ESTIMATION OF CD4+ AND CD8+ T-LYMPHOCYTES IN HUMAN IMMUNODEFICIENCY VIRUS INFECTION AND ACQUIRED IMMUNODEFICIENCY SYNDROME PATIENTS IN MANIPUR

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1 Indian 126 Journal of Medical Microbiology, (2007) 25 (2): 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 Abstract Purpose: To estimate and stratify CD4 + and CD8 + T-lymphocyte levels in human immunodeþciency virus (HIV) infected (asymptomatic) and acquired immunodeþciency syndrome (AIDS) patients (symptomatic) and correlate the clinical features of the patients with CD4+ and CD8+ lymphocyte level. Methods: Between April 2002 and September 2003, a total of 415 HIV seropositive adult patients (297 males and 118 females) attending Regional Institute of Medical Sciences (RIMS) hospitals were tested for CD4+ and CD8+ T-lymphocytes by ßuorescent activated cell sorter (FACS) counter (Becton Dickinson). Symptomatic patients were diagnosed as per NACO clinical case deþnition. Results: Ranges of 0-50, , , , , and above 500 CD4+ T-lymphocyte per microlitre were seen in 68, 52, 101, 73, 47, 31 and 43 patients respectively whereas CD8+ T-lymphocyte ranges of 0-300, , , , , per microlitre were seen in 29, 84, 92, 145, 40 and 25 patients respectively. One hundred and Þfty patients were asymptomatic and 265 were symptomatic. CD4/CD8 ratio in asymptomatics and symptomatics were and respectively. Tuberculosis and candidiasis occurred in CD4+ T-lymphocyte categories between cells per µl in symptomatics. However, cryptosporidiosis, toxoplasmosis, herpes zoster, cryptococcal meningitis, Pneumocystis carinii pneumonia, penicilliosis and cytomegalovirus retinitis were seen in patients having CD4+ T-lymphocyte less than 200 per µl. Conclusions: CD4+ T-lymphocyte was decreased in both asymptomatic and symptomatic HIV patients, The decrease was greater in symptomatics while CD8+ T-lymphocyte was increased in both except advanced stage symptomatics. CD4: CD8 ratio was reversed in both groups. Opportunistic infections correlated with different CD4+ T-lymphocyte categories. Key words: CD4+ T-lymphocytes, CD8+ T-lymphocytes, human immunodefi ciency 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, 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 per thousand blood samples screened against the all India Þgure *Corresponding author ( <dr_rebachandra@yahoo.com>) Department of Microbiology, Regional Institute of Medical Sciences, Imphal , Manipur, India Received : Accepted : of Since it was reported that the initial stage of HIV infection involves speciþc 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 subpopulations. 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 126 CMYK

2 April-June 2007 Singh et al CD4+ and CD8+ T-Lymphocytes in HIV Infection and AIDS 127 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 Þnd 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 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 confi rmation of HIV infection Voluntary conþdential 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., Pajang-dong, Jangan-ku, Suwon-si, Kyonggi-do, korea , 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 (Rec) manufactured by Biokit, S.A,08186 dlissa d anount, Barcelona, Spain. Identifi cation of the AIDS cases (for cases above 12 years of age) For identiþcation of AIDS (symptomatic cases) patients, clinical case deþnition 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) signiþcant 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 conþrmed HIV/AIDS 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 ). The FACS count instrument is a compact cell counter with a built-in computer. When whole blood is added to the reagent, ßuorochrome labelled antibodies in the reagent bind speciþcally to lymphocyte surface antigen. After a Þxative 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 ßuorochrome labelled cells to ßuoresce. The ßuorescent light provides the information necessary for the instrument to count the cells. The software identiþes 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 127 CMYK

