Studies On The Co-Infectivity Of HIV And Atypical Mycobacteria

Project and Seminar Material for Microbiology

Studies On The Co-Infectivity Of HIV And Atypical Mycobacteria


The increasing global incidence of tuberculosis and nontuberculous mycobacteria (NTM) has been attributed to immuno suppresion due to the Human immunodeficiency virus (HIV) epidemic. The paucity of information on the contribution of NTM to mycobacterial infections in Africa including Nigeria has however been closely associated with limited laboratory culture for its isolation and identification. This study investigated the coinfectivity of HIV and Atypical Mycobacteria in Nsukka L.G.A. Two hundred cases (100 HIV negative: 56 males and 44 females and 100 HIV positive- 39 males and 61 females), between the ages of 15 and 71 years with mean age of 37.5 years participated in the research. HIV antibodies were screened using two HIV test kits: the Determine (Abbot Co Ltd, Japan) for preliminary test and the Gold (Unigold) which was used to check for consistency. CD4+ count was carried out using the cytometry (CyFlow®), while acid fast bacilli (AFB) were identified by the Ziehl-Neelson staining technique. AFB positive samples were subjected to nested PCR for species identification. T-test was employed to check for statistical significance between the mean prevalence in test and control groups and CD4 count of HIV single infection and co infection with TB. Chi square correlation was also employed to check for relationship between the demographic characteristics and the distribution of the disease. A preponderance of HIV infection was observed among the age group 21-50 years (72.5%) with overall HIV prevalence of 19.4%. The highest AFB prevalence of 26.6% was observed among patients aged 21-30 years, with overall prevalence of 24%. About 79.1% of TB infection occurred at CD4 count less than 400 cells/µl. Molecular analysis of the samples (using nested PCR) showed 97 (78.9%) M. tuberculosis, 14 (11.4%) M. bovis and 10 (8.1%) NTM. The NTM identified was M. avium complex (MAC). The prevalence rate of TB/HIV co-infection was 24 (24%) out of which 14 (53.8%) were M. tuberculosis, 5 (20.8%) were M. bovis and 3 (12.5%) were NTM. The highest NTM prevalence of 66.7% was observed among patients aged 21-30 years in the HIV positive group while the highest prevalence of 42.8% was observed among 41-50 years in the HIV negative group. TB co-infection was significantly associated with CD4+ cell count (P<0.05). Individuals with lower level of education and the rural settlers were observed to be at higher risk of TB co-infection with HIV (RR =1.40, P =0.002) and (RR = 3.17, P = 0.01). This study establishes the significant role of nontuberculous AFB organisms in pulmonary TB and HIV co-infection and subsequently enhanced progression to the active disease, AIDS. Furthermore, it underscores the importance of introduction of molecular screening assays for the rapid and efficient detection of NTM infections in high burden, and resource limited settings such as Nigeria for effective management and control of HIV/TB co-infectivity.

Chapter One

1.1 Introduction

Tuberculosis (TB) is an infectious disease that primarily affects lungs causing pulmonary tuberculosis but can also affect meninges, intestine, bones, lymph nodes, skin and other tissues of the body. Worldwide, tuberculosis causes 2.9 million deaths annually (WHO, 1998). An estimated two billion persons are currently infected with Mycobacterium tuberculosis and other Mycobacterium species. The rates of increase are even greater in developing countries, primarily because of increased immigration of people from regions of high endemicity, declining socio-economic conditions in densely populated cities and the increasing number of human immunodeficiency virus (HIV) infected individuals (Szabo, 1990). The total number of tuberculosis cases in the world is increasing and the HIV epidemic is implicated for this increased incidence and an estimated three million persons with tuberculosis worldwide also have AIDS. Over 125 Mycobacterium species have been characterized and identified (Griffith et al, 2007). There are numerous species of Mycobacterium and because of recently developed molecular methods, more are being recognized (Tortoli, 2003), although regional variation in species isolation has been shown (Griffith et al, 2007). Historically M. tuberculosis and M. leprae have caused the preponderance of human disease. However in recent years, other mycobacteria have become more widely appreciated as potential pathogens. Most other mycobacteria are present in the environment as saprophytes. These organisms in the past have been called atypical mycobacteria, the term first coined by Pinner (1935) but species have been described with different nomenclatures such as anonymous, nontuberculous mycobacteria (NTM), environmental, opportunistic and mycobacteria other than tubercle bacilli (MOTT). While there has not been an international consensus on the nomenclature, the American Thoracic

Society (ATS) has endorsed the name NTM (Wallace et al, 1990; Katoch and Mohan, 2001).

