Biochemical profiling of tuberculosis patients co-infected with hepatitis C virus

Abstract

The present research work was planned to investigate the biochemical parameters in tuberculosis (TB) patients in comparison with healthy individual and TB patients co-infected with hepatitis C virus (HCV). For the study, we determined liver enzymes, serum proteins, hemoglobin photometrically, and erythrocyte sedimentation rate (ESR) by the Westergren method. The results showed a significant (P <0.05) increase in the level of alanine aminotransferase in the Test group (TB + HCV patients) and the TB control group when compared with the healthy control group. A non-significant (P >0.05) increase in the level of aspartate aminotransferase was observed in the Test group while a non-significantly (P >0.05) reduced level was observed in the TB control group when compared with healthy controls. Alkaline phosphatase was increased significantly (P <0.05) in the Test group but non-significantly (P >0.05) in the TB control group compared to healthy control group. Significant (P <0.05) variation in the concentration of serum proteins among different groups in the studied population was also observed. Significant (P <0.05) variation in hemoglobin concentration was observed only on comparing the Test group with healthy individuals (P = 0.013). No significant (P >0.05) variation in serum calcium level was observed among study groups. ESR was found to be lower in the healthy control group while significantly (P <0.05) increased in the Test and TB control groups. The HCV co-infected patients were investigated through HCV screening by the immunochromatographic method. We concluded that variation in biochemical and hematological parameters observed in the studied population help us to diagnose HCV co-infected TB patients.

Introduction

Tuberculosis (TB) is a chronic infectious disease resulting from many strains of mycobacteria, especially Mycobacterium tuberculosis. ¹ TB can infect lungs and various other body parts like bones, joints, central nervous system (CNS), lymphatic system, and circulatory system. Transmission is through air droplets due to coughing and sneezing. The main symptoms include prolonged cough, fever, bloody sputum, chills, night sweats, and weight loss. ² According to the World Health Organization (WHO), 13% of all TB cases are co-infected with HIV. ³ In terms of expected TB cases, Pakistan is considered one of the 22 high burden countries. ⁴

Hepatitis C is an inflammation of the liver caused by hepatitis C virus (HCV) which is transmitted through blood transfusions, contact with infected body fluids, sharing needles, and other personal items with infected persons. The main symptoms include loss of appetite, fatigue, fever, abdominal pain, swelling, jaundice, and dark urine. ⁵ Among infectious diseases, TB is considered the main cause of death. ⁶ Regarding some biochemical parameters, liver enzyme fluctuates representing hepatic dysfunction. It is suggested that HCV infection should be ruled out before anti-TB therapeutic regimen. The present research was planned to study the selected biochemical parameters including liver enzymes, serum proteins, hemoglobin concentration, and erythrocyte sedimentation rate (ESR) in the enrolled human participants.

Material and methods

This research work was designed to compare and evaluate specific biochemical and hematological markers in healthy controls, TB patients, and HCV co-infected TB patients. Informed consent has been taken from all volunteers before inclusion into study population. A total of 45 adult male volunteers (age range, 25–40 years) were selected for the study and then divided into three groups: Control 1 (healthy individuals, n = 5); Control 2 (TB patients, n = 5); and Test (HCV co-infected TB patients, n = 35). The volunteers were enrolled from different hospitals of Faisalabad including District TB hospital, General Hospital, and Civil Hospital. Blood samples were collected from study participants and screened for HCV infection by the immunochromatographic method. Sample processing and analysis was carried out in Bio-Medical Research Lab, Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan.

Determination of hemoglobin and erythrocyte sedimentation rate

Hemoglobin in blood was determined by the method illustrated by Bhaskaram et al. ⁷ Potassium ferricyanide oxidizes hemoglobin into methaemoglobin which is further converted into cyanmethaemoglobin by potassium cyanide and absorbance was measured at 540 nm. ESR was determined by the Westergren method described by Lee et al. ⁸ in which red corpuscles settled down due to their differential densities and medium and measured in mm/h.

