3. Results
Overall, a total of 39,107 samples of Tuberculosis suspects were processed for upfront Xpert MTB/RIF testing between January 2022 and December 2022. Of 29,114(74.45%) pulmonary samples tested, 21.42 %( n=6,236) were positive for tuberculosis and 7.68 %( n=479) were rifampicin drug resistant (
Figure 1). Among 8,581(21.95%) extra-pulmonary samples processed for tuberculosis diagnosis using upfront Xpert MTB/RIF assay, 10.72 %( n=920) and 3.26 % (n=30) were resistant to the rifampicin drug. Thirty-four thousand eight hundred fourteen samples (92.36%) were referred from public sector facilities, and only 7.64% (n=2881) were referred from the private sector (
Figure 2). Among 37,695 Tuberculosis suspect samples tested, 18.98% (n = 7156) were positive for
M.tuberculosis, and 7.11 % (n = 509) were resistant to rifampicin drug using the Xpert MTB/RIF test. A total of 1,412 (3.6%) Xpert MTB/RIF cartridges were wasted in this complete study. A total of 32,825 patients with presumptive TB were enrolled for the study, out of which 15.01 %( n = 4927) were positive for
M.tuberculosis, and 4.22% (n = 208) were rifampicin drug resistant. Among 4870 Presumptive DR-TB (Pulmonary) patients, 45.77 %( n = 2229) were positive for
M.tuberculosis, and 13.50% (n = 301) were rifampicin drug-resistant (
Table 1).
Of 2,374 samples received from PL-HIV patients, 7.54% (n = 179) were positive for
M.tuberculosis, and 5.59% (n = 10) were rifampicin-resistant. Among 2,257 paediatric samples processed for diagnosis of TB, 2.22% (n = 50) were positive for
M.tuberculosis. 20.53% (n = 2,306) were positive for
M.tuberculosis among all smear-negative presumptive TB cases, either from previously treated patients or new suspects, and MDR suspect (n=11, 233) samples processed for diagnosis of tuberculosis and 4.90% (n =113) were resistant for rifampicin drug using Xpert MTB/RIF test. A total of 1,820 samples were received from other vulnerable () for diagnosis of tuberculosis using Xpert MTB/RIF assay, out of which 14.45% (n = 263) were positive for
M.tuberculosis and 3.80% (n =10) were rifampicin drug resistant. Out of 595 Contacts of TB & DR-TB patient samples tested, 16.97 %( n=101) were positive for
M.tuberculosis, and 23.76 %( n=24) were rifampicin drug-resistant. Among 7,639 EPTB (extra-pulmonary tuberculosis) samples tested, 9.78 %( n =747) were
M.tuberculosis, positive, and 3.48 %( n=26) were rifampicin resistant. Of 4,026 samples offered for the upfront molecular test, 14.88% (n=599) were
M.tuberculosis positive, and about 1% were rifampicin-resistant (
Table 1). Out of the total 3,846 presumptive adults (new) notified TB patients who have provided sputum samples for
M.tuberculosis diagnosis,40.90%(n=1,573) were
M.tuberculosis positive, and 17.04%(n=268) were rifampicin resistant. Of 462 notified pre-treated TB patient samples tested for
M.tuberculosis diagnosis, 4048 % (n=187) were positive for
M.tuberculosis, and 10.16 %( n=19) were rifampicin drug resistant. Among 562 non-responders (Drug sensitive tuberculosis-DSTB and isoniazid mono-resistant tuberculosis-H
rTB) tested for
M.tuberculosis diagnosis, 83.45% (n=469) were
M.tuberculosis positive and 2.99% were rifampicin resistant. Xpert sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using the concentrated smear microscopy (Fluorescence) method.
The sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for PTB were found to be 99.87 % (CI: 0.12-0.07), 99.92%(CI: 0.04-0.03), 99.71%(CI: 0.17-0.11), 99.97%(CI: 0.04-0.01), 21.38%(CI: 0.46-0.48) and 99.91%(0.04-0.03) respectively. For EPTB, the sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay counted for 99.45 % ( CI: 0.72- 0.37), 99.84% (CI: 0.11-0.08) 98.70% (CI: 0.97-0.55) 99.93% (CI: 0.09-0.04) 10.64% (CI: 0.65-0.67) and 99.80% (CI: 0.12-0.08), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Presumptive TB were found to be 99.82%(CI: 0.17-0.10), 99.91%(CI: 0.04-0.03), 99.51% (CI: 0.23-0.16), 99.97% (CI: 0.03-0.01), 14.96% (CI: 0.38-0.39) and 9.93% (CI: 0.04-0.02), respectively (
Table 2). For Presumptive DRTB (Pulmonary), the sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay counted for 99.82% (CI: 0.28-0.13), 99.77% (CI: 0.26-0.15), 99.73% (CI: 0.33-0.15), 99.85% (CI: 0.25-0.09), 45.73% (CI: 1.41-2.41) and 99.92% (CI: 0.10-0.05), respectively.
