Drug Resistance In Mycobacterium Tuberculosis

Project and Seminar Material for Public Health

Drug Resistance In Mycobacterium Tuberculosis

Abstract

This study determined the drug resistance in mycobacterium tuberculosis in Kaduna State, Nigeria using both phenotypic and genotypic methods. A total of 1186 sputum samples were screened for the presence of AFB by Ziehl-Neelsen staining procedure. Two hundred and twenty one (18.6%) of the samples screened were positive for AFB. Of the 221 smear positive sputum samples cultured on Lowenstein Jensen (L-J) medium, 171 (77%) yielded M. tuberculosis complex, 22 (10%) were AFB and culture positive but negative for MPT 64 antigen on SD-bioline, these were considered non tuberculous mycobacteria (NTM), 15 (7%) were smear positive but culture negative while 13 (6%) were contaminated.

All the MTBC isolates (100%) were further characterized as M. tuberculosis genotypically by LPA. Tuberculosis and HIV co-infection was found in 21 (9.5%). One hundred and eighty two (82%) of the subjects were new cases while thirty nine (18%) were retreatment cases. Any drug resistance was found in 21 (52.5%) of all cases; 19 (47.5%) among new cases and 2 (5.0%) were found in retreatment cases. Mono-drug resistance against streptomycin was seen in 9 (22.5%) of the isolates tested. All mono-drug resistant isolates were from the new cases. MDR-TB was detected in 2 (5.0%) of the subjects all of which were found among new cases.

Four (10.0%) isolates of all cases were poly-drug resistant, 3 (7.5%) were new cases, only 1 (2.5%) was retreatment case. One isolate each (2.5%) from new case and retreatment case showed poly-drug resistance to SM EMB, 1 (2.5%) was resistant to INH EMB. Poly resistance to SM INH EMB was found in only 1 (2.5%) new case. No poly-drug resistance to SM INH, RIF EMB, RIF SM or RIF SM EMB was observed. One isolate was characterized as MDR with bands at rpoβ MUT2A region and ihnA MUT2 corresponding to H526Y and A16G mutations respectively. Rifampicin mono resistance with band at rpoβ MUT3 corresponding to S531L was found in one isolate.

Also, isoniazid mono resistance was observed in one isolate with ihnA MUT2 band corresponding to A16G mutation. The comparison of Geno Type MTBDRplus LPA and phenotypic LJ-proportion method showed that one isolate was mono resistant to RIF and one was mono resistant to INH by LPA, one and two MDR-TB isolates respectively were characterized by genotypic and phenotypic methods. The remaining isolates were found to be pan susceptible by both methods. There was association between TB and age (OR=1.72,CI=1.21-2.44, P= 0.002), sex (OR=2.10, CI=1.56-2.84, P<0.0001), case (OR=21.8,CI=13.35-35.51, P<0.0001), contact with TB patient (OR=0.09, CI=0.06-0.14, P<0.0001) and HIV (OR=0.22,CI=0.14-0.35, P<0.0001).

No association was observed between HIV and MDR-TB (OR=6.3, CI=1.64-23.84, p=0.007); however, there was association between MDR-TB and sex (OR=0.25, CI=0.07-0.90, p=0.034). This study has shown an overall high prevalence of TB and TB drug resistance in Kaduna State. It also demonstrated that the prevalence of MDR-TB is high in the State. These need to be urgently addressed. Laboratory facilities for rapid TB culture and DST are needed in Kaduna State and across Nigeria for early and accurate diagnosis of TB and drug resistant cases. This remains an important step in managing TB drug resistance in Nigeria.

Table Of Contents

Preliminary Page(s)

  • Title page
  • Certification page
  • Dedication
  • Acknowledgement
  • Abstract
  • Table of content

Chapter One

1.0 Introduction

  • 1.1 Background of the study
  • 1.2 Statement of the problem
  • 1.3 Justification
  • 1.4 Research Questions
  • 1.5 Research Hypothesis
  • 1.6 Aim of the study
  • 1.7 Objectives

Chapter Two

2.0 Literature Review

  • 2.1 History of Tuberculosis
  • 2.2 The genus Mycobacterium
  • 2.2.1 Nutritional requirements and growth
  • 2.2.2 Susceptibility to physical and chemical agents
  • 2.2.3 Habitats
  • 2.3 The Mycobacterium tuberculosis complex (MTBC)
  • 2.4 Pathogenesis and clinical presentations of TB
  • 2.5 Global TB burden
  • 2.6 Drug resistant tuberculosis
  • 2.7 TB drug susceptibility testing

Chapter Three

3.0 Materials And Methods

  • 3.1 Ethical approval
  • 3.2 Study design
  • 3.3 Study area
  • 3.4 Sample Size
  • 3.5 Inclusion and exclusion criteria
  • 3.6 Sample collection
  • 3.7 Analyses of samples

