Effects of Duration of Treatment, HIV and HCV Co-Infection on Hematological and Hepatic Functions in Libyan Patients with Pulmonary Tuberculosis

Background: Pulmonary tuberculosis (TB) remains a major global health problem despite the availability of effi cient treatment over the last decades. TB is the most common opportunistic infection among HIV patients and complicated the outcome of treatment globally. Aims: The prevalence of HCV infection among TB patients has not fully been investigated and limited data on rates of HCV co-infection in TB patients exist. Therefore, this study was aimed to investigate the effects of duration of treatment with fi rst line anti-TB drug, HIV, HCV and co-infection on haematological and hepatic functions in Libyan patients with pulmonary TB. Methods: A total of 120 Libyan newly diagnosed pulmonary TB patients (74 males and 46 females) with age range of 26 to 41 years old were enrolled in this study. They were selected for a regular follow up on the basis of inclusion and exclusion criteria. Pulmonary TB was confi rmed by chest X-ray and sputum smear in all the patients. Patients were divided into three groups; the fi rst group of 75 patients with no HIV and HCV infections (positive control), the second group of 35 patients who had TB and HIV co-infection, before starting anti-microbial therapy, while the third group consists of 20 patients who had TB and HCV coinfection before starting anti-HCV treatment. All patients received a confi rmation of the 1st line anti-TB drug (isoniazid, rifampin, ethambutol and pyrazinamide for fi ve consecutive weeks) simultaneously. Results: The results showed a signifi cant decrease in white blood cells (WBCs) in all the groups of patients and signifi cant changes in other haematological parameters, there were also signifi cant increases in the hepatic enzyme activities, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), in all the groups which indicate hepatic toxicity. Conclusion: Treatment with fi rst line anti-TB drug simultaneously produced hepatotoxicity after two weeks which is more in HIV and HCV co-infection patients. Research Article Effects of Duration of Treatment, HIV and HCV Co-Infection on Hematological and Hepatic Functions in Libyan Patients with Pulmonary Tuberculosis Fadya A Menesi1, Mabroka A ElMajdob2, Saleh E Mghil3, Isam Denna4, Ghazala Othman2, Mustafa YG Younis5, Faraj El -Shari5, Abdulkader H El-Debani3, Fathi M Sherif6* and Awad G Abdellatif2 1Department of Internal Medicine, Nephrology Unit, Faculty of Medicine, University of Tobruk, Al-Batha, Libya 2Department of Pharmacology, Faculty of Public Health, University of Benghazi, Libya 3Department of Internal Medicine, Faculty of Public Health, University of Benghazi, Libya 4Department of Nutrition, Faculty of Public Health, University of Benghazi, Libya 5Department of Biochemistry, Faculty of Medicine, University of Benghazi, Libya 6Department of Pharmacology and Clinical Pharmacy, University of Tripoli, Tripoli, Libya Dates: Received: 04 January, 2017; Accepted: 27 January, 2017; Published: 30 January, 2017 *Corresponding author: Fathi Mohamed Sherif, Professor. Ph.D., Department of Pharmacology and Clinical Pharmacy, University of Tripoli, P.O. Box: 82 757, Tripoli, Libya, Tel: +00218 91 211 7258, E-mail:


