Nonrespiratory Specimens Research Articles

Evaluation of GeneXpert MTB/RIF for detection of Mycobacterium tuberculosis complex and rpo B gene in respiratory and non-respiratory clinical specimens at a tertiary care teaching hospital in Saudi Arabia.

ObjectivesTo assess the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), against smear microscopy and culture method for diagnosis of MTB infection.MethodsThis is a retrospective analysis of 103 respiratory and 137 non-respiratory patient specimens suspected of tuberculosis at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia performed between April 2014 and March 2015. Each sample underwent smear microscopy, mycobacterial culture, and GeneXpert MTB/RIF test.ResultsFifteen out of 103 respiratory samples were smear and culture positive, whereas 9 out of 137 non-respiratory samples were smear positive. Out of 9 smear positive specimens, 8 were also culture positive. All 15 culture positive respiratory samples were detected by Xpert MTB/RIF (sensitivity and positive predictive value [PPV]=100%). Similarly, all 8 culture positive non-respiratory specimens were identified by Xpert MTB/RIF (sensitivity 100%; PPV 88.8%). The Xpert MTB/RIF detected only one false positive result in 88 smear negative respiratory specimens (specificity 98.9%; negative predictive value [NPV]= 100%). All 125 smear negative non-respiratory specimens tested negative by culture and Xpert MTB/RIF (sensitivity, specificity, PPV, NPV= 100%).ConclusionThe performance of Xpert MTB/RIF was comparable to the gold standard culture method for identification of MTB in both respiratory and non-respiratory clinical specimens.

Performance of the BD ProbeTec ET direct detection assay for the analysis of Mycobacterium tuberculosis in respiratory and non-respiratory clinical specimens.

ObjectivesEarly detection of Mycobacterial tuberculosis infection (MTB) is pivotal for the treatment of tuberculosis (TB).BackgroundThis study was performed to evaluate the performance of BD ProbeTec ET direct detection assay (DTB) against the gold standard culture technique for confirmation of MTB infection.MethodsA total of 266 consecutive and non-duplicate clinical specimens for detection of MTB were included in this study. There were 118 respiratory and 148 non-respiratory samples. All samples were tested by microscopy for acid-fast bacillus (AFB), MTB culture and biochemical identification with simultaneous testing by DTB.ResultsA total of 88 samples (33%) were culture-positive for MTB including 39/118 respiratory, 29/99 fluid and 20/49 tissue samples. DTB sensitivity for respiratory samples was 97% and specificity was 96% with a positive predictive value (PPV) of 93% and negative predictive value (NPV) of 99%. Sensitivity of DTB in fluid samples was 80%, specificity 88%, PPV 69% and NPV 93% whereas sensitivity of DTB for tissue samples was 25%, specificity 90%, PPV 63% and NPV 63%. Of the 50 (56.8%) smear-positive samples, DTB sensitivity was 100% for respiratory, 85% for fluid and 100% for tissue samples.ConclusionDTB performed within acceptable limits for the rapid detection of MTB in respiratory samples compared to fluid and tissue specimens.

Diagnosis of Concurrent Pulmonary Tuberculosis and Tuberculous Otitis Media Confirmed by Xpert MTB/RIF in the United States.

Tuberculosis (TB) remains an important cause of infectious morbidity in the United States (US), necessitating timely and accurate diagnosis. We report a case of concurrent pulmonary and extrapulmonary TB presenting as tuberculous otitis media in a hospitalized US patient admitted with cough, night sweats, and unilateral purulent otorrhea. Diagnosis was made by smear microscopy and rapidly confirmed by Xpert MTB/RIF-a novel, automated nucleic acid amplification test for the rapid detection of drug-susceptible and drug-resistant TB. This case adds to the growing body of evidence validating Xpert MTB/RIF as an effective tool for the rapid diagnosis of extrapulmonary TB, even in low TB-prevalence settings such as the US, when testing is performed on non-respiratory specimens.

Paediatric Tuberculosis at a Referral Hospital in Istanbul: Analysis of 250 Cases.