Chapter Four

4.0 Results

  • 4.1 Prevalence of TB by microscopy in Kaduna State, Nigeria
  • 4.2 Prevalence of TB by microscopy in Kaduna State, Nigeria according to age group
  • 4.3 Prevalence of TB by microscopy in Kaduna State, Nigeria according to sex
  • 4.4 Prevalence of Tuberculosis by Senatorial area in Kaduna State, Nigeria
  • 4.5 The distribution of TB by sex across the three Senatorial areas
  • 4.6 Mycobacterial culture result
  • 4.7 Tuberculosis/HIV co-infection among the study population in Kaduna State, Nigeria
  • 4.8 Prevalence of tuberculosis by case definition in Kaduna State, Nigeria
  • 4.9 Demographic characteristics and possible risk factors associated with TB
  • 4.10 Resistance patterns to first-line anti-TB drugs in Kaduna State
  • 4.11 Demographic characteristics and risk factors for drug resistance
  • 4.12 Banding patterns of mutations associated with rifampicin and isoniazid resistance
  • 4.13 Comparison of Geno Type MTBDRplus LPA and phenotypic LJ-proportion method

Chapter Five

Discussion of Findings, Conclusion and Recommendation

  • 5.1 Discussion
  • 5.2 Conclusion
  • 5.3 Limitations
  • 5.4 Recommendation
  • References

Chapter One

1.0 Introduction

1.1 Background Of The Study

The discovery of anti-tuberculosis drugs in the 1940s followed by combination chemotherapy made tuberculosis a curable disease. In the developed countries, effective treatment and surveillance reduced tuberculosis dramatically with high hopes of total eradication (Raviglione et al., 1992; Raviglione et al., 1995). However, in the 1980s, it was realized that tuberculosis had not only ceased to decline in the developed countries, notably the USA, but was actually increasing, particularly in major cities (Raviglione et al., 1995). It was also soon realized that the disease was out of control and increasing at an alarming rate across most of the poorest regions of the world especially Africa due to HIV/AIDS (Raviglione et al., 1992; WHO, 2009).

Despite aggressive international efforts, tuberculosis remains a leading infectious cause of death, with an estimated 8.6 million incident cases per year. In 2012, an estimated 1.3 million people died from the disease. These death rates, however, only partially depict the global TB threat; more than 80% of TB patients are in the economically productive age of 15 to 49 years (WHO, 2013).

Global tuberculosis control efforts have been threatened by the emergence of multidrug resistant tuberculosis (MDR-TB). MDR-TB is defined as strains of Mycobacterium tuberculosis which show high level resistance to both isoniazid and rifampicin, with or without resistance to other anti TB drugs (WHO, 2013). MDR-TB is estimated to cause 4% of new tuberculosis cases in the developing world. Patients infected with MDR strains are not only difficult to cure but also more likely to remain sources of infection for a longer period of time than those with drug susceptible organisms. MDR-TB requires longer duration of treatment (up to 2 years) to achieve cure, in comparison with 6 month treatment for drug susceptible TB, lower cure rates and even higher default rates.

The cost of drugs to treat an MDR-TB case can be up to 100 times more expensive than the cost of treating a drug susceptible TB case (Leimane and Leimans, 2006). Because of its increasing prevalence MDR-TB is now subdivided into basic MDR-TB, with resistance only to rifampicin and isoniazid, and extensively drug resistant TB (XDR-TB), with a similar resistance pattern but with resistance to one or more additional first and/or second line drugs.Various perturbations in the individual drug target genes are responsible for the genesis of anti-TB drugs resistance. Rifampicin resistance has been shown to be caused by a change in the β-subunit of DNA dependent RNA polymerase, which is encoded by the rpoβ gene. More than 95% of rifampicin resistant strains are associated with mutations within an 81-base pair region of the rpoβ gene, which is termed rifampicin resistance determinant region (Telenti et al., 1993; Traore et al., 2000; Sharma and Mohan, 2006).

On the contrary, resistance to isoniazid is due to mutations at one of two main sites, in either the katG or inhA genes (Zhang et al.,1992; Piatek et al., 2000). These mutations are not directly connected, and so separate mutations are required for organisms to change from a drug susceptible isolate to MDR-TB. Furthermore, rifampicin resistance has been considered to be a surrogate marker for checking multidrug resistance in clinical isolates of M. tuberculosis since rifampicin resistance is often accompanied by resistance to isoniazid (Traore et al., 2000; Sharma and Mohan, 2006).