Among 32,825 presumptive TB enrolled for this study, the sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for PL-HIV out of presumptive TB (n=2374) were 99.44% (CI: 2.53-0.55), 99.91% (CI: 0.24-0.08), 98.88% (CI: 3.20-0.84), 99.95% (CI: 0.27-0.04), 7.50% (CI: 1.03-1.13) and 99.87% (CI: 0.24-0.10), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for Paediatric out of presumptive TB (n=2257) were found to be 97.96% (CI: 8.81-1.99), 99.91% (CI: 0.24-0.08), 96.00% (CI: 10.28-2.27), 99.95% (CI: 0.26-0.04), 2.17% (CI: 0.56-0.69) and 99.87% (CI: 0.26-0.10), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Smear Negative, X-ray suggestive TB (n=11233) were found to be 100% (CI: 0.16-0.0), 99.99% (CI: 0.05-0.01), 99.96% (CI: 0.27-0.03) 100% (CI: 0.04-0.00), 20.52% (CI: 0.74-0.76) and 99.99 % (CI: 0.04-0.01), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for Other Vulnerable group TB (n=1820) were found to be 99.62% (CI: 1.73-0.37), 99.87% (CI: 0.33-0.11), 99.24% (CI: 2.21-0.57), 99.94% (CI: 0.39-0.05), 14.40% (CI: 1.59-1.69) and 99.84% (CI: 0.32-0.13), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Contacts of TB & DRTB patients (n=595) were found to be 100% (CI: 3.69-0.00), 99.40% (CI: 1.15-0.48), 97.03% (CI: 5.67-1.99), 100% (CI: 0.74-0.00), 16.47% (CI: 2.89-3.23) and 99.50% (CI: 0.97-0.40), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for extra-pulmonary TB (n=7639) were found to be 99.60% (CI: 0.78-0.32), 99.90% (CI: 0.11-0.06), 99.06% (CI: 1.00-0.39), 99.96% (CI: 0.09-0.03), 9.73% (CI: 0.66-0.68), and 99.87% (CI: 0.11-0.07), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Upfront Molecular test offered(n=4026) were found to be 99.83% (CI: 0.76-0.17), 99.97% (CI: 0.13-0.03), 99.83% (CI: 1.00-0.15), 99.97% (CI: 0.18-0.03), 14.88% (CI: 1.09-1.54) and 99.95% (CI: 0.13-0.04), respectively.
Among 4,870 the Presumptive DR-TB (Pulmonary) enrolled for this study, the sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Notified TB patients (New)- UDST (n=3846)were 99.94% (CI: 0.34-0.06), 99.87% (CI: 0.25-0.10), 99.81% (CI: 0.40-0.13), 99.96% (CI: 0.27-0.03), 40.85% (CI: 1.56-1.57) and, 99.90% (CI: 0.17-0.07), respectively. The sensitivity, specificity, PPV, NPV, Diseases prevalence and accuracy of Xpert MTB/RIF assay for Notified TB patients (Previously treated) -UDST (n=462)were found to be 99.47% (CI: 2.41-0.52), 99.64% (CI: 1.65-0.35), 99.47% (CI: 3.13-0.45), 99.64% (CI: 2.15-0.31), 40.48% (CI: 4.51-4.63)and 99.57% (CI: 1.12-0.38), respectively (
Table 2). The sensitivity, specificity, PPV, NPV, Diseases prevalence, and accuracy of Xpert MTB/RIF assay for Non-responders (n=562) were found to be 99.57% (CI: 1.10-0.38), 97.85% (CI: 5.40-1.89), 99.57% (CI: 1.23-0.32), 97.85% (CI: 5.91-1.60), 83.45% (CI: 3.33-2.98) and, 99.29% (CI: 1.10-0.62), respectively.