The frequency of pulmonary disease from nontuberculous mycobacteria (NTM) is reportedly on the rise in Europe, North America, Asia and Southern Africa (Marras and Daley, 2002). In sub-Saharan Africa, information on the extent of the burden of pulmonary disease from nontuberculous mycobacteria (NTM) is lacking due to limitations in tools for mycobacterial species identification. However, studies conducted as far back as the late 1950s and early 1960s using traditional tools for identifying mycobacterial groups based on certain characteristics like speed of growth and morphology, have reported the isolation of NTM from both tuberculosis patients and the general public in some African countries including Nigeria (Zykov et al, 1967; Zykov and Roulet, 1967). They have been increasingly recognized to cause pulmonary and non pulmonary infections (Pinner, 1935; Wolinsky, 1979; Good, 1985; Smith and Grange, 1989; Wayne and Sramek, 1992), which is in part explained by the increase in the number of susceptible/immunocompromised individuals such as those suffering from acquired immune-deficiency syndrome (AIDS) and is also due to better recognition of their role through more sensitive and specific techniques (Wallace et al, 1990). Mycobacterium infections are frequent opportunistic pathogens associated with the acquired immunodeficiency syndrome (AIDS). Its relative virulence and potential for person-to-person transmission distinguishes Mycobacteriumtuberculosis. Persons infected with the human immunodeficiency virus (HIV) are particularly susceptible to tuberculosis, either by the reactivation of latent infection or by a primary infection with rapid progression to active disease (CDC, 1991; Daley et al, 1992; Edlin et al, 1992). In addition, disseminated infections with Mycobacterium avium complex are increasingly common in advanced human immunodeficiency virus (HIV) infection and cause substantial morbidity (Horsburgh, 1991; Hoover et al, 1993). Persons with HIV infection and CD4 lymphocyte counts less than 100 cells/mm3 have a probability of 10% to 20% per year of developing M. avium complex disease or bacteremia (Chaisson et al, 1992; Nightingale et al, 1992). Indeed, NTM are being identified as causative agents of human disease with increasing frequency. They were not traditionally considered a threat to public health, as person-to-person transmission occurs rarely if at all, yet these organisms can produce serious morbidity. In addition, cases of mycobacterial disease are becoming more difficult to diagnose or treat, especially when fastidious NTM or drug-resistant strains are involved (Wallace et al, 1990). The diseases caused by these mycobacteria have varied manifestations but have been broadly grouped as mycobacterioses (Katoch, 2004). In the immunocompromised individuals the infections due to NTM have been observed to be an important cause of morbidity and mortality in western countries (Wallace et al, 1990).

The distribution of NTM and the incidence of disease caused by them are perhaps not fully understood in most parts of the world. NTM are widely distributed in nature and have been isolated from natural water, tap water, soil, water used in showers and surgical solutions (Kazda, 1983). In United States most isolates were M. avium, M. kansasii and M. fortuitum (O’ Brien et al, 1987). There have been some reports from Japan (Tsukamura et al, 1988), UK (Public Health Bull 1993) and India (Sachdev et al, 2002). They are considered opportunistic pathogens, and several species which include M. avium Complex (MAC), M. intracellulare, M. kansasii, M. paratuberculosis, M. scrofulaceum, M. fortuitum, M. chelonae, M. ulcerans, ((Katoch and Mohan, 2001), are associated with human disease which is typically pulmonary, skin/soft tissue, lymphatic, or disseminated in presentation. It is a common observation that environmental mycobacteria cause disease in individuals who offer some opportunity due to altered local or systemic immunity (Pinner, 1935; Wolinsky, 1979; Wallace et al, 1990; Katoch and Mohan, 2001). NTM cause pulmonary disease that is similar to M. tuberculosis and is often chronic and occurs in older women or those with underlying lung disease (Aksamit, 2002; Oliver et al, 2003). They also cause skin/soft-tissue infections of varying severity in both sporadic and epidemic form. They have been linked to nosocomial infections (Knackmuhs et al, 2004), and outbreaks in nail salons (Winthrop et al, 2002). Disseminated disease due to NTM is primarily associated with AIDS and other forms of severe immunosuppression (Horsburgh et al, 2001).