Drug resistance in M. tuberculosis occurs by random, single step, spontaneous mutation at a low but predictable frequency, in large bacterial populations. The accurate diagnosis of MDR-TB requires a positive culture of M. tuberculosis and drug susceptibility testing. Previous drug treatment is the largest single risk factor for the presence of MDR-TB. There is a strong suspicion of drug resistance, including MDR-TB, in persons with a history of prior treatment or in treatment failure cases.

The emergence of drug resistance in M. tuberculosis has been associated with a variety of management, health provider and patient related factors. These include

  1. Deficient or deteriorating TB control programmes resulting in inadequate administration of effective treatment;
  2. Poor case holding, administration of sub-standard drugs, inadequate or irregular drug supply and lack of supervision;
  3. Ignorance of health care workers in epidemiology, treatment and control; (iv) improper prescription of regimens;
  4. Interruption of chemotherapy due to side effects; (vi) non-adherence of patients to the prescribed drug therapy;
  5. Availability of anti-TB drugs across the counter, without prescription;
  6. (viii) massive bacillary load;
  7. Illiteracy and low socio economic status of the patients; (x) the epidemic of HIV infection; (xi) laboratory delays in identification and susceptibility testing of M. tuberculosis isolates;
  8. Use of nonstandardized laboratory techniques, poor quality drug powders and lack of quality control measures; and
  9. Use of anti-TB drugs for indications other than tuberculosis (Paramasivan and Venkataraman, 2004).

In most countries, MDR-TB has increased in incidence and interferes with TB control programs, particularly in developing countries, where prevalence rates are as high as 48% (Iseman and Sbarbaro, 1992; Cohn et al., 1997). The high infection and death rates pose an urgent challenge to rapidly detect cases. The extent of the problem of MDR-TB has been examined by the World Health Organization (WHO) in cross sectional surveys of drug resistance in either clinical series or whole country cohorts (Espinal et al., 2001). Cross sectional surveys almost certainly under estimate the burden and number of cases of MDR-TB because they do not take into account the numerical burden of TB in the high burden countries.

When the exercise is repeated with a mathematical modelling design using drug resistance estimates and the number of cases of TB, a more accurate picture of the global MDR-TB burden is claimed (Dye et al., 2002b). However, even this has been criticized as under estimating the global burden for the following reason. The stated number of cases per year from a country often includes up to 20% of cases which are actually on retreatment (i.e. have had a previous course of first line drugs). The prevalence of MDR-TB in retreatment cases is between 30% and 80% depending on the country.

In Gujerat, India, for example, where there are about 400,000 new cases annually, if it is assumed that 20% are being retreated and there is an MDR-TB rate of 30 – 80% in retreatment cases, this would include 24,000 – 64,000 cases of MDR-TB [i.e. (400,000 × 0.2 × (0.3 – 0.8)]. The estimate of the global burden obtained by modelling could be wrong by a factor of 2 – 4. In the USA, HIV positive MDR-TB cases initially had a 100% mortality (Small et al., 1993), but with greater awareness and early diagnosis an improvement in initial survival rates up to 50% has been reported (Salomon et al., 1995). HIV negative cases in the USA have had better response rates of between 56% (Goble et al., 1993) and 69% (Telzak et al., 1995). Nosocomial outbreaks, often in an HIV setting, are well documented in other countries as well as the USA. An outbreak in Spain between 1991 and 1995 killed 47 of 48 patients infected (Herrera et al., 1996), and in two outbreaks in London (Chelsea and Westminster Hospital and St Thomas‟s Hospital) the mortality was over 50% in HIV positive patients (Breathnach et al., 1998).

Understanding the scientific basis of short course 6 month chemotherapy for tuberculosis helps to explain why the loss of sensitivity to both isoniazid and rifampicin, even without resistance to additional drugs, has such major effects on outcome. Numerous controlled trials have shown that a 6 month regimen of rifampicin and isoniazid, supplemented by pyrazinamide and streptomycin or ethambutol for the first 2 months, will provide a cure in > 95% of cases if the medication is taken correctly. Such a regimen also renders infectious cases non infectious in 2 weeks
(Ormerod, 1997). Each drug varies in its ability to kill tubercle bacilli (bactericidal ability), to deal with persistent organisms which are only occasionally metabolically active (sterilizing ability) and to prevent the emergence of drug resistance (Ormerod, 1997).

Isoniazid is the best bactericidal drug and if mono resistance to this occurs, treatment with rifampicin and ethambutol has to be extended for 9 – 12 months, in addition to 2 months initial pyrazinamide. Rifampicin is the best sterilizing drug, and mono resistance to this drug requires treatment with isoniazid and ethambutol for 18 months, with 2 months initial pyrazinamide. Therefore loss of response to both the main bactericidal drug and the main sterilizing drug means that patients remain infectious for much longer, both in the community and in hospital, that treatment is required for at least 12 and possibly more than 24 months, and that less effective and more toxic second line drugs have to be used (Joint Tuberculosis Committee of the British Thoracic Society, 1998).
Although some individuals who have not had previous TB treatment are infected by MDR-TB, this is not the case for most patients. Many new cases of MDR-TB are created each year by a combination of physician error and poor patient compliance with treatment, which turn fully susceptible organisms, or those with less complex resistance patterns, into MDR-TB.

