¹ Department of Internal Medicine, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar 190011, India;

² Advanced Centre for Human Genetics, SKIMS, Srinagar 190011, India;

³ Immunology and Molecular Medicine, SKIMS, Srinagar 190011, India

Corresponding Author: Dr. GhulamNabi Dhobi, MD, Professor, Department of Internal Medicine (Infectious Disease unit), Sher-i-Kashmir Institute of Medical Sciences, Srinagar 190011, J&K, India. Tel: 0194-2401014; Fax: 0194-2401013; E-mail: bagdadifarhana6@gmail.com.

Note: Dr. Arshad A. Pandith and Dr. Mohmad H. Mir contributed equally.

	ABSTRACT
	INTRODUCTION
	MATERIALS & METHODS
	RESULTS
	DISCUSSION
	ACKNOWLEDGMENTS
	CONFLICT OF INTEREST
	REFERENCES

	ABSTRACT

Background/Purpose: Tuberculosis (T.B) an infectious disease caused by Mycobacterium tuberculosis is still one of the biggest killers among the infectious diseases. Immunological deficiencies of various magnitudes have been reported in TB which include CD4 + T and CD8 + T cells. The aim of the study was investigating the at the occurrence of CD4+ T cell lymphopenia in patients with active pulmonary tuberculosis and the response of this abnormality to ATT in HIV negative patients. Methods: The study subjects included three groups in which 30 patients were with sputum positive pulmonary tuberculosis without any evidence of HIV infection and second group of10 healthy controls. Third group included10 patients with pulmonary tuberculosis co-infected HIV sero-positivity. Zeil Nelson (ZN) method was used to diagnose pulmonary tuberculosis, ELISA method was adopted to confirm the HIV status and CD4 and CD8 count was done by flow cytometry. Results: The CD­4 + T cell lymphopenia was observed significantly associated (p=0.000) in Non-HIV pulmonary tuberculosis cases as compared to controls (370.87 ± 209.00 cells/µl v/s 673.60 ± 120.30 cells/µl).CD4 + T cell lymphopenia was significantin HIV positive pulmonary TB patients compared to healthy controls (p=0.000). CD4 + T cell lymphopenia was more severe in HIV co-infected pulmonary tuberculosis patients compared to Non-HIV pulmonary TB cases (p=0.029). Conclusion: We conclude that the pulmonary tuberculosis is an important cause of non HIV CD4, CD8 lymphopenia, with reversal of CD4/CD8 ratio, and this is reversible with effective treatment with antitubercular therapy (ATT).

	INTRODUCTION

   Tuberculosis (T.B) an infectious disease caused by Mycobacterium tuberculosis (MTB) is not only one of the leading causes of death from any infectious disease world wide (1) but also a severe public health problem. Its severity has lead declared as “a global health emergency by the World Health Organization” (1).

   Approximately one third of the world’s population is infected with Mycobacterium tuberculosis (MTB), with 6-8 million new active cases reported each year thereby accounting for 2–3 million deaths annually in developing countries (2).

   Rising trend in HIV infection in some countries together with the emergence of multi-drug resistant (MDR) strains of tuberculosis pose an additional threat (3). India is the highest TB burden country in the world and accounts for nearly one-fifth (20 percent) of global burden of tuberculosis, 2/3^rd of cases in SEAR. According to 2006 estimates India has the number one global rank by estimated number of total cases of tuberculosis. Around 80% of all new cases of T.B. are limited to the lungs. However, up to 2/3^rd of HIV-infected patients with tuberculosis may have both pulmonary and extra-pulmonary disease and extra-pulmonary alone (4).

   Active T.B is associated with an increase in the number of absolute lymphocytes and decrease in T₄-lymphocytes. Of the various subsets of lymphocytes, the importance of CD4 + T cells is clearly brought out in HIV infection, where their depletion renders the patient exquisitely sensitive to tuberculosis. CD8 + T cells also play a part in protective immunity against MTB infection, forming a cuff at the periphery of epitheliod cell granulomas. Despite the pivotal importance of CD4+ and CD8+ T lymphocytes in mycobacterial immunity, very few researchers have studied changes in peripheral blood counts of these cells in the setting of T.B (5).

