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The Truenat MTB Plus assay is a rapid molecular test that has been recommended by the World Health Organization since 2020 as an initial test to detect tuberculosis (TB). The WHO highlighted the need to further evaluate assay performance to inform future recommendations, including in people living with HIV and compared to the Xpert MTB/RIF assay. We conducted a prospective evaluation of the diagnostic accuracy of the Truenat assay in Cameroon, a country with a high burden of HIV/TB. Adult outpatients were recruited at four hospitals; demographic information and medical history were collected, and participants produced two sputum specimens. Truenat and Xpert testing was performed on the same specimen, and performance was compared to TB culture as the reference standard. From November 2019 to December 2020, 945 participants were enrolled and included in the analysis. Among 251 participants with culture-positive TB, the sensitivity of Truenat MTB Plus was 91% (95% confidence interval [CI], 86 to 94%), similar to Xpert (90%; 95% CI, 86 to 93%). Among 74 HIV-positive participants with culture-positive TB, the sensitivity of Truenat MTB Plus was 85% (95% CI, 75 to 92%) compared to 81% for Xpert (95% CI, 70 to 89%). Among 47 participants with smear-negative TB, the sensitivity of Truenat MTB Plus was 55% (95% CI, 40 to 70%), similar to Xpert (53%; 95% CI, 38 to 68%). The specificity of Truenat MTB Plus was 96% (95% CI, 94 to 97%) compared to 99% (95% CI, 97 to 99%) for Xpert. For TB detection compared to the reference standard of TB culture, the performance of the Truenat MTB Plus assay was similar to that of Xpert in this population, including among people living with HIV.

Rapid and accurate identification of staphylococcal pneumonia is crucial for effective antimicrobial stewardship. We performed a meta-analysis to evaluate the diagnostic value of nucleic acid amplification tests (NAAT) from lower respiratory tract (LRT) samples from suspected pneumonia patients to avoid superfluous empirical methicillin-resistant Staphylococcus aureus (MRSA) treatment. PubMed, Scopus, Embase, Web of Science, and the Cochrane Library Database were searched from inception to 2 September 2020. Data analysis was carried out using a bivariate random-effects model to estimate pooled sensitivity, specificity, positive likelihood ratio (PLR), and negative likelihood ratio (NLR). Of 1,808 citations, 24 publications comprising 32 data sets met our inclusion criteria. Twenty-two studies (n = 4,630) assessed the accuracy of the NAAT for methicillin-sensitive S. aureus (MSSA) detection, while 10 studies (n = 2,996) demonstrated the accuracy of the NAAT for MRSA detection. The pooled NAAT sensitivity and specificity (with 95% confidence interval [CI]) for all MSSA detection were higher (sensitivity of 0.91 [95% CI, 0.89 to 0.94], specificity of 0.94 [95% CI, 0.94 to 0.95]) than those of MRSA (sensitivity of 0.75 [95% CI, 0.69 to 0.80], specificity of 0.88 [95% CI, 0.86 to 0.89]) in lower respiratory tract (LRT) samples. NAAT pooled sensitivities differed marginally among different LRT samples, including sputum, endotracheal aspirate (ETA), and bronchoalveolar lavage (BAL) fluid. Noticeably, NAAT pooled specificity against microbiological culture was consistently ≥88% across various types of LRT samples. A meta-regression and subgroup analysis of study design, sample condition, and patient selection method could not explain the heterogeneity (P > 0.05) in the diagnostic efficiency. This meta-analysis has demonstrated that the NAAT can be applied as the preferred initial test for timely diagnosis of staphylococcal pneumonia in LRT samples for successful antimicrobial therapy.

International audience; Young children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Efforts to control transmissible infectious diseases rely on the ability to screen large populations, ideally in community settings. These efforts can be limited by the requirement for invasive or logistically difficult collection of patient samples, such as blood, urine, stool, sputum, and nasopharyngeal swabs. Oral sampling is an appealing, noninvasive alternative that could greatly facilitate high-throughput sampling in community settings. Oral sampling has been described for the detection of dozens of human pathogens, including pathogens whose primary sites of infection are outside of the oral cavity, such as the respiratory pathogens Mycobacterium tuberculosis and SARS-CoV-2. Oral sampling can demonstrate active infections as well as resolving or previous infections, the latter through the detection of antibodies. Its potential applications are diverse, including improved diagnosis in special populations (e.g., children), population surveillance, and infectious disease screening. In this minireview, we address the use of oral samples for the detection of diseases that primarily manifest outside the oral cavity. Focusing on well-supported examples, we describe applications for such methods and highlight their potential advantages and limitations in medicine, public health, and research.