1.2 Statement Of The Problem

The global burden of TB remains enormous. In 2012, there were an estimated 8.6 million incident cases of TB and 1.3 million people died from the disease. Among these deaths there were an estimated 170,000 from MDR-TB. General problem of MDR-TB with an estimated 450,000 incidence cases worldwide annually has been recognized since the first World Health Organization (WHO) global survey on drug resistance in the late 1990s (WHO, 2013). MDR-TB has reached alarming levels worldwide with the emergence of strains that are virtually untreatable with the existing drugs. Drug-resistant strains, along with HIV/AIDS, are causing the biggest challenge to efficient management and control of TB. The report of an outbreak of extensively drug resistant TB (XDR-TB) in South Africa (Gandhi et al., 2006), with its extremely high case fatality rate, has drawn wide attention.

It has been indicated that MDR-TB is likely to be more prevalent in Africa than previous reports indicated. The latest WHO global report on anti-tuberculosis drug resistance in the world was produced in 2008 and published in a 2010 WHO report on MDR-TB epidemic. A systematic literature review of evidence about mortality associated with MDR-TB was commissioned by WHO in 2013. The results have been used to produce global estimates of MDR-TB incidence and mortality in 2012. The estimate of mortality due to MDR-TB is slightly higher than before, while the incidence is similar to the previous estimate (WHO, 2013).

1.3 Justification

Five of 13 countries with the highest incidence rates of TB per capita are in Africa. According to the WHO global report on anti-tuberculosis drug resistance in the world, MDR-TB strains have emerged in all regions of the world (WHO, 2013). The overwhelming burden of MDR-TB is in high burden resource poor countries. The diagnosis depends on confirming the drug susceptibility pattern of isolated organisms, which is often only possible in resource rich settings. Lack of comprehensive national DRS data from all countries in Africa is a barrier to understanding the magnitude of prevalence and incidence of MDR-TB.

WHO (2013) reported Nigeria as the thirteenth in the list of the 22 highest incidence countries on the basis of numbers of new cases of TB. Also, Nigeria is considered as having moderate rate of MDR-TB. According to the National drug resistance survey in 2012, the prevalence rate of MDR-TB in Nigeria is about 2.9%. However, MDR-TB in Africa, including in Nigeria, is more prevalent than previously reported. Given the limited health care funding and substantial incidence of HIV in Nigeria, even a relatively low but increasing tide of MDR-TB can lead to disastrous consequences for the country. The availability of drugs on the open market and a private sector that delivers drugs to the population in an unregulated fashion in Nigeria could also be factors that might favour development of MDR-TB. There is very little information on the prevalence of MDR-TB in Nigeria and specifically in Kaduna State.

1.4 Research Questions

  1. Is there MDR-TB in Kaduna State?
  2. If there is, what is the prevalence of MDR-TB in Kaduna State?
  3. What socio-demographic factors predispose to MDR-TB in Kaduna State?

1.5 Research Hypothesis

  • Null hypothesis: MDR-TB is not prevalent in Kaduna State
  • Alternative hypothesis: MDR-TB is prevalent in Kaduna State

1.6 Aim Of The Study

The aim of the study is to determine the existence and prevalence of MDR-TB in Kaduna State, Nigeria.

1.7 Objectives

The objectives of the study are:

  1. To determine some demographic and risk factors that may be associated with tuberculosis among the study population
  2. To screen sputum samples for Acid Fast Bacilli (AFB) using microscopy
  3. To isolate and characterize M. tuberculosis from smear positive sputum samples
  4. To determine the anti-tuberculosis drug susceptibility of the isolates
  5. To determine the patterns of mutations in the resistant isolates

Chapter Five

Discussion of Findings, Conclusion and Recommendation

5.1 Discussion

In recent years, there has been an increasing concern on the threat of tuberculosis to public health, especially in developing countries where its alliance with HIV/AIDS is making the situation worse. From this study, a prevalence rate of 18.6% AFB positive was found in Kaduna State. This high rate could be attributable to a combination of high poverty levels, poor socioeconomic status, poor education, associated with poor knowledge of TB risks of infection and dissemination as well as poor access to health care facilities. The comparative high HIV prevalence rate (5.1%) among the general population is also fuelling the TB epidemic in the State. The presence of many Federal institutions of higher learning in the State attracts people from within and outside the country to the State which could in part contribute to the high rate of infection in the State. Similar prevalence of 16.83% in Abia (Nwachukwu et al., 2009) and 14.7% in Kano (Imam and Oyeyi, 2008) were reported. However, higher rates were reported in Nasarawa (44.8%) and Lagos (24.8%) by Onubogu et al. (2010) and Umeh et al. (2007) respectively.

