Contribution to the Study of Resistance to Anti-Tuberculosis Drugs in the Mycobacterium tuberculosis Complex Isolated at the National Laboratory of Clinical Biology and Public Health in Bangui in the Central African Republic in 2022: Case of Rifampicin ()
1. Introduction
Multidrug-resistant tuberculosis is defined as an infection caused by a mycobacterium of the Mycobacterium tuberculosis complex, resistant to at least isoniazid and rifampicin, the two standard anti-tuberculosis drugs. Resistance to anti-tuberculosis drugs is a public health problem worldwide [1] . The infection is mainly transmitted through the air from a patient suffering from pulmonary tuberculosis. Mycobacterium tuberculosis is transmitted between humans. The bacilli are excreted by coughing and sneezing. The often small particles (<5 µM) can remain in suspension for several hours and can be carried over long distances by air flow [2] . These are small droplets of bronchial secretion which can remain suspended in the air and which are likely to be inhaled by contact subjects. The symptoms of tuberculosis are observed by cough, hemoptysis, weight loss and fever [3] . The unreasoned use of anti-tuberculosis drugs leads to the emergence of resistant strains of Mycobacterium tuberculosis [2] . The Central African Republic is one of the countries in the world most affected by tuberculosis with an incidence of 423 cases per 100,000 inhabitants. However, a recent hospital study by Tekpa et al. reports a hospital prevalence of 10.99% in 2019 [4] . In 2015, the Pasteur Institute in Bangui (C.A.R) carried out a study on the monitoring of rifampicin resistance in strains of Mycobacterium tuberculosis using the GeneXpert MTB/RIF technique and published a first result after three years of activities [5] . Despite the efforts of the National Tuberculosis Control Program (PNLT), the fight against this pandemic remains insufficient in the country. The successive military-political crises have brought the health system in CAR into decline; it is not able to provide an adequate diagnosis and sufficient care for the population; which has the corollary of late detection and insufficient monitoring of patients delaying their initiation of treatment.
Thus, faced with the problem of resistance to anti-tuberculosis drugs in strains of Mycobacterium tuberculosis in the world and in particular the Central African Republic, it was very important to also carry out this study at the National Laboratory which now has this platform (GeneXpert).
1.1. Main Objective
Contribute to the study of resistance to anti-tuberculosis drugs in strains of Mycobacterium tuberculosis isolated at the National Laboratory of Clinical Biology and Public Health in Bangui in the Central African Republic: Case of Rifampicin.
1.2. Specific Objectives
➢ Identify Mycobacterium tuberculosis;
➢ Determine the resistance profile to Rifampicin;
➢ Identify the variables associated with resistance to Rifampicin.
2. Patients and Methods
This study was carried out at the National Laboratory of Clinical Biology and Public Health in Bangui in the Central African Republic. This is a six (6) month cross-sectional study running from February to July 2022. The study sample consisted of 755 patients with microscopy-positive pulmonary tuberculosis (TPM+) with 80 cases of resistance. The microscopic examination (Bacilloscopy) is based on the analysis of sputum in one day using Ziehl Neelsen staining. The coloring is done in three stages: Coloring with Carbol Fushine, decolorization with a sulfuric acid-alcohol mixture and counter-coloring with Methylene Blue. The stained slides are then dried. The colored slides are read under a microscope with a 100× objective. The bacilli are colored red by Fushine on a blue background. The test is negative if there is no AFB on 300 fields. The test is rare positive AFB if there are 1 to 10 per 100 fields, positive 1+ if there are 10 to 99 AFB per 100 fields, positive 2+ if there are 1 to 10 AFB per field and positive 3+ if there are more than 10 AFB per field. Sputum microscopic examination techniques have variable specificity depending on the quality of the microscopist and the quantity (> or = 10 mL) of sputum to be analyzed.
The GeneXpert molecular method was applied to detect Mycobacterium tuberculosis and also rifampicin resistance. 2 ml of sputum for 4 ml of Reagent sample were homogenized and left for 2 to 5 minutes then homogenized again and left for 30 minutes. 4 ml of the solution was introduced into the MTB/RIF cartridge and the result comes out in less than two hours.
Quality Checks Were Carried out as Follows
For the microscopic examination after Ziehl Nielsen staining, microscopists regularly trained in the recognition of AFB were subjected daily to the test of a colored slide and an unstained slide which served as an internal control.
For analysis by GeneXpert, 5 positive fluorescences (taking into account the set Cycle Threshold value) should be obtained to validate a positive sample while a positive sample should when the positive external controls were amplified.
The results recorded in a register should corroborate those returned to patients via the computerized system protected by an access code.
Study participants gave their consent and their anonymity was guaranteed by signing the consent form. Sociodemographic data were thus collected and collected while respecting patient confidentiality.
3. Results
3.1. Sociodemographic Characteristics of Patients
3.1.1. Distribution of Patients by Age
In total, we analyzed data from 755 patients with microscopy-positive pulmonary tuberculosis with 80 cases of resistance. The patients were aged 4 to 72 years with a mean age of 32 ± 7. The age group from 20 to 39 was predominant (45.82%). Figure 1 shows the distribution of patients by age.
