Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 25, Num. 2, 2007, pp. 126-132

Indian Journal of Medical Microbiology, Vol. 25, No. 2, April-June, 2007, pp. 126-132

Original Article

Estimation of CD4+ and CD8+ T-lymphocytes in human immunodeficiency virus infection and acquired immunodeficiency syndrome patients in Manipur

HR Singh, NGB Singh, TB Singh

Department of Microbiology, Regional Institute of Medical Sciences, Imphal - 795 004, Manipur

Correspondence Address: Department of Microbiology, Regional Institute of Medical Sciences, Imphal - 795 004, Manipur, India (email: dr_rebachandra@yahoo.com)

Date of Submission: 27-Sept-2005
Date of Acceptance: 01-July-2006

Code Number: mb07036

Abstract

Keywords: CD4+ T-lymphocytes, CD8+ T-lymphocytes, human immunodeficiency virus, opportunistic infection, symptomatic

The HIV/AIDS epidemic is a major health concern worldwide with an estimated 39.4 (range 35.9 to 44.3) millions people including women 17.6 (range 16.3 to 19.5) millions and children under 15 years 2.2 (range 2.0 to 2.6) millions affected by HIV virus and more than 3.1 (range 2.8 to 3.5) million deaths due to AIDS as of December, 2004. ^[1] The major share of this devastation occurs in the developing countries and the number of people living with HIV has been rising in every region. ^[11] India alone recorded an estimated 5.1 millions infected people with HIV/AIDS. ^[2] Manipur, a small north-eastern state of India with hardly 0.2% of India′s population, is contributing nearly 8% of India′s HIV positive cases only next to Maharashtra and Tamil Nadu. However, with respect to seroprevalence rate per one million population, Manipur is six times higher than Maharashtra and twenty times higher than that of Tamil Nadu. ^[3] As on July, 2005 a total of 20,524 HIV positive cases (including 4,012 female) and 3490 AIDS cases (497 deaths) were reported out of 1,26,973 blood samples screened giving seropositivity rate of 161.64 per thousand blood samples screened against the all India figure of 40.93. ^[4] Since it was reported that the initial stage of HIV infection involves specific interaction of the virus with the CD4 molecule on the T-lymphocyte surface, the role of CD4 ⁺ T-lymphocytes in HIV/AIDS patients has been extensively studied. A complex interaction between HIV and CD4 ⁺ T-lymphocytes ensues in the HIV infected persons to control the viral replication on the one hand and immune recognition and elimination of the virus infected cells on the other. The present knowledge concerning the staging of disease, monitoring of progression and initiation of therapeutic regimen depend heavily on determination of peripheral lymphocyte sub-populations. ^[5]

Estimation of CD4 ⁺ T-lymphocyte is one of the measures of ascertaining the immune competence of the HIV-infected individual throughout the broad spectrum of HIV-disease and it should be obtained in the initial evaluation of all HIV infected patients for staging purposes and re-checked depending on the level of CD4 ⁺ T-lymphocyte count. Early in HIV infection, the number of leucocytes and lymphocytes, including T cells and their subsets are normal. However, the number and percentage of CD8 ⁺ T-lymphocyte subset begins to increase dramatically soon after seroconversion in the initial few months. These cells may operate by killing the infected CD4 ⁺ T-lymphocytes thereby partially controlling the infection, while simultaneously contributing to the destruction of the immune system. ^[6] Later, as AIDS develops, the absolute lymphocyte number falls resulting in a decrease in absolute CD8 ⁺ T cell count. In the last stage, almost all the remaining T cells are CD8 ⁺ T cells. ^[7] The present study was carried out to estimate the levels of CD4 ⁺ and CD8 ⁺ T-lymphocyte among HIV/AIDS patients with an attempt to stratify the HIV infected patients based on the CD4 ⁺ and CD8 ⁺ counts and to find out relations between CD4 ⁺ and CD8 ⁺ T-lymphocyte counts and clinical presentations of HIV/AIDS patients.

Materials and Methods

A prospective study was carried out in the Department of Microbiology, Regional Institute of Medical Sciences (RIMS), Imphal. The study was carried out between April 2002 and September 2003. A total of 415 HIV infected and AIDS patients (adults above 12 years of age) were included in the study (n= 415) consisting of 297 males (71.6%) and 18 females (28.4%) within the age group 13 to 67 years. A predesigned proforma was used for the study and detailed signs and symptoms of the patients were recorded. The individuals included in the study were from patients attending OPD RIMS, Imphal, patients admitted in the RIMS hospitals and patients attending National Reference Laboratory (NRL) and VCCTC, Department of Microbiology, RIMS, Imphal.