In most instances it cannot be determined why a particular person does or does not develop tuberculosis after becoming infected with tubercle bacilli. However, a multitude of factors have been identified which increase the risk of progression from sub-clinical infection with M. tuberculosis to overt tuberculosis (Rieder, 1999).

The prevalence of TB in Kaduna from this study showed that the disease is more prevalent among the economically productive age group (15-44 years) confirming the known fact that TB is more prevalent among this group. About 78.3% of the study population falls within this age group. Exposure to the risk factors like smoking, alcoholism, imprisonment, migration is more common among these subjects; other co-morbid factors such as HIV also peak within the group. Children are being administered TB vaccine (BCG) at birth and this vaccine is said to have above 80% protection against TB in children for as long as 15years if administered before first exposure (Bannon and Finn, 1999; Crofton et al., 1999). The average life expectancy of Nigerians is 53 years and 55 years for males and females respectively (WHO, 2012). These may in part explain the low rate of TB cases observed in children and in elderly.

There was significant association (OR=1.72; 95% CI: 1.21-2.44; P=0.002) between TB and age.

Several studies have supported the strong confounding effects of demographic factors such as age and gender on the incidence of TB globally (Holmes et al., 1998; Crampin et al., 2004).

The findings in this study revealed a statistically significant association (OR=2.10; 95% CI: 1.56-2.84; P=0.0001) in the rate of occurrence of TB between males and females subjects. The risk ratio of males to females is 1.41 (95% CI: 1.25-1.60; P=0.0001). This could be due to differences in access to health care and in health seeking behaviour of people due to stigma associated with TB. Women tend to have a more far reaching psycho-social consequences of stigmatizing ailments and may consequently avoid self exposure inherent in presenting at health facilities. Moreso, majority of women especially in the Northern and Central parts of the State are mostly full time housewives and are thereby hardly exposed to the risk of infection from contact with infected people. Although, most studies are in keeping with this finding, Nwachokor and Thomas (2000), in a 30 year review of tuberculosis in lbadan, Nigeria reported that more females were infected with TB which reached twice the rate of males. It could be because lbadan being in the Southern part of Nigeria, more women are working than the North where the reverse is the case. The social roles of men and cultural habits that influence risk of exposure have also been implicated as possible reasons. Social factors such as smoking, alcoholism, imprisonment and poor nutrition may also play a role. Several studies have reported higher prevalence of pulmonary TB in males than females (Borgdorff et al., 2000; Al-Hajoj et al., 2013; Gambo et al., 2013a; WHO, 2014).

There was no significant difference(χ2 = 0.192; p=0.9085) in the rate of occurrence of TB across the three areas. However, the highest prevalence rate was observed in the southern part of the State. High poverty levels and relatively high HIV prevalence rates is fuelling the TB epidemic, especially in the Southern part of the State where the burden of HIV (11.4%) is disproportionately higher than the other two areas (Central = 1.9% and Northern = 0.7%) (Gidado and Ejembi, 2009).

Of the 221 smear positive sputum samples cultured on LJ slants, 7% were smear positive but culture negative. The 4% NaOH used for decontamination of the sample could kill up to 70% of tubercle bacilli this may lower the number thus may not be able to grow when cultured. The samples might have been exposed to sunlight; TB are rapidly destroyed when exposed to sunlight. Moreso, LJ medium does not support the growth of some mycobacterial species such as M. bovis which are also AFB, all these may partly explain why some samples were smear positive but culture negative. Contamination rate was found to be 6%. This could be attributed to the nutritionally rich medium (LJ medium) used for the isolation as well as the prolong incubation period. Also, the contaminating microorganisms which are abundant as normal flora in the respiratory tract may cling to food particles commonly found in the sputum thereby evading killing during decontamination. Mycobacteria other than members of the MTBC are abundant in the environmental, this could be responsible for the 10% NTM found in this study.

Similar finding was reported in two different studies (5.7% and 15%) by Gambo et al. (2013a; 2013b) in Northern Nigeria, while Al-Hajoj et al. (2013) reported 11.5% NTM in Saudi Arabia.

