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ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 499-503
 

Pre-transplant cytomegalovirus immunoglobulin G antibody levels could prevent severe cytomegalovirus infections post-transplant in liver transplant recipients: Experience from a tertiary care liver centre


1 Department of Clinical Virology, Institute of Liver and Biliary Sciences, New Delhi, India
2 Department of Transplantation and HPB Surgery, Institute of Liver and Biliary Sciences, New Delhi, India
3 Department of Pathology, Institute of Liver and Biliary Sciences, New Delhi, India
4 Department of Clinical Research, Institute of Liver and Biliary Sciences, New Delhi, India

Date of Web Publication1-Feb-2018

Correspondence Address:
Dr. Ekta Gupta
Department of Clinical Virology, Institute of Liver and Biliary Sciences, Sector D1, Vasant Kunj, New Delhi - 110 070
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_201

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 ~ Abstract 

Background: Humoral immune responses in cytomegalovirus (CMV) are not studied well. Pre-transplant CMV immunoglobulin G (IgG) antibody levels (Pre-Tx IgG) could influence the occurrence of post-transplant CMV infections. Objective: Correlation between pre-Tx IgG and post-Tx risk of acquiring CMV infection was investigated. Materials and Methods: A total of 146 liver Tx recipients, not on CMV prophylaxis, were included. Pre-Tx IgG in donor (D) and recipient (R) were estimated and all the recipients were followed up for 1 year for CMV infections. Results: D+ R+ serostatus was seen in 142 (97.3%) and D− R+ in 4 (2.7%). A total of 113 (77.4%) recipients had pre-Tx IgG of ≥250 AU/ml. Overall, post-Tx CMV infections were seen in 54 (36.9%) recipients. In 32 (59.2%) patients, CMV infection was seen during the 1st month after TX. Incidences of post-Tx CMV infection in recipients with pre-Tx IgG <250 AU/mL and ≥250 AU/mL were 42.4% and 34.5%, respectively (P = 0.99). Median viral load was significantly higher in patients with pre-Tx IgG <250 AU/ml: 4log10 (R: 2.8–6.6 log 10) copies/ml than those with ≥250 AU/ml: 2.2 log10 (R: 1.6–3.8 log10) copies/ml, P = 0.04. There was no difference in the time of occurrence of CMV infection in both the groups. Concurrent occurrence of rejection and CMV infection was seen in significantly more patients 18/54 (32.7%) than in patients without CMV infection (12/99, 12%, P = 0.002). Conclusions: Higher pre-Tx CMV IgG levels might prevent severe CMV infections post-Tx. Recipients with low pre-Tx CMV titre might be benefitted by CMV prophylaxis or aggressive pre-emptive treatment.


Keywords: Cytomegalovirus infection, infections, liver transplant


How to cite this article:
Gupta E, Pamecha V, Verma Y, Kumar N, Rastogi A, Hasnian N, Bhadoria AS. Pre-transplant cytomegalovirus immunoglobulin G antibody levels could prevent severe cytomegalovirus infections post-transplant in liver transplant recipients: Experience from a tertiary care liver centre. Indian J Med Microbiol 2017;35:499-503

How to cite this URL:
Gupta E, Pamecha V, Verma Y, Kumar N, Rastogi A, Hasnian N, Bhadoria AS. Pre-transplant cytomegalovirus immunoglobulin G antibody levels could prevent severe cytomegalovirus infections post-transplant in liver transplant recipients: Experience from a tertiary care liver centre. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 11];35:499-503. Available from: http://www.ijmm.org/text.asp?2017/35/4/499/224427



