Clinical, molecular and drug sensitivity pattern of mycobacterial isolates from extra-pulmonary tuberculosis cases in Addis Ababa, Ethiopia

In conjunction to HIV, tuberculosis remains a major global health problem. It causes ill-health among millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). According to the latest estimates, globally 8.6 million people develop TB and 1.3 million died from the disease in 2012. The South-East Asia and Western Pacific Regions collectively accounted for 58 % of the world’s TB cases in 2012 and the African region had approximately one quarter of the world’s cases [1].

In Ethiopia, the number of caseloads reported in all forms of TB in 2012 was 143 503 which makes it one of the largest reported cases in the world [1]. There are 22 high burden countries (HBCs), which accounts 81 % of the estimated number of new TB cases (all forms) arising worldwide each year. Of these Ethiopia ranks in the 7^th position after India, China, Indonesia, Pakistan and South Africa. According to WHO (2013) estimate; the prevalence, incidence and mortality in all forms of TB were 224, 247 and 18 per 100,000 respectively [1] Based on the national surveillance data in the year 2010/11 a total of 159,017 TB cases were notified and the proportion of new smear-positive, smear negative and Extra Pulmonary Tuberculosis (EPTB) among all new cases is 32.7 %, 34.8 %, and 32.5 % respectively.

With the advent of HIV/AIDS the incident rate of EPTB is also on the rise and becomes worldwide problem [2–5]. The disease is highly pronounced in countries where HIV infection and incident TB rate is higher. Accordingly, EPTB accounts for 15 to 20 % of all forms in people who do not have HIV but 53 to 62 % in HIV co-infected individuals [6,7]. The case notification of extra pulmonary tuberculosis is in Ethiopia is also higher and almost equal to that of smear positive and negative pulmonary tuberculosis cases with a percentage of 33 each [8].

The development of DNA finger printing (genotyping) of Mycobacterium tuberculosis isolates has provides a better insight in understanding of the transmission of tuberculosis [9]. Genotyping outcome of the isolates together with epidemiological data assists researchers to identify recent transmission and trace outbreaks. The techniques can be utilized as a tool for epidemiological studies to determine the overall diversity of Mycobacterium tuberculosis strain that can help to address important epidemiological questions such as the origin of an infection in patient’s household or community, and the spread. It also assists in an early detection of organisms with acquired drug resistance [9, 10]. Although a number of molecular techniques are available, spoligotyping is selected for a number of reasons. It is relatively simple, rapid and good for strains with low copy numbers of IS6110. Moreover it helps to identify and type strains simultaneous [11].

It has been indicated that strain variation associated with clinical manifestation of tuberculosis [12, 13] and explained that some strain possess superior ability than others and becomes highly virulent to causes extra thoracic tuberculosis and disseminated out of the lung [12, 14–16]. Moreover, recent studies reported the variation in the genetic diversity of tuberculosis causing pulmonary and extra pulmonary tuberculosis in different cities of Ethiopia [17–22]. However, data on extra pulmonary tuberculosis in Addis Ababa was not reported. Therefore, in this study we tried to evaluate the genetic diversity, clinical manifestation and drug sensitivity of mycobacterial isolates causing extra pulmonary tuberculosis is Addis Ababa city.

Before the beginning of the HIV epidemic, ∼85 % of reported tuberculosis cases were limited to the lungs [31]. Latter, this proportional distribution was considerably different among persons with HIV infection and then EPTB came out of the shadow and becoming a growing problem [29]. At present, Extra-pulmonary tuberculosis is on the increase globally including Ethiopia [32, 33]. The global increase is believed to be increased by HIV related immune incompetence. In addition to this global picture, the national report also shows that it covers almost one third of the total TB case reports [33].

In agreement to these observations, our result showed that among the total suspected cases, 62 (31 %) were HIV positive. We have also found that 21 (35 %) were HIV positive from culture confirmed EPTB cases. However, this figure is higher and accounts for 53 to 62 % in HIV co-infected individuals whereas only 15 to 20 % of all forms of TB in HIV negative patients [34, 35]. Different studies indicated that EPTB increased significantly with the pandemic of HIV and the co-infection rate is higher in developing countries where the prevalence is higher. It has been well established that EPTB is higher among HIV positives. Earlier reports of Abate et al. (2003) from Tikur Anbessa hospital in Addis Ababa showed that the proportion of newly diagnosed EPTB patients among HIV patients was more than those of smear positive and smear negative pulmonary TB [36] and other studies also showed that HIV is associated with Extra pulmonary tuberculosis [37, 38]. HIV, poor living condition and inadequate health care contribute to the high level of TB in developing countries (Fanning 1999; Sharma and Mohan, 2004).

The higher proportions of HIV within the age range 25–35 and the higher frequency of HIV and EPTB in female patients was also higher than in males. As suggested in other studies, this might be due to differential exposure of women to infectious TB patients, and medical care compared with men [6, 39].

EPTB has different manifestations based on the organ affected and the distribution varies in different studies. Mohammed et al., (2003) reported that lymph node tuberculosis constituted 40 % of all TB cases and 72.8 % of the clinical suspects in Southern Ethiopia [40]. In agreement to the above observations, in our study lymphadenitis, disseminated TB and Pleural TB were the three major types. As shown in Fig. 1, 65 % of the confirmed cases were lymph node tuberculosis including axillary, inguinal and cervical. This is also similar to the study reported from Germany [5]. In contrary, the most common sites involved were the bone/joints and lymph nodes in the United States [4], while the genitourinary system and skin were the common sites in a report from Hong Kong [41]. These differences may be attributable to either host or pathogen related factors as well as access to patient sampling in the clinical settings. Though, strong statistical association is not established, our study showed that there is dominance of certain strains (Fig. 5) and clusters of CAS found only lymphadenitis (Fig. 6).