Tuberculosis/HIV co-infection was found in 21 (9.5%) while 200 (95.5%) were infected with TB only. The TB/HIV co-infection rate in this study is lower than the 14.2% rate among tuberculosis patients reported in the National drug-resistant tuberculosis prevalence survey of 2012 (National Drug-resistant TB prevalence survey report, Nigeria, 2012). Similar rates reported in other studies include 9.6% in Edo State (Okodua et al., 2012), 12.0% in Ile-Ife, Osun State (Onipede et al., 1999), 10.5% and 14.9% among children and adults respectively in Sagamu, Oyo State (Daniel et al., 2005), 6.1% among those aged 20-40 years in Jos, Plateau State (Anteyi et al., 1996). According to the United States Embassy in Nigeria, Nigeria Tuberculosis Fact Sheet (2012), about 8% of the HIV-positive persons in Nigeria are tuberculosis positive; this is in keeping with the finding in this study. The low TB/HIV co-infection rate in this study could be as a result of the well established fact that HIV-mediated immunosuppression impairs granuloma formation, resulting in both ineffective containment of M.tuberculosis bacilli and diminished formation of pulmonary cavities (Klautau and Kuschnaroff, 2005; Murray, 2005). These effects manifest clinically as frequent extrapulmonary disease (Poprawski et al., 2000) and lower concentrations of bacteria in sputum (Colebunders and Bastian, 2000). Most TB/HIV coinfection patients are smear negative and most of the tuberculosis patients who are HIV positive will have pauci bacillary or smear negative disease. However, the HIV infection rate among TB patients in this study is almost twice that in the general population (5.1%) in the State (Federal Ministry of Health, Nigeria, 2010). This further buttresses the fact of TB/HIV co-morbidity. A higher prevalence of 26.9% was reported in Northern Nigeria by Gambo et al. (2013a); a much lower rate of 1.9% was however reported in Saudi Arabia by Al-Hajoj, et al. (2013). Chi-square analysis indicated a significant difference (χ2= 37.246; pꞌ0.0001) in the rate of occurrence of TB among HIV positive and negative subjects.

Our findings showed that the majority (82%) of the TB study participants were newly diagnosed cases whereas the remainder (18%) were previously treated cases. This could be explained by the fact that a pre-existing infection provides some protection against re-infection compared to acquisition of new infection. Thus, if infection occurs, those with pre-existing infection will have, on average, better defense mechanisms than those who have not been previously infected (Rieder, 1999). This is in keeping with previous findings of 82.1% new cases and 17.9% retreatment cases in Nigeria (National Drug-resistant TB prevalence survey report, Nigeria, 2012); and 88.3% new cases and 11.7% previously treated cases in Cameroon (Meriki et al., 2013).

Drug-resistant TB ultimately develops from the inadequate treatment of active pulmonary TB. This may result from poor prescription practices among medical doctors with poor drug selection and insufficient treatment duration (Sharma and Mohan, 2006). Systemic problems, through inadequate public health resources and unpredictable drug supplies or supply of sub standard drugs, also play a role (Mukherjee et al., 2004). Erratic or selective compliance to treatment and default among patients is another key factor as it causes M. tuberculosis to be exposed to sublethal doses for insufficient durations. These could thus, result in treatment failure and foster emergence of drug-resistant TB (Pablos-M´endez et al., 1997).

The resistance to first line anti-TB drug was found to be 52.5% of all cases of pulmonary TB in Kaduna State. Mono-drug resistance was seen in 9 (22.5%) of the isolates tested and was against streptomycin. The highest resistance to streptomycin was not surprising because it is used for the treatment of other disease conditions such as brucellosis and cross resistance with other aminoglycosides such as gentamicin which are commonly used in our settings for many years (Houang and Greenwood, 1977). Moreso, similar results of high resistance to streptomycin were previously reported; 30.6% in the national DR survey in Nigeria (National Drug-resistant TB prevalence survey report, Nigeria, 2012), 34% in Sudan (Hassan et al., 2012) 18.8% in Cameroon (Meriki et al., 2013) and 28.1% in India (Sethi et al., 2013).

The most important measure of TB drug resistance is the number of new cases that are MDR-TB (Dye et al., 2002a). According to the Global TB control, WHO report (2012), the estimated prevalence of MDR-TB in Nigeria among new cases is 3.1%. This study showed a prevalence rate of 5.0% (2/40) among new cases. Occurrence of MDR-TB in drug naïve patients is due to the fact that the infecting mycobacteria may not be drug naïve, or acquired resistance might have developed due to natural selection which is a function of ineffective treatment and non compliance to drug prescription by the previous host, when such resistant strains are transferred to a susceptible host, primary drug resistance develops (Johnson et al., 2009). The availability of drugs on the open market and a private sector that delivers drugs to the population in an unregulated fashion in Nigeria could also be factors that might favour development of MDR-TB.