 ~ Introduction Top


Cytomegalovirus (CMV) is a ubiquitous, double-stranded DNA virus of Herpesviridae family. It causes morbidity and occasional mortality in solid organ transplant (SOT) recipients. Seroprevalence of CMV in the general population varies from 30% to 70% in the developed countries and about 100% in the developing countries.[1] In India, a seroprevalance of >95% has been reported. CMV is a major viral pathogen complicating organ transplantation. Major advances in the management of CMV infection in transplant patients have been achieved through the development of new diagnostic techniques and use of antivirals.[2],[3] The incidence of CMV infection among patients undergoing liver transplantation ranges from 23% to 85%, and symptomatic disease develops in 15%–40% of these patients.[3] There are three distinct patterns of CMV infection seen after liver transplantation: primary infection, reactivation and super infection. The reactivation of latent CMV is common following liver transplantation, especially in seropositive recipients.[1],[4],[5]

The most important risk factor for the development of CMV reactivation is the pre-transplant (Tx) serological status of the donor and recipient.[6],[7] Seropositivity is used as a marker for exposure in donors and recipients. CMV immunoglobulin G (IgG) is the standard tool for pre-Tx assessment of serological status in donor and recipient. There is a paucity of literature regarding the influence of pre-Tx CMV IgG antibodies levels (pre-Tx IgG) on the post-Tx CMV reactivations/infection.[8],[9],[10]

Objective

This prospective study was done to investigate the incidence and timeline of CMV infection in seropositive liver transplant (LTx) recipients and correlate the risk of infection with pre-Tx IgG levels.


 ~ Materials and Methods Top


Study population and design

All patients who underwent LTx at the Institute of Liver and Biliary Sciences from March 2012 to April 2015 were included in this prospective study. Exclusion criteria consisted of patients who received simultaneous other organ transplant, patients not on immunosuppression, recipients of organs from cadaveric donors and patients who were unwilling to participate in the study. The study was approved by the Institutional Ethical Review board. Pre-Tx blood samples were collected from all the recipients and their respective donors for CMV IgG determination. After the transplant, all the recipients were followed up weekly for a month and later monthly for 1 year post-Tx by CMV DNA polymerase chain reaction (PCR), for the presence of an active CMV infection. CMV disease was diagnosed and defined as per recommendations.[4] CMV DNA positivity in blood (DNAemia) was considered as the evidence of CMV infection. CMV disease i.e., CMV infection with attributable symptoms was further categorised as: CMV syndrome (fever, malaise, leucopenia and/or thrombocytopenia) and CMV tissue-invasive disease (detection of CMV in the tissue specimen).

Treatment and follow-up

All patients received triple regimen immunosuppression (tacrolimus + mycophenolate mofetil + steroid). The trough level of tacrolimus was maintained at 5–10 ng/ml. Steroids were tapered in majority by the end of 3 months. Recipients were not on anti-CMV prophylaxis. Patients were clinically examined for signs and symptoms of CMV infection every month. Patients were given pre-emptive treatment if the CMV viral load was ≥500 copies/ml regardless of clinical manifestations as per the Institute's protocol. Pre-emptive therapy consisted of intravenous ganciclovir (5 mg/kg, 12 hourly) or oral valganciclovir (900 mg twice daily) until the patient became negative by CMV DNA PCR. Therapy was stopped once two consecutive samples taken 2 weeks apart were negative for CMV DNA.

Immunoglobulin G antibody to cytomegalovirus

Quantitative determination of IgG antibodies to CMV in plasma was done by the Architect CMV IgG assay (Abbott Laboratories, Chicago, IL, USA) which is a chemiluminescent microparticle immunoassay as per the manufacturer's instructions. The cut-off values were calculated based on each individual run to determine each sample as reactive or non-reactive. Results were expressed as AU/ml. Samples with concentration values ≥6.0 AU/mL were considered reactive for IgG antibodies to CMV. The assay calculated the results till 250 AU/ml and samples with values ≥250 AU/ml were reported as ≥250 AU/ml. To analyse all the patients, we characterised the study population in two groups: patients with titres ≥250 AU/ml and those with titres <250 AU/ml. This value provided the best sample size balance for result analysis.