Analysis of the diversity of Mycobacterial isolates and data base comparison is essential in determining the distribution of M. tuberculosis complex in different geographical setting and worldwide. It also gives us an insight to understand the dynamics of current transmission and dominant strains associated with the disease pattern. In line with this, we described the strain diversity of 59 M. tuberculosis isolates from extra pulmonary tuberculosis patients in Addis Ababa.

There is geographical sub structuring within the Euro-American lineage and most other areas were also associated with only one or two lineages. However, all six main lineages were represented in Africa [42]. In addition, there is an observation of Indo-Oceanic lineage, the most ancestral of the six lineages, which was associated with East Africa. In agreement to this justification, our isolates from EPTB cases were categorized in to five lineages with the absence of Mycobacterium africanum and M. caprae. Accordingly Euro American lineage (71.1 %), Indo Oceanic (5.1 %), East Asian (3.4 %) East Africa Indian (18.6 %) and M. bovis (1.6 %) (Fig. 4).

In this study, the distribution of strains showed that the T1 family is the dominant followed by CAS, T3 and Harlem with a percentage of 40.7, 16.2, 13.6 and 10.2 respectively. In line with this finding it was reported that the T family and other which were categorized under Euro-American and Indo-Oceanic were predominantly found in extra pulmonary cases [43] and also 42 % of isolates from tuberculous lymphadenitis were belonging to the Harlem genotype [44].

The data base comparison using SpolDB4 to assign into Shared International Number, spoligotyping pattern showed that the largest cluster, ST53 comprised 12 (20.3 %) isolates, followed by ST149 (10.1 %) and ST25 (8.47 %) with 6 and 5 isolates, respectively. ST910 and ST21 comprised of 3 (5.8 %) of isolates for each. The rest were unique (non-clustered) spoligotype patterns in 24 (40.7 %) of the isolates. In addition, all the spoligotypes not found in SpolDB4 data base (orphans), were also assigned to families and sub lineage using SPOTCLUST ID web based algorithm model SpolDB3 (http://tbinsight.cs.rpi.edu/run_spotclust.html). Accordingly most of the new strains obtained from extra-pulmonary cases were assigned to T1 family with a probability of 0.99. Based on these assignments, the T1 lineage was dominant and accounted for 21 (35 %). This might be due to clonal association and an active transmission of this family with in the society as it has been indicated on a number of research it is an indication of active transmission in the community [45–49].

Evidence suggests that there is strain-specific difference in virulence and immunogenicity. Moreover, strain variation might be attributed to the variation in clinical appearances and some strains are more virulent and possess greater ability to disseminate than others [50]. In this study, the isolates were categorized into different sub lineages. Accordingly, the T1, H, CAS, ETH2/U and T3 cover more than 60 % of the EPTB isolates; the T1 is the dominant, widely distributed and isolated from different parts of the body whereas CAS strains are clustered with close similarity and isolated from cervical and axillary samples only. Out of the total 59 isolates 10 were categorized under CAS1 family, of these 9 were found in lymph node tuberculosis. Showing there is dominance of CAS1 high in TBLN. In line with this, studies from Pakistan showed that these strains were also prevalent and accounted for 39 % of isolates form both pulmonary and extra pulmonary cases [51]. Similarly, Lari et al. (2009) reported that there is association between the CAS strains and extra-pulmonary TB [52] which might in part explained by the long established link of extra-pulmonary tuberculosis TB with South Asian of African origin patients that has been also reported earlier. On the other hand, two strain families, LAM3/F11 and W-Beijing, predominated in the South African study [53]. However, in our study we found only a single isolate of Beijing strains which is similar to studies conducted on the same area [54].

Earlier study conducted by Abate etal, (1998) on retreatment cases in Addis Ababa showed that drug resistance of pulmonary tuberculosis to one or more drugs was 50 % and that of MDR TB was 12 % for that period [36] Nevertheless, the overall primary drug resistance to pulmonary tuberculosis was 15.6 % [55] and 21 % [56] in studies conducted in Addis Ababa. According to the national survey (2003–2006), the percentage of MDR TB in Ethiopia is 1.6 and 11.8 of new and retreatment cases respectively. Similarly WHO reported that the % of new and retreatment cases with MDR-TB were 1.6 and 12 respectively [57].

Moreover studies revealed that the frequency of single drug resistance in Extra pulmonary tuberculosis was higher (13.3 %) and that of multiple drug resistance was 15.9 % [58] which is much higher than that reported by the World Health Organization (3.6 %) [59]. Our study showed that the proportion of resistance to rifampicin (21.6 %) was higher and single drug resistance was lower than the above study. However, another study conducted in the eastern part of Ethiopia reported that 9.5 % resistance against isoniazid and the rate of MDRTB was 1.1 % [60]. An additional study also reported that the proportion of resistance to Rifampicin (RMP) and isoniazid were 17 (2.8 %) and 15 (2.5 %) respectively [61]. However, the resistance in our study to other first line drugs were comparatively similar to these studies.

The study showed that the disseminated and lymphoid tuberculosis is the most frequent forms of extra pulmonary tuberculosis in Addis Ababa. Development of drug resistance in the study isolates was low except for rifampicin and multi drug resistance was not found in the study strain.

The Euro American and East African Indian Lineages are the dominant lineage and SIT 53 strains were also. It has also indicated that CAS appeared to be highly clustered in TBLN than in other forms. A moderate level of cluster formation suggested the presence of recent transmission of TB in the area. The dominance and distribution of the T lineage might be delay in diagnoses of EPTB cases, which will create a condition for dissemination or might suggest higher virulence of these strains as compared to others. Whereas, the localization of CAS strain within cervical areas needs further investigation.