A Study on the Timeline of Onset of Opportunistic Cytomegalovirus: Infection in Renal Transplant Recipients and Comparison of Various Methods of Its Detection  

 Talat Masoodi 1 ,  Raman Sardana 1 ,  Ashfaq ul Hassan 3 ,  Sanjiv Jasuja 2 ,  D.K. Aggarwal 2 ,  Reetika Dawar 1 ,  Gowhar N. Mufti 4 ,  Leena Mendiratta 1 ,  Khalid Zaffar Masoodi 5
1. Department of Microbiology, Indraprastha Apollo Hospitals, New Delhi, India;
2. Department of Nephrology and Transplant Medicine, Indraprastha Apollo Hospitals, New Delhi, India;
3. Department of Anatomy/Histology, SKIMS Medical College, Bemina, India;
4. Department of Paediatric Surgery MAMC, New Delhi, India;
5. Department of Urology Research, Laboratories University of Pittsburgh, India;
Author    Correspondence author
International Journal of Molecular Medical Science, 2013, Vol. 3, No. 6   doi: 10.5376/ijmms.2013.03.0006
Received: 08 Apr., 2013    Accepted: 22 Apr., 2013    Published: 20 Jun., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Masoodi et al., 2013, A Study on the Timeline of Onset of Opportunistic Cytomegalovirus, International Journal of Molecular Medical Science, Vol.3, No. 6 pp.41-49 (doi: 10.5376/ijmms.2013.03.0006)

Abstract

Infectious complications are still a significant cause of morbidity and death in recipients of organ transplant. Among them Cytomegalovirus is the single most important agent affecting these patients, with at least two-thirds of these patients developing CMV infection 1~4 months after transplantation. This study was aimed to bring out the best and effective methodology or combination to detect CMV disease in post renal transplant Indian patients. We compared Quantitative CMV–DNA  Real time PCR with pp65 Antigenemia, serology (anti-CMV IgM/IgG antibody assay) and Anti-CMV IgG avidity. We tried to evaluate the time line of onset of Cytomegalovirus disease post renal transplant. Fifty consecutive renal transplant recipients with clinical suspicion of CMV disease were studied prospectively. CMV Disease was defined clinically based on some criteria. These patients were subjected to a detailed viral diagnostic workup. The clinical response to treatment was considered as a strong clinical evidence of CMV disease in retrospect. The time course of onset of CMV disease post-transplant in majority of patients was found to be between 8 to 14 weeks. Five tests were done in all 50 patients and 20 healthy subjects (control group).Out of the 50 patients who formed our study population, 32 (64%) were found to have CMV disease. All the diseased cases were picked by the Real-Time PCR giving a sensitivity of 100% and specificity of 88.8%. The sensitivity and specificity of pp-65 antigenemia was found out to be 53% and 100%. 32% of patients were found positive for CMV IgM antibody showing a sensitivity 47.05%. Out of the 32 positive patients, 9 had low avidity showing recent infection, 11 patients had high avidity showing long term infection and 12 patients had avidity in the indeterminate range. The Quantitative  Real Time PCR stood out as a sensitive test and detected all CMV infections which were considered to be clinically significant and went on to receive treatment whereas pp-65 antigenemia was found to be a more specific and confirmatory test. CMV DNA copy numbers of >500 detected by the Real-time PCR correlate 100% with the presence of CMV disease. We found CMV-IgM antibody assay was not sensitive enough for diagnosis and monitoring of active CMV disease since there is a prolonged presence of IgM antibodies after recent CMV infection. Anti-CMV IgG antibody avidity was found to have a marginal role in diagnosis of this disease. 

Keywords
Renal transplantation; Cytomegalovirus; Real-time PCR detection

1 Background
The major types of infections observed in transplant recipients are categorized according to the time period post-transplant in which they occur. Post-surgical bacterial infections occur in the first month after transplantation, opportunistic infections with cytomegalovirus during one to four months post-transplant and other conventional infections in the late post-transplant period as reported by Rubin et al. (1981).

The human Cytomegalovirus (CMV), a member of the betaherpesviridae family is characterized by certain typical biological characteristics like restricted cell tropism and an extended replicative cycle (Ricard, 2007; Britt, 2005). It is the most common virus causing opportunistic infections in  transplant recipients  in whom T cell lymphocyte-mediated immunity is compromised as a result of pharmacological immunosuppression (antithymocyte globulin) (Rubin et al., 1981; Britt, 2005). Presentation of CMV Infection in Solid Organ Transplant Recipients may range from CMV virus syndrome (Fever, neutropenia, leucopenia, hepatitis, nephritis), Myelosuppression to Gastrointestinal invasion (colitis, ulcers or perforation) (Kotton and Fishman, 2005; Simon and Levin, 2001; Hibberd et al., 1995; Parasuraman et al., 2006). 