Transrenal urine cell-free DNA (cfDNA) is a promising tuberculosis (TB) biomarker, but is challenging to detect because of the short length (

A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone., A nonsputum triage test to rule out tuberculosis (TB) disease is a WHO high-priority diagnostic, and a combinatory score based on a 3-gene host signature has shown promise in discriminating TB from other illnesses. We evaluated the accuracy of an early-prototype cartridge assay (“Xpert MTB Host Response” or Xpert-MTB-HR-Prototype) of this 3-gene signature on biobanked blood samples from people living with HIV (PLHIV) against a comprehensive microbiological reference standard (CMRS) and against Xpert MTB/RIF on the first sputum sample alone. We depict results based on performance targets set by the WHO in comparison with a laboratory-based C-reactive protein (CRP) assay. Of 201 patients included, 67 were culture positive for Mycobacterium tuberculosis. The areas under the concentration-time curve (AUCs) for Xpert-MTB-HR-Prototype were 0.89 (confidence interval [CI], 0.83 to 0.94) against the CMRS and 0.94 (CI, 0.89 to 0.98) against Xpert MTB/RIF. Considering Xpert-MTB-HR-Prototype as a triage test (at the nearest upper value of sensitivity to 90%), specificities were 55.8% (CI, 47.2 to 64.1%) compared to the CMRS and 85.9% (CI, 79.3 to 90.7%) compared to Xpert MTB/RIF as confirmatory tests. Considering Xpert-MTB-HR-Prototype as a stand-alone diagnostic test, at a specificity near 95%, the test achieved a sensitivity of 65.7% (CI, 53.7 to 75.9%), while the CRP assay achieved a sensitivity of only 13.6% (CI, 7.3 to 23.4%). In this first accuracy study of a prototype blood-based host marker assay, we show the possible value of the assay for triage and diagnosis in PLHIV.

The cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens., The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine–sodium hydroxide (NALC–NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.

Mycobacterium septicum is a rarely identified nontuberculous mycobacterium capable of causing infections in both healthy and immunocompromised individuals. Only a few cases of M. septicum infections have been reported, which makes recognizing corresponding clinical disease more challenging for clinicians. Antimicrobial susceptibility profiles for this organism are not well described, and corresponding optimal therapeutic regimens have not been established. We report a tertiary care center’s experience with M. septicum from 2014 to 2020. Twelve adult patients with positive cultures for M. septicum were identified. Most cases were identified from sputum samples of individuals with underlying lung disease. Most cases involving M. septicum isolation in culture were not felt to be clinically significant. Two cases were considered possible infections, while only one case was considered a definite infection that required antimicrobial treatment. All M. septicum isolates were susceptible in vitro to amikacin, ciprofloxacin, imipenem, linezolid, moxifloxacin, and trimethoprim-sulfamethoxazole. Isolates were universally resistant to clarithromycin and doxycycline. The isolation of M. septicum in culture is uncommon and requires clinical correlation to determine its clinical relevance and need for treatment. Susceptibility testing should be performed to guide therapy.

The diagnosis of tuberculosis (TB) in HIV-infected patients is challenging. Both a urinary lipoarabinomannan (LAM) test (Alere TB LAM) and GeneXpert-MTB/RIF (Xpert) are useful for the diagnosis of TB. However, how to optimally integrate Xpert and LAM tests into clinical practice algorithms remain unclear. We performed a post hoc analysis of 561 HIV-infected sputum-expectorating patients (median CD4 count of 130 cells/ml) from a previously published randomized controlled trial evaluating the LAM test in hospitalized HIV-infected patients with suspected TB. We evaluated 5 different diagnostic strategies using sputum culture as a reference standard (Xpert alone, LAM alone, sequential Xpert followed by LAM and vice versa [LAM in Xpert-negative patients and Xpert in LAM-negative patients], and both tests concurrently [LAM + Xpert]). A cost-consequence analysis was performed. Strategy-specific sensitivity and specificity, using culture as a reference, were similar with the Xpert-only and sequential and concurrent strategies. However, when any positive TB-specific test was used as a reference, the incremental yield of LAM over Xpert was 29.6% (45/152) and that of Xpert over LAM was 75% (84/11). The incremental yield of LAM increased with decreasing CD4 count. The costs per TB case diagnosed were similar for the sequential and concurrent strategies ($1,617 to $1,626). In sputum-expectorating hospitalized patients with advanced HIV and access to both tests, concurrent testing with Xpert and LAM may be the best strategy for diagnosing TB. These data inform clinical practice in settings where TB and HIV are endemic.