A total of 44 HIV seronegative healthy controls were also tested. For controls, the study group consisted of normal healthy adults (>18 years of age) consisting of doctors-5, technicians-12, nurses-five, other staff-11 and known outsiders-11.

Screening and laboratory confirmation of HIV infection

Voluntary confidential HIV antibody testing was carried out for the patients after adequate pretest counseling and consent from the patients. Strategy III of testing by ELISA/rapid/simple (3E/R/S) test was followed as recommended by NACO, Government of India. ^[8]

The different combinations of the tests were taken from HIV 1+2 Immunodot test combaids-RS (Span Diagnostic, Surat, India), HIV1/2 Stat pak (Chembio diagnostic systems, Inc, 3661 horseblock road, medford, NY, USA, SD Standard Diagnostic, Inc, 575-34 Pajang-dong, Jangan-ku, Suwon-si, Kyonggi-do, korea-440-290, Genedia HIV 1/2 ELISA 3.6 (manufactured by Greencross life science corp, 227-3/227-3 Gugalli biheung emp, yonginshi,Kyunggi-do,Korea.) and Bioelisa HIV 1/2 0 (Rec) manufactured by Biokit, S.A,08186 dlissa d′anount, Barcelona, Spain.

Identification of the AIDS cases (for cases above 12 years of age)

For identification of AIDS (symptomatic cases) patients, clinical case definition for AIDS (NACO, India, 1999) ^[9] was presence of two positive tests for HIV infection (E/R/S) and any one of the following criteria: a) significant weight loss (>10% of body weight within the last one month/cachexia, not known to be due to a condition other than HIV infection and chronic diarrhoea (intermittent or continuous) more than one month or prolonged fever (intermittent or continuous) of more than one month, b) extensive pulmonary tuberculosis - disseminated, miliary or extrapulmonary, c) neurological impairment - preventing independent daily activities, not known to be due to the conditions unrelated to HIV infection, d) candidiasis of the oesophagus (diagnosable by oral candidiasis with odynophagia), e) pneumonia - clinically diagnosed life-threatening or recurrent episodes of pneumonia, with or without etiological confirmation, f) Kaposi′s sarcoma, g) other conditions: Cryptococcal meningitis, neurotoxoplasmosis, CMV retinitis, Penicillium marneffei infection , recurrent herpes zoster and multidermatomal, disseminated molluscum contagiosum etc.

Enumeration of CD4 ⁺ , CD8 ⁺ T-Lymphocytes and CD4/CD8 ratio

The confirmed Hiv/Aid0 S patients were registered and advised to report at FACS count section of the immunology laboratory. Blood (2 mL) was collected aseptically from the antecubital vein with the help of sterile, disposable needle and syringe. The blood was processed as per the manufacturer′s instructions and subjected to FACS.

Blood (4 mL) was collected from all the healthy persons aseptically using sterile, disposable needle and syringe. Half of the blood was transferred to a sterile vial for HIV serology and the other half to K3EDTA vacutainer for CD4 ⁺ , CD8 ⁺ T- cell count. Screening test for HIV of all the normal samples was done with E/R/S tests.

The estimation of CD4 ⁺ , CD8 ⁺ T lymphocytes and CD4 /CD8 was done by FACS count system (Becton Dickenson Immunocytometry system, San Jose, CA 95131 -1807.). The FACS count instrument is a compact cell counter with a built-in computer. When whole blood is added to the reagent, fluorochrome labelled antibodies in the reagent bind specifically to lymphocyte surface antigen. After a fixative solution is added to the reagent tubes, the sample is run in the instrument. The cell comes in contact with the laser beam, which causes the fluorochrome labelled cells to fluoresce. The fluorescent light provides the information necessary for the instrument to count the cells. The software identifies T-lymphocyte subpopulations and correlates with the absolute count. Results provide absolute counts of CD4 ⁺ , CD8 ⁺ , CD3 ⁺ and CD4/CD8 ratio.

