5-year period at our institution. Underlying conditions included hematologic malignancy, solid tumor, bone marrow and solid organ transplantation, and immunosuppressive therapy. The majority of patients (94%) presented with respiratory symptoms and abnormal pulmonary chest radiography; only 40% had neutropenia at time of infection. Invasive aspergillosis was suspected in only 21 cases (60%). Concomitant infections were present in 83% of patients. Half of patients had pathogenic or potentially pathogenic microorganisms other than Aspergillus spp. isolated from pulmonary specimens at time of aspergillosis. Aspergillus spp. were recovered from sputum in 75% of patients and from bronchoalveolar lavage in only 52%. Invasive aspergillosis is an unexpectedly unrecognized disease with poor outcome; overall mortality was 94% in our series. The lack of sensitivity of diagnostic procedures, together with the high frequency of concomitant infections, delays the time of diagnosis. Early diagnostic tests are needed, and presumptive antifungal therapy among high-risk patients is mandatory.

Author(s): Trollip, AP; Moore, D; Coronel, J; Caviedes, L; Klages, S; Victor, T; Romancenco, E; Crudu, V; Ajbani, K; Vineet, VP; Rodrigues, C; Jackson, RL; Eisenach, K; Garfein, RS; Rodwell, TC; Desmond, E; Groessl, EJ; Ganiats, TG; Catanzaro, A | Abstract: ObjectiveTo establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay.SettingA multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa.DesignIn the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum.ResultsBreakpoints for MFX (0.5 μg/ml), OFX (1 μg/ml), AMK (2 μg/ml), KM (5 μg/ml) and CPM (2.5 μg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low.ConclusionMODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

Mucormycosis is increasingly common in patients with risk factors such as diabetes mellitus, neutropenia, and corticosteroid therapy. However, mucormycosis seems to be less common in patients with human immunodeficiency virus (HIV) infection compared to patients with other risk factors. Despite their lower virulence, Lichtheimia species should be regarded as emerging pathogens among Mucoralean fungi. We report a fatal case of pulmonary mucormycosis due to Lichtheimia ramosa in a 52-year-old man with an end-stage HIV infection. He had a cachectic appearance and his CD4 count was 8 cells/mm(3). The fungal infection was diagnosed based on a positive sputum culture with histopathologic confirmation. The fungus was resistant to caspofungin, anidulafungin, and voriconazole [minimum inhibitory concentration (MCI) >32 µg/ml], whereas the E test MIC values of itraconazole, posaconazole, and amphotericin B were 0.38, 0.38, and 0.5 µg/ml, respectively. Although intravenous drug use is the main risk factor for the development of mucormycosis in HIV-infected patients, it may also develop in patients with low CD4 count, opportunistic infections and/or additional diseases, such as Kaposi's sarcoma or severe immunodeficiency, as in our case.

BACKGROUND: Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. METHODS & FINDINGS: 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3-99.8%), 98.3% (97.5-98.8%), 95.8% (94.0-97.1%), and 99.7% (99.3-99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9-13) days for conventional MODS and 8.5 (7-11) for MODS Kit (p

Drug susceptibility testing (DST) is often needed in patients clinically failing tuberculosis (TB) therapy. Most studies of phenotypic direct drug susceptibility tests, such as microscopic observation drug susceptibility (MODS) tests, have been performed in patients not receiving TB treatment. The effect of ongoing TB treatment on the performance of MODS direct DST has not been previously explored, but patients failing such therapy constitute an important target group. The aim of this study was to determine the performance of MODS direct rifampicin and isoniazid DST in patients clinically failing first-line TB treatment, and to compare MODS direct DST with indirect proportion method DST. Sputa from 264 TB patients were cultured in parallel in Lowenstein–Jensen (LJ) and MODS assays; strains were tested for rifampicin and isoniazid susceptibility by the proportion method at the national reference laboratory. Ninety-three samples were culture-positive by LJ and MODS (concordance of 96%; kappa 0.92). With conventional MODS plate DST reading (performed on the same day as the sample is classified as culture-positive), the isoniazid DST concordance was 96.8% (kappa 0.89), and the concordance for rifampicin susceptibility testing was 92.6% (kappa 0.80). Reading of MODS DST plates 1 week after cultures had been determined to be culture-positive improved overall performance marginally—the isoniazid DST concordance was 95.7% (kappa 0.85); and the rifampicin DST concordance was 96.8% (kappa 0.91). Sensitivity for detection of multidrug-resistant TB was 95.8%. MODS testing provided reliable rifampicin and isoniazid DST results for samples obtained from patients receiving TB therapy. A modified DST reading schedule for such samples, with a final reading 1 week after a MODS culture turns positive, marginally improves the concordance with reference DST.