Rapid diagnosis of infections caused by carbapenem-resistant Enterobacteriaceae (CRE) is crucial for proper treatment and infection control. The Xpert Carba-R assay is a qualitative multiplex real-time PCR method that qualitatively detects and differentiates five common carbapenemase genes (bla(KPC), bla(NDM), bla(VIM), bla(OXA-48), and bla(IMP)) directly from rectal swabs or purified colonies within approximately 1 h. We performed a multicenter evaluation of the investigational use of the Carba-R assay for detection and differentiation of carbapenemase genes from sputum specimens in patients with a clinical diagnosis of pneumonia. The intra- and interassay coefficients of variation values for the Carba-R assay were 0.2% to 2.0% and 1.4% to 2.3%, respectively. A total of 301 sputum specimens were collected and tested. Compared to bacterial culture followed by PCR identification of resistance genes from colonies, the Carba-R assay reduced turnaround time from 56 to 84 h to less than 2 h. Carbapenemase genes were detected by the Carba-R assay in Klebsiella pneumoniae (n = 236), Escherichia coli (n = 22), Enterobacter cloacae (n = 23), Klebsiella oxytoca (n = 8), Serratia marcescens (n = 6), Citrobacter freundii (n = 4), and Klebsiella aerogenes (n = 2). The Carba-R assay detected 112 bla(KPC) (33.5%), 70 bla(NDM) (21.0%), 8 bla(IMP) (2.4%), and 2 bla(VIM) (0.6%) genes, with positive percent agreement, negative percent agreement, and concordance rates of 92.9%, 86.7%, and 88.3%, respectively, for the dominant bla(KPC) and 85.0%, 87.8%, and 87.4%, respectively, for the bla(NDM) genes. Neither method detected the bla(OXA-48) carbapenemase gene. The convenient, rapid, and simple characteristics of the Xpert Carba-R assay make it a potential tool for CRE detection and identification directly in sputum specimens.

COVID-19-associated pulmonary aspergillosis (CAPA) was recently reported as a potential infective complication affecting critically ill patients with acute respiratory distress syndrome following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with incidence rates varying from 8 to 33% depending on the study. However, definitive diagnosis of CAPA is challenging. Standardized diagnostic algorithms and definitions are lacking, clinicians are reticent to perform aerosol-generating bronchoalveolar lavages for galactomannan testing and microscopic and cultural examination, and questions surround the diagnostic sensitivity of different serum biomarkers., COVID-19-associated pulmonary aspergillosis (CAPA) was recently reported as a potential infective complication affecting critically ill patients with acute respiratory distress syndrome following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with incidence rates varying from 8 to 33% depending on the study. However, definitive diagnosis of CAPA is challenging. Standardized diagnostic algorithms and definitions are lacking, clinicians are reticent to perform aerosol-generating bronchoalveolar lavages for galactomannan testing and microscopic and cultural examination, and questions surround the diagnostic sensitivity of different serum biomarkers. Between 11 March and 14 July 2020, the UK National Mycology Reference Laboratory received 1,267 serum and respiratory samples from 719 critically ill UK patients with COVID-19 and suspected pulmonary aspergillosis. The laboratory also received 46 isolates of Aspergillus fumigatus from COVID-19 patients (including three that exhibited environmental triazole resistance). Diagnostic tests performed included 1,000 (1-3)-β-d-glucan and 516 galactomannan tests on serum samples. The results of this extensive testing are presented here. For a subset of 61 patients, respiratory specimens (bronchoalveolar lavage specimens, tracheal aspirates, and sputum samples) in addition to serum samples were submitted and subjected to galactomannan testing, Aspergillus-specific PCR, and microscopy and culture. The incidence of probable/proven and possible CAPA in this subset of patients was approximately 5% and 15%, respectively. Overall, our results highlight the challenges in biomarker-driven diagnosis of CAPA, especially when only limited clinical samples are available for testing, and the importance of a multimodal diagnostic approach involving regular and repeat testing of both serum and respiratory samples.

The objective of this prospective cross-sectional study, conducted at a national referral hospital in Kampala, Uganda, was to determine diagnostic performance of serum C-reactive protein (CRP) as a triage test for tuberculosis (TB) among HIV-seronegative inpatients. We calculated the sensitivity, specificity, positive and negative likelihood ratios, and positive and negative predictive values to determine the diagnostic performance of a CRP enzyme-linked immunosorbent assay (ELISA) (Eurolyser) in comparison to that of a reference standard of Mycobacterium tuberculosis culture on two sputum samples. We constructed receiver operating curves and reported performance in reference to the manufacturer’s cutoff and also to a threshold chosen to achieve sensitivity of >90%, in accordance with the WHO’s target-product profile for a triage test. Among 119 HIV-seronegative inpatients, 46 (39%) had culture-positive pulmonary TB. In reference to M. tuberculosis culture, CRP had a sensitivity of 78% (95% confidence interval [CI], 64 to 89%) and a specificity of 52% (95% CI, 40 to 64%) at the manufacturer’s threshold of 10 mg/liter. At a threshold of 1.5 mg/liter, the sensitivity was 91% (95% CI, 79 to 98%) but the specificity was only 21% (95% CI, 12 to 32%). Performance did not differ when stratified by illness severity at either threshold. In conclusion, among HIV-seronegative inpatients, CRP testing performed substantially below targets for a TB triage test. Additional studies among HIV-seronegative individuals in clinics and community settings are needed to assess the utility of CRP for TB screening.