Although it has been shown that most culture-positive Mycobacteria are M. tuberculosis in regions where tuberculosis is highly prevalent (Idigbe et al, 1986; Olusoji et al, 2011), NTM isolates have been increasing gradually. There are several reasons for this increase in disease due to NTM, including improved diagnostic techniques, increased recognition of NTM in immunocompetent patients, increasing life expectancy and increased numbers of, particularly, older women, increased incidence of NTM associated with HIV/AIDS and other forms of immune compromise and altered host defense (Jones and Havlir, 2002; Griffith et al, 2007). These organisms trigger diseases and true infections and thus can be important clinically (Wagner and Young, 2004). The prevalence of NTM and NTM-associated hospitalization has been on the increase in several industrialized countries (Martin-Casabona et al, 2004; Billinger et al, 2009; Park et al, 2010). Some of the countries have reported an NTM prevalence rate as high as 50% among cultured mycobacteria (Martin-Casabona et al, 2004; Billinger et al, 2009; Park et al, 2010). However, there is paucity of data from developing countries largely due to lack of laboratory infrastructure for culture and specie identification. In Africa, the contribution of NTM to such disease has been examined on a small scale only (Buijtels et al, 2009). In Nigeria, 9.1% NTM have been reported in Oshun (Olusoji et al, 2011), 11% in Lagos (Idigbe et al, 1986) and as high as 26.6% in Jos (Mawak et al, 2006).

Diagnosis and treatment of TB patients in most Sub-Saharan African countries including Nigeria is based solely on the results of microscopic smear positivity. As such, all sputum smear positive diagnosed patients are indiscriminately placed on Directly Observed Treatment Short course (DOTS), the current international TB treatment strategy.

Consequently, many of the pulmonary diseases caused by NTM are not identified but rather treated with conventional anti-TB which eventually fails because majority of the NTM are resistant to conventional anti-TB treatment (Philalay et al, 2004; Nasha et al, 2006). The implication is that NTM is inappropriately managed with first-line antituberculous drug (Koh and Kwon,2004, Yim and Han, 2005), worsening the patient’s condition and raising the risk of drug resistance.

Nigeria has one of the highest burdens for TB in the world and remains a major target in the global control of the disease (WHO, 2010). In 2011 an estimated 280,000 cases of TB (68% incident cases) were reported from Nigeria which corresponds to a prevalence rate of 280 per 100,000 population according the WHO global tuberculosis report of 2012.The in country prevalence of pulmonary TB due to species other than Mycobacterium tuberculosis like mycobacterium bovis and Mycobacterium africanum is reportedly on the rise, (Cadmus et al, 2006; Mawak et al, 2006). However, this evidence is inconclusive, and data are insufficient on the prevalence of other mycobacterial species raising question about the importance of the different species of Mycobacterium causing TB in Nigeria.

1.1.1 Statement Of The Problem

Historically, all tuberculosis cases have been attributed to Mycobacterium tuberculosis (Tuberculous mycobacteria). However, recent increasing level of immune suppression resulting from HIV/AIDS and other related infections has led to the emergence of other nontuberculous mycobacterial (NTM) infections. Furthermore, poorly diagnosed NTM among people living with HIV/AIDS (PLWHA) complicates treatment protocol and subsequently exacerbate their condition and hastens progression to AIDS

1.1.2 Aims

The aim of this research is to study the prevalence of atypical mycobacteria among HIVseropositive individuals with a view to achieving early diagnosis of mycobacterioses and make reliable discrimination from tuberculosis as this is essential for effective therapeutic regimen and appropriate management of HIV infected individuals.

1.1.3 Objective Of The Research

  1. To determine the prevalence of atypical mycobacteria infection among HIV-
    seropositive patients.
  2. To characterize and identify the different species To determine the distribution of each species among the study groups.
  3. To compare the prevalence of atypical mycobacteria in HIV-seropositive and
    seronegative patients.
  4. To determine the effect of the socio-demographic factors on the distribution of the different species.

