• Bleach sedimentation: an opportunity to optimize smear microscopy for tuberculosis diagnosis in settings of high prevalence of HIV

      Bonnet, M; Ramsay, A; Githui, W; Gagnidze, L; Varaine, F; Guerin, P J; Epicentre, Paris, France; Médecins Sans Frontières, Paris, France; Liverpool School of Tropical Medicine, Liverpool, United Kingdom; Centre for Respiratory Diseases Research, Kenya Medical Research Institute, Nairobi, Kenya (Infectious Diseases Society of America, 2008-06-01)
      BACKGROUND: The purpose of the study was to evaluate the performance and feasibility of tuberculosis diagnosis by sputum microscopy after bleach sedimentation, compared with by conventional direct smear microscopy, in a setting of high prevalence of HIV. METHODS: In a community-based study in Kenya (a population in which 50% of individuals with tuberculosis are infected with HIV), individuals with suspected pulmonary tuberculosis submitted 3 sputum specimens during 2 consecutive days, which were examined by blind evaluation. Ziehl-Neelsen-stained smears were made of fresh specimens and of specimens that were processed with 3.5% household bleach followed by overnight sedimentation. Two different cutoffs for acid-fast bacilli (AFB) per 100 high-power fields (HPF) were used to define a positive smear: >10 AFB/100 HPF and 1 AFB/100 HPF. Four smear-positive case definitions, based on 1 or 2 positive smears with the 1 AFB or 10 AFB cutoff, were used. RESULTS: Of 1879 specimens from 644 patients, 363 (19.3%) and 460 (24.5%) were positive by bleach sedimentation microscopy, compared with 301 (16.0%) and 374 (19.9%) by direct smear microscopy, with use of the 10 AFB/100 HPF (P < .001) and 1 AFB/100 HPF (P < .001) cutoffs, respectively. Regardless of the case definition used, bleach sedimentation microscopy detected significantly more positive cases than did direct smear microscopy: 26.7% (172 of 644) versus 21.7% (140 of 644), respectively, with the case definition of 1 positive smear and the 1 AFB/100 HPF cutoff (P < .001), and 21.4% (138 of 644) versus 18.6% (120 of 644), respectively, with the case definition of 1 positive smear and the 10 AFB/100 HPF cutoff (P < .001). Inter- and intrareader reproducibility were favorable, with kappa coefficients of 0.83 and 0.91, respectively. Bleach sedimentation was relatively inexpensive and was not time consuming. CONCLUSIONS: Bleach sedimentation microscopy is an effective, simple method to improve the yield of smear microscopy in a setting of high prevalence of HIV. Further evaluation of this method, under operational conditions, is urgently needed to determine its potential as a tool for tuberculosis control.
    • Cetyl-pyridinium chloride is useful for isolation of Mycobacterium tuberculosis from sputa subjected to long-term storage.

      Pardini, M; Varaine, F; Iona, E; Arzumanian, E; Checchi, F; Oggioni, M R; Orefici, G; Fattorini, L; Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. (2005-01)
      Recovery of Mycobacterium tuberculosis from sputa treated with cetyl-pyridinium chloride (CPC) and stored for 20 +/- 9 days was significantly higher than that from sputa that were untreated and processed by the N-acetyl-L-cisteine-NaOH method. Addition of CPC is useful for isolation of M. tuberculosis from sputa subjected to long-term storage received from remote areas of the world.
    • Evaluation of FASTPlaqueTB to diagnose smear-negative tuberculosis in a peripheral clinic in Kenya

      Bonnet, M; Gagnidze, L; Varaine, F; Ramsay, A; Githui, W; Guerin, P J; Epicentre, Paris, France; Liverpool School of Tropical Medicine, Liverpool, UK; United Nations Children’s Fund/United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training for Tropical Diseases (TDR), Geneva, Switzerland; Centre for Respiratory Diseases Research, Kenya Medical Research Institute, Nairobi, Kenya (2009-09-01)
      OBJECTIVE: To evaluate the performance and feasibility of FASTPlaqueTB in smear-negative tuberculosis (TB) suspects in a peripheral clinic after laboratory upgrading. DESIGN: Patients with cough > or=2 weeks, two sputum smear-negative results, no response to 1 week of amoxicillin and abnormal chest X-ray were defined as smear-negative suspects. One sputum sample was collected, decontaminated and divided into two: half was tested with FASTPlaqueTB in the clinic laboratory and the other half was cultured on Löwenstein-Jensen medium in the Kenyan Medical Research Institute. Test sensitivity and specificity were evaluated in all patients and in human immunodeficiency virus (HIV) infected patients. Feasibility was assessed by the contamination rate and the resources required to upgrade the laboratory. RESULTS: Of 208 patients included in the study, 56.2% were HIV-infected. Of 203 FASTPlaqueTB tests, 95 (46.8%) were contaminated, which interfered with result interpretation and led to the interruption of the study. Sensitivity and specificity were respectively 31.2% (95%CI 12.1-58.5) and 94.9% (95%CI 86.8-98.4) in all patients and 33.3% (95%CI 9.9-65.1) and 93.9% (95%CI 83.1-98.7) in HIV-infected patients. Upgrading the laboratory cost euro 20,000. CONCLUSION: FASTPlaqueTB did not perform satisfactorily in this setting. If contamination can be reduced, in addition to laboratory upgrading, its introduction in peripheral clinics would require further assessment in smear-negative and HIV co-infected patients and test adaptation for friendlier use.
    • Evaluation of Tuberculosis Diagnostics in Children: 1. Proposed clinical case definitions for classification of Intrathoracic Tuberculosis Disease. Consensus from an expert panel