The crucial aspect in the diagnosis of Cytomegalovirus is the distinction between “CMV infection” which is defined as the isolation of CMV virus or detection of viral proteins or nucleic acids in any body fluid or tissue specimen and “CMV disease” (Pneumonia, Colitis, Hepatitis etc) which is defined by the presence of signs/or symptoms of tissue injury combined with virus isolation and/or positive viral nucleic acid detection assays (molecular) including quantification either alone or in combination with pp65 antigenemia (Hernando et al., 2005; Nouza and Korcáková, 1996). Also CMV is a great confounder as it co-infects and is known to predispose and modulate other opportunistic pathogens (Rao et al., 2000).

The various modalities available currently for diagnosis include conventional cell cultures, shell vial cultures, serological tests which includes antibody reactivity to IgG and IgM, CMV IgG avidity, antigenemia assay and the molecular assays (Nouza and Korcáková, 1996).The impact of CMV infection on post renal transplant recipients is great. New treatment modalities are availability for CMV, which if instituted early in the course of infection prove to be effective (Ricard, 2007; Britt, 2005; Hirsch, 2008; Charavarty et al., 2009). This underlines the importance of rapid and reliable diagnostic tests, prompting the present study. In the  present study we also tried to evaluate the time line of onset of Cytomegalovirus disease post renal transplant in the Indian Patient population as there is limited literature available in these patients.

2 Results
Out of the 50 patients who formed our study population (Table 1), 32 (64%) were found to have CMV disease. The time course of onset of CMV disease post transplant in majority of patients was found to be between 8 to 14 weeks with a mean time of onset of 13 weeks. Earliest presentation of CMV disease was at 6 weeks and Latest presentation was at 24 weeks. Of the study population of 50 patients, 34 (68%) tested positive for CMV DNA with CMV Real Time PCR out of which 32(64%) were later confirmed to have CMV disease. All the diseased cases were picked by the Real-Time PCR giving a sensitivity of 100%, specificity of 88.8%, positive predictive value of 94.1%, negative predictive value of 100% and an overall accuracy of 100% (Table 2). In our study we found that CMV DNA copy numbers of >500 detected by the Real-time PCR correlated 100% with the presence of CMV disease. The thresholds of PCR copy numbers was taken from 500 to 500,000. Of the 50 patients studied, 17 (34%) tested positive for pp65 antigenemia assay and all of these patients had CMV disease. However, of the 33 patients who tested  negative on pp65 antigenemia assay, 15 (30%) later were found to have disease. Thesensitivity, specificity, positive predictive value, negative predictive value and overall accuracy of pp-65 antigenemia was found out to be 53%, 100%, 54.5% and 70% respectively. A good correlation was found between the Real Time PCR copy numbers and pp-65 antigenemia positivity. At greater copy numbers pp-65 antigenemia positivity increased. In the control group of 20 people both PCR and antigenemia assay were negative.  A total of 16 (32%) patient were found positive for CMV IgM antibody. The sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy of this test was found out to be 47.05%, 100%, 100%, 2.94% and 68%  respectively. Out of the 32 positive patients, 9 had low avidity showing recent infection, 11 patients had high avidity showing long term infection and 12 patients had avidity in the indeterminate range. Out of the 32 diseased patients, 12 had CMV virus syndrome, 8 had gastrointestinal involvement, 5 had allograft dysfunction, 3 had hepatitis, 3 had nephropathy and 1 had pneumonitis.
 

 
Table 1 Age and Sex distribution of the patients with CMV Disease (n=32)

 

 
Table 2 Comparative assessment of various diagnostic modalities


3 Discussion
Cytomegalovirus is ubiquitous among renal transplant recipients and is the single most frequent cause of infectious complications in the early post renal transplant period. It has an effective treatment modality which if instituted early helps greatly in preventing the morbidity and mortality and hence the importance of rapid and reliable diagnostic tests. This study was conducted with the objective of comparing the various methods of detection for the early detection of CMV infection and disease in post renal transplant patients.

