Socio-demographic characteristics, GenoTypic and phenotypic drug sensitivity test
The greater proportion of male patients 55.5% (213/384) to females might be due to biological, social and economic activities with many people which is in comparable proportion to 56.2% [17]. The disease was also more common within an active age group of the society which could be due to their vigorous movement from place to place for economic purpose that leads to greater risk of exposure. The lower proportion of LJ-culture positivity (29.2%) in this study might be due to an electric interruption at the health institutes where the collected sputum samples were temporarily stored in the refrigerator which affect the survival of bacteria. In addition, the bacteria were not cultured on the spot due to lack of active regional TB laboratory around the study area. Such inconvenient circumstances affect the survival of Mycobacterium in the collected sputum samples which is comparable to 32.2% of culture positivity result in northwest Ethiopia [18]. On the contrary, many studies showed greater proportion of culture positivity from smear positive samples than this growth rate [19, 20, 21].
The additional detection of drug resistant isolate using MTBDRplus assay and BACTEC MGIT 960 test was due to single detection of resistance by the Xpert (only RIF resistant cases). The GenoTypic LPA can detect an additional INH resistance where as the phenotypic MGIT can detect even a more additional drugs such as STM and EMB to RIF and INH. This could enhance the chance of detecting more resistant isolates. The lower proportion of MGIT sub-culture positivity detection 61.6% (69/112) for DST test from LJ-culture positive Mycobacterium sample than the heat killed LPA isolates 84.8% (95/112) might be due to the existence of bacterial DNA in the later one. In the case of freezed live Mycobacterium, its survival could be interrupted through time until it was sub-cultured using the MGIT instrument. The higher sensitivity result 94.2% of LPA and its lower specificity value 30.2% is comparable with similar settings done in Kenya where its sensitivity and specificity was 99.2 and 26.9%, respectively [22]. This showed that LPA has a good performance in detecting the true positivity of Mycobacterium and in a fair agreement (Kappa = 0.276; P < 0.001) with the BACTEC MGIT 960 performance.
The overall genotypic drug sensitivity test in this study detected 16.8% (16/95) as drug resistant to either of RIF and/or INH. This is higher than 7.7% (10/130) of similar studies done by using LPA in Arsi South central Ethiopia [23], 8.7% (14/161) and 13.3% (37/279) reported from central Ethiopia [21]. In contrast, our finding is in less proportion than a study report from northwest Ethiopia [19] and southwest Ethiopia [17] having a rate of 18.0% (20/111) and 39.3% (44/112), respectively. Studies from Chad [24] and Punjab state of India [25] also reported greater proportion of resistance as 23.4% (73/312) and 58.4% (163/279), respectively. The possible explanation for such variations of drug resistant proportion could be due to difference in sample size, study sites and subjects where the samples were collected.
The rate of MDR in this study was 8.4% (8/95) by genotypic MTBDRplus assay which is greater than 1.1% (3/279)[21], 1.8% (2/111) [19], 3.1% (5/161) [26] and 6.7% (11/165 [27] similar studies. On the contrary, our MDR finding is less than, 17.5% (33/189) [28], 25.8% (72/279)[25] and 27.7% (31/112)[17]. Regarding the detection of RIF 6.3% (6/95) and INH 2.1% (2/95) mono-resistance, the current prevalence in line with a number of studies [18, 29, 30] that rifampicin mono-resistant is greater than isoniazid mono-resistant with varying degrees of proportion. Such RIF resistance is an important implication for higher risk of multi-drug resistance as it is a surrogate marker of MDR [28]. Nevertheless, there are also studies [19, 21] that show isoniazid mono-resistance as the leading mono-resistant than RIF.
The 15.9% (11/69) drug resistant detection rate using BACTEC MGIT 960 in this study was at greater proportion than similar studies [31, 32]. On the contrary, less proportion of drug resistances were reported in this study than other research reports by using the same detection system [20, 33] which might be due to different study areas and sample size. Most of the isolates detected as resistant by LPA were also resistant by MGIT. In 2 of the samples which were detected as resistant by MGIT but not by LPA could be due to its extra drug sensitivity detection test. All the MDR resistant samples by MGIT in this study were not restricted to isoniazid and rifampicin, but had an additional resistance to EMB, STM or both. This implied sensitivity detection by MGIT posses an extra benefit to detect other first line TB drugs.
The high sensitivity of the MTBDRplus assay (100%) in detecting RIF resistance in this study is the same with similar study settings in Southern [34] and northwestern [35] Ethiopia, but with little variation to the isolates from central Ethiopia [21]. The specificity of the assay (98.3%) in detecting RIF resistance is comparable to 99.2% [34], 99.8% [21] and 100% [35] in detecting RIF resistant isolates. There is a great variation of MTBDRplus assay sensitivity to INH in our study 75% and a study from the Southern Ethiopia 33.3% [34]. The sensitivity of the assay to INH was 82.7 in a study of isolates from central [21] and 91.7% in isolates collected from northwestern part of the country [35]. The susceptibility of MTBDRplus to INH in this finding 98.2% corresponds to similar reports (97.2–100%) in the country. Although the assay's sensitivity result to detect MDR have some discrepancy, the susceptibility result accounts 100% which coincides exactly with other studies reported from the country [21, 34, 35].
The study also found that there was an excellent agreement between BD BACTEC MGIT 960 and MTBDRplus assay in detecting RIF and MDR with a Kappa value of 0.925 and 0.901, respectively. However, it was found as a good agreement (Kappa value, 0.774) between the two testing methods in detecting INH. Similarly, it was also reported as an excellent agreement in detecting MDR (Kappa value, 1) but with good (0.663) and moderate (0.494) agreement in detecting RIF and INH resistance, respectively [34]. Such difference between the two findings might be due to the difference in number of drug resistant isolates. Further, the agreement between Culture MGIT and LPA in detecting katG, inhA and both (katG and inhA) resistance was almost perfect and agrees with a study from Kenya [22].