   CD4+ T cell lymphopenia is a pivotal immune aberration in HIV positive patients and important changes in CD8+ T cell counts also occur in this disease. However, the independent effect of tuberculosis in general and radiological extent of disease in particular on CD4+ and CD8+ T cell counts has been shown by very few studies, the effect of ATT on these counts has not been well studied. There is however equivocal contention that tuberculosis is a cause of non HIV associated CD4+ T cell lymphopenia and such lymphopenia is reversible with the treatment (6). Predominant T4 lymphopenia is a common finding in pulmonary T.B (7, 8) .This T4 cell depletion appears to be secondary to increased recruitment of sensitized T4-cells to the site of granuloma formation in lungs and in glands etc. Such a sequestration of the cells is demonstrated in infected lymph nodes of patients with tubercular lymphadenitis (9) and in tubercular pleural effusion (10). The intensity of lymphopenia is unrelated to the extent of lesions, seen radiologically, and the tuberculin test response (11, 12). CD4 T cell lymphocytopenia had been previously shown in both smear positive (13-16) and smear negative pulmonary tuberculosis (14). It has been seen that patients with pulmonary tuberculosis had lower CD4 counts than control groups with more severe disease (17, 18).

   There has not been a single study about CD4 counts and pulmonary tuberculosis in this region so far. In this back drop the present study was carried out in this Institute to look at the occurrence of CD4+ T cell lymphopenia in patients with active pulmonary tuberculosis (sputum positive) and the response of this abnormality to ATT in HIV negative patients.

	MATERIALS & METHODS

   The present study was conducted over a period of 2 years in the Department of Internal Medicine and Department of Immunology and Molecular Medicine, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, J&K, India. It was a retrospective and prospective comparative study. Clearance from local SKIMS ethical committee was taken prior to study.

   Study Group

   The following groups of patients/subjects were included in the study after a written informed consent.

   Group 1 (Prospective Study Group). A total of 30 patients with sputum positive pulmonary tuberculosis without any evidence of HIV infection were registered in our study whose CD4+ T-cell and CD8+ T-cell count was determined before starting ATT and after 3 months of ATT intake. Pulmonary tuberculosis was established by sputum positivity for AFB and HIV was ruled out by absence of high risk factors and negative serology for HIV 1 and 2.

   Detailed history was recorded and complete physical examination was performed in all these patients. Routine investigations were done on all patients, in addition to specific investigations for mycobacterium tuberculosis (sputum for AFB, chest x-ray and Monteux test) and CD4+ T-cell and CD8+ T-cell counts. These patients were followed up to the period of completion of ATT. CD4+ and CD8+ T-cell count was again determined at 3 months of ATT. CD4+ and CD8+ T-Cell counts were measured by taking blood samples in K3 EDTA BD vacutainers and processed on a BD Flowcytometer (FAC Scan). From CD4+ and CD8+ T-cells, ratio of CD4+/ CD8+ T cells was derived. Patients receiving any immunosuppressive therapy or with HIV sero-positive/AIDS were not included in this study group.

   Group 2 (Control Group). A total of 10 age, sex, ethnically matched healthy controls who had never been treated for any form of tuberculosis or taken any immunosuppressant drugs were taken in this group. CD4+, CD8+ T-cell counts were determined in this group as well as CD4+/ CD8+ ratio.

   Group 3 (Retrospective & Prospective Study Group). A total of 10 patients were included in this retrospective groups with HIV sero-positivity and pulmonary tuberculosis studied from the records available in the Department of Immunology and Molecular Medicine, SKIMS, Srinagar. New patients with HIV seropositivity and evidence of pulmonary tuberculosis were also included in this study group. CD4+, CD8+ T-cell counts and CD4+/ CD8+ ratio were compared with the patients belonging to the prospective group (Group 1).