3.1 Time line of onset of CMV infection
In the present study we tried to evaluate the time line of onset of Cytomegalovirus disease post renal transplant. There are very few studies which have dealt with this aspect and most of them are western studies. No published data is available from Indian patient population amongst the post renal transplant recipients according to the pubmed search. The mean time to detection of CMV post transplant was found out to be 13 weeks in our study. Maximum number of patients presented in the 8-14 week period.  This is in agreement with Rubin RH et al who set forth a time-table that characterizes when in the post transplant course, a particular infection is more likely to occur and has found that the Cytomegalovirus is the single most important infectious agent affecting recipients of organ transplants, with two-thirds of these having CMV infection 1~4 months after transplantation (Rubin, 1990). Kotton CN et al while studying viral infections in renal transplant recipients found that CMV disease both reactivational and primary infections tend to appear 1 to 4 months after transplantation which is again in agreement to our results (Kotton, 2005).  The important reasons for this are that the immunosuppressive drug therapy is at its peak during this period and also due to the cessation of antiviral prophylaxis.

In our study we tried to find the efficacy of various diagnostic modalities in predicting the severity of the CMV disease in post renal transplant recipients with signs and symptomatology suggestive of CMV disease.

3.2 Role of PCR and pp-65 antigenemia
Of the 50 patients who fitted into our inclusion criteria, 34 were positive with CMV Real Time PCR giving a total positivity of 68 %. Out of these 34 CMV Real Time PCR positive cases, 32 went on to develop active CMV disease and the 2 other had non CMV illness. All the diseased cases were picked by the Real Time-PCR. Out of these 32 PCR positive CMV diseased patients, only 17 were positive for pp-65 antigenemia (34%). Out of the 33 patients who were pp-65 antigenemia negative, 15 turned out to have CMV disease and none of the pp-65 antigenemia negative patients developed disease. Thus PCR test detected all CMV infections which were considered to be clinically significant.  The p value was not found to be statistically significant.

3.3 On comparing PCR with antigenemia assay
For the diagnosis of CMV infection in post renal transplant recipients, Bhatia et al. (2004) found that out of 73 patients with clinical suspicion of CMV disease, 37 were confirmed to have CMV disease, all of which were PCR positive giving a total positivity of 100%. The antigenemia assay was positive in 33 patients giving a positivity of 89.18%. Thus they found PCR to be a sensitive assay. This is in agreement with our study which has shown a greater positivity for PCR.

Hernando et al. (2005) and Gouarin et al. (2007) in their respective studies on transplant recipients also found Real Time PCR assay to be more sensitive than the pp65 antigenemia for the detection and the quantitation of CMV load in transplant patients and hence they concluded that the Real Time PCR was an efficient and reliable tool for rapid diagnosis of CMV infection in this patient group.This is consistent with the results in our study. Sola et al. (2005) in his study compared the efficiency of pp65 antigenemia with reverse transcription-polymerase chain reaction for the diagnosis of CMV in  recipients of renal transplant between weeks 5 and 12. All samples positive by antigenemia were also positive by PCR. However in a good number of,cases only PCR was positive. They concluded that detection of DNA in serum is a more sensitive method than antigenemia for the diagnosis of CMV infection. This is also in agreement with our study. 

Lo et al. (1997) in his study tried to determine the optimum diagnostic test for CMV disease in renal allograft recipients in a locality with a high CMV seropositive rate prior to transplant. They compared pp65 antigenemia assay with the detection of DNAemia by a nested PCR. He found a sensitivity and specificity of 100% and 96% for antigenemia assay. On the other hand DNAemia by nested PCR was found in many asymptomatic patients and correlated poorly with clinical manifestations. Tanabe et al. (1997) also had similar results. Both the above studies are in disagreement to our results where PCR proved to be a superior assay. This discrepancy could be explained by the fact that ours being a Real Time PCR has an edge since it is a quantitative assay as compared to the conventional PCR which is a qualitative assay. The threshold values of 500 copies and above was taken as positive and helped us in differentiating the diseased from undiseased patients. Further, in our study we found a good correlation between the RT-PCR copy numbers and pp-65 antigenemia positivity. The pp-65 positivity was seen in cases which had higher PCR copy numbers and hence was a good indicator towards the higher sensitivity of RT-PCR in picking up the disease earlier.

3.4 Role of Serology
In our study  46 patients were positive for CMV IgG antibody (92%) and 16 were positive for CMV IgM antibody (32%).The sensitivity of serological assays was very low as compared to the other modalities used. Mean time of detection of CMV IgM antibody was found to be 11 weeks post transplant.