Childhood tuberculosis (TB) presents significant diagnostic challenges associated with paucibacillary disease and requires a more sensitive test. We evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Ultra) compared to other microbiological tests using respiratory samples from Ugandan children in the SHINE trial. SHINE is a randomized trial evaluating shorter treatment in 1,204 children with minimal TB disease in Africa and India. Among 352 samples and one cervical lymph node fine needle aspirate, one sample was randomly selected per patient and tested with the Xpert MTB/RIF assay (Xpert) and with Lowenstein-Jensen medium (LJ) and liquid mycobacterial growth indicator tube (MGIT) cultures., Childhood tuberculosis (TB) presents significant diagnostic challenges associated with paucibacillary disease and requires a more sensitive test. We evaluated the diagnostic accuracy of Xpert MTB/RIF Ultra (Ultra) compared to other microbiological tests using respiratory samples from Ugandan children in the SHINE trial. SHINE is a randomized trial evaluating shorter treatment in 1,204 children with minimal TB disease in Africa and India. Among 352 samples and one cervical lymph node fine needle aspirate, one sample was randomly selected per patient and tested with the Xpert MTB/RIF assay (Xpert) and with Lowenstein-Jensen medium (LJ) and liquid mycobacterial growth indicator tube (MGIT) cultures. We selected only uncontaminated stored sample pellets for Ultra testing. We estimated the sensitivity of Xpert and Ultra against culture and a composite microbiological reference standard (any positive result). Of 398 children, 353 (89%) had culture, Xpert, and Ultra results. The median age was 2.8 years (interquartile range [IQR], 1.3 to 5.3); 8.5% (30/353) were HIV infected, and 54.4% (192/353) were male. Of the 353, 31 (9%) were positive by LJ and/or MGIT culture, 36 (10%) by Ultra, and 16 (5%) by Xpert. Sensitivities (95% confidence intervals [CI]) were 58% (39 to 65% [18/31]) for Ultra and 45% (27 to 64% [14/31]) for Xpert against any culture-positive result, with false positives of

The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum., The global coronavirus (CoV) disease 2019 (COVID-19) pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) RNA in specimen transport media under various storage conditions. Transport media tested included UTM, UTM-RT, ESwab, M4, and saline (0.9% NaCl). Specimen types tested included nasopharyngeal/oropharyngeal swabs in the above-named transport media, bronchoalveolar lavage (BAL) fluid, and sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various medium types. Aliquots of samples were stored at 18°C to 26°C, 2°C to 8°C, and −10°C to −30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean cycle threshold differences of

Diagnostic testing for COVID-19 is central to controlling the global pandemic. Recently, To and colleagues reported that 20 of 23 (87%) patients who had SARS-CoV-2 detected by reverse transcriptase PCR (RT-PCR) in nasopharyngeal swabs (NPS) or sputum also had SARS-CoV-2 detectable in saliva ([1][1

A multicenter study was performed to evaluate the value of testing gastric aspirate (GA) with Xpert MTB/RIF Ultra assay (Ultra) for childhood tuberculosis (TB) detection in China. In total, 129 children with active TB and 173 children without TB were enrolled. The sensitivity of Ultra in bacteriologically confirmed TB and probable TB cases was 87.5% (42/48) and 44.4% (36/81), respectively. The specificity of Ultra was high (99.4%, 172/173). When Ultra, culture, and acid-fast bacilli outcomes were integrated as a composite reference standard, the percentage of children with definite TB increased from 37., A multicenter study was performed to evaluate the value of testing gastric aspirate (GA) with Xpert MTB/RIF Ultra assay (Ultra) for childhood tuberculosis (TB) detection in China. In total, 129 children with active TB and 173 children without TB were enrolled. The sensitivity of Ultra in bacteriologically confirmed TB and probable TB cases was 87.5% (42/48) and 44.4% (36/81), respectively. The specificity of Ultra was high (99.4%, 172/173). When Ultra, culture, and acid-fast bacilli outcomes were integrated as a composite reference standard, the percentage of children with definite TB increased from 37.2% (48/129) to 67.4% (87/129). The sensitivity of Ultra is 80.0% (40/50) in children aged

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

The ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. Current diagnostic testing options include culture, molecular testing, and antigen detection. These methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. This study assessed the performance of the BioFire FilmArray Pneumonia Panel (PN panel) and Pneumonia Plus Panel (PNplus panel), an FDA-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage [BAL] fluid)., The ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. Current diagnostic testing options include culture, molecular testing, and antigen detection. These methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. This study assessed the performance of the BioFire FilmArray Pneumonia Panel (PN panel) and Pneumonia Plus Panel (PNplus panel), an FDA-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage [BAL] fluid). Semiquantitative results are also provided for the bacterial targets. This paper describes selected analytical and clinical studies that were conducted to evaluate performance of the panel for regulatory clearance. Prospectively collected respiratory specimens (846 BAL and 836 sputum specimens) evaluated with the PN panel were also tested by quantitative reference culture and molecular methods for comparison. The PN panel showed a sensitivity of 100% for 15/22 etiologic targets using BAL specimens and for 10/24 using sputum specimens. All other targets had sensitivities of ≥75% or were unable to be calculated due to low prevalence in the study population. Specificity for all targets was ≥87.2%, with many false-positive results compared to culture that were confirmed by alternative molecular methods. Appropriate adoption of this test could provide actionable diagnostic information that is anticipated to impact patient care and antimicrobial stewardship decisions.