Liver enzymes, serum proteins, calcium, and inorganic phosphate

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by photometric method as stated by Rietman and Frankel. ⁹ Alkaline phosphatase (ALP) was determined following the method illustrated by Schlebusch et al. ¹⁰ Total protein was measured by the biuret method according to the protocol described by Henry et al. ¹¹ while serum albumin was measured through the Bromocresol purple method described by Pinnell and Northam. ¹² The globulin concentration was calculated by subtracting albumin from total protein. The A/G ratio was also calculated by dividing albumin with globulin. Serum calcium and phosphate was determined using the photometric method described by Mahmood et al. ⁵

Statistical analysis

The data obtained were analyzed by ANOVA to determine the significance of results using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). Mean ± SD was calculated using Microsoft Excel.

Results

The results showed significantly (P = 0.038) increased ALT in the Test group when compared to the Control 1 group while a non-significant increase in ALT was also observed in the Test group compared with the Control 2 group. A highly significant (P = 0.016) increase in ALT was observed in the TB control group compared to healthy controls. AST was found to be increased non-significantly (P = 0.518) in the Test group in comparison to healthy as well as TB patients. AST was reduced non-significantly (P = 0.317) in TB patients compared to healthy individuals. A statistically significant (P = 0.002) increase in ALP was observed in the Test group when compared to the healthy individuals while non-significantly (P = 0.530) increased when compared to TB patients. On comparing healthy individual with TB patients, a significant (P = 0.004) increase in ALP was observed in TB patients. Total protein concentration was increased significantly (P = 0.028) in the Test group compared to healthy individuals while a significantly (P = 0.004) lower concentration was observed in the Test group compared to TB patients. Variation in serum albumin and globulin was also observed in different groups under study. No significant variation of albumin concentration was observed between the healthy individuals and the Test group while a significantly (P = 0.007) decreased level was observed in the Test group compared to TB patients. A significant (P <0.05) variation in serum globulin was also observed in different groups. No significant variation in serum calcium was observed among individuals in the study. A significantly high level of serum phosphate was observed in the Test group compared to healthy individual and TB patients. A significant variation in hemoglobin concentration was observed only on comparing the Test group with the healthy control group (P = 0.013) whereas a highly significant (P <0.05) variation in ESR was observed among individuals of different study groups. The ESR was found to be lower in healthy individuals compared to TB patients and the Test group. The results are shown in Figures 1 and 2.

Figure 1.

Overall comparison of the mean ± SD of studied biochemical parameters among different groups in the studied population.

Figure 2.

Overall comparison of the mean ± SD of liver enzymes and ESR among different groups in the studied population.

Discussion

Enzyme activities in tissue and body fluids can be measured for disease investigation and diagnosis. Enzymes do not originate from serum but can be derived from disintegrated, metabolized turnover tissues and blood cells. ¹³ Sun et al. ¹⁴ reported elevation in aminotransferases five times greater than normal limits indicating hepatitis in patients receiving anti-TB treatment. According to Kwon et al. ¹⁵ HCV sero-positive patients receiving TB treatment showed elevated liver transaminases. Moses et al. ¹⁶ reported significantly lower serum total protein and albumin in pulmonary TB patients compared to controls while globulin was significantly higher in TB patients. Previous studies reported that patients receiving anti-TB treatment may develop hepatotoxicity. Drug-induced hepatotoxicity (DIH) patients showed lower serum albumin. Studies suggested that serum albumin <3.5 g/dL was the risk factor for DIH patients. According to Morris et al., ¹⁷ 72% of selected TB patients showed hypoalbuminemia. Our study also revealed lower levels of serum albumin in the Test group. Emokpae et al. ¹⁸ found hypercalcemia associated with chronic granulomatous diseases like TB. In our study, we observed no significant variation in serum calcium level among different study groups. Okogun et al. ¹⁹ analyzed serum calcium and inorganic phosphate in TB patients and found significantly increased inorganic phosphate in TB patients than in healthy control. Hemoglobin concentration was only significantly decreased in the Test group when compared with healthy individuals. We also found an elevation in the ESR of the Test group. Our results therefore coincide with the findings of Okafor et al. ²⁰ as they also reported an elevation in the ESR of TB patients rather than that of control participants.

The study concluded that significant variations in hematological and biochemical parameters of HCV co-infected TB patients from healthy individuals and TB patients suggests the determination of these parameters along with determination of HCV antibodies to rule out HCV infection before starting the anti-TB therapy.