3.1.2. Distribution of Patients by Sex
Among the 755 patients screened for positive TPM, the male gender was more represented (65.16%) as shown in Figure 2.
3.2. Prevalence of Resistance to Anti-Tuberculosis Drugs
3.2.1. Overall Prevalence of Resistance
Among the 755 patients registered, 80 tested positive by GeneXpert ® for resistance to first-line anti-tuberculosis drugs. The overall prevalence of resistance was 10.60% (Figure 3).
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Figure 1. Distribution of patients according to age group.
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Figure 2. Distribution of patients by sex.
3.2.2. Type of Resistance to Anti-Tuberculosis Drugs
Primary resistance to rifampicin accounted for 73.75% and secondary resistance was 26.25%. Figure 4 shows the distribution of TPM+ resistance according to the type of resistance.
3.2.3. Distribution of Resistance Cases According to Patient Characteristics
1) Distribution of cases of resistance according to type and age
According to the multivariate analysis, age was not associated with the type of resistance (p-value > 0.05). Table 1 presents the distribution of patients who developed resistance according to age group.
2) Distribution of resistance cases according to type and sex
The male gender was predominant among patients with TPM+ resistance 72.50%. The occurrence of resistance was not associated with sex (p = 0.89) as shown in Table 2.
3) Distribution of cases of resistance according to type and marital status
Primary resistance and secondary resistance predominate in these patients who live as a couple as shown in Table 3.
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Figure 3. Prevalence of resistance to anti-tuberculosis drugs.
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Figure 4. Distribution of rifampicin resistance by type.
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Table 1. Distribution of cases of resistance according to type and age group.
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Table 2. Distribution of resistance cases according to type and sex.
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Table 3. Distribution of resistant cases according to type and marital status.
4) Medical and clinical history of patients
Among the cases of resistance to anti-tuberculosis drugs, 11 patients had experienced treatment failure due to anti-tuberculosis drugs (13.75%) and 11 cases of treatment relapse (13.75%). The notion of contagion was found among patients (28.75%). A history of tobacco and alcohol was found respectively in 40% and 61.25% of cases in patients with resistant TPM+. Primary resistance was more common in patients who did not experience treatment failure and in those who did not experience a relapse. Treatment failure and relapse were the variables associated with the occurrence of resistant TPM+ (p < 0.05). The risk of developing resistant TB was 21.37 times higher in patients who experienced a relapse (OR = 21.37 [4.07 - 31.71]). Table 4 presents the distribution of patients’ medical and clinical histories.
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Table 4. Distribution of history of patients with resistant TPM+.
NA = not applicable (p-value > 5%).
4. Discussion
The study carried out at the National Laboratory of Clinical Biology and Public Health of Bangui aimed to evaluate the resistance to anti-tuberculosis drugs of the Mycobacterium tuberculosis complex in patients with microscopy-positive pulmonary tuberculosis from February to July 2022. The reliability of these results was based on the quality of the set of methodologies used. This exclusivity made it possible to have as a target population both male and female patients coming for the diagnosis of tuberculosis at the LNBCSP in Bangui. This cross-sectional study made it possible to analyze fairly recent data in order to have an idea of the epidemiological situation of multidrug-resistant tuberculosis in the Central African Republic. During this study, 755 patients with microscopy-positive pulmonary tuberculosis with 80 cases of resistance. The 80 patients were aged 6 to 68 years. Their average was 35 years. The age group of 20 to 39 was predominant with 57.50%. The minimum age of patients in our study was lower than the minimum age of patients in a study carried out in England, Norway, Ireland, Burkina Faso, Djibouti, Ethiopia and South Africa including Minimum age was 18 years [6] [7] . The maximum age is higher than that of studies carried out in Europe [6] , in Tunisia [8] [9] , and lower than that of the study carried out in South Africa [10] . Our study did not demonstrate an association between age and the occurrence of TPM+ resistant to anti-tuberculosis drugs (p > 5%). In contrast, a study carried out in Tunisia showed that the age of patients (elderly or not) is a risk factor associated with resistance [8] . The male gender was predominant among patients with TPM+ resistance. A study carried out in Niger in 2016 showed that the male sex was dominant. Primary resistance and secondary resistance were predominant in males. The predominance of the male sex among tuberculosis cases had been the subject of several studies carried out in CAR [3] [11] . The predominance of resistant TPM+ in men in CAR has also been observed in other studies carried out in other countries [9] [12] . Unlike our data which did not highlight a link between sex and resistant tuberculosis, male sex was identified as a risk factor associated with resistance to anti-tuberculosis drugs in Tunisia [8] . In this study, marital status is not a risk factor for contracting tuberculosis (p-value = 0.31). In a study carried out by Laoulet and colleagues in Bangui, the p-value is greater than 0.05 [13] . This p-value is in line with the p-value obtained in this study.
