• Evaluation of OMNIgene® SPUTUM and ethanol reagent for preservation of sputum prior to Xpert and culture testing in Uganda.

      Ardizzoni, E; Orikiriza, P; Ssuuna, C; Nyehangane, D; Gumsboga, M; Taremwa, IM; Turyashemererwa, E; Mwanga-Amumpaire, J; Langendorf, C; Bonnet, M (American Society for Microbiology, 2019-10-16)
      Background: Xpert MTB/RIF (Xpert) and culture are the most reliable methods for tuberculosis diagnosis but are still poorly accessible in many low resource countries. We aimed to assess the effect of OMNIgene® SPUTUM (OM-S) and ethanol in preserving sputum for Xpert and OM-S for mycobacteria growth indicator tube (MGIT) testing over a period of 15 and 8 days respectively. Methods: Sputum were collected from newly diagnosed smear-positive patients. For Xpert, pooled samples were split into 5 aliquots: 3 for Xpert on day 0, 7 and 15 days without additive and 2 with either OM-S or ethanol at day 15. For MGIT, 2 aliquots were tested without preservative and 2 with OM-S at 0 and 8 days. Results: A total of 48 and 47 samples were included in the analysis for Xpert and culture. With Xpert, using Day 0 as reference, untreated samples stored for 7 and 15 days showed concordance of 45/46 (97.8%) and 46/48 (95.8%). For samples preserved with OM-S or ethanol for 15 days compared with untreated samples processed at day 0 or after 15 days, OM-S concordance was 46/48(95.8%) and 47/48(97.9%), while ethanol was 44/48 (91.7%) and 45/48 (93.8%). With MGIT, concordance between untreated and OM-S treated samples was 21/41(51.2%) at Day 0 and 21/44(47.7%) at day8. Conclusions: Xpert equally detected TB in OM-S treated and untreated samples up to 15 days but showed slightly lower detection in ethanol treated samples. Among OM-S treated samples, MGIT positivity was significantly lower compared to untreated samples at both time-points.
    • Field evaluation of a simple fluorescence method for detection of viable Mycobacterium tuberculosis in sputum specimens during treatment follow-up.

      Schramm, B; Hewison, C; Bonte, L; Jones, W; Camélique, O; Ruangweerayut, R; Swaddiwudhipong, W; Bonnet, M; Epicentre, Paris, France; Médecins Sans Frontières (MSF), Paris, France;International Organization for Migration (IOM), Bangkok, Thailand; Epicentre, Geneva, Switzerlandf (2012-08)
      Simple tuberculosis (TB) treatment monitoring tools are needed. We assessed the performance of fluorescein-diacetate (FDA) smear microscopy for detection of viable Mycobacterium tuberculosis in sputum specimens (n = 288) of TB cases under treatment compared to culture (17.4% culture positivity). FDA sensitivity was moderate (83.7% [95% confidence interval {CI}, 70.3 to 92.6]), and specificity was low (66.1% [59.5 to 72.2]). The good negative predictive value (94.8% [90.1 to 97.8]) and negative likelihood ratio (0.2) suggest using this method to rule out treatment failure in settings without access to culture.
    • Implementation of the thin layer agar for the diagnosis of smear-negative pulmonary tuberculosis in a high HIV prevalence setting in Homa Bay, Kenya.

      Martin, A; Munga Waweru, P; Babu Okatch, F; Amondi Ouma, N; Bonte, L; Varaine, F; Portaels, F; Institute of Tropical Medicine, Antwerp, Belgium; Médecins Sans Frontières, Paris, France; Homa Bay District Hospital, Kenya. (2009-06-03)
      The objective of this study was to evaluate the performance of a low-cost method, the Thin Layer Agar (TLA), for the diagnosis of smear-negative patients. This prospective study was performed in Homa Bay district Hospital in Kenya. Out of 1584 smear-negative sputum samples, 212 were positive by Löwenstein-Jensen (LJ) (13.5%) and 220 positive by TLA (14%). The sensitivity of LJ and TLA was 71% and 74 % respectively. TLA could become an affordable method for the diagnosis of smear-negative tuberculosis in resource-limited settings with results available within 2 weeks.
    • Lowenstein-Jensen Selective Medium for Reducing Contamination in Mycobacterium tuberculosis Culture