Introduction
Pulmonary tuberculosis (TB) is a serious health problem in Libya [1]. World Health Organization (WHO) estimated TB incidence in Libya for the period of 1990 to 2010 as 40 per 100000 [2]. This incidence rate is considered too high according to the recommendation of the National Institute for Health and Clinical Excellence (NICE) for vaccination and screening in England and Wales [3]. In the late 60s and early 70s, a large number of people from neighbouring countries come to Libya to seek for employment. This increased the TB cases in Libya as large number of TB patients entered the country. In 1973, based on the advice of the Central TB Committee, two legislative actions were implemented by the government. First was to screen all foreigners and local workers for TB before seeking employment or in the course of employment for those already employed. The second concerned anti-TB drugs where TB hospitals and centres became the only dispensing organizations for anti-TB drugs. A study from 1971 to 1976 revealed that a signifi cant decrease in the prevalence of primary and acquired Citation: Menesi  drug resistance [4]. The authors indicated that the decline in new and retreatment cases was mainly due to introduction of the two legislations in 1973.
Anti-TB drugs have been altered throughout several years according to their effi cacy, safety and emergence of resistant strains of mycobacterium TB. For this reason, the 1 st anti-TB drug "streptomycin" is no longer considered because of the toxicity and elevated incidence of drug resistance [5]. Therefore, according to WHO, the current 1st anti-TB drug regiment consists of isoniazid, rifampin, ethambutol and pyrazinamide. These drugs act by inhibiting cell membrane synthesis. Agreeing to WHO [6], the risk factors for adverse reactions for anti-TB drugs include, age (> 60 years), hepatic and/or renal diseases, human immune defi ciency virus (HIV) or hepatitis C virus (HCV) co-infection as well as sodium or albumin defi ciency. Adverse drug reactions of anti-TB drugs can be classifi ed as gastrointestinal tract (GIT), neurologic, hepatic, immune mediated and some other adverse drug reactions. Isoniazid, pyrazinamide and rifampin have hepatotoxic potential and can lead to such reactions during anti-TB chemotherapy. Most of the hepatotoxic reactions of anti-TB drugs are dose related and usually occur in the initial few weeks of intensive therapy [7].
Till now, the exact mechanism of anti-TB drug hepatotoxicity is not completely understood but the toxic metabolites are suggested to play a crucial role, at least in case of isoniazid [8]. The degree of severity of hepatotoxicity induced by anti-TB drugs is identifi ed according to the WHO toxicity classifi cation standards [9,10] as grade-I: mild hepatotoxicity is defi ned as alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) elevation of < 3X the upper limit of normal activities (< 120 IU/L) and resolved spontaneously despite continued anti-TB therapy. Grade II: moderate hepatotoxicity occurs as both enzymes elevated 3-5X the upper limit of normal activities (121-200 IU/L). Grade III: very severe hepatotoxicity exists where enzymes elevation > 10X the upper limit of normal activities (201-400 IU/L) and grade IV: very severe hepatotoxicity > 10X the upper limit of normal of ALT and/or AST activities (> 400 IU/L) or more than 250 IU/L, if accompanied by symptoms such as nausea, vomiting, abdominal pain, and jaundice. Previous studies also revealed that hepatotoxicity induced by anti-TB drugs is increased four-to fi ve-fold in patients with HIV and HCV coinfection, respectively [11,12].
Tuberculosis is considered to be the most common opportunistic infection among HIV patients and complicated the outcome of treatment [13]. In adults, it is estimated that about 15% of all new TB patients are attributed to the HIV infection [14]. HIV infection signifi cantly increases risk of progression from latent to active TB [15,16]. The fi rst priority for HIV-positive TB patients is to initiate TB treatment followed by anti-retroviral drugs and cotrimoxazole [9]. It is well known that HIV infection causes a slow decline in cluster of differentiation 4 (CD4+) cells in most patients and consequently, the prevalence leukopenia is increased with declined CD4+ cell counts [17]. For long period of time, CD4+ cell counts were considered to be the best predictor of the disease stage and risk of developing AIDs related complication. HCV has also emerged as an important global health problem.
WHO estimated that 3% of the world population is infected with HCV and more than 170 million chronic carriers are at risk of developing liver cirrhosis and/or liver cancer. Globally, the prevalence of HCV infection among patients with TB has extensively been investigated, however, with a very limited data on rates of HCV co-infection among patients with TB exists [9,18]. To the best of our knowledge, no previous studies have been reported regarding the effects of duration of treatment and co-infections with HIV and HCV on patients with pulmonary TB. In Libya, the prevalence of HCV infection among TB patients has also not been investigated at all, and inadequate data on rates of HCV co-infection in TB patients exist. Thus, the aim of the present study was to explore the effects of duration of treatment with 1 st line anti-TB drug, HIV and HCV co-infection on haematological and hepatic functions in Libyan patients suffering from pulmonary TB.