Frequency of gene mutations associated with rifampicin and isoniazid resistant isolates by GenoType MTBDRplus assay
The wild-type rpoB probe hybridization band pattern showed an omission of the bands at WT3, WT4, WT6, WT7 and WT8 probes. Of these, the miss of WT8 probe predominates and accounted for 71.4% (10/14) among the RIF resistant isolates in a small region of amino acids located between position numbers 530–533 of the rpoB gene. Omission of all the rpoB gene probes was without any gain of probes in the MUT region and depicted as "unknown" mutation. Such lack of binding of a WT probe without simultaneous binding of a mutant probe is likely caused by the presence of a resistant mutation. Similarly, missing of probes without any gain of the corresponding MUT was reported from northwestern Ethiopia [19]. A report from central Ethiopia also in line with our finding that no gain of any mutant band was identified for the non-hybridized WT8 probe [21]. Similar findings were also reported for the absence of MUT band from other countries [36, 37]. Despite such reports, there were research findings where gain of MUT probes was identified in a part of the rpoB gene [17].
Greater frequency of resistance 8/16 (50%) to INH occurred due to mutation of the katG gene, whereas lower frequency of resistance 5/16 (31.3%) was caused by the mutations in the promoter region of the inhA gene. Greater frequencies of the katG gene WT omission at codon 315 was also reported from other studies in Ethiopia that agrees with this finding [17, 19, 21]. A miss of WT probe at Codon 315 in the katG gene without the presence of specific MUT band accounted 75% (6/8) in this study. The remaining 25% of the strains had mutations in the katG gene at codon 315 with amino acid change of S315T1 (AGC→ACC). The inhA promoter region showed a miss of -15/-16 codon that was detected in 5/16 (31.3%); and 1/16 (6.3%) at -8 gene region without conferring any addition of specific mutational band in both cases. This mutation also agrees with the finding from central Ethiopia and also considered as 'unknown' mutation [21].
Genetic diversity of the drug resistant Mycobacterium tuberculosis
The higher proportion of Euro-American lineages among drug resistance strains could be due to its higher prevalence 63.4% (59/93) under the interpretable spoligotyping results of the LPA done isolates in this study. This in line with findings that reported greater proportion of the same most frequently reported major lineage from other parts of Ethiopia [19, 38]. The clustered strains of the study also showed higher frequency and a statistically significant association with any anti-TB drug resistance than the unique ones revealing an implication for the highest risk of drug resistance among recent TB transmission.
Tuberculosis patients awareness towards the disease
Majority of the TB patients 70.4% (243/345) heard about the disease where as lower proportion didn't hear until they went to the health institutes for treatment. This is in less proportion as compared to 99.6% (245/264) of a high school students study in southern Ethiopia [39] which implies students have a great opportunity to hear about the disease either through their education at school or through different media. Small proportion of the TB patients 21.3% (72/338) under study heard about drug resistant bacteria which might be due to lack of awareness either through healthcare workers or other different sources of information. This is likely supported by low proportion (36.4%) of university students that knew about drug resistant M. tb [40]. The implication of such low proportion toward drug resistance bacteria is that a lot of work is required in creating awareness to minimize the alarming drug resistant TB against the "End TB strategy" of WHO.
The etiologic agent of TB was known by 57.3% (122/213) of the patients. This is in less proportion to 81.7% (201/246) of the high school students in southern Ethiopia. On the contrary, there were low proportions of study participants to determine the correct etiologic agent as bacteria/germ among prisoners in northern [41], community participants in central [42] and East Shao Zone of Ethiopia [43] with a proportion of 37.7%, 34% and 31%, respectively. The final year university students in Iran [44], community studies in Malaysia [45], TB patients interviewed in South Sudan [46] and patients at primary health care in South Africa [47] detect the right etiologic agent of TB with greater percentage than our finding as 92.9% (130), 88.2% (90), 80.4% and 60.2% (305); respectively. Nevertheless, there is low proportion of community participants 10.6% (215) that knew the causative agent of TB in India [48] and 11.5% among population based study in Lesotho [49]. Such variations showed educated participants have more awareness to tell the exact causative agent of TB. The contagious nature of the disease and its curability was well known by majority of the TB patient participants. This also in line with the highest proportion of other relevant studies [39, 46, 47, 49].
Greater than half 61.1% (207/339) of the study participants in our study check for TB when they have prolonged cough (> 2 weeks). This is in less proportion to 66.7% (68) of a study in Malaysia [45] and 76.9% (1563) of a study among respondents in India [48] which might be due to lack of awareness. Some of the TB patients’ understudy 10.6% (34/320) reported that they had a repeated history for TB which needs great consideration for drug resistance. Majority of the TB cases in this study 60.4% (204/338) were not aware of the likelihood of being infected by drug resistant strains which is comparable to 63.7% among study prisoners in North Ethiopia [41]. In addition, most of the TB patients 79.1% (269/340) didn't know the required treatment duration for drug resistant TB contrary to 98% (246) of the drug resistant patients who knew the correct treatment duration. Such differences could be due to awareness disparity between drug resistant TB patients and others [50]. The KAP assessment also revealed that majority of the respondents 72.0% replied TB is not specific and anybody can be infected by the disease. In fact, about 8.2% of the TB patients under study didn't know who are susceptible to the disease which might be due to limited knowledge to susceptibility of the disease.