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Ethical committee of the All India Institute of Medical Sciences (AIIMS), New Delhi approved the study protocol in accordance with National Guidelines by Indian Council of Medical Research. All the subjects were recruited with their signed consent on ethically approved consent form informed in both Hindi and English after explaining the purpose and implications of the study by the well trained field investigator.
Study Design and Subject Recruitment: The study was conducted between 2006 and 2010 at the TB Laboratory, Clinical Microbiology Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi in collaboration with designated microscopy centers (DMC) and DOTS centers of South Delhi region (Khanpur, Dakshinpuri, Madangir, Safdarjung, and Shahpur Jat). After approval of the study from the central TB control division of Government of India, we approached the DMCs of the respective area to identify the smear positive patients diagnosed at their respective DMC within last two weeks. All the sputum smear positive patients were contacted at their place of residence, their detailed clinical history was noted and after written consent, 508 index PTB cases and 1792 family contacts were recruited. However, after further work-up 62 PTB patient refused consent for inclusion in the study, and 18 had no regular house hold and thus these were excluded. Similarly out of 1792 recruited family contacts, 961 refused to give blood sample and 42 were found to have co-prevalent TB and thus grouped into the PTB group (please see Figure 1). Finally a total of 1259 subjects were enrolled in the study. All PTB cases, whether untreated, relapse, or under treatment (but not responding to treatment) were included in the study. All the demographic details and relevant clinical symptoms, signs and duration were documented in predesigned subject information form.
TB patients were defined as PTB cases where infection of lungs, pleural cavities or respiratory tracts with M. tuberculosis occurs and the disease is diagnosable with chest X-ray, smear microscopy, culture or had favourable response to antitubercular treatment. Household contacts in this study were defined as all the family members/tenants/groups generally living together in the same shelter with same front door and who live in prolonged/intense contact with the PTB patient [32]. Among the household contacts only contacts who had no symptoms of TB infection in preliminary investigation were included in the present study.
Preliminary diagnosis of PTB was made at local designated microscopy centers (DMC) after examining patient’s morning or spot sputum samples. After obtaining information of smear positive patients we noted the contact details of the patients and field worker contacted the patient’s family and fixed the appointment for sampling. A repeat sputum (1 morning plus 1 spot) sample and 5 ml blood were collected (before doing Mantoux test) in sterile containers and samples transported on the same day to TB Laboratory, Clinical Microbiology Division at AIIMS for further processing. The asymptomatic healthy contacts who could not produce good quality sputum, even the saliva samples were accepted for the study. The sputum/saliva samples were processed after decontamination by modified Petroff’s (NALC/NaOH) method [33]. The processed sputum samples were inoculated in MGIT (Mycobacterium Growth Indicator Tube) of automated BACTEC™ MGIT 960 culture system following manufacturer’s instructions (Becton Dickinson, USA). Ziehl-Neelson (ZN) staining followed by microscopy was done on both direct and decontaminated sputum samples for acid fast bacilli (AFB). Serum was separated from the blood samples by centrifugation and stored at −20°C for further use in ELISA avoiding repeated freezing and thawing.
Tuberculin skin test (TST) or Mantoux test was carried out by intradermally injecting 0.1 ml of 5TU (Span Diagnostics Ltd, India) purified protein derivative (PPD) into the volar surface of the forearm. While injecting PPD it was ensured that level of tuberculin syringe needle was facing upward so that a pale elevation of the skin (a wheal), 6 to 10 mm in diameter, was formed. Mantoux test was done only after withdrawing blood sample. The patients were instructed not to apply any soap/detergent or wash the area to avoid itching and scratching for the next 48 hours. The injection site was encircled by permanent marker and reaction induration (palpable, raised, hardened area or swelling) was measured in millimeter (mm) after 48–72 hours [32], [34]. The test was performed by well trained field investigators.
Recording the Details of BCG Vaccination: BCG status was determined using visual inspection of scars. The subjects with clearly visible scar were considered as BCG vaccinated and remaining without scar as non-vaccinated.
We screened published literature for performance of dozens of commercial serological tests offered for sale in Indian market and selected Pathozyme® Myco IgG, IgA and IgM, EIA kits manufactured by Omega Diagnostic Limited, Scotland, UK [35]. These kits were selected because of more/or widespread use, comparative better performance as available on the public domain and combination of antigens used in it [24]. These kits are based on two highly purified immunodominant antigens, the cell wall lipoarabinomannan (LAM) antigen which, and a 38-kDa mycobacterial recombinant antigen [35]. The kits claimed to be having 91% specificity and 72% sensitivity [36]. The EIA tests were performed according to the instructions provided in kits’ manual (Omega diagnostics limited, Scotland, UK). All three EIA kits were evaluated simultaneously with the same serum samples aliquots stored at −20°C.
For proper analysis of performance, ELISA tests were evaluated first on asymptomatic household contacts and then on confirmed PTB cases. Sensitivity and specificity was determined using confirmed PTB cases and asymptomatic contacts as positive and negatives references. Other statistical analysis, such as Positive predictive values (PPV), negative predictive value (NPV) and likelihood ratio for positive (LRP) test were also calculated with 95% CI (confidence intervals). PPV (also called precision rate) is the proportion of subjects with positive test results who are correctly diagnosed for infection. NPV is defined as the proportion of subjects with negative test result who are correctly ruled out of infection. Higher PPV and NPV denote more correct assessment. Likelihood ratio of positive (LRP) test helps to predict the likelihood of true positive result allowing the clinician to better interpret the results of the diagnostic test. A LRP of greater than 1 indicates the test result is associated with the presence of disease and less than 1 means the test result is associated with the absence of the disease. Percentage agreement was assessed between the results of Mantoux test and EIAs. To rule out the proportion of agreement by chance, Cohen’s kappa test was used. To check the effect of BCG vaccination on the performance of EIAs and Mantoux test, Pearson Chi-square test and exact mid-p test were used. P-value <0.05 was considered statistically significant. STATA SE.9 software was used for all statistical analysis.
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