Rifampicin-resistance Pattern of Mycobacterium Tuberculosis and Associated Risk Factors Among Presumptive Pulmonary and Extra Pulmonary Tuberculosis Patients at Madda Walabu University Goba Referral Hospital, Southeast Ethiopia

Background: Drug resistant M.tuberculosis is one of the serious public health trouble that intimidating progress made in tuberculosis cases and control in several countries. Early detection of drug-resistant tuberculosis is crucial for patient management and infection control. Methods: Hospital based cross-sectional study design was conducted from October 2020 to February 2021. Detection of Mycobacterium tuberculosis and resistance to Rifampicin pattern was determined by using GeneXpert MTB/RIF assay. Data were entered and analyzed by SPSS version 23.0. Bivariate and multivariate analyses were used to examine the relationship between dependent and independent variables. Less than 0.05 P-value was used to show signicance. Results: A total of 301 presumptive tuberculosis patients were included in the study; of these, 46 (15.3%) of them were identied as having Mycobacterium tuberculosis, 2/46 (4.35%) were resistant to Rifampicin and 4/46 (14.8%) patients were TB/HIV co-infected. From the total of M. tuberculosis detected 40 (16.7%) were identied in pulmonary and 6 (9.8%) were in extra-pulmonary presumptive patients. Rifampicin-resistant M. tuberculosis was detected in 2 patients who had a history of taking Anti-tuberculosis drugs. Conclusion: Previous history of tuberculosis treatment and having close contact history with tuberculosis patients were found as important associated factors that enhance the prevalence of tuberculosis among presumptive tuberculosis patients. This indicates the mandate to make better and oversee the treatment protocol to control the burden of drug resistant M.tuberculosis.


Background
Tuberculosis (TB) is potentially fatal contagious bacterial disease caused by Mycobacterium tuberculosis (most common cause) that can affect any part of the body, but principally associated with lung diseases (Pulmonary Tuberculosis) and it can also affect other parts of the body, Extra Pulmonary Tuberculosis (EPTB) and remains as an important infectious disease and public health concern worldwide (1).
At the end of 2019, the world as a whole, most WHO regions and many high TB burden countries were not on track to reach the 2020 milestones of the End TB Strategy. Globally, an estimated 10.4 million people fell ill with TB in 2019, (10% among HIV co-infected individuals), 1.67 million deaths and 490,000 MDR-TB plus an additional 110,000 RR-TB cases was reported by WHO. Geographically, most people who developed TB in 2019 were from south-East Asia (44%), Africa accounts for (25%) and the Western Paci c shared (18%) (2).
Rifampicin resistant tuberculosis is caused by tubercle bacilli that display in vitro resistance to Rifampicin (3) and a good indicator of MDR-TB, because it discloses the existence of more than 90% Isoniazid and Rifampicin resistance, and regarded as an alternative method for detection of MDR-TB (4).
Drug-resistant tuberculosis arises due to improper and irrational use of anti-tubercular drugs (ATDs) in treatment of drug-susceptible TB patients, a genetic mutation of bacilli, an inadequate administered treatment regimen and weak services program that lead to delay detection and ineffective treatment of drug resistance and unequipped to support patients to keep adherence to treatment (5, 6).
Drug-resistant TB continues to be a public health threat Worldwide including our country Ethiopia. The challenge to the control of DR-TB starts with a delay in diagnosis and the low rate of detection of drug susceptible TB by microscopy. For better patient management and infection control in tuberculosis cases, early detection of drug resistance TB is the most crucial approach (7,8). Therefore, GeneXpert is an automated real-time polymerase chain-reaction assay designed for the rapid and simultaneous detection of MTB and rifampicin resistance. The present study was designed to assess the prevalence and associated risk factors for RIF resistant TB at Madda Walabu University Goba Referral Hospital, Southeast Ethiopia.