INTRODUCTIONDespite the widespread use of prophylactic antibiotics in high-risk individuals, Pneumocystis jirovecii remains an important cause of pneumonia in immunocompromised patients. During the peak of the AIDS epidemic, many hospitals and outpatient clinics were very proficient at collecting induced sputum specimens for the diagnosis of Pneumocystis jirovecii pneumonia (PJP). With the dramatic reduction in the occurrence PJP in the current era of highly effective antiretroviral therapy, many centers no longer collect induced sputum samples. Thus, the diagnosis of PJP requires bronchoalveolar lavage (BAL) specimens or a decision to treat the patient empirically without a definitive diagnosis. Sputum or BAL specimens are tested for P. jirovecii using special stains or molecular assays, which require highly trained staff that may not be available with a rapid turnaround time. Given the invasive nature of collecting BAL specimens and the expertise needed for interpreting PJP test results, there is interest in using serum 1,3-β-d-glucan (BDG) testing for the diagnosis of PJP. In this point-counterpoint, Luis Ostrosky-Zeichner and Gabriela Corsi-Vasquez discuss the pro view of using BDG testing for the diagnosis of PJP, while Paul E. Sax and Edward F. Pilkington III present the con view of using BDG testing for the diagnosis of PJP.

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 . An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species.

South Africa is a country with a high incidence of tuberculosis (TB), complicated by coinfection with human immunodeficiency virus (HIV). The Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) is used in South Africa as the test for the initial diagnosis of TB, and other molecular platforms such as the m 2000 (Abbott Molecular, Des Plaines, IL, USA) are widely used for molecular monitoring of HIV load. The latter platform is now also equipped with the RealTi m e (RT) MTB and RealTi m e MTB RIF/INH assays for TB and first-line drug resistance screening but has not been evaluated in settings of HIV and TB coinfection. A prospective clinical validation study was conducted at a community health center in Johannesburg, South Africa, and consenting individuals with presumptive pulmonary TB were enrolled. The performance of the Abbott assays was compared with those of the Xpert MTB/RIF, liquid culture, drug susceptibility testing, and clinical case definitions. A statistical analysis was performed on 206 individuals (73% were HIV positive). The sensitivity and specificity of the RT MTB were 82.5% (confidence interval [CI], 67.2 to 92.7) and 93.1% (CI, 86.2 to 97.2) on raw sputum and 77.5% (CI, 61.5 to 89.2) and 95.1% (CI, 88.9 to 98.4) on concentrated sputum, respectively, compared with those from liquid culture. The RT MTB correctly identified 17/35 more smear-negative culture-positive specimens than the Xpert MTB/RIF. Both the RT MTB and the Xpert MTB/RIF displayed sensitivities >70% and specificities >90% in HIV-positive individuals. The available drug resistance results concurred with MTBDR plus and drug susceptibility profiles. The RT MTB assay has similar diagnostic performance to the Xpert MTB/RIF and is suited to testing presumptive TB patients coinfected with HIV. The existing laboratory information system connectivity, training, and technical support make this a viable polyvalent option to scale up TB alongside HIV laboratory testing services in South Africa.

Achromobacter species are increasingly being detected in cystic fibrosis (CF) patients, with an unclear epidemiology and impact. We studied a cohort of patients attending a Canadian adult CF clinic who had positive sputum cultures for Achromobacter species in the period from 1984 to 2013. Infection was categorized as transient or persistent (≥50% positive cultures for 1 year). Those with persistent infection were matched 2:1 with age-, sex-, and time-matched controls without a history of Achromobacter infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency and lung function decline. Isolates from a biobank were retrospectively assessed, identified to the species level by nrdA sequencing, and genotyped using pulsed-field gel electrophoresis (PFGE). Thirty-four patients (11% of those in our clinic), with a median age of 24 years (interquartile range [IQR], 20.3 to 29.8 years), developed Achromobacter infection. Ten patients (29%) developed persistent infection. Persistence did not denote permanence, as most patients ultimately cleared infection, often after years. Patients were more likely to experience PEx at incident isolation than at prior or subsequent visits (odds ratio [OR], 2.7 [95% confidence interval {CI}, 1.2 to 6.7]; P = 0.03). Following persistent infection, there was no difference in annual lung function decline (−1.08% [95% CI, −2.73 to 0.57%] versus −2.74% [95% CI, −4.02 to 1.46%]; P = 0.12) or the odds of PEx (OR, 1.21 [95% CI, 0.45 to 3.28]; P = 0.70). Differential virulence among Achromobacter species was not observed, and no cases of transmission occurred. We demonstrated that incident Achromobacter infection was associated with a greater risk of PEx; however, neither transient nor chronic infection was associated with a worsened long-term prognosis. Large, multicenter studies are needed to clarify the clinical impact, natural history, and transmissibility of Achromobacter .

