Patients
This study was designed as a pair matching case-control study. Between October 2007 and December 2008, 60 newly recruited sputum-positive TB patients (cases) over 15 years of age were enrolled at the principal anti-TB center of Antananarivo, the capital of Madagascar, where the annual incidence of newly diagnosed sputum smear-positive patients is about 80 cases per 100, 000 inhabitants according to the National TB Control Programme (NTCP) [4]. The patients were treated according to the NTCP strategy and were followed up by the clinical physicians.
The 60 subjects selected for the patients group had a median age of 35.5 years (age range, 15-59 years) and 54 (45%) were males, 66 (55%) were females.
Control group
The control group consisted of 60 individuals selected randomly from apparently healthy household or family contacts of TB patients, and matched for sex and age with case subjects (difference between birthdays less than 5 years). All members of control group had no previous history of TB, no signs or symptoms suggestive of pulmonary TB, no evidence of TB on chest X rays. The household contacts (HCs) of the included index cases were visited at home by the study physicians and were invited to join the study. They were included if they were ≥15 year old and had been living in the same house as the case patient for at least 6 months.
Household contacts controls underwent a PPD skin test (10 units; tuberculin purified protein derivative; Aventis Pasteur). Induration was recorded after 72 h.
All subjects selected for the control group were HIV and PPD skin test negative.
Serum samples were obtained from almost all patients before initiation of antituberculosis treatment and stored at -20°C until tested. All patients and controls participated in the study were vaccinated with Mycobacterium bovis BCG. Individuals with a diagnosis of TB disease were referred to the antituberculosis center for treatment.
The study was approved by the National Ethics Committee to the Ministry of Health in Madagascar.
Samples collection
All TB cases were screened for TB independently by microscopy, culture and SD Rapid TB to evaluate the performance of the test under field conditions. A questionnaire was filled for each patient with basic clinical and demographic information after taking written consent. Three sputum samples were obtained from each of the 60 patients using routine techniques, and the samples were processed in the Chest Clinic according to established norms [5]. Venous blood was drawn from the study participants and placed into one 5 ml dry vacutainer tube without preservative or anticoagulant for separation of serum. Each serum sample was divided into two parts: part was used for HIV testing and the other was frozen at -20°C for SD Rapid TB testing.
SD Rapid TB interpretation
The SD Rapid TB test (Standard Diagnostic, Inc; Cat. No. 08FK10) is a chromatographic immunoassay for qualitative detection of antibodies to M. tuberculosis in human serum. Briefly, the test consists of a cardboard folding device containing a nitrocellulose strip and absorbent pads. Antigen secreted by M. tuberculosis during active infection is immobilized on a line across the strip. When serum (100 μl) is applied, it flows past the antigen line. If specific antibody to the antigen is present, it binds to the line. Bound antibody is detected by a goat anti-human immunoglobulin G antibody conjugated to colloidal gold particles which give a pink band when bound to human antibody. Test card has one control line to indicate the validity of the test procedure and its working condition. Control and test lines appeared within 15 minutes in a reading window. SD Rapid TB was performed and the results were interpreted according to the instructions of the manufacturer by two independent readers who were blind to the clinical status of the subjects and to the TB diagnosis results. The presence of two bands (control and test line) indicates a positive result (Figure 1). Each SD Rapid TB was saved as documentation for future reference. Only tests from one batch were used (Manufacture June 07, expiry January 09 batch no. 046022).
Clinical and laboratory evaluation
Two spot and one overnight sputum specimens were collected from each patient.
The sputa were decontaminated by the sodium dodecyl sulfate method of Tacquet and Tison [5]. Fluorescence microscopy was performed for the direct detection of mycobacteria by the auramine staining method [6]. One drop of the decontaminated sputum was fixed on a slide, stained with auramine-phenol, and examined under a fluorescence microscope (objective, ×40) for acid-fast bacilli (AFB). Sputum smear microscopy was considered positive when AFB were seen in at least two out of the three specimens. The remaining decontaminated specimen was inoculated into two tubes of standard LJ medium (Diagnostics Pasteur, Paris, France) and on one tube of LJ medium without glycerol but supplemented with 0.5% pyruvate. Culture said to be positive when at least one colony of M. tuberculosis was grown, and negative if there was no growth after 8 week. The numbers of colony forming unit (CFU) growing in each LJ tube were counted. Mycobacterial isolates were identified according to growth on LJ medium, colony morphology, and biochemical tests for the following: niacin production, catalase, urease, and nitrate reductase [6]. All subjects included were serotested for HIV.
Data analysis
The performance of SD Rapid TB was expressed by calculating the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) for TB separately taking culture results as gold standard (According to Grange and Laszlo 1990 [7]). Data were double entered, validated and analysed using Epi Info™ 3.3.6 software (CDC Atlanta GA, USA). Proportions were compared using the chi-square test.