   Detection of M. Tuberculosis (MTB)

   Sputum Smear for AFB. Sputum smear were stained by Zeil Nelson (ZN) method after proper procedures and stained smears were visualized under direct microscope for the detection of MTB. MTB appears as bright red rod like structure under microscope. The sputum smear microscopy has a sensitivity of ~64% and specificity of ~98%.

   HIV Testing ELISA. Blood samples were collected in vacutainers and sera were separated on the same day and tested for HIV antibodies on the same or following day of collection, by WHO approved ELISA/Rapid kits supplied by NACO, New Delhi, following its testing guidelines11. All samples were tested by the same kit manufactured by the same company.

   CD4+ and CD8+ T-cell Count Determination by Flowcytometer. CD4+ and CD8+ T-Cell counts were measured by taking blood samples in K3 EDTA BD Vacutainers and processed on a BD Flowcytometer (Becton Dickinson Pvt. Ltd., Mountain View, CA)) using BD true count tubes with beads and BD Tri Test (CD3, CD4, CD8 Cocktail) antibody following a lyse-no-wash protocol. The blood samples were processed immediately within 2 h of collection, for determining the absolute counts of CD4+, CD8+, CD3+ cells and their ratios by two color Immunophenotyping on the single platform fluorescence activated cell sorting (FACS) count system, using fluorochrome labeled monoclonal antibodies to CD4+/CD3+ and CD8+/CD3+ T-cells, strictly following manufacturer’s instructions. The same batches of antibodies were used for testing the samples of subjects of all the groups. The batches, however, differed from time to time. FACS count protocol software versions 1.2 (3/95), 1.3 (2/00) and 1.4 (4/02) (Becton Dickinson) were used for data acquisition and analysis.

	RESULTS

   A total of 30 cases of non HIV pulmonary tuberculosis of patients were studied between 2007 and 2009. The mean age of patients was 44.03 years in which there were 16 (53.3%) males and 14 (46.7%) females (Table 1). A total of 10 patients of Pulmonary Tuberculosis with HIV co infection (HIV-TB co infected) were from records available at ART clinic of SKIMS, (J&K, India). Both retrospective and prospective cases were taken which included 9 males (90%) and 01 female (10%).

   Age, sex, and ethnicity related 10 healthy controls were taken into study for comparison of CD4 + T cell counts including 07 (70%) males and 03 (30%) females.

   The constitutional symptoms were common in our cases. Fever and cough were the most common symptoms each in 90.0% of cases, followed by decreased appetite in 53.3% and weight loss in 36.7% while as in HIV co-infected pulmonary tuberculosis all patients presented with fever were (100.0%), poor appetite 40.0% and weight loss in 10.0%. None of the controls had any family or personal history of ATT intake.

   In non-HIV pulmonary TB Mantoux test was positive (> 10 mm) in 70.0% of cases with two sputum smears for AFB being positive in 36.7% of cases and three sputum smears positive for AFB in 63.3% of cases. In contrast to HIV infected TB, Montoux test was positive in only 01 patient (10.0%) with two sputum smears positive for AFB in 70.0% of patients and 3 sputum smears positive in 30.0% of patients.

   Non- HIV pulmonary tuberculosis patients showed good survival (96.7%) as against 70% in pulmonary tuberculosis patients with HIV co infection during follow up of one year.

   The CD4 + T cell lymphopenia was observed significantly associated (p=0.000) in Non-HIV pulmonary tuberculosis cases as compared to controls. The mean CD4 + T cell counts at the time of diagnosis were 370.87 ± 209.00 cells/µl v/s 673.60 ± 120.30 cells/µl in healthy controls (Table 2). Similar situation of CD4 + T cell lymphopenia was observed in HIV positive pulmonary TB patients compared to healthy controls with significant association(p=0.000) (mean CD4 + T cell in HIV positive pulmonary TB cases were 224.60 ± 101.44 cells/µl vs. 673.60 ± 120.30 cells/µl in healthy controls). CD4 + T cell lymphopenia was more severe in HIV co-infected pulmonary tuberculosis patients compared to Non-HIV pulmonary TB cases (mean CD4 + T cells 224.60 ± 101.44 cells/µl vs. 370.87 ± 209.00 cells/µl; p=0.029) (Table 2).

