This was a cross sectional analytical study.
The study was done at the Mulago National Referral Hospital in Kampala, the capital city with a day population of 2.5 million, HIV sero-prevalence of 8.5% among adults, HIV sero-prevalence of 2.2% in children under five and BCG vaccination coverage of 94.6%. This hospital with a 1700-bed capacity and an annual turnover of 100,000 patients also serves as the primary health care facility for the surrounding areas. The children were recruited from the paediatric emergency unit between February and June 2011. This paediatric emergency unit has an annual turnover of 20,000 patients with pneumonia admissions accounting for about 20% of the admissions.
Participants and procedures
Children aged 2 months -12 years who fulfilled the WHO case definition for severe or very severe pneumonia and whose caretaker gave informed consent were eligible to participate in the study. The children above 8 years of age provided assent. Children already on treatment for TB were excluded. The enrolment was consecutive after at least a 24-hour admission period to limit the misclassification of the cases that fulfil the WHO criteria for severe pneumonia but are not pneumonia for example asthma and severe anaemia.
Severe pneumonia was defined as a presentation with cough or difficulty in breathing, fast breathing and chest in drawing. A very severe pneumonia case fulfilled the case definition for severe pneumonia plus either cyanosis, convulsions or inability to feed
. The potential participants were identified at the triage by the triage nurse or the research assistant.
After obtaining informed consent, specific clinical history that included duration of cough, TB contact, weight loss, BCG vaccination, previous pneumonia episodes was obtained from the caregiver or parent. The children were examined for BCG scar, peripheral lymphadenopathy and their weights and height obtained. The specific investigations done included; Full blood count, blood culture for mycobacterium, one sputum induction, tuberculin skin test (TST) placement by the Mantoux method and chest radiography. The HIV serology results were obtained from the Hospital Routine HIV counselling and testing (HCT) register. The caregivers or parents were encouraged to have the children tested for HIV within the hospital HCT program. The children found to be HIV positive were linked to the HIV care centre at the Mulago Hospital for further management. The Chest radiography and placement of TST were done on the day of enrolment. The TST was read within 48 – 72 hours by the same trained nurse who placed the skin test. The one sputum induction was done early in the morning within 24 hours of admission. The older children provided at least 2 expectorated sputum samples. The sputum samples were placed in a cool box with ice packs and taken to the - Mycobacteriology Laboratory, Department of Microbiology, Makerere University College of Health sciences for processing within 1 hour of collection.
Complete blood count was done using a coulter Act Diff 5 machine. Blood (2-5 ml) for Mycobacterial culture was aseptically inoculated into BD BACTEC-MYCO/F-Lytic media (Becton and Dickson, Franklin Lakes, NJ USA) for up to six weeks at the Mycobacterialogy (BSL-3) laboratory. Cultures in which the machine flagged positive were unloaded and a Ziehl Nelseen (ZN) smear for Acid Fast Bacilli (AFB) was done. A portion was also inoculated onto a Blood Agar Plate (BAP) to rule out contaminants. Cultures that revealed AFB on ZN smear were subjected to Capilia TB NeoTM (TAUN, Numazu, Japan) for identification of Mycobacterium tuberculosis complex (MTBC). However, cultures which were ZN negative and revealed growth on BAP were considered contaminated.
Sputum samples were prepared for culture according to laboratory standard operating procedures
. A volume 0.5 ml of the re-suspended deposit was inoculated in a BACTEC BBL MGITTM 960 tube (Becton and Dickson, Franklin Lakes, NJ USA). Additionally, two drops were inoculated in LJ tubes (Becton and Dickson, Franklin Lakes, NJ USA) and cultured. A smear for fluorescent microscopy was also done according to standard procedures
Cultures were incubated at 37°C for 6 weeks in MGIT and 8 weeks on LJ media. For purity, any MGIT-positive culture was sub-cultured at 37°C on blood agar for 24 hours and a Ziehl Neelsen (ZN) smear done. Cultures which were ZN-positive and pure (i.e. no growth on blood agar) were subjected to Capilia TB NeoTM (TAUN, Numazu, Japan) for identification of the MTB complex
. Cultures with growth on blood agar but also ZN-positive were sub-cultured again as described above; the persistent ZN-negative cultures with growth on blood agar were considered contaminated. All ZN-positive, blood agar negative and Capilia TB NeoTM negative samples were considered as having Mycobacteria Other Than Tuberculosis (MOTT). Otherwise absence of colonies on LJ or fluorescence on MGIT was considered negative. The data were entered into a computerized laboratory access database linked with patient records on sample reporting form.
The main outcome measure was the diagnosis of pulmonary TB. A case of Pulmonary TB is one that fulfilled either the confirmed case or probable case definition
. A confirmed case is one with culture confirmed MTBC while a probably Pulmonary TB case is one with at least one of the signs and symptoms suggestive of TB AND Chest radiography consistent with intrathoracic disease due to MTBC AND there is at least one of the following; Documented exposure to MTBC infection, Immunological evidence of MTBC infection. The chest radiography reading for features of TB was done by 2 independent blinded radiologists with the third acting as a tie-breaker.
The study was approved by the School of Medicine Research and Ethics committee, Makerere University College of Health Sciences. Written informed consent was given by the parents/caretakers if they agreed to their children participating in the study and assent was obtained from older children. Children that were diagnosed with Pulmonary TB were linked to the National TB and Leprosy control program for care and treatment.
Using the Kish-Leslie formula, a minimum sample size of 196 participants was sufficient to determine the burden of Pulmonary TB among children admitted with severe pneumonia while a minimum sample size of 257 with at least 43 TB cases was sufficient to explore associated factors.
The data was captured using Epidata version 3.1 and analyzed using SPSS (version 14.0). The proportion with Pulmonary TB was determined with its 95% CI. The continuous variables were summarized as means (SD) or medians (IQR). Any associations with Pulmonary TB for categorical variables were explored using the χ2 test whereas normally distributed continuous variables were tested using student’s t-test and skewed data using the Mann-Whitney U test. Multivariate analysis using logistic regression was carried out on variables with p- value of less than 0.2 at bivariate to adjust for confounding. P value <0.05 was considered significant.