Pseudomonas aeruginosa is an important opportunistic pathogen. P. aeruginosa strains secrete several virulence factors, in the form of extracellular proteins. Adhesins, pyocyanin, proteases, hemolysins, exotoxin A and exoenzyme S are some of the virulence factors found in P. aeruginosa strains. In this study, the presence of siderophore, total matrix protease and elastase activities were investigated in a total of 157 P. aeruginosa strains isolated from lower respiratory tract (n: 81; sputum, bronchoalveolar lavage, tracheal aspirate) and extrarespiratory sites (n: 76; urine, wound, blood) of hospitalized pati ents. Chrome azurol S (CAS) agar plates were used for detection of siderophore activity. Hide powder azure was used for the investigation of total matrix protease activity and elastin congo red was used to test elastase activity. All strains gave positive reaction on CAS agar. Enzyme activities of the test strains were compared with the activity of P. aeruginosa PAO1 positive control strain. Mean total matrix protease and elastase activities were less than P. aeruginosa PAO1 activity in the test strains, however, some strains exhibited activity higher than PAO1. There was no significant difference for mean protease and elastase activities between the strains isolated from lower respiratory tract samples and the others (p > 0.05) [corrected] as well as no difference with respect to antibiotic resistance (p > 0.05) [corrected] It was found that ceftazidime and cefoperazone were the most resistant agents in both groups (67.9% and 57.9% for ceftazidime and 49.3% and 48.7% for cefoperazone, respectively). It was concluded that further in vivo studies are necessary to clarify the role of virulence factors of P. aeruginosa in the establishment of infection in different body sites.

Serum resistance is one of the major virulence factors of Gram negative bacteria. The aim of this study was to investigate the serum resistance of Pseudomonas aeruginosa and Acinetobacter baumannii strains isolated from various clinical samples (19 tracheal aspirates, 14 urine, 7 sputum, 7 wound and one peritoneal fluid specimens). Forty-eight P. aeuginosa and 48 A. baumannii strains were tested for human serum bactericidal effect by using Benge's method. Thirty-five (72.9%), 9 (18.7%), and 4 (8.3%) of P. aeruginosa strains were found to be resistant, intermediate sensitive and sensitive to serum, respectively. These rates were detected as 81.2% (39/48), 14.5% (7/48), and 4.1% (2/48) for A. baumannii strains, respectively. It can be concluded that, high serum resistance rates of P. aeruginosa and A. baumannii isolates might have an important role in the pathogenesis of infections of these bacteria.