Background. Tuberculosis (TB) still remains a growing public health problem globally. TB in children is often diagnosed clinically. Methods. We conducted a retrospective chart review of children with TB from November 2004 through December 2010 to determine the appropriateness of using contact history and diagnostic testing. Results. A total of 250 children with TB were identified. One hundred and sixty-two children had only pulmonary disease while 39 had features of both extrapulmonary and pulmonary TB. Mean age was 7.8 years. Thirty-six patients had known contacts. The index case/cases were first-degree relatives in 75%. Sixteen patients who were symptomless were yielded by contact investigation of newly identified TB cases. Tuberculin skin test positivity was 53.3%. Acid-fast bacilli smear positivity was 13.1%, and culture positivity was 18.7%. Twenty-six patients had histopathology of nonrespiratory specimens (lymph nodes and other tissues) showing granulomatous inflammation and caseous necrosis consistent with TB. Conclusions. Presence of contact history directed us to search for TB in children with nonspecific symptoms even if physical examinations were normal. Some children who were close contacts to TB cases were identified to have TB before development of symptoms.

Sensitivity and specificity of Cobas TaqMan MTB real-time polymerase chain reaction for culture-proven Mycobacterium tuberculosis: meta-analysis of 26999 specimens from 17 Studies.

Since 2010, studies on the diagnostic accuracy of COBAS TaqMan MTB (CTM) have been frequently reported with an unignorable discrepancy. The key inclusion criterion for this systematic review was original studies that could provide sufficient data for calculating the sensitivity and the specificity of CTM for M tuberculosis (TB) or M tuberculosis complex. The reference test was Mycobacterium culture. We used bivariate model for meta-analyses. Of the 201 candidate articles, we finally identified 17 eligible articles.Concerning the respiratory specimens, 1900 culture positive specimens and 20983 culture negative specimens from 15 studies were assessed. This provided the summary estimate sensitivity of 0.808 (95% CI 0.758–0.850) and the summary estimate specificity of 0.990 (95% CI 0.981–0.994). The area under curve was 0.956. The diagnostic odds ratio was 459 (95% CI 261–805, I2 26%). For the smear positive respiratory specimens, the sensitivity was 0.952 (95% CI 0.926–0.969) and the specificity was 0.916 (95% CI 0.797–0.968). For the smear negative respiratory specimens, the sensitivity and the specificity were 0.600 (95% CI 0.459–0.726) and 0.989 (95% CI 0.981–0.993), respectively. The diagnostic accuracy was poorer for the non-respiratory specimens, than for the respiratory specimens, but was acceptable. We believe that the information obtained from this study will aid physicians’ decision making.

Underestimation of the True Specificity of the Urine Lipoarabinomannan Point-of-Care Diagnostic Assay for HIV-Associated Tuberculosis.

Underestimation of the True Specificity of the Urine Lipoarabinomannan Point-of-Care Diagnostic Assay for HIV-Associated Tuberculosis.

Evaluation of the Cobas TaqMan MTB test for detection of Mycobacterium tuberculosis complex

Background: The Cobas TaqMan MTB assay is used for rapid detection of the Mycobacterium tuberculosis complex (MTC) in clinical samples. It is only validated for respiratory samples, but is often requested by physicians for non-respiratory specimens. The aim of this study was therefore to evaluate the performance of this assay in clinical praxis in a country with low prevalence of tuberculosis (TB). Methods: The results from 2388 respiratory and 1005 non-respiratory human specimens were analyzed by this real-time PCR technique. Using culture results as the gold standard, the sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) of the PCR results were calculated. Results: For smear-positive respiratory specimens all the four values investigated were 100%. The number of smear-positive non-respiratory specimens was only eight, and all of these eight specimens reacted positively. Sensitivity was 51% for smear-negative respiratory specimens, and 47% for smear-negative non-respiratory specimens. When all non-respiratory specimens were analyzed together the sensitivity was 51%. Specimens from 16 patients were PCR-positive but culture-negative and these cases are discussed. In one case, TB DNA was still present in sputum 2 years after a successful treatment. Conclusion: The study shows that the Cobas TaqMan MTB assay performs well in smear-positive samples, while the sensitivities are unsatisfactory for both respiratory and non-respiratory smear-negative specimens. Furthermore, the analyses emphasize that this assay cannot be used to evaluate treatment or contagiousness, or to detect relapses or screen for TB.