Guidelines for performance of the test, biosafety practices, troubleshooting and maintenance of equipment were strictly followed as recommended by the manufacturer for maintaining accuracy, reproducibility and comparability of the estimates.

Results

This study included a total of 415 HIV infected individuals of which 297 (71.6%) were males and 118 (28.4%) were females (all above the age of 12 years) in different age groups and their age range was 13-20 years (two patients), 21-30 years (131 patients), 31-40 years (226 patients) and 41-67 years (56 patients).

There were 150 (36.14%) asymptomatic HIV infected individuals of whom 89 were males and 61 females. The absolute CD4 ⁺ T-lymphocyte count ranges per microlitre were 0-50 (nil), 51-100 (nil), 101-200 (nil), 201-300 (35 patients), 301-400 (42 patients), 401-00 (31 patients) and> 500 (42 patients) as shown in [Table - 1].

There were 265 (63.85%) symptomatic or AIDS patients that included 208 males and 57 females. The CD4+ T-lymphocyte range per mL of blood were 0-50 in 68 patients, 51-100 in 52 patients, 101-200 in 101 patients, 201-300 in 38 patients, 301-400 in five patients, 401-500 in none and above 500 in one patient. The ranges of absolute CD8 T-lymphocytes in AIDS/symptomatic patients (n = 265) per μL of blood were 0-300 in 29 patients, 301-600 in 70 patients, 601-900 in 63 patients, 901-1500 in 74 patients, 1501-2000 in 19 patients and 2001-3500 in 10 patients [Table - 2], [Table - 3]. The ranges of absolute CD3+ T-lymphocytes or total T-lymphocytes per mL of blood ranged from 118 to 3500 cells in symptomatics and 673 to 3500 cells in asymptomatics.

The ranges of CD4/CD8 ratio in asymptomatic patients were from 0.13 to 1.69 while in symptomatic AIDS patients it ranged from 0.01 to 0.93 [Table - 4].

The sources of infection found in this study were intravenous drug abuse in 223 (53.7%), sexual route in 149 (35.9%), blood transfusion in 27 (6.5%), mother to child in one and unknown in 15(3.6%). Of all patients, 288 (69.4%) were married. The mean CD4+, CD8+, CD4/CD8 ratio and CD3+ T-lymphocytes among healthy adults is shown in [Table - 5].

Signs and symptoms of all the patients in different CD4 ⁺ T-lymphocyte count categories were examined. Weight loss was the commonest finding and occurred mainly in patients with CD4 ⁺ T- cell count less than 200 cells/μL. Fever, asthenia, cough, skin infections and diarrhoea in descending order of frequency occurred mainly in counts below 200 cells/μL and also in ranges between 201-300 cells/mL [Table - 6].

Frequency of opportunistic infections (OIs) in different CD4 ⁺ T cell count categories were correlated with CD4 ⁺ T- lymphocyte count categories in an attempt to correlate the OIs of the patients with CD4 ⁺ T-lymphocyte counts [Table - 7].

Among various classification systems, the Centers for Disease Control and Prevention (CDC), Atlanta, USA suggest a classification system using CD4 ⁺ T-lymphocytes as a marker of relative risk of developing HIV related OIs viz. stage-I: Acute (primary) infection (seroconversion), stage-II: early disease (asymptomatic) CD4 ⁺ T- lymphocyte usually> 500 cells/mL, stage-III: intermediate HIV infection (symptomatic) CD4 ⁺ T-lymphocyte usually 200-500 cells/μL , stage -IV: late stage HIV disease (symptomatic) CD4 ⁺ T-lymphocyte count is 50-200 cells/mL and, stage-V: Advanced HIV disease (symptomatic) CD4 ⁺ T-lymphocyte < 50 cells/mL . ^[10]

In our study, there were 108 (72%) patients within the CD4 ⁺ T-lymphocyte range 201-500 cells/μL and asymptomatic which should have been in the stage III/intermediate HIV infection symptomatic if the above classification system was followed. This may be explained by the fact that CD4 ⁺ T-lymphocytes count varies in ethnic groups and in groups with inherently low CD4 ⁺ T-lymphocytes count, the CDC classification system of HIV- infected individual may not be appropriate. ^[11],[12] A new criterion for consideration of therapy as suggested by a Chinese study group compared the CDC classification system commonly followed to monitor disease progression of HIV - infected individuals suggests CDC cut-off values for CD4 ⁺ T-lymphocyte count with increasing disease progression of> 500, 200-500 and < 200 cells per μL should be > 220, 100-200 and < 100 cells/ mL . ^[11]

Likewise, a new prognostic staging criteria of CD4 ⁺ T-lymphocyte count of> 300, 81-300 and < 80 cells/ μL was suggested by a south Indian study group. Therefore, it is important to study the maturational and developmental changes in lymphocyte subpopulations in Indian subjects from infancy to adulthood and to compare these data with those of the Caucasians.