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

A novel selective agar (RGM medium) has been advocated for the isolation of rapidly growing mycobacteria from the sputa of cystic fibrosis (CF) patients. The aim of this study was to compare RGM medium to Burkholderia cepacia selective agar (BCSA) and a standard acid-fast bacillus (AFB) culture method for the isolation of nontuberculous mycobacteria (NTM) from patients with CF. The applicability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for the identification of NTM isolated on RGM medium was also assessed. Respiratory samples ( n = 869) were collected from 487 CF patients and inoculated directly onto RGM medium and BCSA. Cultures were incubated at 30°C and examined for up to 28 days. A subset of 212 samples (from 172 patients) was also cultured by using a mycobacterial growth indicator tube (MGIT) and on Lowenstein-Jensen medium following dual decontamination. By using a combination of all methods, 98 mycobacteria were isolated from 869 samples (11.3%). The sensitivity of RGM medium (96.9%) was significantly higher than that of BCSA (35.7%) for the isolation of mycobacteria ( P < 0.0001). The sensitivity of RGM medium was also superior to that of standard AFB culture for the isolation of mycobacteria (92.2% versus 47.1%; P < 0.0001). MALDI-TOF MS was effective for the identification of mycobacteria in RGM medium. RGM medium offers a simple and highly effective tool for the isolation of NTM from patients with CF. Extended incubation of RGM medium for 28 days facilitates the isolation of slow-growing species, including members of the Mycobacterium avium complex (MAVC).

The utility of a line probe assay (Genotype MTBDRplus) performed directly on 2-month sputa to monitor tuberculosis treatment response is unknown. We assessed if direct testing of 2-month sputa with MTBDRplus can predict 2-month culture conversion and long-term treatment outcome. Xpert MTB/RIF-confirmed rifampin-susceptible tuberculosis cases were recruited at tuberculosis diagnosis and followed up at 2 and 5 to 6 months. MTBDRplus was performed directly on 2-month sputa and on all positive cultured isolates at 2 and 5 to 6 months. We also investigated the association of a positive direct MTBDRplus at 2 months with subsequent unsuccessful tuberculosis treatment outcome (failure/death during treatment or subsequent disease recurrence). A total of 279 patients (62% of whom were HIV-1 coinfected) were recruited. Direct MTBDRplus at 2 months had a sensitivity of 78% (95% confidence interval [CI], 65 to 87) and specificity of 80% (95% CI, 74 to 84) to predict culture positivity at 2 months with a high negative predictive value of 93% (95% CI, 89 to 96). Inconclusive genotypic susceptibility results for both rifampin and isoniazid were seen in 26% of MTBDRplus tests performed directly on sputum. Compared to a reference of MTBDRplus performed on positive cultures, the false-positive resistance rate for direct testing of MTBDRplus on sputa was 4% for rifampin and 2% for isoniazid. While a positive 2-month smear was not significantly associated with an unsuccessful treatment outcome (adjusted odds ratio [aOR], 2.69; 95% CI, 0.88 to 8.21), a positive direct MTBDRplus at 2 months was associated with an unsuccessful outcome (aOR 2.87; 95% CI, 1.11 to 7.42). There is moderate utility of direct 2-month MTBDRplus to predict culture conversion at 2 months and also to predict an unfavorable outcome.

Chronic colonization by opportunistic environmental bacteria is frequent in the airways of cystic fibrosis (CF) patients. Studies of Pseudomonas aeruginosa evolution during persistence have highlighted the emergence of pathoadaptive genotypes and phenotypes, leading to complex and diversified inpatient colonizing populations also observed at the intraspecimen level. Such diversity, including heterogeneity in resistance profiles, has been considered an adaptive strategy devoted to host persistence. Longitudinal genomic diversity has been shown for the emergent opportunistic pathogen Achromobacter , but phenotypic and genomic diversity has not yet been studied within a simple CF sputum sample. Here, we studied the genomic diversity and antimicrobial resistance heterogeneity of 132 Achromobacter species strains (8 to 27 strains of identical or distinct colonial morphotypes per specimen) recovered from the sputum samples of 9 chronically colonized CF patients. We highlighted the high within-sample and within-morphotype diversity of antimicrobial resistance (disk diffusion) and genomic (pulsed-field gel electrophoresis) profiles. No sputum sample included strains with identical pulsotypes or antibiotic susceptibility patterns. Differences in clinical categorization were observed for the 9 patients and concerned 3 to 11 antibiotics, including antibiotics recommended for use against Achromobacter . Within-sample antimicrobial resistance heterogeneity, not predictable from colonial morphology, suggested that it may represent a selective advantage against antibiotics in an Achromobacter persisting population and potentially compromise the antibiotic management of CF airway infections.

New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold ( C T ) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk importedMycobacterium tuberculosisstrains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR BeijingM. tuberculosisstrains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

Unmanned aerial vehicles (UAVs) could potentially be used to transport microbiological specimens. To examine the impact of UAVs on microbiological specimens, blood and sputum culture specimens were seeded with usual pathogens and flown in a UAV for 30 ± 2 min. Times to recovery, colony counts, morphologies, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identifications of the flown and stationary specimens were similar for all microbes studied.