Methods
Study design, area and period A Hospital based cross-sectional study design was conducted from October 2020 to February 2021 at Madda Walabu University Goba Referral Hospital (MWU GRH). MWU GRH is located 446 km Southeast of Addis Ababa. The hospital has more than 120 beds offering different specialized services and serves more than 1.5 million peoples which come from catchment area and different district of the Bale zone.

Study population and sampling techniques
Presumptive tuberculosis patients who visited MWU GRH and who ful lled the inclusion criteria were involved in the study. By using the non-probability consecutive sampling technique, 301 presumptive tuberculosis patients who visited the hospital during the study period were involved in the study.

Variables
Prevalence of M. tuberculosis and Rifampicin-resistant M. tuberculosis were the dependent variables whereas demographic factors, HIV sero status and treatment related history were the independent variables.

Sample Size
The sample size was calculated by using a single proportion formula by considering the prevalence of 23.2 %, 95% level of con dence (α=0.5), with the tolerable error of 5% (d=0.05) and Assuming 10% nonresponse rate.

Specimen collection and processing
From each study participant presumptive of TB, 4 ml of clinical specimens (either Sputum, Gastric lavage, Lymph node aspirates, or pleural uid) were collected in a 50 ml falcon tube based on site of presumptive/infection. Clinical specimens were diluted and GeneXpert MTB/RIF assay was carried out as stated by the manufacturer's company. Accordingly, One ml of clinical sample was treated with 2 mL of sample reagent containing NaOH and isopropanol. Then it was shaked gently, homogenized and incubated for 15 minutes at room temperature. Two mL of the treated samples were transferred into the cartridge and then loaded into the GeneXpert instrument. Delayed samples were stored in the deep freezer -20° C until processed.
GeneXpert MTB/RIF Assay test principle A semi quantitative nested real-time PCR designed to amplify the 81-bp hot-spot region of the rpoβ gene within Rifampin-resistance determining region (RRDR) and probing the region subsequently for RIFresistance related mutations. The assay tests for MTB and mutations associated with Rifampicin resistance directly from clinical samples in less than 2 hours.

GeneXpert MTB/RIF Quality assurance
Both Sample processing control and Probe check control internal controls were used. Sample processing control ensures the sample was correctly processed and Probe check control veri es that the steps of the tests taken place correctly.

Data Quality Assurance
Data quality was ensured through the use of structured questionnaires and intensive supervision during data collection. The patient's mouth was rinsed twice with water before sputum was collected in a sterile container. The GeneXpert MTB/RIF cartridges were stored at 2-28 °C, the cartridge was used within 4 hours after opening the cartridge lid, and powder-free glove was used because powdered gloves interfere with molecular detection.

Data analysis and interpretation
Data were entered and analyzed by using SPSS version 23.0 software. Frequency and percentage was calculated to summarize the results. Bivariate and multivariate logistic regressions were performed to compute the effect of independent variables on the dependent variable. Those results with p-value < 0.05 at 95% con dence interval were regarded as statistically signi cant.

Rifampicin resistant M. tuberculosis
Of the 46 GeneXpert detected Mycobacterium tuberculosis cases, the overall prevalence of rifampicin resistant Mycobacterium tuberculosis was 2 (4.35%). The rifampicin resistant Mycobacterium tuberculosis cases were detected in pulmonary presumptive drug resistant tuberculosis patients and in patients who had a previously history of TB treatment (Table 1).