The Xpert assay test provides semi-quantitative
M. tuberculosis detection based on the probe’s Cycle Threshold (Ct)-the number of PCR cycles required for amplifying MTB DNA to detectable levels. Xpert assay cycle threshold values (CT) is a semi-quantitative measure of bacillary burden in the specimen. Out of 509 RR-TB detected results, 36(7.07%) are reported as “High” (Ct<16), 98(19.25%), 163(32.02%), 212(41.65%) are reported as “Medium” (Ct16-22),” Low” (Ct 22-28) and “Very Low” (Ct >28) respectively (Figure 3). Delta Ct (ΔCt) max was calculated as the difference between the earliest and latest Ct across the five molecular beacon (A-E) probes (
Table 3). Out of 509 rifampicin-resistant, 243(47.74%) were reported as “dropout” (no hybridization), and 266(52.25%) cases were reported as “delayed” (ΔCt >4), as represented in
Table 3. The most common probes for RIF resistance detection were E (n=68, 13.36%), D (n=57, 11.20%), and B (n=55, 10.81%). The probe with the most delayed binding Ct value was categorized as ΔCt 4.1-4.9(49, 20.16%) and ΔCt >5(194, 79.84%).
Among 7,156 (18.33%)
M.tuberculosis positive cases, 7.11 % (n=509) were rifampicin-resistant, and 92.89 % (n=6647) were rifampicin-sensitive tuberculosis (
Table 1). Of 157(30.84%) RR tested for MDBDRsl assay, 16.56 % (n=26) were pre-XDR, and 0.64 % (n=1) were XDR tuberculosis. 69.16 % (n=352) were not tested for MDBDRsl assay due to the non-availability of samples. Of 6,647(92.89%) RS-TB cases, 92.1% (n=6,122) were tested for the MDBDRplus assay, and 525(7.9%) were not tested for the MDBDRplus assay due to non-availability of samples. Among 6,122 RS TB were tested for MDBDRplus assay, 8.13% (n=498) were HR-TB and 91.87% (n=5624) were HS tuberculosis. Of 498(8.13%) HR tested for MDBDRsl assay, 4.02% (n=20) were pre-XDR tuberculosis (
Figure 1). Of 47 fluoroquinolone-resistant, 16.56% (26/157) of Multidrug-resistant tuberculosis isolates and 4.02% (20/498) isoniazid-resistant are fluoroquinolone-resistant, a characteristic distribution leading to about 17.2% of fluoroquinolone resistance events and relevant marker
gyr-A mutations in MDR tuberculosis isolates (
Table 4).
4. Discussion
Tuberculosis is the most dreadful infectious disease in the world, with high morbidity and mortality among people. Thus, early detection is of utmost importance for reducing deaths and transmission. The lack of rapid and accurate diagnostic tests hampers global TB control. Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA, USA) is the semi-quantitative real-time polymerase chain reaction (PCR) to rapidly detect the M.tuberculosis complex and rifampicin resistance by amplifying a DNA fragment containing the 81bp hotspot region of the rpoB gene (codons 507–533) that is then hybridized to five molecular beacon probes.
Overall, this study's MTB and Rifampicin Resistant TB frequency was 18.33% and 7.11%, respectively. The incidence rate of
M. tuberculosis (18.33%) in this study was higher than the previous study reported 6.5% [
14] and 7.9% [
15] in Ethiopia, 13.8% [
16] in Nepal, and 12% [
17] in India. The incidence rate of
M. tuberculosis infection among total suspects (n=39107) tested for Xpert MTB/RIF was 18.33% (7156/39107), which is lower than the previous study reported 38.77% [
18] in China, 22.65 % [
19], 22.9%[
20] in Nigeria and 23.82% [
21] in India. The 3.26% positivity among the 8581 Extra-pulmonary samples tested for tuberculosis using Xpert MTB/RIF assay in this study is lower than the previous study (13%) conducted by Anwar et.al. [
22].
Elbrolosy et al. [
23] reported in their study that the sensitivity specificity of Xpert MTB/RIF assay for PTB was found to be 90.2 % and 86.9 %, respectively, while for EPTB, the sensitivity specificity of Xpert MTB/RIF assay counted for 81.6 % and 78.9 %, respectively. Mulengwa et al. [
24]reported sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MTB/RIF test was 91.6%, 95.3%,83%, and 97.80%, respectively. But in this study, the sensitivity, specificity, PPV, NPV, Disease prevalence, and accuracy of Xpert MTB/RIF assay for PTB were found to be 99.87 %,99.92%, 99.71%, 99.97%, 21.38%, and 99.91% respectively and for EPTB, the sensitivity, specificity, PPV, NPV, Disease prevalence, and accuracy of Xpert MTB/RIF assay counted for 99.45 %, 99.84%, 98.70%, 99.93%, 10.64%, and 99.80%, respectively compared to concentrated smear (Fluorescence Microscopy) method. Raina et al. [
25]reported that the sensitivity, specificity, PPV, and NPV of Xpert MTB/RIF were 100%, 99.5%, 97.5%, and 100%, respectively, compared to the gold standard culture method.