The turnaround times for conventional methods used to detect Mycobacterium tuberculosis in sputum samples and to obtain drug susceptibility information are long in many developing countries, including Panama, leading to delays in appropriate treatment initiation and continued transmission in the community. We evaluated the performance of a molecular line probe assay, the Genotype MTBDR plus version 2.0 assay, in detecting M. tuberculosis complex directly in respiratory specimens from smear-positive tuberculosis cases from four different regions in Panama, as well as the most frequent mutations in genes conferring resistance to isoniazid ( katG and inhA ) and rifampin ( rpoB ). Our results were confirmed with the nitrate reductase assay and genomic sequencing. M. tuberculosis complex was detected by the Genotype MTBDR plus 2.0 assay with 100% sensitivity and specificity. The sensitivity and specificity for rifampin resistance were 100% and 100%, respectively, and those for isoniazid resistance were 90.7% and 100%. Isoniazid monoresistance was detected in 5.2% of new cases. Genotype MTBDR plus 2.0 is highly accurate in detecting M. tuberculosis complex in respiratory specimens and is able to discriminate isoniazid-monoresistant cases from multidrug-resistant cases within 2 days.

There are several convenient and accurate molecular assays to detect respiratory bacterial infection. The NeoPlex RB-8 detection kit (NeoPlex RB-8) is a new multiplex real-time PCR assay that simultaneously detects Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, Haemophilus influenzae, Bordetella pertussis, Bordetella parapertussis, and Moraxella catarrhalis in a single test. This study compared the clinical concordance of NeoPlex RB-8 with another method, Seeplex PneumoBacter ACE detection assay (Seeplex PB ACE), which simultaneously detects S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis. We tested 2,137 nasopharyngeal swab and sputum specimens using both assays. For discordant Bordetella parapertussis and M. catarrhalis specimens, we also performed bidirectional sequencing. For S. pneumoniae, M. pneumoniae, C. pneumoniae, L. pneumophila, H. influenzae, and B. pertussis, which are detected by both NeoPlex RB-8 and Seeplex PB ACE, the positive and negative agreement between the two assays ranged from 91.7 to 100% (κ = 0.918 to 1). S. pneumoniae and H. influenzae were the most discordant targets and measured with higher sensitivity and specificity by NeoPlex RB-8 than Seeplex PB ACE. For Bordetella parapertussis and M. catarrhalis, which are not detected by Seeplex PB ACE, NeoPlex RB-8 sensitivity and specificity were >99%. Overall, NeoPlex RB-8 was highly comparable to Seeplex PB ACE, but NeoPlex RB-8 was more clinically accurate, with higher throughput and more convenience.

The objective of this study was to compare the diagnostic yield of the Kudoh-Ogawa (K-O) swab method for the culturing of Mycobacterium tuberculosis from clinical samples with the standard Petroff–Lowenstein-Jensen (P-LJ) procedure. A total of 2,287 sputum samples and 685 extrapulmonary clinical specimens were processed with both decontamination methods and compared for M. tuberculosis detection rate, recovery of M. tuberculosis colonies, and culture contamination. Overall, 23.9% and 23.5% of the samples, processed with, respectively, the K-O swab method and the P-LJ procedure, yielded M. tuberculosis after 8 weeks of incubation. The K-O swab method and the P-LJ procedure provided comparable diagnostic yields for extrapulmonary clinical specimens (P = 0.688), but the K-O method showed a slightly but statistically significantly higher diagnostic yield for pulmonary samples (P = 0.002). No significant difference for culture contamination or colony recovery was found for either method. The turnaround time for the isolation of M. tuberculosis was significantly shorter for the K-O swab method, with 77% of the M. tuberculosis cultures being positive within 3 weeks of incubation, and only 6.1% positivity for the P-LJ method. Concerning the workload, the K-O swab method needs a minimum sample manipulation and takes less than 4 min per sample, as the samples are not centrifuged in this procedure. The K-O swab method is an efficient and fast (in terms of sample processing and culture growth) alternative for culturing M. tuberculosis from either pulmonary or extrapulmonary clinical specimens. The method is particularly suitable for laboratories with a high workload and for laboratories lacking a special infrastructure.

Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise., Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% for smear-positive, culture-positive samples and 88% for smear-negative, culture-positive samples with a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93%, and the positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.