   Likewise CD8 + T cells were also low in our patients at the time of diagnosis before start of treatment. In Non-HIV pulmonary TB cases the mean CD8 + T cell counts were 399.55 ± 282.12 cells/µl, as against 546.90 ± 242.67 cells/µl in healthy controls (p=0.132) (Table 2). The mean CD8 + T cell counts in HIV co-infected pulmonary TB cases were 915.40 ± 215.11 cells/µl compared to 546.90 ± 242.67 cells/µl in controls with significant difference (p=0.003). The high CD8 T cell counts in HIV co-infected pulmonary tuberculosis as compared to HIV negative pulmonary tuberculosis was statistically significant (p=0.000) (Table 2).

   The CD4/CD8 T cell ratio in HIV negative pulmonary tuberculosis cases at the time of diagnosis was insignificant as compared to normal healthy controls (mean CD4/CD8 T cell ratio 1.18 ± 0.72 vs. 1.39 ± 0.44, p = 0.344). However the CD4/CD8 T cell ratio was very low in HIV co-infected pulmonary tuberculosis patients compared to controls (mean CD4/CD8 T cell ratio 0.25 ± 0.12 vs. 1.18 ± 0.72; with p value of 0.000). The ratio of CD4/CD8 T cell in HIV co-infected patients was again significant to HIV negative pulmonary TB (p=0.000) (Table 2).

   The mean CD4 + T and CD8 + T cell counts were higher after 3 months of treatment in both Non-HIV and HIV co-infected pulmonary TB and compared to their initial counts, and the increase was found significant in Non-HIV (p<0.05) than in HIV co-infected group (p>0.05) (Table 3). However the rise in CD4/CD8 T cell ratio after treatment in both the groups of patients was not significant compared to before the treatment ratios (p=0.208 vs. 0.145) (Table 3). Table 4 shows the CD4 and CD8 cell count response in pulmonary tuberculosis before and after treatment.

	DISCUSSION

   Tuberculosis (TB) remains one of the leading causes of death from any infectious disease worldwide (19). The problem of TB is so severe that this time it is the only infectious disease to be declared as “a global health emergency by the World Health Organization (1). India is the highest TB burden country in the world where almost 0.37 million people die every year of TB (20, 21).

   Mycobacterium Tuberculosis (MTB) is a classic example of a pathogen for which the protective response relies on cell mediated immunity. Both CD4+ and CD8+ T Cells are important for successful immunity to TB. They have many effecter functions such as cytolysis and release of potent antimycobacterial cytokines like Interferon-gamma and tumor necrosis factor- alpha (22).

   The CD4+ T Cells are considered to be primary T-Cell subset responsible for regulating the immune response of MTB (2). The importance of CD4+ T Cells is clearly brought out in HIV infection, where their depletion renders the patient exquisitely sensitive to TB. Predominant CD4 + T-Lymphopenia is a common finding in pulmonary (7, 8). Active pulmonary TB appears to be associated with marked CD4 + T lymphopenia leading to reversal of T4 / T8 ratio. CD4 + T lymphopenia had been previously shown in both smear positive and smear negative pulmonary TB (13-16).