Drug-resistant tuberculosis in HIV-infected patients in a national referral hospital, Phnom Penh, Cambodia

Background and objectiveThere are no recent data on the prevalence of drug-resistant tuberculosis (DR TB) in Cambodia. We aim to describe TB drug resistance amongst adults with pulmonary and extra-pulmonary TB and human immunodeficiency virus (HIV) co-infection in a national referral hospital in Phnom Penh, Cambodia.DesignBetween 22 November 2007 and 30 November 2009, clinical specimens from HIV-infected patients suspected of having TB underwent routine microscopy, Mycobacterium tuberculosis culture, and drug susceptibility testing. Laboratory and clinical data were collected for patients with positive M. tuberculosis cultures.ResultsM. tuberculosis was cultured from 236 HIV-infected patients. Resistance to any first-line TB drug occurred in 34.7% of patients; 8.1% had multidrug resistant tuberculosis (MDR TB). The proportion of MDR TB amongst new patients and previously treated patients was 3.7 and 28.9%, respectively (p<0.001). The diagnosis of MDR TB was made after death in 15.8% of patients; in total 26.3% of patients with MDR TB died. The diagnosis of TB was established by culture of extra-pulmonary specimens in 23.6% of cases.ConclusionsThere is significant resistance to first-line TB drugs amongst new and previously treated TB–HIV co-infected patients in Phnom Penh. These data suggest that the prevalence of DR TB in Cambodia may be higher than previously recognised, particularly amongst HIV-infected patients. Additional prevalence studies are needed. This study also illustrates the feasibility and utility of analysis of non-respiratory specimens in the diagnosis of TB, even in low-resource settings, and suggests that extra-pulmonary specimens should be included in TB diagnostic algorithms.

Accuracy of polimerase chain reaction for the diagnosis of pleural tuberculosis

Polymerase chain reaction (PCR)-based techniques to detect Mycobacterium tuberculosis DNA in respiratory specimens have been increasingly used to diagnose pulmonary tuberculosis. Their use in non-respiratory specimens to diagnose extrapulmonary tuberculosis is, however, controversial. In this study, we estimated the accuracy of three in-country commercialized PCR-based diagnostic techniques in pleural fluid samples for the diagnosis of pleural tuberculosis. Patients underwent thoracenthesis for diagnosis purposes; pleural fluid aliquots were frozen and subsequently submitted to two real time PCR tests (COBAS(®)TAQMAN(®)MTB and Xpert(®)MTB/Rif) and one conventional PCR test (Detect-TB(®)). Two different reference standards were considered: probable tuberculosis (based on clinical grounds) and confirmed tuberculosis (bacteriologically or histologically). Ninety-three patients were included, of whom 65 with pleural tuberculosis, 35 of them confirmed. Sensitivities were 29% for COBAS(®)TAQMAN(®)MTB, 3% for Xpert(®)MTB/Rif and 3% for Detect-TB(®); specificities were 86%, 100% and 97% respectively, considering confirmed tuberculosis. Considering all cases, sensitivities were 16%, 3% and 2%, and specificities, 86%, 100%, and 97%. Compared to the 95% sensitivity of adenosine deaminase, the most sensitive test for pleural tuberculosis, the sensitivities of the three PCR-based tests were very low. We conclude that at present, there is no major place for such tests in routine clinical use.

Performance of the Xpert MTB/RIF Assay on Nonrespiratory Specimens and Accuracy of This Assay for Detection of Rifampin Resistance in a Low-Prevalence Setting

With interest, we read the paper “Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area” by Deggim et al. ([1][1]). Based on the failed detection of Mycobacterium tuberculosis complex (MTB) by the Xpert assay (

Reply to “Performance of the Xpert MTB/RIF Assay on Nonrespiratory Specimens and Accuracy of This Assay for Detection of Rifampin Resistance in a Low-Prevalence Setting”

With interest, we read the paper “Integrating the Xpert MTB/RIF Assay into a Diagnostic Workflow for Rapid Detection of Mycobacterium tuberculosis in a Low-Prevalence Area” by Deggim et al. ([1][1]). Based on the failed detection of Mycobacterium tuberculosis complex (MTB) by the Xpert assay (

Evaluation of GeneXpert MTB/RIF for the detection of Mycobacterium tuberculosis and resistance to rifampin in clinical specimens