Protocol of the study
This study was planned as unicenter study and conducted at Alquefi a Chest Hospital, Benghazi, Libya (2015). The protocol was approved by the Ethical Research Committee of University of Benghazi (2014). All the participants of the study gave informed consent to the doctor for using their data determined during the course of treatment at the hospital for this purpose. The protocol was designed as retrospective study.

Retrospective study
The study included pulmonary TB patients with negative HIV and HCV infection (positive control), pulmonary TB patients with positive HIV and pulmonary TB patients with positive HCV who were subjected to 1 st line anti TB drug when admitted to the hospital and before start of the study. For all the patients, the effi cacy and safety of anti-TB drugs were evaluated.

Patients
A total of 120 Libyan newly diagnosed pulmonary TB patients were enrolled in this study. They were selected for a regular follow up on the basis of inclusion and exclusion criteria. The number of male and female patients were 74 and 46, respectively. Patients who participated in this study were aged between 26 to 41 years old and had sputum smear positive AFB. All the patients were interviewed for their medication history, concomitant diseases before their participation in the study. Patient's name, gender, age, fi le number, date of admission and discharge from the hospital were recorded.

Exclusion criteria
Non Libyan pulmonary TB patients, extra pulmonary TB patients, pulmonary TB patients returning after defaulting or relapsing from their fi rst treatment course (previously treated by anti-TB drugs), MDR-TB, alcoholic, drug abusers, smokers, liver diseases except in the third group, HIV infection except in the second group, renal and cardiovascular diseases, patients receiving other potentially hepatotoxic drugs (methotrexate, Citation: Menesi  phenytoin, valproate, acetaminophen, fl uconazole), diabetes mellitus, thyroid and connective tissue diseases.

Methods
The patients were divided into three groups according to the presence or absence of co-infection (Table 1)

Data collection
All data was collected from the fi les of the patients: patients

Effi cacy and safety evaluations
Effi cacy outcome: patients were observed for improvement of the primary effi cacy variables (WBC and ESR) reduction and rise of Hb at two and fi ve weeks of treatment relative to the baseline. Sputum was also examined for follow up.
Safety outcome: Safety was assessed on the basis of adverse events reported during the study and it was measured by the effect of drugs on hepatic and renal functions.

Statistical analysis
All data were expressed as mean ± S.E.M. Data were analysed by analysis of variance test (one-way ANOVA). If this analysis indicated a signifi cant difference among the means then multiple comparisons between the individual groups were tested by post-hoc test (LSD) or unpaired t-test. A value of *p < 0.05 is considered signifi cant, **p < 0.01 is highly signifi cant and ***p < 0.001 is very highly signifi cant.  Table 2 shows values of white blood cells (WBC) determined at the baseline, two and fi ve weeks after the start of anti-TB treatment. The duration of treatment (after 2 weeks and 5    Figure 4 shows that duration of anti-TB treatment (5     Table 3 shows duration of treatment (2 and 5