Last Chapter

Discussions And Conclusion

In this study, 8.1% of patients who sought clinical treatment for tuberculosis in Nsukka and environs were caused by NTM infection. This is comparable though slightly lower than 9.1% and 11% earlier reported in south west region of Nigeria (Idigbe et al, 1986; Olusoji et al, 2011), but much lower than 16% and 15% reported in Cross River State and northern Nigeria respectively (Pokam and Asuquo, 2012; Aliyu et al, 2013), and 23.1% reported in Jos (Mawak et al, 2006). NTM and MTB infections share clinical radiographic similarities. They tend to be more common among older age groups, people with pre existing lung conditions, cases of advanced HIV disease and may take long to treat, often with poor outcome compared to MTB (Sakatani, 1999; Griffith et al 2007; Singh et al, 2007). However, in this study, we found that both NTM and MTB infected cases were older in age. This is contrary to earlier report by Aliyu et al (2013), although it agrees in part with their report in that the disease was more common among younger patients with HIV. This difference may be because of the lower number of patients who presented to the clinic in this age category. We did not find any association between NTM and population environment (rural or urban dwellers). Given the very low sensitivity of the standard of smear diagnostic test and absolute specificity of the newly WHO recommended point-of-care diagnostic (GeneXpert) for the NTM, a mechanism for the routine identification of NTM infections in high burden resource limited areas of the world is urgently needed (Marlowe et al, 2011).

In this study, majority of Mycobacterium tuberculosis complex (MTBC) cases were caused by M. tuberculosis with few cases caused by M. bovis. The 11.4% prevalence of M. bovis reported in our study is comparable though lower than recent report in Ibadan (Cadmus et al, 2006). Our finding agrees with recent evidence that strains of M. bovis different from the cattle strains infect humans (Cadmus et al, 2006). This further strengthens the speculation on the possibility of human-to-human airborne transmission of bovine tuberculosis and its relative contribution to new infections in humans (Cosivi et al, 1995). This suggests that in addition to ingestion, an inhalational route of transmission from cattle to human may occur with those working with infected livestock on farms or slaughter houses. Closeness of people in the rural areas to domestic and wild animals might be the reason for the high prevalence of M. bovis in the rural areas as reported in our study.

The atypical mycobacterium isolated from the sputum specimen was Mycobacterium avium complex (MAC). This organism has been reported in literature as significant NTM that is responsible for PTB-like infections in humans (Massenkeil et al, 1992; Chaisson et al, 1992; Nightingale et al, 1992; Chin et al, 1994; von Reyn et al, 1994; Hoover et al, 1995; Henry et al, 2004; Cassidy et al, 2009). Currently, many species of NTM are recognized as potential pathogens. With AIDS epidemic, atypical mycobacteria have taken a new importance with the recognition that the M. avium complex (MAC) generally infect AIDS patients more so when their CD4+ cell count decreases below 200/mm3. 90% of mycobacterial infections in patients with AIDS involve either MTBC or MAC while the remaining 10% of infections is accounted for by various NTM (Sivasankari et al, 2006).