GeneXpert (GX) is a novel real-time polymerase chain reaction assay for the simultaneous detection of Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF). We evaluated the performance of GX for direct detection of MTB in respiratory and non-respiratory specimens and assessed the ability of the assay to detect resistance to RIF in non-tuberculosis (TB) endemic country. We analyzed 595 clinical samples in a 1550-bed tertiary hospital in Madrid, Spain. Specimens were processed using GX, auramine smear, conventional culture, and drug phenotypic susceptibility testing (DST) with MGIT SIRE (BD). Of the 595 clinical samples, 305 (51.3%) were non-respiratory and 290 (48.7%) were respiratory. In total, MTB was isolated in 81 specimens, 71 of which were positive with GX. The sensitivity, specificity, PPV, and NPV of the GX were: 97.1%, 98.6%, 95.7% and 99.1% for respiratory samples and 33.3%, 99.7%, 80.0% and 97.3% for non-respiratory. GX detected 8 RIF-resistant samples, but only 5 were confirmed by DST. GX is an accurate, easy-to-apply and rapid test to detect MTB, especially in smear-positive respiratory samples. Although is a useful tool for the rapid identification of RIF-resistant MTB strains, the rifampin resistant results should be confirmed by DST.

Potential of two nucleic acid amplification assays for quantifying mycobacterial load in respiratory and non-respiratory specimens: a prospective study

Quantification of bacillary load plays a critical prognostic role in tuberculosis patients. This study evaluated the potential of the Cepheid GeneXpert (GX) MTB/RIF and the BD ProbeTec system as quantitative assays. The time to positivity (TTP) measured by the Mycobacterial Growth Index Tube system was compared to the cycle threshold (Ct) of GX MTB/RIF and the Metric Other than Acceleration (MOTA) scores generated by the ProbeTec system. Out of 714 samples examined, 44 culture confirmed cases were identified. The Ct values in 21 respiratory samples showed a high linear fit with the TTP in liquid culture (Spearman's correlation coefficient r = 0.9, P value < 0.0001), which was not the case in 23 non-respiratory samples. In both types of specimens, the MOTA scores did not correlate with the TTP in liquid culture. This indicates the suitability of GX as a quantitative measurement of mycobacterial load in respiratory but not non-respiratory specimens.

Comparison of PNA probe-based real-time PCR and Cobas TaqMan MTB for detection of MTBC

We compared the sensitivities and specificities of peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR™ TB/NTM) and Cobas TaqMan MTB assays for detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. A total of 425 clinical specimens including 360 respiratory specimens and 65 non-respiratory specimens were evaluated for comparative analysis. In respiratory specimens, the sensitivity of TaqMan MTB and PNAqPCR assay for detection of MTBC was 82.9% and 91.5%, respectively. In non-respiratory specimens, the sensitivity of the TaqMan MTB and PNAqPCR assay was 23.1% and 76.9%, respectively. Overall, the sensitivity and specificity of the TaqMan MTB assay for detection of MTBC was 76.9% and 100%, respectively. The PNAqPCR assay had a sensitivity and specificity of 90% and 99.7%, respectively.

Rapid Detection and Monitoring Therapeutic Efficacy of Mycobacterium tuberculosis Complex Using a Novel Real-Time Assay

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 nonrespiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was < or =35 and the ratio of real-time RT-PCR and real-time PCR load was > or =1.51. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including nonrespiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

Can a Simple Flotation Method Lower the Limit of Detection of Mycobacterium tuberculosis in Extrapulmonary Samples Analyzed by the GeneXpert MTB/RIF Assay?

The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.

Multi-drug-resistant tuberculosis: the experience of an urban tertiary care hospital in South India using automated BACTEC 460 TB

The emergence of multi-drug-resistant (MDR) strains has been a major obstacle in the tuberculosis (TB) control programme. In the present study we looked into the prevalence of MDR-TB in an urban tertiary care hospital in South India over four years (2007-2010). During this period, 641 clinical specimens (317 respiratory specimens and 324 non-respiratory specimens) were received for mycobacteriological culture and drug susceptibility testing for first-line drugs, using the BACTEC 460 TB system. Mycobacterium tuberculosis (MTB) was isolated in 34.8% (n = 223) specimens. Of the total 223 MTB isolates 83 (37.2%) were MDR. Forty-two percent of the pulmonary MTB isolates (n = 72) and 20.4% of the extra-pulmonary isolates (n = 10) were MDR. Although we observed a high percentage of drug resistance, the prevalence of MDR was not observed to vary significantly within the four years which suggested good management.

