Distribution and transmission of Mycobacterium tuberculosis complex lineages among children in peri-urban Kampala, Uganda

Children in contact with adults who had pulmonary TB in peri-urban Kampala were studied. The study considered MTBC isolates that were collected from children aged ≤ 15 years using a well characterized TB household contact study conducted in the Kawempe area of Kampala, the Kawempe Community Household study (KC) [20]. The KC study focused on TB and its risk factors among adult patients (≥18) and their household contacts. Culture positive TB patients were consecutively enrolled as index cases (patient who reported first at the TB clinic with pulmonary TB) and their housemates (contacts) whom had stayed in the index case household for at least 7 consecutive days during the 3 months prior to the diagnosis of TB. Household contacts were classified as co-prevalent cases if active TB was present at baseline or during three months of household follow-up and as incident cases if active TB developed after three months of follow-up.

At baseline, data of enrolled children, including age, sex, HIV status and presence of BCG scar (deltoid scar) were recorded. Clinical features such as persistent cough for more than 3 weeks, night sweats and hemoptysis were documented. The radiological features such as extent of lung involvement classified as (i) normal/moderate [mild lung disease], where lesions appeared as infiltrates of slight to moderate densities affecting a small portion of one or both lungs with no cavitation (ii) advanced/far advanced [advanced lung disease], where lung lesions were more extensive than in mild lung disease category with cavities ≤ 4 cm, were documented. Tuberculin skin test (TST) was performed by Mantoux method (where a positive TST was defined as an induration of ≥ 10 mm if a child was HIV negative and ≥ 5 years of age or ≥ 5 mm if a child was HIV positive or < 5 years of age). Sputum or gastric lavage samples collected at baseline were microscopically examined and processed for culture at the Joint Clinical Research Centre (JCRC) TB Laboratory in Kampala following standard procedures [22]. Isolates were confirmed as MTBC using the BACTEC® para-nitro-acetyl amino-hydroxy-propiophenone (NAP) susceptibility method [23] after 4 weeks and stored at – 80 °C in 7H9 broth supplemented with OADC and glycerol for future analysis. In this study, a total of 761 children from 351 households were enrolled. Of these, 69 (9 %) were culture positive and had their bacilli isolated and stored for future use. In an earlier study, a total of 1746 isolates from adult (>15 years) patients within the same cohort were genotyped as described [13].

A total of 69 isolates were stored in replicates at – 80 °C. Isolates corresponding to an individual child were selected for genotyping. To extract DNA, the selected isolates were thawed overnight at – 20 °C and later at room temperature for 12 hours. The vials were centrifuged at 15,000 g for 30 min and the pellet washed twice with 500 μl of Qiagen PCR-grade water. The final pellet was re-suspended in 100 μl of Qiagen PCR-water, heated at 95 °C for 30 minutes to kill and lyse the bacilli and later sonicated for 15 min at room temperature. The extracted crude genomic DNA in the supernatant was recovered by centrifugation at 15,000 g for 30 min; the latter was used immediately in the real time PCR (RT-PCR) assay or stored at −20 ° C for future use.

To ascertain the MTBC lineage to the 69 isolates, three single nucleotide polymorphism (SNP) markers were used. These are specific for identifying MTBC Uganda family (MTB L4-U), MTBC Lineage 4 and MTBC Lineage 3 (MTBC L3). Corresponding primers and probes were used in a LightCycler real time PCR SNP assay (LRPS) to identify the MTBC lineages on the basis of differences in melting temperature (Tm) as described by Wampande et al. [13]. In all the assays, we used MTB L4-U genomic DNA from our laboratory, H37Rv genomic DNA (Lineage 4) and MTB Lineage 3 (Central Asian strain) genomic DNA (Courtesy of Mark Nicol) as positive control DNA.

The baseline characteristics of children with TB were compared with MTBC lineage using the chi-square test for binary data. A series of univariate and multivariable logistic regression models were fitted to evaluate the relationship between MTBC lineage (main exposure variable) and extent of lung involvement of TB disease (either Normal/moderate [mild lung disease]; or advanced/far advanced [advanced lung disease] on chest radiograph as the outcome [24]. The extent of lung involvement i.e. normal/moderate (mild lung disease) or advanced/far advanced (advanced lung disease) was used as a measure of disease severity and hence transmission [10, 12, 25]. The covariates age, sex, HIV status, presence of BCG scar, night sweats, hemoptysis, tuberculin skin test, and cough were used as adjusters. All analyses were performed using STATA version 12.

The institutional review boards and ethics committees at Case Western Reserve University, Makerere University, Uganda AIDS Research Council, and the Uganda National Council for Science and Technology approved the study protocols. Patients/guardians gave written consent for the children and children aged ≥ 8 years provided additional assent. Pre- and post-HIV test counseling was provided.

A total of 761 children (from 351 household) were surveyed from 2002 to 2010 in a household study. Of these, 69 (9 %) children (from 62 household) were culture positive, this finding is similar to earlier studies carried out in the same study cohort [21]. Seventy-one percent (49/69) were children aged up to 5 years, 54 % (37/69) were females, 14 % (9/66) were HIV positive, 67 % (46/69) had a BCG scar, 12 % (8/69) reported signs of night sweating, 29 % (19/65) showed advanced lung disease on radiography, 42 % (29/69) had cough, 62 % (41/66) had a positive tuberculin skin test and 85 % (47/55) were smear negative (Table 1). Furthermore, 91 % (59/62) had no cavitation, and 97 % (67/69) also had no hemoptysis.

All 69 MTB isolates corresponding to an individual child were genotyped using LRPS as described by Wampande et al. [13]. Overall, 71 % (49/69) were MTBC L4-U (MTBC Uganda family), 17 % (12/69) MTBC L4-NU (Lineage 4 strains other than Uganda family) and 12 % (8/69) Linage 3 (MTBC L3). This data was comparable with MTBC lineages in the adult (≥18 years) patients published by Wampande et al. [13] from the same study cohort. Given that finding, further analysis was done to seek for possible association between MTBC lineages and the available clinical and epidemiological variables.

The additional data analysis (this analysis has combined MTB L4-NU and MTB L3 in one group: from now onwards this shall be referred to as MTBC non-Uganda) showed that children infected with MTBC non-Uganda lineage were more likely to have cough (P = 0.048) and advanced extent (Advance/far advanced) of lung involvement (P = 0.041) compared to those infected with MTBC Uganda family. Children infected with MTBC Uganda were more likely to have smear-positive TB (P = 0.041). No significant differences (P > 0.05) were observed between MTBC lineages with respect to age, sex, HIV/TB co-infection, presence of a BCG scar, night sweats and tuberculin skin test status (Table 1).

Since advanced lung disease is positively correlated with severity of disease and hence a measure of transmissibility [10‐12, 25, 26], we looked for differences in transmissibility among MTBC lineages, using advanced extent of lung involvement (mild or advanced lung disease) as the outcome in the logistic model. Multivariate analysis was performed to look for risk factors associated with advanced lung disease while using MTBC lineage as the main predictor. After adjusting for age, sex, HIV status, presence or absence of BCG scar, a child having or not having night sweats, children who cough and those who do not, data showed that the odds of a child having advanced lung disease did not differ significantly (OR = 0.304, CI: 95 %; 0.039-2.326, P = 0.251) between MTBC lineages and no other risk factor was associated with advanced lung disease (Table 2).