Of 2,374 PLHIV samples enrolled for this study, the sensitivity, specificity, PPV, NPV, Disease prevalence, and accuracy of Xpert MTB/RIF assay for PLHIV out of presumptive TB were 99.44%, 99.91%, 98.88%, 99.95%, 7.50%, and 99.87%, respectively. In countries with low endemicity rates, the sensitivity and the specificity of Xpert MTB/RIF vary between 82, 95, and 98% for sensitivity and 96 and 99% for specificity. However, in countries with high endemicity, these rates vary between 80 and 88% for sensitivity and between 95 and 98% for specificity [
26,
27,
28].In another study, Faria et al. [
29] reported Xpert MTB/RIF assay’s sensitivity ranging from 68% to 100%. Specificity ranged from 91.7% to 100%, the positive predictive value from 79.2% to 96.1%, and the negative predictive value from 84.6% to 99.3%.
In their study, Cox et al. [
30] reported 76% sensitivity and 98% specificity for 5,717 smear-negative samples processed for Xpert MTB/RIF. In our study, the sensitivity of Xpert MTB/RIF was also high 100% among smear-negative, and specificity was 99.99% with 99.99% accuracy. Rimal et al. [
31] reported the sensitivity, specificity, positive predictive value, and negative predictive values of Xpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. In this study, 11,223 sputum samples were collected and processed by microscopy followed by Xpert MTB/RIF assay. The sensitivity, specificity, PPV, NPV, Disease prevalence, and accuracy of Xpert MTB/RIF assay for Smear Negative, X-ray suggestive TB (n=11,233) were found to be 100%, 99.99%, 99.96%,100%, 20.52%, and 99.99 %, respectively.
Hebte et al. [
32] reported 89.1% (n=106) positivity and 4.2% (n=5) rifampicin-resistant TB out of 119 index TB cases enrolled for Xpert assay. The positivity rate among the 494 contacts of TB and drug-resistant tuberculosis patients registered for this study was 16.97%. Its sensitivity, specificity, positive predictive value, negative predictive value, Disease prevalence, and Xpert MTB/RIF assay accuracy were 100%, 99.40%, 97.03%, 100.00%, 16.47%, and 99.50, respectively. Our study reported lower positivity than that reported by Hebte et al. and higher than that reported by Gebretsadik et al.[
33]. He said 8.98% (n=38) positivity and 5.3% (n=3) rifampicin-resistant TB out of 423 index TB cases enrolled for Xpert assay. In their recent study, Gurung et al. [
34] reported 4.5 % (n=770) positivity out of 17114 index TB cases registered for Xpert assay. Kalra et al. [
35] reported 6.6% positivity (n=6270) and 8.7% (n=545) rifampicin-resistant, of the total 94,415 presumptive pediatric TB cases diagnosed on Xpert MTB/RIF assay. However, the positivity rate among the 2257 pediatric cases enrolled for this study was 2.22%. Its sensitivity, specificity, positive predictive value, negative predictive value, Disease prevalence, and Xpert MTB/RIF assay accuracy were 97.96%, 99.91%, 96.00%, 99.95%, 2.17%, and 99.87%, respectively.
In a previous study, Ibrahim et al. [
19] reported the incidence of
M.tuberculosis and rifampicin resistance was 22.68% and 4.50%, respectively, out of 2451 samples tested on Xpert assay. Of total 990 presumptive tuberculosis tested [
16], the estimated prevalence of
M.tuberculosis in presumptive TB patients was 13.8% (95%CI: 11.88%–16.16%), and the estimated prevalence of rifampicin resistant in
M.tuberculosis confirmed patients was 10.2% (4.97%–15.1%). Of 132 notified new TB cases enrolled in a previous study [
36], the positivity on Xpert MTB/RIF assay was 78.79%. The sensitivity, specificity, PPV, and NPV of GXP in diagnosing and detecting rifampicin resistance in pulmonary TB were 95%, 93%, 98%, 84% and 96%, 100%, 100%, and 96% respectively. The positivity rate among the 3846 notified new TB cases enrolled for this study was 40.90%. Its sensitivity, specificity, positive predictive value, negative predictive value, Disease prevalence, and accuracy of Xpert MTB/RIF assay were 99.94%, 99.87%, 99.81%, 99.96%, 40.85%, and 99.90%, respectively. Our study reported a higher (40.90%) incidence of
M.tuberculosis and rifampicin-resistant (17.04%) rate than the previous studies mentioned here.