Cytomegalovirus DNA polymerase chain reaction

Quantitative real-time PCR for CMV was performed by LightMix ® Kit on Roche Light cycler 480 Instrument (Roche Diagnostics, Manheim, Germany) in accordance with the manufacturer's instructions. This assay targets viral glycoprotein gene, 226 bp in size using Light DNA master hybridisation probe. Results were expressed as copies of virus/ml plasma. The linear range of the test is 102 copies to 106 copies/ml. Lower limit of detection of the assay is 500 copies. Samples with values ≥500 copies/ml were reported in exact copies/ml and others as <500 copies/ml.

Statistical analysis

Statistical analysis was done using SPSS version 20 (SPSS Inc., Chicago, IL, USA). Quantitative variables were expressed as median with interquartile ranges (IQRs), and qualitative variables as percentages. P < 0.05 was considered statistically significant. Numerical data were calculated using Microsoft Excel and analyzed. The cumulative incidence of CMV reactivation after transplant with respect to pre-transplant CMV titre was estimated with Kaplan–Meier methodology. A univariate cox proportional hazard model was used to estimate the risk of CMV infection after Tx and its association with various variables.


 ~ Results Top


Characteristics of the study population

Overall, 155 LTx and their respective donors were enrolled in the study. Nine recipients died during the 1-year follow-up; hence they were excluded from the study. The final analysis included 146 patients (132 adults and 14 paediatric patients) [Table 1]. Mean age of the paediatric patients was 5.9 (range: 2–12) years and none were <12 months of age. The pre-Tx recipient (R) seropositivity in the study group was 100% whereas both donor (D) and recipient seropositive status (D+/R+) was 97% in adults and 100% in the paediatric population. None were in the category of high-risk group i.e., D+/R−. In the study group, out of the various causes, the most common aetiology for end-stage liver disease was cryptogenic liver disease (33%) followed by alcoholic liver disease (22.5%) [Table 2].
Table 1: Baseline characteristics of the study population (n=146)

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Table 2: Underlying end-stage liver disease in recipients (n=146)

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Post-transplant cytomegalovirus infection

Post-Tx CMV DNA positivity in blood (DNAemia) was considered as the evidence of CMV infection. Out of 146 LTx recipients, post-Tx CMV infection was seen in 54 (36.9%) recipients consisting of 60 episodes. Median CMV viral load was 3.5 log10 (R: 2.5–6.6 log10) copies/ml. Forty-five (29%) recipients had a CMV viral load of ≥500 copies/ml. In 14 (9.5%) patients, demonstrable CMV disease was seen, none of the recipients developed CMV invasive disease based on negative histopathology and immunohistochemistry markers in tissue biopsy specific for CMV infection. CMV infections with viral load of ≥500 copies/ml were successfully treated with ganciclovir/valganciclovir. No evidence of clinical drug resistance was recorded as all responded to the treatment and cleared off the infection. Clearance of CMV from blood in them was seen in a median time of 6.4 days (IQR: 2.4–13 days) of therapy. Post-Tx occurrence of CMV infection was higher in paediatric patients (10 [71.4%]) than adults (44 [31.2%]) (P = 0.004).

Timing of cytomegalovirus infection

On estimating the time of occurrence of CMV infection after Tx, maximum number of infections 32 (59.2%) were seen in the 1st month followed by 13 (24%) infections in the 2nd month [Table 3]. Median time of occurrence of CMV was 22 days (IQR, 10.5–48 days). There was a significant difference in the time of occurrence of CMV infection between adult and paediatric patients. Time of occurrence was earlier in children than adults (13.4 ± 6.9 vs. 42.4 ± 3 days, respectively, P = 0.001).
Table 3: Monthly kinetics of cytomegalovirus infections post-transplant

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Pre-transplant cytomegalovirus immunoglobulin G titres and cytomegalovirus infection