A 56 year old man with a history of smoking related COPD was admitted to our hospital with a one week history of worsening chronic cough with the recent development of haemorrhagic phlegm and a subfebrile state. Chest x-rays showed a cavitary infiltrate in the left upper lobe, a right pleural effusion and bilateral mediastinal partially calcified enlarged lymph nodes. Direct sputum examination was positive for M. tuberculosis and was confirmed by culture. Eight days after initiation of a standard four drug antituberculosis treatment regime the patient presented with repeated and major haemoptysis of up to 200 ml/event. Chest x-ray showed a new air fluid level in the left superior cavity. A CTscan of the thorax disclosed a voluminous Rasmussen’s pseudoaneurysm (figure 1). Selective catheterization using a femoral approach to the left apical superior pulmonary artery showed the pseudoaneurysm arising from a subsegmental branch (figure 2). The branch was selectively embolized with 3 coils (0.035 inches 5–3 mm in diameter [COOK, Bloomington, Indiana, USA]) resulting in occlusion of this segmental branch and no filling of the pseudoaneurysm (figure 2). Haemoptysis stopped and over the next few days sputum became negative for acid fast bacilli. No further surgical intervention was performed. Chest x-ray after one year follow-up showed resolution of the cavitary lesion and no recurrence of haemoptysis. Standardized tuberculosis treatment with a four drug regime (isoniazid – rifampicin – pyrazinamide – ethambutol) over two months followed by four months of a two drug regime (isoniazid – rifampicin) was successfully completed and the patient was declared cured at the end of the treatment (1 direct exam and 2 negative cultures before the end of the treatment) [1].

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most common human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum samples from patients with clinicopathological evidence of tuberculosis and sarcoidosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, amplifying a 243 bp fragment of the gene encoding the immunogenic protein MPB 64, whereas the second traces members of the Mycobacterium avium (M. avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp region of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chain reaction assay identified mycobacterial DNA of 10(-3) colony-forming units (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacteria positive samples in the first case and in the second we found 95 M. tuberculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria positive samples. The sensitivity of the assay was significantly higher than that of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacterial DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved. Mod Pathol

Fifty-eight cases of bacteremia due to Moraxella catarrhalis, including seven that occurred in patients treated at our facilities, are analyzed. The host's medical history plays a major role in the presentation and outcome of M. catarrhalis bacteremia. Bacteremia is typically accompanied by pneumonia in adults with underlying respiratory disease. Many neutropenic patients do not manifest a focus of infection; in contrast, the source identified in healthy, immunocompetent patients is usually the upper airway or the ears. In the recent literature, it has been reported that a rash is typically absent in adults with bacteremic pneumonia and in immunocompetent hosts and that only some neutropenic patients have a rash. The prognosis is grave for patients with endocarditis and for patients with immunoglobulin deficiency or neutropenia not related to a hematologic malignancy. In addition, mortality is substantial among bacteremic patients with respiratory conditions or other chronic debilities, especially when respiratory copathogens are present. The prognosis is good for febrile neutropenic patients with underlying leukemia or lymphoma when the neutropenia resolves. When healthy, immunocompetent individuals are affected with M. catarrhalis bacteremia, their presentations range from self-limited febrile illness to life-threatening disease. Clin Infect Dis

Background Routine tuberculosis culture remains unavailable in many high-burden areas, including Tanzania. This study sought to determine the impact of providing mycobacterial culture results over standard of care [unconcentrated acid-fast (AFB) smears] on management of persons with suspected tuberculosis. Methods Adults and children with suspected tuberculosis were randomized to standard (direct AFB smear only) or intensified (concentrated AFB smear and tuberculosis culture) diagnostics and followed for 8 weeks. The primary endpoint was appropriate treatment (i.e. antituberculosis therapy for those with tuberculosis, no antituberculous therapy for those without tuberculosis). Results Seventy participants were randomized to standard (n = 37, 53%) or intensive (n = 33, 47%) diagnostics. At 8 weeks, 100% (n = 22) of participants in follow up randomized to intensive diagnostics were receiving appropriate care, vs. 22 (88%) of 25 participants randomized to standard diagnostics (p = 0.14). Overall, 18 (26%) participants died; antituberculosis therapy was associated with lower mortality (9% who received antiuberculosis treatment died vs. 26% who did not, p = 0.04). Conclusions Under field conditions in a high burden setting, the impact of intensified diagnostics was blunted by high early mortality. Enhanced availability of rapid diagnostics must be linked to earlier access to care for outcomes to improve.