      Graham, S M; Ahmed, T; Amanullah, F; Browning, R; Cardenas, V; Casenghi, M; Cuevas, L E; Gale, M; Gie, R P; Grzemska, M; et al. (Oxford University Press, 2012-03-22)
      There is a critical need for improved diagnosis of tuberculosis in children, particularly in young children with intrathoracic disease as this represents the most common type of tuberculosis in children and the greatest diagnostic challenge. There is also a need for standardized clinical case definitions for the evaluation of diagnostics in prospective clinical research studies that include children in whom tuberculosis is suspected but not confirmed by culture of Mycobacterium tuberculosis. A panel representing a wide range of expertise and child tuberculosis research experience aimed to develop standardized clinical research case definitions for intrathoracic tuberculosis in children to enable harmonized evaluation of new tuberculosis diagnostic technologies in pediatric populations. Draft definitions and statements were proposed and circulated widely for feedback. An expert panel then considered each of the proposed definitions and statements relating to clinical definitions. Formal group consensus rules were established and consensus was reached for each statement. The definitions presented in this article are intended for use in clinical research to evaluate diagnostic assays and not for individual patient diagnosis or treatment decisions. A complementary article addresses methodological issues to consider for research of diagnostics in children with suspected tuberculosis.
    • Hepple - Microscopy compared to culture for the diagnosis of Tuberculosis in Induced Sputum samples; a systematic review

      Hepple, P; Ford, N; McNerney, R; Manson Unit, Médecins Sans Frontières, London, UK. pamela.hepple@london.msf.org (2012-03-07)
      Resource-limited settings rely on sputum examination using microscopy to diagnose tuberculosis (TB); however, the sensitivity of the test is poor and case detection rates are low. Sputum induction is proposed as a way to improve sample collection and enhance test sensitivity.
    • Meningitis Serogroup W135 Outbreak, Burkina Faso, 2002.

      Nathan, N; Rose, A M C; Legros, D; Tiendrebeogo, S R M; Bachy, C; Bjørløw, E; Firmenich, P; Guerin, P J; Caugant, D A; Epicentre, Paris, France. (Published by Centers for Disease Control (CDC), 2007-06)
      In 2002, the largest epidemic of Neisseria meningitidis serogroup W135 occurred in Burkina Faso. The highest attack rate was in children <5 years of age. We describe cases from 1 district and evaluate the performance of the Pastorex test, which had good sensitivity (84%) and specificity (89%) compared with culture or PCR.
    • Reducing the Number of Sputum Samples Examined and Thresholds for Positivity: An Opportunity to Optimise Smear Microscopy.

      Bonnet, M; Ramsay, A; Gagnidze, L; Githui, W; Guerin, P J J; Varaine, F; Epicentre, Paris, France. maryline.bonnet@geneva.msf.org (International Union Against TB and Lung Disease, 2007-09)
      SETTING: Urban health clinic, Nairobi. OBJECTIVE: To evaluate the impact on tuberculosis (TB) case detection and laboratory workload of reducing the number of sputum smears examined and thresholds for diagnosing positive smears and positive cases. DESIGN: In this prospective study, three Ziehl-Neelsen stained sputum smears from consecutive pulmonary TB suspects were examined blind. The standard approach (A), > or = 2 positive smears out of 3, using a cut-off of 10 acid-fast bacilli (AFB)/100 high-power fields (HPF), was compared with approaches B, > or = 2 positive smears (> or = 4 AFB/100 HPF) out of 3, one of which is > or = 10 AFB/100 HPF; C, > or = 2 positive smears (> or = 4 AFB/100 HPF) out of 3; D, > or = 1 positive smear (> or = 10 AFB/100 HPF) out of 2; and E, > or = 1 positive smear (> or = 4 AFB/100 HPF) out of 2. The microscopy gold standard was detection of at least one positive smear (> or = 4 AFB/100 HPF) out of 3. RESULTS: Among 644 TB suspects, the alternative approaches detected from 114 (17.7%) (approach B) to 123 cases (19.1%) (approach E) compared to 105 cases (16.3%) for approach A (P < 0.005). Sensitivity ranged between 82.0% (105/128) for A and 96.1% (123/128) for E. The single positive smear approaches reduced the number of smears by 36% compared to approach A. CONCLUSION: Reducing the number of specimens and the positivity threshold to define a positive case increased the sensitivity of microscopy and reduced laboratory workload.
    • Thin layer agar compared to BACTEC MGIT 960 for early detection of Mycobacterium tuberculosis

