The role of Mycobacterium tuberculosis antigen specific cytokines in determination of acid fast bacilli culture status in pulmonary tuberculosis patients co-infected with human immunodeficiency virus

Introduction The interaction between Mycobacterium tuberculosis and HIV leads to rapid progression of tuberculosis (TB) and human immunodeficiency virus (HIV)-induced immunosuppression. Diagnosis of TB in these patients is more difficult due to its atypical presentations giving contradicting results. The overall aim of this study was to evaluate the ability of pro-inflammatory cytokine (Th1) and anti-inflammatory cytokine (Th2) to discriminate between culture-positive and -negative smear status in HIV-TB co-infected patients. Methods In a prospective cohort, a total of 86 study participants were recruited: 46 culture-negative and 40 culture-positive. Blood and sputum samples were collected from all participants. The blood was then analyzed using FACSCalibur flow cytometer to immunophenotype the cells and ELISA performed for cytokine profiles. Sputum samples were analyzed to determine smear status using direct microscopy and Lowenstein Jensen medium. Statistical analyses were performed using R software. Independent samples t-test was used to compare means between the two groups, while the medians were compared using two-sample Wilcoxon rank sum test. Pearson's Chi-square test was used to compare the proportion of male and female participants across the culture and AFB smear status. In order to determine the predictive power of Th1 and Th2 in discriminating Pulmonary Tuberculosis status (PTB) (culture status was used as a confirmatory test), binary logistic regression models were fitted for Th1 covariates [IFN-γ, TNF-α, IL-2 and IL-12(p70)] and Receiver Operating Characteristic (ROC) curves plotted. Results The overall mean age of the participants was 39 years (SD=12), 42% being male. Although, lymphocytes counts were higher in culture-positive relative to culture-negative, the CD8, CD19, and CD16/CD56 were comparable in the two groups. The CD4 counts differed between the two groups (P=0.012). The Th1 showed a better discrimination between culture-positive and -negative PTB individuals; IFN-γ (P=0.001), TNF-α (P=0.001), IL-2 (P=0.001) and IL-12(p70) (P=0.016). The Th2 cytokines (IL-4, IL-6 and IL-10) were comparable between the culture-positive and -negative groups. However, when the combination of Th1 cytokines [IFN-γ, TNF-α, IL-2 and IL-12(p70)] was fitted in binary logistic regression models, the predictive power was high with area under curve (AUC) being 89.7% in discriminating PTB. Conclusion This study provides evidence for the ability of a combination of Th1 cytokines in discriminating against culture-positive and culture-negative PTB.


Introduction
Tuberculosis (TB) is one of the most common opportunistic infections in people infected with human immunodeficiency virus or acquired immunodeficiency syndrome (HIV and AIDS). Moreover, it is the leading cause of mortality and morbidity amongst persons with HIV and AIDS [1]. In 2014, an estimated 9.6 million people were infected with TB according to WHO-2015 global TB report, with 1.5 million people dying from the disease; 1.1 million were HIV-negative and 0.4 million HIV-positive [1]. Previous studies have revealed that one in every three person who died of AIDS was due to TB infection [2]. As such, delayed diagnosis of TB and initiation of treatment after more than three weeks of presentation was responsible for 45-85% of deaths reported among HIV-infected patients [3]. The clinical presentation of TB is often unmodified in HIV-infected immune-competent individuals. As HIV infection progresses, the immune responses weaken resulting in attenuation of host tissue-damaging responses and subsequent failure of Mycobacterial containment. This increases the likelihood of atypical presentation in these patients with greater proportions of extra-pulmonary and disseminated disease being reported [4]. This necessitates the need for further sputum samples examination even if the chest radiograph was normal. This observation is due to the fact that pulmonary cavitations are less common in HIV-positive patients consequently reducing the sensitivity of sputum microscopy for acid-fast bacilli (AFB) significantly [3]. Furthermore, it has been demonstrated that diagnosis by culture is time-consuming [5]. It is thus critical that emphasis is put on early detection and accurate tuberculosis disease [6]. The Th1 CD4 T cells play an important role in protection against TB by promoting activation of macrophages through the production of IFN-γ, IL-2 and Tumor Necrosis Factor alpha (TNF-α) [7,8]. Thus, it has been postulated that perturbation in Th1 activation mechanism may lead to enhanced development of TB [9]. The CD4 depletion during the course of HIV or functional neutralization of TNF-α as a result of anti-TNF-α antibodies increases the risk of an individual to develop TB [10]. In addition, the differential expression of the Th1 cytokines (such as TNF-α, IFNγ, IL-2, IL-8 and IL-12p70) as well as Th2 cytokines (like IL-10, IL-6 and IL-4) are associated with active TB [11]. Therefore, there is need to have distinct cytokine profiles in order to diagnose active TB. Hence, the present study was designed to evaluate the relationship between Th1 [IFN-γ, TNF-α, IL-2, IL-8 and IL-12(p70)] and Th2 (IL-4, IL-6, and IL-10) cytokine responses in culture smearpositive and -negative PTB patients co-infected with HIV.