      Kassaza, K; Orikiriza, P; Llosa, A; Bazira, J; Nyehangane, D; Page, A-L; Boum, Y (2014-07)
      We compared Mycobacterium tuberculosis sputum culture recovery and contamination rates between Lowenstein-Jensen medium (LJ) containing the following decontaminants and LJ alone: (i) PANTA (n = 299), (ii) Selectatab-MB (n = 299), and (iii) penicillin G (n = 234). The contamination rate for LJ alone was approximately 31%, versus 5.0% for PANTA-containing, 2% for Selectatab-containing, and 9% for penicillin-containing media (P < 0.001). M. tuberculosis isolation rates were 9.8%, 17%, 18%, and 12% for standard LJ, PANTA, Selectatab, and penicillin cultures, respectively.
    • Rapid detection of Mycobacterium tuberculosis resistance to second-line drugs by use of the manual mycobacterium growth indicator tube system.

      Martin, A; von Groll, A; Fissette, K; Palomino, J C; Varaine, F; Portaels, F; Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium. amartin@itg.be (2008-12)
      The objective of this study was to evaluate the manual mycobacterium growth indicator tube (MGIT) system for the testing of Mycobacterium tuberculosis susceptibility to second-line drugs compared to the proportion method. One hundred eighty-eight M. tuberculosis isolates were tested for susceptibility to ofloxacin, kanamycin, ethionamide, and capreomycin by the manual MGIT, and results were compared to those obtained with the proportion method on 7H11 agar, considered a reference method. Results for ofloxacin and capreomycin were excellent, with 100% accuracy, and a result of 99.4% accuracy was achieved for kanamycin. For ethionamide, accuracy was lower, with a result of 86.7% compared to that of the proportion method. We proposed the following critical concentrations for the drugs: for ofloxacin, 2.0 microg/ml; for kanamycin, 2.5 microg/ml; for ethionamide, 5 microg/ml; and for capreomycin, 2.5 microg/ml. The time required to obtain results was an average of 8 days by the manual MGIT and 3 weeks by the reference method. Our results show that the manual MGIT is an accurate method for the rapid susceptibility testing of M. tuberculosis to second-line drugs. There is no need for a machine when using the manual MGIT, and results can be read with a simple UV lamp or with a semiquantitative reader, which considerably reduces the cost of the method.
    • Use of Colorimetric Culture Methods for Detection of Mycobacterium Tuberculosis Complex Isolates from Sputum Samples in Resource-Limited Settings

      Boum, Y; Orikiriza, P; Rojas-Ponce, G; Riera-Montes, M; Atwine, D; Nansumba, M; Bazira, J; Tuyakira, E; De Beaudrap, P; Bonnet, M; et al. (2013-07)
      Despite recent advances, tuberculosis (TB) diagnosis remains imperfect in resource-limited settings due to its complexity and costs, poor sensitivity of available tests, or long times to reporting. We present a report on the use of colorimetric methods, based on the detection of mycobacterial growth using colorimetric indicators, for the detection of Mycobacterium tuberculosis in sputum specimens. We evaluated the nitrate reductase assay (NRA), a modified NRA using para-nitrobenzoic acid (PNB) (NRAp), and the resazurin tube assay using PNB (RETAp) to differentiate tuberculous and nontuberculous mycobacteria. The performances were assessed at days 18 and 28 using mycobacterium growth indicator tube (MGIT) and Löwenstein-Jensen (LJ) medium culture methods as the reference standards. We enrolled 690 adults with suspected pulmonary tuberculosis from a regional referral hospital in Uganda between March 2010 and June 2011. At day 18, the sensitivities and specificities were 84.6% and 90.0% for the NRA, 84.1% and 92.6% for the NRAp, and 71.2% and 99.3% for the RETAp, respectively. At day 28, the sensitivity of the RETAp increased to 82.6%. Among smear-negative patients with suspected TB, sensitivities at day 28 were 64.7% for the NRA, 61.3% for the NRAp, and 50% for the RETAp. Contamination rates were found to be 5.4% for the NRA and 6.7% for the RETAp, compared with 22.1% for LJ medium culture and 20.4% for MGIT culture. The median times to positivity were 10, 7, and 25 days for colorimetric methods, MGIT culture, and LJ medium culture,respectively. Whereas the low specificity of the NRA/NRAp precludes it from being used for TB diagnosis, the RETAp might provide an alternative to LJ medium culture to decrease the time to culture results in resource-poor settings.