      Martin, A; Fissette, K; Varaine, F; Portaels, F; Palomino, J C; Institute of Tropical Medicine, Mycobacteriology Unit, Antwerp, Belgium; Médecins Sans Frontières, Paris, France (2009-07-05)
      We compared the sensitivity and time to detection of growth of Mycobacterium tuberculosis in the thin layer agar (TLA) compared to BACTEC MGIT960. The average time for growth of M. tuberculosis in TLA and BACTEC MGIT960 was 10.6 and 9.6 days, respectively. The sensitivity of detection of M. tuberculosis was 97.3% on TLA and 97% on BACTEC MGIT960 for smear positive samples. TLA showed comparable results to BACTEC MGIT960 and could be an alternative method for low-income countries.
    • Thin-layer agar for detection of resistance to rifampicin, ofloxacin and kanamycin in Mycobacterium tuberculosis isolates

      Martin, A; Paasch, F; Von Groll, A; Fissette, K; Almeida, P; Varaine, F; Portaels, F; Palomino, J-C; Institute of Tropical Medicine, Mycobacteriology Unit, Antwerp, Belgium; Laboratory of Mycobacteriology, Universidade Federal do Rio Grande, Rio Grande do Sul, Brazil; Médecins Sans Frontières, Paris, France (2009-10-01)
      BACKGROUND: In low-income countries there is a great need for economical methods for testing the susceptibility of Mycobacterium tuberculosis to antibiotics. OBJECTIVE: To evaluate the thin-layer agar (TLA) for rapid detection of resistance to rifampicin (RMP), ofloxacin (OFX) and kanamycin (KM) in M. tuberculosis clinical isolates and to determine the sensitivity, specificity and time to positivity compared to the gold standard method. METHODS: One hundred and forty-seven clinical isolates of M. tuberculosis were studied. For the TLA method, a quadrant Petri plate containing 7H11 agar with RMP, OFX and KM was used. Results were compared to the Bactec MGIT960 for RMP and the proportion method for OFX and KM. RESULTS: The sensitivity and specificity for RMP and OFX were 100% and for KM they were 100% and 98.7%, respectively. The use of a TLA quadrant plate enables the rapid detection of resistance to the three anti-tuberculosis drugs RMP, OFX and KM in a median of 10 days. CONCLUSION: TLA was an accurate method for the detection of resistance in the three drugs studied. This faster method is simple to perform, providing an alternative method when more sophisticated techniques are not available in low-resource settings.
    • Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities.

      McNerney, R; Maeurer, M; Abubakar, I; Marais, B; McHugh, T D; Ford, N; Weyer, K; Lawn, S; Grobusch, M P; Memish, Z; et al. (2012-05-15)
      Tuberculosis is unique among the major infectious diseases in that it lacks accurate rapid point-of-care diagnostic tests. Failure to control the spread of tuberculosis is largely due to our inability to detect and treat all infectious cases of pulmonary tuberculosis in a timely fashion, allowing continued Mycobacterium tuberculosis transmission within communities. Currently recommended gold-standard diagnostic tests for tuberculosis are laboratory based, and multiple investigations may be necessary over a period of weeks or months before a diagnosis is made. Several new diagnostic tests have recently become available for detecting active tuberculosis disease, screening for latent M. tuberculosis infection, and identifying drug-resistant strains of M. tuberculosis. However, progress toward a robust point-of-care test has been limited, and novel biomarker discovery remains challenging. In the absence of effective prevention strategies, high rates of early case detection and subsequent cure are required for global tuberculosis control. Early case detection is dependent on test accuracy, accessibility, cost, and complexity, but also depends on the political will and funder investment to deliver optimal, sustainable care to those worst affected by the tuberculosis and human immunodeficiency virus epidemics. This review highlights unanswered questions, challenges, recent advances, unresolved operational and technical issues, needs, and opportunities related to tuberculosis diagnostics.
    • Use of filter paper as a transport medium for laboratory diagnosis of cholera under field conditions

      Page, Anne-Laure; Alberti, Kathryn P; Guénolé, Alain; Mondongue, Vital; Lonlas Mayele, Sylvaine; Guerin, Philippe J; Quilici, Marie-Laure; Epicentre, Paris, France; Institut Pasteur, Centre National de Reference des Vibrions et du Cholera, Unite des Bacteries Pathogenes Enteriques, Paris, France; Ministry of Health, Kinshasa, Democratic Republic of Congo; Medecins Sans Frontieres, Brussels, Belgium; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom (American Society for Microbiology, 2011-06-22)
      Confirmation of a cholera epidemic is based on bacteriological identification of the agent and requires the sending of samples to a culture laboratory, often in countries with limited resources. Comparison of the use of filter paper with the use of Cary-Blair reference medium for stool transport showed that this simple transport medium is appropriate for the recovery of Vibrio cholerae.