Risk factors among tuberculosis presumptive
Multivariate analysis showed that M. tuberculosis infection was signi cantly associated with participants aged between 15 and 39 years (AOR = 6.67; CI 0.54-2.0), previous history of TB treatment (AOR = 3.1, CI 0-1.29), Contact with TB patient (AOR = 3.38, CI 0.17-0.85) and frequent use of congested transportation (AOR = 1.1, CI 0.01-0.52) ( Table 2).   (11). However, result from our study was lower than studies done in India 32.9% (12), Egyptian 19% (13), Gambo 22.4% (14) and Debre Markos 23.2% (15). The possible reasons attributable for such inconsistency were variation in subset of targeted population, setting and locations where the data was collected, and framework of study design. In contrast, it is higher than studies conducted in other parts of Ethiopia like Ataye 8.98% (16) and Tigray 7.9% (17). This lower prevalence and discrepancy in estimation with the current study might be due to dissimilarities in nature of study subjects and geographical area.  (21) and Gambo 0.3% (14). This might be due to majority of TB suspected were new cases and differences in TB control and prevention program among countries. To the contrary, there was a lower reported prevalence of rifampicin-resistant M. tuberculosis from Eastern Uttar Pradesh 26.1% (12), Debre Markos 10.3% (15) and Gondar 15.8% (22). This lower prevalence and inconsistency could be due to difference in history of taking Anti-tuberculosis drugs, possibility of HIV/AIDS acquisition and TB, TB/HIV and MDR-TB prevention and control program.

Associated factors of tuberculosis infections among TB presumptive
The prevalence of M. tuberculosis was 17% in males and 13.2% in females but no statistically signi cant association was observed. This study is in agreement with WHO 2020 report (23), India (12), Nigeria (21), St. Peter Tuberculosis Specialized Hospital (24), and Felege Hiwot and Debre Tabor (11). This Variation could be due to difference in community cultural expectations on attempt to get healthcare services, having habit of smoking and alcoholism, high level exposure of males to surrounding.
The results of our study showed that the assessed TB/HIV co-infection rate was 8.7%, which is comparable with studies done at Ataye 7.89% (16), Addis Ababa 6.7% (9) and WHO 2020 report (8.2%) (23).However, high rate of TB/ HIV co-infection was reported from southwest Ethiopia 35.5% (25), Felege Hiwot and Debre Tabor 41.9 % (11), and Gondar 23.8 % (22). This inconsistency could be due to difference in the high burden of HIV setting area, Lack of awareness of TB/HIV co-morbidity among communities and the extent of performed HIV testing.
The chance of getting M. tuberculosis was 6.67 fold in patients between ages 15-39 when compared to those age > 60 years, the difference being statistically signi cant with p value 0.038. This signi cance difference may be due to the extent contact of productive ages to the surrounding for different activities and movability from one location to another may be results in chance of getting tuberculosis bacilli. Our result is in agreement with similar studies in Pakistan (26), Nigeria (21) and Debre Berhan (19), and Gambella (25).
The prevalence of M. tuberculosis among previously treated patients was higher 24.1% than treatment new patients 13.4%, with statistical signi cant difference (P < 0.050). Our nding agrees with studies done at Nepal (27), Hiwot Fana (10) and St. Peter Tuberculosis Specialized Hospital (24). High prevalence of rifampicin resistant M. tuberculosis was also detected among patients who had a history of previously taking anti-tuberculosis drugs (15.4 %) which is comparable with studies from Eastern Uttar Pradesh (12), Nepal 13.79% (27) and Addis Ababa 27.4% (9).This might be due to lack of success from previous tuberculosis treatment history and patients with previously history of taking anti-tuberculosis drugs were more likely to harbor drug-resistant bacilli because of previous exposure of bacilli to TB agents.
In this study, high prevalence of pulmonary tuberculosis 16.7 % was detected when compared with extrapulmonary tuberculosis which was 9.8%. This result agrees with a study done at Pakistan (26), Egyptian (13) and Hiwot Fana (10). This shows that pulmonary tuberculosis infection has in ated chance of transmission from pulmonary tuberculosis patients during coughing, sneezing and others way of bacilli transmission.
The result of this study also showed frequent utilization of congested transportation had a signi cant association (p < 0.05) with the prevalence of drug susceptible tuberculosis and drug resistance tuberculosis. The possible reason for such signi cance could be there is chance of acquiring bacilli from the index cases because of denied of opening window for different reasons and lack of ventilation during voyage. This study agrees with study done at Ataye (16).