   The present study is the first from this region to look for the CD4 + T cell lymphopenia in HIV negative sputum positive pulmonary TB patient and to see its reversibility with anti tubercular treatment (ATT), and to compare it with healthy controls and HIV positive pulmonary TB co-infected patients. Also we looked at CD8+T cell counts and CD4 + / CD8 + ratio in HIV negative sputum positive pulmonary tuberculosis patients and the effect of ATT on it and compared it also with the controls and HIV-TB co-infected cases. It has been seen that patients with pulmonary TB had lower CD4 + T cell counts than control groups. It has also been shown that patients of pulmonary TB with lower CD4 + T cell counts have more severe disease. The results from present study support these findings. This CDT+4 depletionappear to be secondary to increased recruitment of sensitized T4 cells to the site of granulomas formation in lung and in glands etc. The other explanation is that genetically susceptible patients probably mount more vigorous immunological response to MTB infection, leading in turn to more severe depletion of antigen responsive T cells (due to phenomenon of compartmentalization and selective increased programmed cell death of activated T-cell). This depletion of CD4+ T cells thus leads to spread of the disease (intra or extra pulmonary), because of lack of production of interferon-gamma, interleukin-2 and cytokines known for helping in granuloma formation and thus limiting spread of the disease (2). Studies from many parts of the world have shown higher incidence of TB among HIV infected individuals, ranging from 5 to 10% per year of observations, which is in sharp contrast to the life time risk of 10% among people without HIV22. TB is the commonest opportunistic infection occurring among HIV positive persons in India and it has been estimated that 60 to 70% of HIV positive patients will develop TB in their lifetime (1).

   Our study showed a significant CD4 + T cell lymphopenia in HIV negative pulmonary TB patients at the time of diagnosis as compared to controls (P = 0.000) with CD8 + T cell also on lower side but with insignificant association (P>0.05). But on comparison CD4 + T cell and CD8 + T cell counts and CD4+ /CD8+ T cell ratio of HIV negative pulmonary TB patients with HIV positive pulmonary TB cases, the differences were significant (P<0.05). Our study showed that significant difference between the mean CD4+ T cell counts in HIV positive pulmonary TB cases were lower than in HIV negative patients of pulmonary TB (P<0.05). CD8+ T cell counts in HIV positive pulmonary TB were significantly higher compared to HIV negative pulmonary TB (p<0.05) (Table 2). The CD4 + / CD8+ T cell ratio was very low in HIV positive pulmonary TB cases compared to HIV negative pulmonary TB patients (mean CD4+ /CD8+ T cell ratio of 0.25 ± 0.12 Vs 1.18 ± 0.72) and it was also statistically significant (P=0.000). Beck et al (8) in their study have found that T4 cells were fewer in patients (mean 748/mm³) than in controls (mean 1,043/mm3) however T8 cells were not lowered significantly. The CD+T4: CD+T8 ratio was less than 1.6 in 11 patients and was less than 1.2 (the lower limit of normal) in six patients but no controls. The results of CD+T4 lymphopenia are consistent with our study. Moreover results of the present study are in agreement with the results ofMenon et al (21) who showed an increase in the total lymphocyte count while there is a significant decrease in CD+T4 cells leading to reversal of CD+T4/CD+T8 ratio (23) found that active MTB infection can lead to CD4+ T lymphopenia with counts as low as 300 × 10⁶ /L¹⁶ in their study found that CD4 counts and percentage of CD4 were significantly lower, but CD8 values were normal, in patients with pulmonary tuberculosis (HIV negative) when compared with values obtained in normal blood donors. The CD4/CD8 ratio was significantly lower in patients with tuberculosis. The results of our study are consistent with it except for that the CD8 + T cell counts were also reduced (Table 2). Our results also support the findings ofAziz, et al (2) who found that active MTB infection is associated with profound CD4 + T cell lymphopenia in HIV negative patients and those with CD4+ T cell count < 150 × 10⁶/L are likely to have high mortality and showed reversibility of CD4 T lymphopenia with anti tubercular treatment. Our study is also consistent with the study conducted byDavoudi et al (24) that showed that mean CD4 and CD8 counts of patients with pulmonary tuberculosis was significantly lower than control groups which they thought as a sign of suppressed cellular immunity. The severe decrease of CD4+ T cells in HIV positive pulmonary tuberculosis patients compared to HIV negative pulmonary tuberculosis is because of additive effect of HIV and pulmonary tuberculosis co-infection (25) in their study found that the mean CD4 counts in HIV/TB co-infected patients were significantly lower than those of patients with HIV infection alone which is in complete agreement with our study where we observed profound CD4+ T cell lymphopenia in same group.