TPM+ is a disease essentially transmitted between humans, it is a cosmopolitan infectious disease caused by mycobacteria of the Tuberculosis complex. But at the end of the 1980s the HIV pandemic led to a resurgence of tuberculosis in many countries and the hope of rapid eradication was definitively abandoned. This disease constitutes a historical evil and still remains a growing global scourge, especially in developing countries, despite the control actions implemented for several years. Resistant tuberculosis is the most worrying due to the cost of its treatment. This study initially included 755 patients screened for TPM+ at the LNBCSP in Bangui. This number includes both patients under treatment and control and new screening cases. Of the 755 patients, 80 developed resistance to anti-tuberculosis drugs; which represents a prevalence of 10.60%. A lower prevalence of resistant tuberculosis was found in England with a rate of 7.86%, in Norway 4.12%, in Northern Ireland 4.40% [5] and in Schwaziland a percentage of 6.3% [14] . In Africa, a prevalence more or less high than that of our study was found in Burkina Faso (15.25%), Djibouti (11%), Ethiopia (5%) and South Africa (17.80%) [6] [7] . This is not surprising since upstream in the 1990s in the United States, the problem posed by multi-resistant strains (MDR or Multi Drug Resistant tuberculosis) of tuberculosis appeared inevitable. Due to the severity of the epidemic in South Africa, the threat has escalated with ultra-resistant forms or XDR (Extreme drug resistant tuberculosis). The ultra-resistant form is characterized by its rapid spread, co-infection with HIV, and multi-resistance [7] . It is known from the literature that the prevalence of tuberculosis in Sub-Saharan Africa is higher than that in Europe [15] . This justifies the high frequency of this resistance in Africa. The prevalence of resistant tuberculosis is high in South Africa compared to other African countries. In many countries, tuberculosis appears to be a specific problem of the disadvantaged classes.
The bacillus already in contact with anti-tuberculosis drugs can mutate and therefore become resistant. Compliance with treatment and HIV status were not addressed in our study.
When anti-tuberculosis treatment is incorrectly prescribed or poorly followed by the patient, it can lead to the selection of resistant mutants (secondary resistance). This is the major cause of therapeutic failure. Strains that have acquired resistance to the most effective first-line anti-tuberculosis drugs are called multi-resistant. Multi-resistant strains which have also acquired mutations leading to resistance to the most effective 2nd line anti-tuberculosis drugs are called ultra-resistant [15] [16] [17] [18] [19] . Among the cases of resistance to anti-tuberculosis drugs, 13.75% of patients had experienced treatment failure with anti-tuberculosis drugs. Primary resistance was more common in patients who did not experience treatment failure (100%). In Ivory Coast the proportion of failure cases revealed by a study was 30.6% [15] . Of the 80 patients in the study, 13.75% were cases of treatment relapses.
Higher proportions of relapse cases were reported in Senegal (21.46%) and Côte d'Ivoire (69.4%) [17] [19] . Treatment failure and relapse were associated with the risk of developing resistant TPM+. This risk was 21.37 times higher in patients who experienced a relapse. Treatment failure as associated with the risk of multidrug-resistant tuberculosis was the subject of analyses in a study carried out in Democratic Congo [20] . The notion of contagion found in 23 patients (28.75%) of our study has already been described in Senegal (31.94%) by Niang and collaborators [17] . History of tobacco and alcohol was found in 40% and 61.25% of cases, respectively, in patients with resistant TPM+ in this study. According to some authors, alcohol consumption and tobacco use are risk factors for drug-resistant tuberculosis [9] . Alcohol consumption is a problem for the metabolism of anti-TB drugs. The negative impact of smoking on the outcome of treatment is not to be questioned.
5. Conclusions
The GeneXpert molecular test was initially developed for the diagnosis of tuberculosis with tuberculosis-resistant bacilli. This test has shown its usefulness in the diagnosis of pulmonary tuberculosis, especially in subjects infected with HIV. It made it possible to reduce the diagnosis time and improve it in subjects infected with HIV. In the Central African Republic, screening for new cases of microscopy-positive pulmonary tuberculosis is based on chest x-rays and microscopic examination of sputum or, failing that, gastric contents by tubing. The aim of the present study was to study the contribution of the molecular test using GeneXpert to the diagnosis of pulmonary tuberculosis resistant to anti-tuberculosis drugs in Bangui. A considerable proportion of the overall resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs has been determined.
GeneXpert not only made it possible to highlight cases of primary resistance; but also to detect primary resistance in a significant proportion. The data from this study also allowed us to understand that some patients with primary resistance would be ignorantly placed on inadequate treatment with the first-line anti-tuberculosis drug, Rifampicin. That said, reform actions regarding TPM+ screening in accordance with WHO recommendations must be implemented. This will not only improve laboratory diagnosis; but also the medical treatment of cases of resistance.
Acknowledgements
We thank Mr. Javan Allan Bengba for help with the translation and Mr. Maurel Annicet Adonis Ouoko Fa-Tigbia for design’s work.
Ethics
This study benefited from ethical clearance from the scientific committee of the Faculty of Health Sciences of the University of Bangui which approved the research protocol before starting. In addition, it is a public utility activity supported by the highest authority in matters of public health.