Naumnik et al. (2007) retrospectively studied renal transplant patients and screened them by CMV-IgM antibody assay and CMV-DNA PCR. All samples negative by CMV-IgM antibody assay were also negative by PCR but some patients were positive for CMV-IgM antibodies and negative for CMV-DNA. Tanabe et al. (1997) also compared the diagnostic role of serology with antigenemia assay, PCR and shell vial cultures. Of the patients who developed symptomatic CMV disease, none showed significant increase in IgG and IgM They concluded that detection of CMV-IgM antibodies was not sensitive enough for diagnosis and monitoring of active disease in these patients since there is a prolonged presence of IgM antibodies after recent CMV infection hence, showing the marginal role of serology in diagnosis of this disease.The results are is in good agreement with our study which has shown a lesser sensitivity for serological assays.In our study out of the 32 post renal transplant recipients who had CMV disease, 9 patients had low CMV IgG Avidity, those having high CMV IgG Avidity were 11 and those with borderline CMV IgG Avidity were 12.

Lafarga et al. (1997) prospectively studied the role of anti-CMV IgG avidity test in patients with microbiological evidence of CMV infection and compared it with detection of CMV viremia by conventional and shell-vial cell culture isolation and antigenemia.They concluded that Avidity index IgG-CMV was a good serological test to diagnose primary CMV disease.

In a similar study Lutz et al. (1994) investigated IgG antibody avidity assay for its ability to distinguish primary CMV from recurrent or long-term infection in immunosuppressed solid organ transplant patients and concluded avidity assay to be a reliable assay in the diagnosis of CMV infection.This is in disagreement to our results.The explanation for this discrepancy in the results is that avidity is a good marker for primary infection and not for recurrent or reactivational infections. Most of the transplant patients in our setting have latent infection which gets reactivated following immunosuppression and a good percentage of patients receive latently infected grafts. Hence avidity can only supplement our diagnosis and cannot be used as a sole diagnostic test.

3.5 Clinical Syndrome associated
Out of the 32 patients who had CMV disease, 12 had CMV virus syndromes (fever, leucopenia, myalgia and fatigue), 8 had gastrointestinal involvement, 5 had allograft dysfunction, 3 had hepatitis, 3 had nephropathy and 1 had pneumonitis. Kotton CN et al in their study found CMV infection presenting most often as an asymptomatic viremia or with fever and neutropenia-often as a “flu-like” illness with myalgias and fatigue. Colitis and chorioretinitis were found by them as presenting late in the post transplant course.

Sen et al. (2003) have found allograft dysfunction and leucopenia as a common presentation in their study population.

Charavarti  et al. (2009) while reviewing CMV infection in the Indian patient population found that the Indian renal transplant recipients with CMV infection present by either CMV syndrome consisting of fever, muscle pain, leucopenia and/or thrombocytopenia (other causes being excluded) or by organ involvement.They further found that the incidence of CMV infection was more  50%~70% when the recipient was CMV antibodies negative and the donor tested positive for CMV antibody.

The patients in our present study, who were proved to be having non CMV illness had other pathologies, some had hepatitis C viral infection, others had fungal infection, a few of them had tuberculosis and a small number had BK and JC virus infection.

3.6 Conclusions and recommendations
The 32 CMV disease positive patients in our study presented with various manifestations. The peak time of onset of CMV disease was found to be between 8th to 14th weeks post transplant. The Real Time PCR stood out as a sensitive test and detected all CMV infections which were considered to be clinically significant and went on to receive treatment whereas pp-65 antigenemia was found to be a more specific and confirmatory test. Serology was found to have a marginal role in the diagnosis of CMV disease.

 We found that CMV DNA copy numbers of >500 detected by the Real-time PCR correlate 100% with the presence of CMV disease. Thus we suggest this cut off value using this assay. Since Real-Time PCR assay was found to be a very sensitive assay and pp-65 as a specific assay, we recommend Real-Time PCR assay either alone or in combination with pp-65 antigenemia for the diagnosis as well as monitoring of CMV disease post transplant.This is agreement to the CARI Guidelines (2004), published in 2004, indicating CMV antigenemia or DNA detection as the methods of choice for the diagnosis and monitoring of active CMV infection. Hence a quantitative DNA test or pp-65 antigenemia is preferred over a qualitative DNA and serological tests.
 
4 Patients and methods
4.1 Study area and study population

The present study was a prospective study conducted over a 2 year period (February 2008 to January 2010 in the Department of Microbiology, Department of Nephrology and Transplant Medicine at our hospital, a super specialty tertiary care referral institute catering to a significant number of renal transplant recipients.