Discussion
Pulmonary TB remains a major global health problem for the last decades despite the availability of effective pharmacotherapy. WHO declared TB a global public health emergency in 1993 [19]. The disease affects mainly third world countries. According to WHO, eight thousand million people are infected by this disease. Pulmonary TB is responsible for 8.9 million new cases each year, and for two million deaths around the world [20]. Pulmonary TB is considered to be high    in Libya despite the enforced legislations to control it, the legislations was fi rst introduced in 1973. These legislations caused decline in primary and acquired resistance [4]. Drug resistance is a major problem in treatment of TB and drug combination is used to minimize this problem. TB is also considered to be the most common opportunistic infection among HIV patients, and complicated the outcome of treatment [13]. There is a lack of data concerning the effect of HIV and al. [22,23], are in line with the present data and showed that patients having higher Hb levels were less likely to develop TB than those with low Hb levels.
The present results also indicated a signifi cant decrease in the counts of WBC with duration of anti-TB therapy in all the groups. This decrease was most signifi cant in HIV groups.
The results indicated eradication of TB infection due to anti-TB therapy, however, HIV co-infected patients had a very low WBC counts compared with the other groups due to immune defi ciency in HIV group. These fi ndings are in line with the previous reported fi ndings [24]. The present data also revealed that number of blood platelets was signifi cantly declined in HIV and HCV co-infected groups in comparison with the control group. Rifampin induces thrompocytopenia which is not common but potentially life threatening complication [25].
It has been known that rifampin-induced thrombocytopenia is caused by the presence of anti-rifampin antibodies [26].
These antibodies fi x a complement on the blood platelets in the presence of rifampin resulting in platelet destruction [27]. Other studies by Rieg et al., and Torre et al. [28,29], showed that thrombocytopenia is a common fi nding among HIV infected patients due to bone marrow suppression. On the other hand, chronic infection with HCV may produce a signifi cant autoimmune reaction to blood platelets, leading to thrombocytopenia [30]. Other mechanisms of thrombocytopenia include a decrease of thrombopietin levels or reduced bone marrow production of platelets [31].  [32]. Other possible explanation for the elevated ESR in HCV infected patients is the host response to HCV infection [33]. In the present study, treatment with 1 st line anti-TB drug caused a signifi cant increase in the levels of total bilirubin in all the patients, and there was no difference in total bilirubin of HIV and HVC groups when compared with control group. This increase may be due to rifampin which may inhibit the major bile salt exporter [34]. Asymptomatic elevated bilirubin may also result from dose dependent competition with bilirubin for clearance at the sinusoidal membrane or from impeded secretion at the canalicular level [35].
In this study, the pattern of alteration of liver enzymes was evaluated. Thus, the normal maximum values in the laboratory are 45 IU/L for ALT and 40 IU/L for AST which were same cutoffs for male and female. The activities of ALT and AST after treatment with anti-TB drugs were signifi cantly elevated above the upper normal limit but remained below the three times the upper normal limit (< 120 IU/L) indicating grade I hepatotoxicity according to WHO classifi cation [6]. These increases in hepatic enzyme activities were transient and resolved with continued use of anti-TB drugs. These fi ndings are in good agreement with previous study [9]. The time between initiation of anti-TB therapy and elevation of liver transaminase enzyme activities of most of our cases occurred from two to fi ve weeks from start of anti-TB drugs. Different previous studies reported comparable fi ndings [36]. The exact mechanism of anti-TB drug induced hepatotoxicity is unknown but toxic metabolites are suggested to play a major role at least in case of isoniazid [8]. Ungo and his colleagues [11] reported that co-infection with HIV and HCV increased the risk of grade I hepatotoxicity which is in line with the present fi ndings. Additionally, the present data indicated that treatment with anti-TB drugs signifi cantly increase ALP activities in all the groups, however, this increase in HIV and HCV co-infected patients was more signifi cant compared with that of the control group. This fi nding is in agreement with the fi ndings by Toppet et al. [37], who reported an increase in the serum ALP levels in pulmonary TB patients on anti-TB therapy.
Elevations of ALP and/or bilirubin with little or no increase in ALT activities indicated cholestasis while increase of ALT and AST activities indicated a sign of hepatic necrosis. Thus, it is concluded that treatment with 1 st line anti-TB drug produced hepatotoxicity after two weeks from start of the treatment and it is more observed in HIV and HCV co-infection patients.
However, clinical signs of jaundice were negative.