The diagnosis of pleural tuberculosis (TB) is problematic. The comparative performance of newer same-day tools for pleural TB, including Xpert MTB/RIF Ultra (ULTRA), has hitherto not been comprehensively studied. Adenosine deaminase (ADA), IRISA-TB (interferon gamma ultrasensitive rapid immunosuspension assay), Xpert MTB/RIF, and ULTRA performance outcomes were evaluated in pleural fluid samples from 149 patients with suspected pleural TB., The diagnosis of pleural tuberculosis (TB) is problematic. The comparative performance of newer same-day tools for pleural TB, including Xpert MTB/RIF Ultra (ULTRA), has hitherto not been comprehensively studied. Adenosine deaminase (ADA), IRISA-TB (interferon gamma ultrasensitive rapid immunosuspension assay), Xpert MTB/RIF, and ULTRA performance outcomes were evaluated in pleural fluid samples from 149 patients with suspected pleural TB. The reference standard was culture positivity (fluid, biopsy specimen, or sputum) and/or pleural biopsy histopathology (termed definite TB). Those designated as having non-TB were negative by microbiological testing and were not initiated on anti-TB treatment. To determine the effect of sample concentration, 65 samples underwent pelleting by centrifugation, followed by conventional Xpert MTB/RIF and ULTRA. Of the 149 patients, 49 had definite TB, 16 had probable TB (not definite but treated for TB), and 84 had non-TB. ULTRA sensitivity and specificity (95% confidence intervals [CI]) were similar to those of Xpert MTB/RIF [sensitivity, 37.5% (25.3 to 51.2) versus 28.6% (15.9 to 41.2), respectively; specificity, 98.8% (96.5 to 100) versus 98.8% (96.5 to 100), respectively]. Centrifugation did not significantly improve ULTRA sensitivity (29.5% versus 31.3%, respectively). Adenosine deaminase and IRISA-TB sensitivity were 84.4% (73.9 to 95.0) and 89.8% (81.3 to 98.3), respectively. However, IRISA-TB demonstrated significantly better specificity (96.4% versus 87.5% [P = 0.034]), positive predictive value (93.6% versus 80.9 [P = 0.028]), and positive likelihood ratio (25.1 versus 6.8 [P = 0.032]) than ADA. In summary, Xpert ULTRA has poor sensitivity for the diagnosis of pleural TB. Alternative assays (ADA and IRISA-TB) are significantly more sensitive, with IRISA-TB demonstrating a higher specificity and rule-in value than ADA in this high-TB-burden setting where HIV is endemic.

We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples)., We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient’s sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI (n = 125, 94%) than with ETOH (n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage (n = 10, 41.7%) than with CPC (n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI (n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle (CT) values significantly decreased compared to those prestorage with ETOH (difference, −1.1; 95% CI, −1.6 to −0.6; P = 0.0001) but not with CPC (P = 0.915) or OMNI (P = 0.33). For one patient’s ETOH- and CPC-stored specimens with a CT of

The study was made possible by funding from the European and Developing Countries Clinical Trials Partnership (EDCTP) – Pan African Biomarker Expansion program (PanBIOME) grant SP.2011.41304.008. Support was also obtained the University of St Andrews School of Medicine research grant. The World Health Organization End tuberculosis (TB) strategy has called for development of- and increased access to- effective tools for diagnosis and treatment of TB disease. Mycobacterium tuberculosis (Mtb), the causative agent of TB is categorized as highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category three laboratory. We have evaluated the use of heat-inactivation to render TB samples safe to work with whilst preserving RNA for downstream molecular tests. Using Mycobacterium bovis Bacillus Calmette Guérin (BCG) cultures and TB positive sputa we show that boiling for 20 min at 80-, 85-, and 95- ºC inactivates all Mtb bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85 and the untreated sputa flagged positive at 3 days of incubation in Mycobacterium Growth Indicator Tube. Quantification of reference genes, 16S rRNA, tmRNA, pre-16S rRNA and rpoB by Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA-species dependent

Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant., Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.

Diagnostic tests for tuberculosis (TB) usually require collection of sputum, a viscous material derived from human airways. Sputum can be difficult and hazardous to collect and challenging to process in the laboratory., Diagnostic tests for tuberculosis (TB) usually require collection of sputum, a viscous material derived from human airways. Sputum can be difficult and hazardous to collect and challenging to process in the laboratory. Oral swabs have been proposed as alternative sample types that are noninvasive and easy to collect. This study evaluated the biological feasibility of oral swab analysis (OSA) for the diagnosis of TB. Swabs were tested from South African adult subjects, including sputum GeneXpert MTB/RIF (GeneXpert)-confirmed TB patients (n = 138), sputum GeneXpert-negative but culture-positive TB patients (n = 10), ill non-TB patients (n = 37), and QuantiFERON-negative controls (n = 34). Swabs were analyzed by using a manual, nonnested quantitative PCR (qPCR) targeting IS6110. Two swab brands and three sites within the oral cavity were compared. Tongue swabbing yielded significantly stronger signals than cheek or gum swabbing. A flocked swab performed better than a more expensive paper swab. In a two-phase study, tongue swabs (two per subject) exhibited a combined sensitivity of 92.8% relative to sputum GeneXpert. Relative to all laboratory-diagnosed TB, the diagnostic yields of sputum GeneXpert (1 sample per subject) and OSA (2 samples per subject) were identical at 49/59 (83.1%) each. The specificity of the OSA was 91.5%. An analysis of “air swabs” suggested that most false-positive results were due to contamination of manual PCRs. With the development of appropriate automated methods, oral swabs could facilitate TB diagnosis in clinical settings and patient populations that are limited by the physical or logistical challenges of sputum collection.