Study setting:
The study was carried out at Academic Model for Providing Access to Healthcare (AMPATH) based in Western Kenya.
AMPATH is a collaboration among Moi University School of Medicine (MuSoM), Moi Teaching and Referral Hospital (MTRH) and a consortium of North American led by Indiana University [12].
Previously, AMPATH focused on the delivery of HIV care, but over the past several years, it has broadened its services to include primary health care and chronic disease management, including prevention, diagnosis and treatment services for TB.

Study population:
A prospective cohort of individuals newly diagnosed with pulmonary TB and HIV attending clinics at MTRH and AMPATH. The inclusion criteria were patients aged over 18 years, without prior history of TB or any TB relevant clinical or radiological findings or relapse. Patients who had one or more TB specific symptoms and signs based on 2013 guidelines for management of TB and leprosy in Kenya which were adopted from WHO guidelines on TB were eligible for the study [13]. Recruited study participants were those who voluntarily accepted to be tested for HIV and were naïve for highly active antiretroviral therapy (HAART) and anti TB treatment. Sputum smear-positive andnegatives patients by Ziehl-Neelsen staining were cultured to determine the presence or absence of AFB. Patients who were pregnant, HIV-negative, diabetic, or otherwise immunologically challenged or harboring an autoimmune disease were excluded from the study. These mentioned medical conditions and diseases are associated with the modification of immune responses, and therefore could alter the study's findings and conclusions. The total sample size required to attain a power of 80% to detect an effect size of 0.65, where the estimated IFN-γ mean for the culture negative is 12.99 ± 5.7 pg/ml, and estimated IFN-γ mean for the Page number not for citation purposes 3 culture positive is 48.69 ± 28.78 [14], is a minimum of 72 i.e. 36 culture-positive and 36 culture-negative. Following this criteria, the total sample size for our study was 86 i.e. 40 in the culture-positive group and the 46 in the culture-negative group.

Laboratory procedures
Diagnosis of TB: Sputum and blood samples collected from the study participants for TB diagnosis were analyzed as follows Ziehl neelsen direct microscopy: Sputum samples collected from the study participants were smeared and stained by Ziehl Neelsen   In the current study, the levels of IFN-γ were significantly high in PTB culture-positive as compared to PTB culture-negative study participants. These findings corroborate the findings of a previous study in which it was indicated that patients with culture-confirmed PTB positives had higher levels of IFN-γ [18]. This was irrespective of HIV status of an individual. These observations indicate the potential of IFN-γ to discriminate between PTB culture-positive and PTB culture-negative after stimulation with MTB-specific antigens.
We further assessed the association between TNF-α and PTB culture status. TNF-α is a Th1 cytokine known to play a key role in the formation of granuloma for the containment and protection against Mycobacterium. Reports from previous studies have shown elevated levels of TNF-α in culture supernatants from stimulated whole blood with TB-specific antigens (ESAT-6, CFP-10 and TB7.7 present in QFT-IT assay), and this was in agreement with the current study [19]. In the current study, there were variations in TNF-α levels between PTB culture-positive and PTB culture- investigations support our current observations [20]. It has been shown, that IL-2 reduces the replication of Mycobacterium through activation of CD8 cytotoxic T lymphocyte and the activation of macrophages to release interferons [22]. In this regard, we hypothesize that higher levels of IL-2 reported in culture-positive individuals could be due to presence of the MTB antigens, hence, the differing levels of this cytokine between the two groups. was stimulated with TB-specific antigens. Despite the differences in the methodology, it was still evident that Th1 cytokines were predominant in discriminating between the two groups.

Conclusion
In conclusion, our study provides evidence that the combination of

Competing interests
The authors declare no competing interests.

Acknowledgments
We would like to acknowledge the AMPATH and AMPATH Reference Lab Staff for their support in this study. Finally, our thanks also go to all study participants who made it possible to conduct the studies by agreeing to participate.