3 128 Indian Journal of Medical Microbiology vol. 25, No. 2 females (all above the age of 12 years) in different age groups and their age range was years (two patients), years (131 patients), years (226 patients) and 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), (nil), (nil), (35 patients), (42 patients), (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, in 52 patients, in 101 patients, in 38 patients, in Þve patients, 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 in 29 patients, in 70 patients, in 63 patients, in 74 patients, in 19 patients and in 10 patients (Tables 2, 3). The ranges of absolute CD3+ T-lymphocytes or total T-lymphocytes per µl 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 Þnding 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 cells/µl (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 Table 1: Range of absolute CD4+ and CD8+ T-lymphocytes per µl of blood in asymptomatic human immunodeficiency virus-infected individuals (n =150) Range of Number Number Total % Range of Number Number Total % CD4+T of of CD8+T of of cells/µl males females cells/µl males females > Total 89 (59.3%) 61 (40.7%) 150 Total 89 (59.3%) 61 (40.7%) 150 The ranges of absolute CD8 + T-lymphocytes per µl of blood in asymptomatic patients (n=150) were in 14 patients, in 29 patients, in 71 patients, in 21 patients and in 15 patients. Table 2: Range of absolute CD4+ and CD8+ T-lymphocytes per µl of blood in symptomatic acquired immunodeficiency syndrome patients (n = 265). Range of Number Number Total % Range of Number Number Total % CD4+T of of CD8+T of of cells/µl males females cells/µl males females > Total 208 (78.5%) 57 (21.5%) 265 Total CMYK

4 April-June 2007 Singh et al CD4+ and CD8+ T-Lymphocytes in HIV Infection and AIDS 129 Table 3: Comparative ranges of absolute CD4+ and CD8+ T-lymphocytes per µl of blood between asymptomatic and symptomatic patients Range of Number Number Total % Range of Number Number Total % CD4+T of males of females CD8+T of males of females cells/µl (A+S) (A+S) cells/µl (A+S) (A+S) (0+57) 11 (0+11) (0+23) 6 (0+6) (0+77) 11 (0+11) (7+57) 20 (7+13) (0+41) 24 (0+24) (17+47) 28 (12+16) (20+29) 24 (15+9) (41+57) 47 (30+17) (27+3) 17 (15+2) (12+16) 12 (9+3) (21+0) 10 (10+0) (12+8) 5 (3+2) > (21+1) 21 (21+0) Total 298 (89+208) 118 (61+57) 415 Total 297 (89+208) 118 (61+57) 415 A=Asymptomatic, S=Symptomatic Table 4: Range of CD4/CD8 ratio in asymptomatic (n = 150) and acquired immunodeficiency syndrome symptomatic patients (n = 265) Range of No. of CD4/CD8 CD4/CD8 Range of No. of CD4/CD8 CD4/CD8 CD4 + T patients ratio ratio CD4 + T patients ratio ratio cells/µl (M+F) (mean value) (range) cells/µl (M+F) (mean value) (range) (57+11) (41+11) (77+24) (20+15) (29+9) (27+15) (3+2) (21+10) > (21+21) 0.62 >500 1 (1+0) 0.93 M=males, F=females Table 5: Mean CD4+, CD8+, CD4/CD8 ratio and CD3+ T-lymphocytes among healthy adults of Manipur (n=44) Male No. of Age CD4/ CD8/ CD4/ CD3/ Female No. of Age CD4/ CD8/ CD4/ CD3/ persons Group µl µl CD8 µl persons group µl µl CD8 µl (in yrs) ratio (in yrs) ratio yrs yrs >30 yrs >30 yrs Total Total Median Median Range Range of the patients with CD4 + T-lymphocyte counts (Table 7). Among various classiþcation systems, the Centers for Disease Control and Prevention (CDC), Atlanta, USA suggest a classiþcation 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/µl, stage-iii: intermediate HIV infection (symptomatic) CD4 + T-lymphocyte usually cells/µl, stage -IV: late stage HIV disease (symptomatic) CD4 + T-lymphocyte count is cells/µl and, stage-v: Advanced HIV disease (symptomatic) CD4 + T-lymphocyte <50 cells/µl. 10 In our study, there were 108 (72%) patients within the CD4 + T-lymphocyte range 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 classiþcation system of HIV- infected individual may not be 129 CMYK