Molecular techniques showed that 3 of the 124 smear positive samples were not members of the genus Mycobacterium, despite the fact that they were isolated from sputum smear positive patients. It may be that these organisms are Nocardia spp. or Tsukamurella spp. The incidence of Nocardiosis especially Nocardia asteroids complex has been on the increase due to increase in the number of immunocompromised patients during the recent decades (Márquez-Diaz et al, 1998). More than 70% of patients with nocardia infections are immunocompromised and disseminated nocardiosis is associated with several immunocompromising conditions. Nocardia spp. and Tsukamurella spp. have also been associated with pulmonary diseases in humans (Ray et al, 1997; Alcaide et al, 2004; Armelle et al, 2009; Ani et al, 2012). All three genera (Mycobacterium, Nocardia and Tsukamurella) belong to the same family Actinomycetales with mycolic acid cell walls (Chun and Goodfellow, 1995; Nam et al, 2003). More recently, HIV infection has been described as a risk factor for disseminated nocardiosis (Malladi et al, 2010).
In the present study, 24% of the HIV-seropositive patients had TB. This is much lower than the 40% and 32.8% prevalence of active TB reported among HIV-seropositive patients in the Nigerian cities of Ilorin (Salami and Katibi, 2006) and Ibadan (Awoyemi et al, 2002) respectively. However, it is higher than 10.5% reported in Northern Nigeria (Zubairu and Musa, 2009) and much higher than that in the United States (Albalak et al, 2007). Evidence from areas with high TB and HIV burden indicates a high incidence of TB/HIV co-infection (Brodt et al, 1997; Mocroft et al, 1998; Race et al, 1998). The differences observed between our centre and other Nigerian centres could be due to selection factors: the other studies were conducted before free antiretroviral drugs were provided in government hospitals. Antiretroviral drugs became free of charge in Nigeria in 2005, before which only those who could afford therapy went to hospitals, sometimes as a last resort, which would have resulted in a pooling of late-stage patients at these hospitals. Several factors have been suggested to contribute to the variation of the smear positivity rate. Among them are the demographic position of the patient (Nun and MeAdam, 1985) and their socio-economic status (Harries, 1990). It has been observed that TB thrives most in communities in which poverty and destitution abound (Onipede et al., 1999). The age distribution reveals highest prevalence to be in the age group 31 – 40 yrs (24.5%) followed by 21- 30yrs (22.5%), which represent the most productive age group economically and correlates with work done by Onipede et al(1999) at Ile-Ife. This can have a serious negative effect on socio-economic status of a country being the economically productive age group.

Out of the 24 co-infected cases, 11 (45.8%) had CD4 count less than 200cells/μl This shows that a low CD4 count may increase the chance of developing TB. In the medical literature, there is no clear cutoff for CD4 count above which the risk for TB development is diminished. However, there is a clear inverse correlation between CD4 count and the risk of opportunistic infections and death (Barnes and Barrows, 1993; Nunn et al, 1997). Smear positivity was significantly associated with severe immune-suppression (CD4 count <200cells/µl) (P <0.05). Markowitz et al (1997) identified 2 major risk factors for TB progression: 1. a positive result on a protein purified derivative test at baseline or during the study, which indicates the importance of the degree of previous or current exposure to Mycobacteriumtuberculosis, and 2. a low CD4 count, which shows the role of immunedepression in the development of active TB. Development of active TB is often prevented by the host’s intact immune system (Schluger and Rom, 1998), but this is the target of HIV infection. In TB/HIV co-infected patients therefore, there will be a steady deterioration in this protective capacity of the cell-mediated immunity till a critical point at which tubercle bacilli begin to proliferate and cause clinical disease (Schluger and Rom, 1998).

Educational status shows that all the species were more prevalent among patients with low literacy level in both HIV positive and negative populations. This might be due to increased level of awareness of the risks, signs and symptoms of TB among population with high literacy level. The association of educational status with awareness of TB signs may be attributed to higher literacy. Higher level of educational attainment is often a factor for better family income. Families that have high income are able to purchase household assets like television, radio and Wi-Fi internet, and other communication appliances that increase their knowledge of health-related matters which are of public concern. Similar studies have also supported our observation that literates were more likely to be aware than the illiterates of signs and symptoms of TB (Malhotra et al, 2002; Ali et al, 2003; Yadav et al, 2006; Sharma et al, 2007; Vukovic et al, 2008).


The data obtained in this study provides some evidence of the role of nontuberculous AFB organisms in pulmonary tuberculosis especially in HIV patients. The comparable ineffectiveness of the standard of smear test in identifying the NTM infection underscores the need for a cheaper, easier, highly sensitive and specific TB screening protocol for effective disease control. The high prevalence of clinical pulmonary tuberculosis due to M. bovis linked to HIV co-infection (20.8%) as reported in our study presents novel public health challenge which needs to be considered when planning prevention and treatment of the disease. The challenge therefore in most African countries and especially Nigeria still remains the introduction in a large scale of laboratory procedure for the specific identification of mycobacteria. Introduction of molecular screening assays that include rapid detection of NTM infections in high burden resource limited settings should be a priority for strengthening the public health response.

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