Several biological mechanisms linking drug-resistant TB to HIV infection have been suggested (Dye et al., 2002b). Drug malabsorption in HIV-infected patients, especially rifampicin and ethambutol, can lead to drug resistance and has been shown to lead to treatment failure. Drugresistant strains may be less virulent and preferentially lead to disease progression in immunocompromised patients, as opposed to immunocompetent individuals. Data supporting this hypothesis has not yet been observed in humans (Anastasis et al., 1997). However, statistical analysis (OR=0.25; 95% CI: 0.07-0.90; P=0.034) in the present study did not reveal any association between HIV infection and anti-TB drug resistance. This is similar to reports from earlier studies in Nigeria. Otu et al. (2013) reported no association between HIV infection and anti-TB drug resistance in Calabar; in Abuja, Lawson et al. (2010) studied 32 TB culturepositive patients and reported no association between HIV infection and anti-TB drug resistance.

Pereira et al. (2005) studied a total of 70 M. tuberculosis isolates, 30 from HIV seropositive, and 40 from HIV seronegative TB patients in India and found that the prevalence of drug-resistant M. tuberculosis isolates among HIV seropositive TB patients was similar to that of HIV seronegative TB patients indicating that HIV infection may not be associated with drug-resistant TB. Three studies in South Africa also found no association between HIV infection and MDRTB. In a retrospective study in Durban, 2.4% of 42 HIV co-infected and 11.5% of 253 HIV negative patients had MDR-TB (Anastasis et al., 1997). A prospective study of hospitalized TB patients in Cape Town found MDR-TB prevalence of 3.2% in 93 HIV co-infected patients, compared to 2.6% in 115 HIV negative patients (Post and Wood., 1997). In gold miners, the MDR-TB rate was 5.3% among 207 HIV co-infected and 6.5% among 215 HIV negative miners (Murray et al., 2000). From the above, it appears that studies from very high TB burden countries like Nigeria, India, and South Africa have consistently reported no association between HIV and TB drug resistance.
The genotypic drug resistance assay revealed that all the resistant isolates were hetero-resistant; a phenomenon where some cells within a population may remain susceptible to the antibiotic, whereas other cells display varying degrees of drug resistance.

This was determined by the simultaneous detection of wild type and mutant molecular susceptibility. In patients infected with a fully susceptible strain, drug resistance can develop gradually during inadequate treatment due to selection of cells with random mutations in sites associated with drug resistance (i.e. secondary resistance). In this case, as the proportion of susceptible cells decrease and resistant cells increase, a hetero-resistant population of cells will be present. These cells are primarily identical throughout the genome but a proportion of the population differs in sites associated with drug resistance. In contrast, patients infected with fully susceptible strains may develop mixed infections if they are co-infected with another strain that is drug resistant, resulting in a mixed population of two genetically distinct strains, one drug susceptible, and the other drug resistant. Also, hetero-resistance likely represents a natural variation in the population of cells of M. tuberculosis (Foundation for innovative new diagnosis, 2012). Prescription of inadequate treatment regimen, irregular drug supply, poor drug quality with low bioavailability, and poor compliance among the study population could be attributed to the development of heteroresistance observed in this study.

The patterns of mutations associated with rifampicin and isoniazid resistance determines the degree of susceptibility to anti TB drugs. Studies determining MICs have shown that high-level RIF resistance is associated with mutations in codons 526 and 531, whereas alterations in codons 511, 514, 515, 516, 518, 521, 522 and 533 result in low-level RIF resistance. Mutations in katG tend to confer high-level resistance to INH, while mutations in inhA generally confer low-level INH resistance (FIND, 2012). Thus, identifying and reporting where the specific mutation was detected is important in the selection of treatment regimen. The two RIF resistant isolates in this study have mutations at codons 526 and 531 respectively, suggesting a high-level RIF resistance. A number of published studies have described that the value of the RIF MIC strongly correlates with the position and nature of the amino-acid substitution in rpoβ RRDR (Yang et al., 1998; Campbell et al., 2001; Ocheretina et al., 2014).
Discordance between genotypic and phenotypic assays was observed in one isolate with RIF mono resistance. The isolate was classified as MDR by the phenotypic assay. This could be due to silent mutation or synonymous single nucleotide polymorphism (sSNP) at the target site. Silent mutations do not result in structural changes in the inhA and so do not interfere with its inhibition by INH. Moreover, findings of silent mutations in inhA are not surprising as SNPs occur every 3 kb of MTB genome (Comas et al., 2011). Ando, et al. (2014) reported a silent mutation in a significant number of INH resistant M. tuberculosis clinical isolates. Mutations conferring INH resistance in other genes such as mabA (G609A) (Ando et al., 2014) aphC (alkyl hydroperoxide reductase), kasA (ß-ketoacyl-ACP synthase) and nadh (NADH dehydrogenase) (Cohen et al., 2003; Balasubramanian et al., 2012) not included in Geno Type MTBDRplus have also been reported. The critical concentration of INH on LJ medium which is being used for over 50 years is 0.2µg/ml (Jamieson et al., 2014). Lower critical concentrations of 0.0312µg/ml for low-level resistance and 0.125µg/ml for high-level resistance were reported by Gumbo, (2010). In addition, no mutation was detected by the genotypic assay in a second MDR isolate. For this discrepant isolate, a mutation either outside or in the rpoβ RRDR or mutations conferring INH resistance in other genes not included in the probes might have occurred. RIF resistance associated mutations outside the rpoβ RRDR V176F (RIF MIC 8 to 32µg/ml) and V146F (RIF MIC 50µg/ml) were reported by Markus et al. (2000) and Jamieson et al. (2014), or a different mechanism of resistance, may account for the resistance.