Pre-Tx IgG levels of the recipients were measured before transplantation. A total of 113 (77.4%) had titres ≥250 AU/ml while the remaining 33 (22.6%) had titres <250 AU/ml. Post-Tx CMV infection rates among those with pre-Tx IgG < 250 AU/mL and ≥250 AU/mL were 42.4% and 34.5% (P = 0.99), respectively. Post-Tx CMV viral load in patients with titre <250 AU/ml was significantly higher, median value was 4.0 log10 (R: 2.8–6.6 log10) copies/ml than in those with titres ≥250 AU/ml: median value was 2.2 log10 (R: 1.6–3.8 log10) copies/ml, P = 0.04. However, on comparing the two groups in the time of occurrence of the CMV infection, we found no difference in both the groups [Figure 1], P = 0.81, log-rank test].
Figure 1: Kaplan–Meier plot of cumulative incidence of cytomegalovirus reactivation after transplant by pre-transplant cytomegalovirus immunoglobulin G titre of <250 versus ≥250 AU/ml

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Other risk factors associated with cytomegalovirus infection

In a univariate analysis, various factors such as age of the recipient, sex and pre-Tx IgG titres were evaluated for their association with the occurrence of CMV infection after Tx, but none of the variables were found to have any significant association with an increased risk of CMV infection.

Rejection and cytomegalovirus infection

Histopathological evidence of acute rejection was seen in 30 (19.5%) recipients. Concurrent occurrence of rejection along with CMV infection was seen in significantly more patients 18/54 (32.7%) than in patients without CMV infection (12/99, 12%, P = 0.002). All the rejection episodes were successfully treated with steroids and optimisation of immunosuppression.


 ~ Discussion Top


The present study explores a very important arm of CMV immunity i.e., CMV-specific humoral immune responses in the transplant settings. Following primary CMV infection, both cellular and humoral immune responses are activated.[11] Sustained control of CMV infection is largely accounted for by cellular immunity, involving various T-cell and B-cell subsets, but humoral responses also interplay between the various host factors contributing to the reactivation of CMV. The role of antibodies in the protection and control of CMV has been debated.[12] Limited literature available across various SOTs favours that CMV IgG antibody titres may have a role in restricting viral dissemination and probably might contribute to minimising the clinical manifestations of the disease, but it has been studied less frequently.[13],[14],[15],[16]

The present study was done on the cohort of LTx recipients who were 100% seropositive to CMV prior to transplant. We studied their pre-Tx CMV IgG titres and its relation to post-Tx occurrence of CMV infection. We found slight increase in the risk of CMV infection in recipients with less pre-Tx titre of CMV IgG, but it was not statistically significant. A similar study done on a very large cohort of seropositive liver Tx recipients had shown a significant association of lower Pre-Tx CMV titres and post-Tx occurrence of CMV infection.[6] Our study demonstrated that post-Tx severity of CMV infection as depicted by CMV viral load was significantly higher in the group that had lower pre-Tx IgG levels, thereby emphasising the role of Pre-Tx CMV IgG estimation. Pre-Tx titre could help in deciding the group of patients to be considered for prophylaxis or in initiation of pre-emptive treatment to prevent further severe CMV infections.

The incidence of post-Tx CMV infection seen in our study is very much in agreement with the published literature but slightly higher from a previously published study from this part of our country.[17] This could be due to the increased sensitivity and specificity of the detection assay used.[2],[18] Variability of CMV viral load testing across laboratories has shown the inconsistency in viral load values for individual samples ranging from 2.0 to 4.3 log10 copies/ml.[19] This may be due to lack of an international reference standard and variation in the assay design. However, now World Health Organization's international reference standard is available to overcome this problem of variability in CMV DNA testing.[20]