   Our study showed that the CD4+ and CD8+ T cell lymphopenia is reversible with treatment by ATT in sputum positive pulmonary tuberculosis who are HIV negative and with combination of anti retroviral therapy and antitubercular in HIV/TB co-infected pulmonary TB cases. With improvement in CD4+ and CD8+ T cells, the ratio of CD4+/CD8+ T cells also improved with the treatment. After starting treatment, at 3 months, the CD4+ and CD8+ T cell counts and the ratio between them was also calculated and it showed a marked improvement (Table 3).

   The mean CD4+ T cell counts at 3 months of treatment were higher in HIV negative pulmonary tuberculosis patients as compared to initial counts. During follow up one patient died due to progressive disease, whose initial CD4+ T cell count was 100 cells/µl and CD8+ T cell count was 161 cells/µl at the time of presentation. The mean CD4+ T cell counts after 3 months of treatment were 612.38 ± 177.44 compared to 383.31 ± 201.07 cells/µl and it was statistically significant (p=0.000) (Table 2). Similarly in HIV/TB co-infected patients the CD4+ T cell and CD8 T counts improved after 3 months of treatment with antitubercular and antiretroviral therapy but could not achieve the significant proportion (p>0.05). Similarity in HIV/TB co-infected pulmonary tuberculosis patients the post treatment CD4+ /CD8+ T cell ratio was 0.34 ± 0.10 compared to 0.23 ± 0.12 before treatment, the increment was not statistically significant (p=0.145). Similar results were obtained in the study byUppal et al (16). They found that the CD4 counts normalized after treatment with ATT in HIV negative pulmonary TB patients. The remaining parameters CD8 cells and CD4/CD8 ratio also improved after treatment. The results in the form of improvement of CD4, CD8 cells and ratio of CD4 / CD8 in HIV negative pulmonary tuberculosis patients after treatment in our study were comparable to results from study conducted by Menon MPS, et al (21). In the study conducted by Aziz, et al (2) who in their study found that all those patients who survived started to recover their CD4+ T cell counts by 3 months which returned to normal within 6 months. This finding showed that CD4+ T lymphopenia produced by MTB infection is reversible with effective treatment of the disease and highlights the importance of cellular immunity (T lymphocytes) in the outcome of tuberculosis (2). The results in the present study are comparable to the above studies.

   Thus from this present study it is clear that pulmonary TB is responsible for T cell lymphopenia. Both CD 4+ and CD8+ T cells get depleted as compared to controls. This got reflected in both pulmonary tuberculosis HIV negative and HIV-TB co-infected patients. However it cannot be conclude whether this depletion was predisposition or a consequence of tuberculosis. But some studies have shown this as a reversible phenomenon which returns to normal with successful anti tubercular treatment, suspecting tuberculosis of causing lymphocytopenia (13, 16, 17, 26). Although the role of effective anti mycobacterial drugs as a major adjunctive cannot be forgotten, the emergence of multi drug resistance mycobacterium poses new problem in controlling this reemerging disease. The need for modalities such as vaccines and immunotherapy is felt more than ever. The more we learn about disease pathogenesis, the faster we can move towards success in battle between human being and mycobacterium tuberculosis (24).

   Our study endorsed the fact that the pulmonary tuberculosis is an important cause of non HIV CD4, CD8 lymphopenia, with reversal of CD4/CD8 ratio, and this is reversible with effective treatment with antitubercular therapy (ATT). Hence it should be looked in all cases of pulmonary tuberculosis to assess the immunological status of patients which may influence the outcome of TB patient.

	ACKNOWLEDGMENTS

   The authors acknowledge the technical help of staff of the Department of Internal Medicine, SKIMS who helped us in procuring blood samples. This work was supported in part by the SKIMS.

	CONFLICT OF INTEREST

   Authors declare that there was no conflict of interest.

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