Clinical and Virological Factors Associated with Viremia in Pandemic Influenza A/H1N1/2009 Virus Infection

BackgroundPositive detection of viral RNA in blood and other non-respiratory specimens occurs in severe human influenza A/H5N1 viral infection but is not known to occur commonly in seasonal human influenza infection. Recently, viral RNA was detected in the blood of patients suffering from severe pandemic influenza A/H1N1/2009 viral infection, although the significance of viremia had not been previously studied. Our study aims to explore the clinical and virological factors associated with pandemic influenza A/H1N1/2009 viremia and to determine its clinical significance.Methodology/Principal FindingsClinical data of patients admitted to hospitals in Hong Kong between May 2009 and April 2010 and tested positive for pandemic influenza A/H1N1/2009 was collected. Viral RNA was detected by reverse-transcription polymerase chain reactions (RT-PCR) targeting the matrix (M) and HA genes of pandemic influenza A/H1N1/2009 virus from the following specimens: nasopharyngeal aspirate (NPA), endotracheal aspirate (ETA), blood, stool and rectal swab. Stool and/ or rectal swab was obtained only if the patient complained of any gastrointestinal symptoms. A total of 139 patients were included in the study, with viral RNA being detected in the blood of 14 patients by RT-PCR. The occurrence of viremia was strongly associated with a severe clinical presentation and a higher mortality rate, although the latter association was not statistically significant. D222G/N quasispecies were observed in 90% of the blood samples.ConclusionPresence of pandemic influenza A/H1N1/2009 viremia is an indicator of disease severity and strongly associated with D222G/N mutation in the viral hemagglutinin protein.

Comparison of Two Nucleic Acid Amplification Assays, the Xpert MTB/RIF Assay and the Amplified Mycobacterium Tuberculosis Direct Assay, for Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

We compared the performance of the Xpert MTB/RIF assay, a new real-time tuberculosis (TB) PCR test, with that of the Amplified Mycobacterium Tuberculosis Direct (MTD) assay using 162 respiratory and nonrespiratory specimens. Based on culture as the gold standard, the overall sensitivity and specificity for all sample types for the Xpert MTB/RIF assay were 90.9 and 89%, respectively, while for the MTD assay, the overall sensitivity and specificity were 97.3 and 87.1%, respectively. A higher proportion of total equivocal results were obtained for the MTD assay, at 10.5% (17/162), while the Xpert MTB/RIF assay generated 5.5% (9/162) of invalid reads.

Evaluation of rapid techniques for the detection of mycobacteria in sputum with scanty bacilli or clinically evident, smear negative cases of pulmonary and extra-pulmonary tuberculosis

The objective of the current study was to compare two rapid methods, the BBL Mycobacteria Growth Indicator Tube (MGIT TM) and Biotec FASTPlaque TB TM (FPTB) assays, with the conventional Löwenstein-Jensen (LJ) media assay to diagnose mycobacterial infections from paucibacillary clinical specimens. For evaluation of the clinical utility of the BBL MGIT TM and FPTB assays, respiratory tract specimens (n = 208), with scanty bacilli or clinically evident, smear negative cases and non-respiratory tract specimens (n = 119) were analyzed and the performance of each assay was compared with LJ media. MGIT and FPTB demonstrated a greater sensitivity (95.92% and 87.68%), specificity (94.59% and 98.78%), positive predictive value (94.91% and 99.16%) and negative predictive value (96.56% and 90.92%), respectively, compared to LJ culture for both respiratory tract and non-respiratory tract specimens. However, the FPTB assay was unable to detect nontuberculous mycobacteria and few Mycobacterium tuberculosis complex cases from paucibacillary clinical specimens. It is likely that the analytical sensitivity of FPTB is moderately low and may not be useful for the direct detection of tuberculosis in paucibacillary specimens. The current study concluded that MGIT was a dependable, highly efficient system for recovery of M. tuberculosis complexes and nontuberculous mycobacteria from both respiratory and non-respiratory tract specimens in combination with LJ media.