Time line of CMV infections in transplant recipients is usually thought to be from 2nd to 4th month after transplant, and is dependent on factors such as levels of immunosuppression and prophylaxis given.[6],[21] There is a clear shift in the timeline of CMV infection seen in the present study with most of the infections seen in the 1st month post-Tx, especially in the 2nd and 3rd weeks post-Tx. The consensus guidelines on timing of CMV monitoring post-Tx are missing and most centres adapt to CMV DNA PCR screening only after the 1st month of transplant, hence may be missing out some of the important infections.[4] A high incidence of CMV infection, especially in the paediatric age group, was seen in our study. Another important observation seen in the present study was paediatric CMV reactivations were earlier and more, as compared to adult patients. Colonisation of CMV starts very early in life due to a relatively more immunodeficit state.[22],[23] In the present study, all the CMV infections were seen during the 1st post-Tx month unlike in adults. This highlights the significance of early screening for CMV in paediatric recipients of SOT. Another observation of the present study is that post-Tx CMV infection also increases the risk of rejection of the transplanted organ. It is known that CMV infection through its various direct and indirect effects posed by CMV DNAemia in the recipients mileu leads to a cascade of reactions affecting the graft survival.[24] Although our data are very limited, they highlight the association of CMV infection to rejection.

The preliminary results presented in our study might be the first step to study CMV humoral responses in a more specific way. A large prospective study with a standardised methodology in multiorgan transplant should be conducted to confirm the results observed in our study.


 ~ Conclusions Top


Pre-Tx CMV IgG titres might influence the course of post-Tx CMV infections. Higher pre-Tx CMV IgG levels might prevent severe CMV infections post-Tx. Recipients with low pre-Tx CMV titre might be benefitted by CMV prophylaxis or aggressive pre-emptive treatment.

Acknowledgements

We would like to acknowledge Mr. Gaurav Singh for his excellent technical support in performance of various virological tests. We would also like to acknowledge Indian Council of Medical Research for providing the financial support for this study.

Financial support and sponsorship

This study is supported by financial grant from Indian Council of Medical Research: Grant number VIR/38/2011/ECD-1.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Razonable RR. Cytomegalovirus infection after liver transplantation: Current concepts and challenges. World J Gastroenterol 2008;14:4849-60.  Back to cited text no. 1
    
2.
Razonable RR, Paya CV, Smith TF. Role of the laboratory in diagnosis and management of cytomegalovirus infection in hematopoietic stem cell and solid-organ transplant recipients. J Clin Microbiol 2002;40:746-52.  Back to cited text no. 2
    
3.
Marcelin JR, Beam E, Razonable RR. Cytomegalovirus infection in liver transplant recipients: Updates on clinical management. World J Gastroenterol 2014;20:10658-67.  Back to cited text no. 3
    
4.
Kotton CN, Kumar D, Caliendo AM, Asberg A, Chou S, Danziger-Isakov L, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 2013;96:333-60.  Back to cited text no. 4
    
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Razonable RR, Humar A; AST Infectious Diseases Community of Practice. Cytomegalovirus in solid organ transplantation. Am J Transplant 2013;13 Suppl 4:93-106.  Back to cited text no. 5
    
6.
Bruminhent J, Thongprayoon C, Dierkhising RA, Kremers WK, Theel ES, Razonable RR, et al. Risk factors for cytomegalovirus reactivation after liver transplantation: Can pre-transplant cytomegalovirus antibody titers predict outcome? Liver Transpl 2015;21:539-46.  Back to cited text no. 6
    
7.
Carbone J, Lanio N, Gallego A, Kern F, Navarro J, Muñoz P, et al. Simultaneous monitoring of cytomegalovirus-specific antibody and T-cell levels in seropositive heart transplant recipients. J Clin Immunol 2012;32:809-19.  Back to cited text no. 7
    
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Kim JM, Kim SJ, Joh JW, Kwon CH, Song S, Shin M, et al. Is cytomegalovirus infection dangerous in cytomegalovirus-seropositive recipients after liver transplantation? Liver Transpl 2011;17:446-55.  Back to cited text no. 8
    
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Singh N, Wannstedt C, Keyes L, Wagener MM, Cacciarelli TV. Who among cytomegalovirus-seropositive liver transplant recipients is at risk for cytomegalovirus infection? Liver Transpl 2005;11:700-4.  Back to cited text no. 9
    
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Fishman JA, Emery V, Freeman R, Pascual M, Rostaing L, Schlitt HJ, et al. Cytomegalovirus in transplantation-challenging the Status Quo. Clin Transplant 2007;21:149-58.  Back to cited text no. 10
    