5 130 Indian Journal of Medical Microbiology vol. 25, No. 2 Table 6: Frequency of different signs and symptoms in different CD4+ T- lymphocyte count categories in symptomatic/ acquired immunodeficiency syndrome patients (n=265) Signs/symptoms Ranges of absolute CD4 + T cells/µl No. % >500 Weight loss (>10% of body weight in the past 1 month) or cachexia Diarrhoea (>1 month duration) Fever (>1 month duration) Asthenia Cough Dermatoses/skin infections Loss of appetite Neurological deþcit* Others (Pallor, headache, Bell s palsy, bleeding gum, jaundice, conjunctivitis, ascites, white vaginal discharge, nausea/ vomiting, dimness of vision) *Dementia/disorientation Table 7: Frequency of OIs in different CD4+ T- lymphocyte count categories in symptomatic/acquired immunodeficiency syndrome patients (n=265) Name of disease Ranges of absolute CD4 + T cells/µl Total % >500 Tuberculosis Candidiasis Cryptosporiodiosis Toxoplasmosis Herpes Zoster Cryptococcal meningitis Pneumocystis carinii pneumonia Penicillium marneffei infection Cytomegalovirus retinitis Other (Bacterial/viral/fungal skin infections oral hairy leukoplakia etc.) appropriate. 11,12 A new criterion for consideration of therapy as suggested by a Chinese study group compared the CDC classiþcation 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, and <200 cells per µl should be > 220, and < 100 cells/ µl. 11 Likewise, a new prognostic staging criteria of CD4 + T-lymphocyte count of >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 ( ) patients had a CD4 + T-lymphocyte count below 200 cells/ µl. 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 reßect 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. 130 CMYK

6 April-June 2007 Singh et al CD4+ and CD8+ T-Lymphocytes in HIV Infection and AIDS 131 Early in HIV-infection, increase in CD8 + T-lymphocyte occurs representing an HIV speciþc 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 Þve 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 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 Þnding 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/µl and also cells/ µl. Acknowledgement The authors thank NACO, Govt. of India and MSACS, Govt. of Manipur, Director, RIMS, Medical Superintendent, RIMS for providing the FACS Count system and necessary reagents and the technicians to successfully carry out the research work. References 1. UNAIDS/WHO. Global summary of AIDS epidemic. AIDS epidemic update. December, p HIV/AIDS estimates. National AIDS Control Organization (NACO). Available from: 0paciÞc%20at%20a%20Glance/India/index.asp. 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In: Specialist s Training and Reference module p National AIDS Control Organization (NACO). Clinical case deþnition for AIDS (NACO, INDIA, 1999). In: Specialist s Training and Reference module p FACS count system user s guide. Manual Part Number: Rev. B Dickinson: p Kam KM, Wang KH, Li PC, Lee SS, Leung WL, Kwok MY. Proposed CD4+ T cell criteria for staging human immunodeþciency virus infected Chinese adults. Clin Immunol Immunopathol 1998;89: Ramalingam S, Kannangai R, Zachariah A, Mathai D, Abraham C. CD4 Counts of Normal and HIV infected south Indian adults: Do we need a new staging system? Natl Med J India 2001;14: Giorgi JV, Nishanian PG, Schmid I, Hulton LE, Chang HL, Detels R. Selective alteration in immuno regulatory lymphocyte 131 CMYK

7 132 Indian Journal of Medical Microbiology vol. 25, No. 2 subsets in early HIV (Human T-lymphotropic virus Type III/ lymphadenopathy associated virus infection. J Clin Immunol 1987;7: National AIDS Control Organization (NACO). Natural History and Clinical Manifestation of HIV/AIDS, Chapter 4. In: Specialist s Training and Reference module p Taylor JM, Fahey JL, Detels R, Giogi JV. CD4 percentage, CD4 number and CD4/ CD8 ratio in HIV infection which to choose and how to use. J Acquir Immune Defi c Syndr 1989;2: Diag Lab Immunol 1996;3: Nag VL, Agarwal P, Venkatesh V, Rastogi P, Tandon R, Agrawal SK. A pilot study on observation on CD4 and CD8 Counts in healthy HIV seronegative individuals. Indian J Med Res 2002;116: Hersh EM, Mansell PW, Reuben JM, Rios A, Newell LG, Goldstein AL, et al. Leukocyte subset analysis and related immunological findings in acquired immunodeficiency disease syndrome (AIDS) and malignancies. Diag Immuno 1983;1: Lawrence J. T-cell subsets in health, infectious disease and idiopathic CD4+ cell lymphocytopenia. Ann Intern Med 1993;119: Kam KM, Leung WL, Kwok MY, Hung MY, Lee SS, Mak WP. Lymphocyte subpopulation reference ranges for monitoring human immunodeþciency virus-infected Chinese adults. Clin 20. Paranjape RS, Thakur MR. Immune response in HIV infection. In: HIV/AIDS in India - Proceedings of the 6 th Round Table Conference. Gupta S, Sood PP, editors. Ranbaxy Science Foundation: New Delhi; p Source of Support: Nil, Conflict of Interest: None declared. 132 CMYK