Members of the MTBC are highly related mycobacteria exhibiting remarkable nucleotide sequence level homogeneity despite variation in pathogenicity, geographic range and certain physiological features (such as colony morphology as well as profiles of resistance and susceptibility to inhibitors, epidemiology, and host preference). M. tuberculosis is the predominant cause of human tuberculosis. However, each of the MTBC subspecies is known to infect humans, and since most laboratories do not fully identify MTBC isolates, the true cause of tuberculosis in these patients and its source often remain undiscovered. An important health concern is the zoonotic transmission of some MTBC subspecies from animals to humans and vice versa. Of particular significance is the transmission of M. bovis to humans from cattle and unpasteurized milk as well as M. bovisBCG infection of immunocompromised individuals. M. bovis is naturally resistant to pyrazinamide, a first-line antituberculosis drug. Therefore, complete identification of MTBC isolates at the subspecies level is required in order to collect information on their epidemiology and also to enable appropriate patient treatment and public health measures (Huard et al., 2003).
All the isolates (100%) were identified as M. tuberculosis. This is in keeping with the well established fact that M. tuberculosis is the principal agent of the disease in humans (Huard et al., 2003). Previous studies on characterization of mycobacterial species causing pulmonary tuberculosis in Nigeria and some West African countries indicated the same trend, 92% in Osun State, 94.4% in Northern Nigeria; 93.4% and 73% in Cote d I voire and Ghana respectively (Alli, et al., 2010; Gambo et al., 2013b).

5.2 Conclusion

This is the first study to demonstrate the prevalence of MDR-TB by both phenotypic and genotypic methods in Kaduna State, Nigeria to our knowledge. The study showed an overall high prevalence of TB (18.6%) and TB drug resistance (52.5% any drug resistance; 12.5% poly-drug resistance and 22.5% Mono-drug resistance). It also demonstrated that the prevalence of MDRTB (5.0%) is high in the State. There was association between TB and age, sex, case, contact with TB patient and HIV. No association was observed between HIV and MDR-TB; however, there was association between MDR-TB and sex. One (1) isolate was mono resistant to RIF by LPA, one (1) was mono resistant to INH by LPA, one (1) and two (2) MDR-TB isolates respectively were characterized by genotypic and phenotypic methods. Also, the remaining isolates were found to be pan susceptible by both methods. All the isolates (100%) were found to be M. tuberculosis.

5.3 Limitations

  1. Difficulty in obtaining ethical approval delayed the early completion of the work
  2. The hospitals (Ahmadu Bello University Teaching Hospital, Zaria, National Tuberculosis and Leprosy Training Centre, Saye, Zaria, Barau Dikko Spacialist, Kaduna, General Hospital Kafanchan) selected for the study might not be representative enough; they were selected based on the large number of attendees, their centrality and high status compared to other medical facilities in the study area
  3. Sequencing was not done due to limited financial support
  4. The accuracy of the responses generated depended on the accuracy of information given by the respondents

5.4 Recommendation

  1. Laboratory facilities for rapid TB culture and DST are urgently needed in selected hospitals in the State and across Nigeria for early and accurate diagnosis of TB and drug resistant cases
  2. Use of robust molecular techniques such as DNA sequencing employed for the detection of occult cases with low-level resistance and other resistance not included in the Geno Type MTBDRplus kit is recommended
  3. Regular supply of standard drugs as well as strict adherence to the DOTS strategy should be ensured by the authorities concerned
  4. Appropriate measures should be taken by the relevant authorities to prevent the indiscriminate and sub-therapeutic use of anti-TB drugs to avert the emergence of resistant strains
  5. Further studies to describe the molecular epidemiology of MTBC in Nigeria; and to identify the predominant genotypes responsible for TB transmission and prevalence should be conducted
  6. Patients recruited in the study were those presenting at the clinics, thus, further studies should be conducted to include active recruitment method for those in communities
  7. Collaboration between academic institutions and health care institutions should be encouraged
  8. Improved health care facilities, standard of living as well as proper education of the public on the knowledge of TB and associated risks of infection and dissemination are also recommended

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