11.
La Rosa C, Diamond DJ. The immune response to human CMV. Future Virol 2012;7:279-93.  Back to cited text no. 11
    
12.
Macagno A, Bernasconi NL, Vanzetta F, Dander E, Sarasini A, Revello MG, et al. Isolation of human monoclonal antibodies that potently neutralize human cytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131A complex. J Virol 2010;84:1005-13.  Back to cited text no. 12
    
13.
Sarmiento E, Rodriguez-Molina JJ, Fernandez-Yañez J, Palomo J, Urrea R, Muñoz P, et al. IgG monitoring to identify the risk for development of infection in heart transplant recipients. Transpl Infect Dis 2006;8:49-53.  Back to cited text no. 13
    
14.
Sarmiento E, Lanio N, Gallego A, Rodriguez-Molina J, Navarro J, Fernandez-Yañez J, et al. Immune monitoring of anti cytomegalovirus antibodies and risk of cytomegalovirus disease in heart transplantation. Int Immunopharmacol 2009;9:649-52.  Back to cited text no. 14
    
15.
Cantisan S, Lara R, Montejo M, Redel J, Rodriguez-Benot A, Gutierrez-Aroca J, et al. Pre transplant interferon- γ secretion by CMV-specific CD8+ T cells informs the risk of CMV replication after transplantation. Am J Transplant 2013;3:738-45.  Back to cited text no. 15
    
16.
Lopez-Oliva MO, Martinez V, Buitrago A, Jimenez C, Rivas B, Escuin F, et al. Pre transplant CD8 T-cell response to IE-1 discriminates seropositive kidney recipients at risk of developing CMV infection post transplant. Transplantation 2014;97:839-45.  Back to cited text no. 16
    
17.
Wadhawan M, Gupta S, Goyal N, Vasudevan KR, Makki K, Dawar R, et al. Cytomegalovirus infection: Its incidence and management in cytomegalovirus-seropositive living related liver transplant recipients: A single-center experience. Liver Transpl 2012;18:1448-55.  Back to cited text no. 17
    
18.
Pang XL, Chui L, Fenton J, LeBlanc B, Preiksaitis JK. Comparison of light cycler- based PCR, COBAS amplicor CMV monitor and pp65 antigenemia assay for quantitative measurement of cytomegalovirus viral load in peripheral blood specimens from patients after solid organ transplantation. JCM 2003;41:3167-74.  Back to cited text no. 18
    
19.
Pang XL, Fox JD, Fenton JM, Miller GG, Caliendo AM, Preiksaitis JK. Inter laboratory comparison of cytomegalovirus viral load assays. Am J Transplant 2009;9:258-68.  Back to cited text no. 19
    
20.
Hayden RT, Preiksaitis J, Tong Y, Pang X, Sun Y, Tang L, et al. Commutability of the first World Health Organization international standard for human cytomegalovirus. J Clin Microbiol 2015;53:3325-33.  Back to cited text no. 20
    
21.
Rubin RH. Cytomegalovirus in solid organ transplantation. Transpl Infect Dis 2001;3 Suppl 2:1-5.  Back to cited text no. 21
    
22.
Breinig MK, Zitelli B, Starzl TE, Ho M. Epstein-Barr virus, cytomegalovirus, and other viral infections in children after liver transplantation. J Infect Dis 1987;156:273-9.  Back to cited text no. 22
    
23.
Russell MY, Palmer A, Michaels MG. Cytomegalovirus infection in pediatric immunocompromised hosts. Infect Disord Drug Targets 2011;11:437-48.  Back to cited text no. 23
    
24.
Stern M, Hirsch H, Cusini A, van Delden C, Manuel O, Meylan P, et al. Cytomegalovirus serology and replication remain associated with solid organ graft rejection and graft loss in the era of prophylactic treatment. Transplantation 2014;98:1013-8.  Back to cited text no. 24
    


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  [Table 1], [Table 2], [Table 3]



 

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