In this study, 265 (63.85%) patients were symptomatic or AIDS cases, which included 208 males and 57 females. The CD4 ⁺ T-lymphocyte was depleted in majority of the patients. Out of 265 patients examined, 221 (68 + 52 + 101) patients had a CD4 ⁺ T-lymphocyte count below 200 cells/ mL . Unlike in asymptomatic patients, the CD4 ⁺ T-lymphocyte count was grossly reduced as expected in most of the patients. One patient had CD4 ⁺ T-lymphocyte count> 500 cells/ μL and symptomatic in spite of the high cell count. Sometimes CD4 ⁺ T-lymphocyte count do not always reflect how someone with HIV feels and functions e.g, some people with high count are sick while others with lower count have medical complications but feel well. This may be a limitation of CD4 ⁺ T-lymphocyte count and too much emphasis should not be placed on a single CD4 ⁺ T-cell count.

Early in HIV-infection, increase in CD8 ⁺ T-lymphocyte occurs representing an HIV specific cytotoxic T-cell response. These cells operate by killing infected CD4 ⁺ T-cells, thereby partially controlling the viral infection while simultaneously contributing for the destruction of the immune system. ^[13]

In our study, absolute CD8 ⁺ T-lymphocytes were increased in majority of the patients. This may be an indication of the T-cell response to counteract the progression of the disease. Comparatively a higher count was noted in asymptomatics than the symptomatics. The conspicuous rise in CD8 ⁺ T-lymphocyte cells/μL as seen in our study was not surprising and was commensurate with the activated cytotoxic T-cell response to combat the progression of the disease and the duration of a patient′s asymptomatic phase would depend on the ability of this response; the better response, the longer will be the asymptomatic period. On an average it is eight to 10 years in Western countries but in India it is five to seven years. ^[14] The lower CD8 ⁺ T-lymphocyte count seen in symptomatic patients may be an indication of the gradual failure of cytotoxic T cell immune response leading to further disease progression. The CD8 ⁺ T-lymphocyte is unable to check the viral replication and when OIs would occur. The CD8 ⁺ T-lymphocyte may show lower counts commensurate with the advanced stage of the disease process and a failing immune response.

A low CD4/CD8 ratio particularly when associated with an absolute decrease in the CD4 ⁺ T-lymphocyte, had been correlated with the clinical diagnosis of AIDS. ^{[15],[16],[17]} CD4/CD8 ratio is not altered in other infectious diseases like hepatitis and/or mycobacterial infections, both of which are highly prevalent and known to depress T helper/inducer cells. ^[18] In our study, lower CD4/CD8 ratio was observed in the symptomatic/AIDS patients with very low CD4 ⁺ T-lymphocyte counts.

Mean values of CD4 ⁺ , CD8 ⁺ T-lymphocyte, CD4/ CD8 ratio and CD3 ⁺ lymphocyte counts per μL of blood among normal healthy HIV-seronegative adult males (n = 24) and females (n = 20) that have been obtained from controls put up during the test showed conformity with the results obtained by Chinese and North Indian studies and others. ^{[12],[17],[18]}

Different clinical features, presenting signs and symptoms and OIs were studied in an attempt to correlate the clinical features and OIs with CD4 ⁺ T-lymphocyte count. Every symptomatic patient (n = 265) presented with one or more of the different signs and symptoms viz, weight loss/cachexia, fever, diarrhoea, asthenia, cough, skin infection, loss of appetite, neurological deficit (dementia/disorientation), headache etc. In our study, weight loss was the commonest finding and occurred mainly in patients with CD4 ⁺ T cell count < 200 cells/μL. Fever, asthenia, cough, skin infection and diarrhoea in descending order of frequency occurred mainly in counts < 200 cells/mL and also 201-300 cells/ mL.

Acknowledgement

References

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