In a recent study, Worku et al.[
37] reported an 11.9% incidence of
M.tuberculosis positivity rate and 2.5% rifampicin resistance out of 1828 smear-negative and re-treatment cases tested on Xpert MTB/RIF assay. Of 462 notified previously treated patients enrolled for this study, the TB positivity rate was 40.98%. Its sensitivity, specificity, positive predictive value, and negative predictive value, Disease prevalence, and accuracy of Xpert MTB/RIF assay were 99.47%, 99.64%, 99.47%, 99.64%, 40.48%, and 99.57%, respectively. Farra et al. [
38,
39] reported 79.1% (488/617)
M.tuberculosis positivity rate and 42.2% rifampicin resistant in confirmed
M.tuberculosis (206/488) in their study out of 617 samples (55.8% relapse; 31.6% failure and 10.2% defaulter) tested using Xpert MTB/RIF assay. Of 562 non-responders (DS TB and INH resistant TB) patients enrolled for this study, the tuberculosis positivity and rifampicin resistance detection rate was 83.45% and 2.99%, respectively and its sensitivity, specificity, positive predictive value, and negative predictive value, Diseases prevalence and accuracy of Xpert MTB/RIF assay was 99.57%, 97.85%, 99.57%, 97.85%, 83.45%, and 99.29%, respectively.
In this study, 16.56% (26/157) of Multidrug-resistant tuberculosis isolates and 4.02% (20/498) isoniazid-resistant are fluoroquinolone-resistant, a characteristic distribution leading to about 17.2% of fluoroquinolone resistance events and relevant marker
gyr-A mutations in MDR tuberculosis isolates. Dreyer et al. [
40] reported 69.2% (703/1016) fluoroquinolone resistance among 1016 Multidrug-resistant tuberculosis isolates tested for resistance. In India, 36% of the Multidrug-resistant tuberculosis isolates are browned to have additional resistance to fluoroquinolone [
41,
42], and about 3% of MDR-TB isolates are estimated to have extensively drug-resistant (XDR-TB). Sharma et al. [
43] reported 3.2% (35/1099) fluoroquinolone resistance among first-line drug-sensitive tuberculosis cases. Furthermore, the increase of fluoroquinolone resistance among isoniazid-resistant tuberculosis suggests that the active adoption of antibiotic stewardship in the community is urgently required.
About 3.6% (n=1412) of samples were not processed due to challenges in getting resamples. Of 1412 samples, 65,301,138,908 were rifampicin indeterminate, invalid, no result, and errors, respectively. The acceptable rate of rifampicin indeterminate, invalid, no result, and error calculations need to be defined. This study reported that 69.16% (352/509) of rifampicin-resistant patient’s samples were not processed to know further the drug susceptibility pattern of fluoroquinolones and second-line tuberculosis drugs. The control of tuberculosis, once unchecked, is extremely difficult to contain and manage, requiring a multidisciplinary, coordinated set of activities. The cornerstones of classic tuberculosis control approaches include:
Early diagnosis.
Novel case-finding methods beyond healthcare facilities.
Shorter and simpler successful treatment regimens for drug-sensitive and drug-resistant tuberculosis.
A greater focus on prevention strategies.
Steps to reduce mortality and transmission in adults and children.
The status quo for many rifampicin-resistant patients is a severe systemic illness characterized by significant lung damage and high mycobacterial burden. Early identification and screening of rifampicin resistance may facilitate better treatment outcomes and less transmission. Similarly, earlier diagnosis would reduce cumulative immunopathological and structural lung damage (morbidity) and potentially reduce mortality. The molecular characteristics of the disease burden and resistant pattern using Xpert MTB/RIF assay, together with Geographic Information System (GIS) mapping of the location where the specimen was received and tested, could be used as a crude epidemiological tool to identify hot spots of tuberculosis transmission and changes in patterns of circulating rifampicin resistance strains.