Fifty consecutive post renal transplant recipients who satisfied our inclusion criteria were included in the study. Patients of all age groups and both sexes were included in the study.The inclusion criteria were:
a) All patients who had undergone renal transplant within the study period and were on immunosuppressive medication.
b) Patients who had a high index of suspicion of signs and symptoms associated with CMV, within the first post transplant year.
Twenty healthy volunteers formed the control group.

4.2 Data collection technique and tools
Patients undergoing renal transplantation in our hospital were followed in our transplant clinic and observed for the development of signs and symptoms or laboratory evidence of CMV associated disease. The clinical details and laboratory parameters of the patients with CMV disease were recorded.

These patients were subjected to a detailed viral diagnostic workup including:
a)Detection and quantification of CMV–DNA by Real time PCR.
b) Detection of pp65 antigenemia by Indirect Immunofluorescence.
c) Detection of anti-CMV IgM/ IgG antibody using ELISA/ELFA
d)Detection of anti-CMV IgG antibody avidity using ELFA/ELISA.                                                             
The method of each test performed was as per standard guidelines for the test.

4.3 Definition of a case
Disease was defined clinically based on one or more of the following criteria:
a) CMV virus syndrome characterized by fever (temp of >38℃), leucopenia (<4 000/mm3) myalgia, fatigue etc.
b) Hepatitis was characterized by transaminitis (ALT >1.5 times the upper limit of normal).
c) Acute allograft dysfunction and nephropathy characterized by deterioration in renal function (increased blood urea /serum creatinine).
d) Gastrointestinal involvement characterized by symptoms of gastritis, gastrointestinal bleed and colitis.
e) Pneumonitis characterized by X-ray findings, hypoxemia etc.
The clinical response to treatment (decrease in immunosuppression or ganciclovir therapy) including settling of fever, rise in total leukocyte count, resolution of pneumonitis, colonic  bleed,  improvements in allograft function; were considered strong clinical evidence of CMV disease in retrospect.

4.4 Samples
5 mL of blood was collected in 2 sterile tubes by venipuncture of peripheral veins only.One tube with EDTA and One tube without additives. After collection, the samples were subjected to the above mentioned tests, strictly following the respective manufacturer’s instructions.

4.5 Quantitative Real-Time PCR assay
It is a real time amplification assay for the quantitative detection of Cytomegalovirus DNA in human plasma. CMV Real-TM Quant Sc (Sacace) Kit was used with Smart cycler system (Cepheid).The gene amplified was the Human Cytomegalovirus Major Immediate Early Gene. CMV DNA was extracted from serum samples using materials  provided in the kit which consists of Lysis solution, washing solutions, Sorbent and  DNA eluent. DNA amplification was carried using primers for HCMV Major Immediate Early Gene.The amplification mixtures comprises of 5 μL of extracted DNA, 5 μL of Taq F polymerase, 5 μL each of Quantitation Standards of CMV, 5 μL of Quantitation Standards for Internal control (IC), 5 μL of PCR mix-1 and 5 μL of PCR mix-2 (PCR mix 1 and 2 contain primers for HCMV Major Immediate Early Gene and each of the deoxynucleoside triphosphates).The amplified products are detected using fluorescent reporter dye probes specific for CMV. The internal control serves as an amplification control for each individually processed specimen and to identify possible PCR inhibition (Figure 1). Monitoring the fluorescence intensities during real time allows the detection and quantification of accumulating product without reopening the reaction tube.
 

 
Figure 1 PCR  amplification graphs of CMV


4.6 CMV antigenemia assay
The CMV antigenemia assay for internal matrix phosphoprotein (protein kinase)  (pp-65) was performed  using CINA rapid antigenemia kit (ARGENE) on peripheral leucocytes.  Reconstituted lysis solution was used to lyse RBC's and to prepare leucocytes. Cell suspension was spotted on slides by  the help of cytospin. For fixation and permeabilisation the slide was immersed in the reconstituted fixative solution provided in the kit. For staining 1 drop of anti-HCMV pp-65 monoclonal antibody was deposited on the slide and it was incubated for 30 minutes at 37℃.This was followed by  an immunofluorescence  staining with 1 drop of Fluorescein isothio cyanate (FITC) secondary antibody.The slide was again incubated for 30 minutes at 37℃ in a humidified dark chamber. Reading was done under 40x of fluorescent microscope. Whole slide was scanned for fluorescent polylobate nuclei. A single stained cell was considered sufficient to indicate antigenemia.
 

4.7 CMV serology
Vidas CMV IgG kit was used with the Vidas instrument for ELISA CMV IgM and IgG Antibody assay.This method combines a two step enzyme linked immunoassay sandwich method with final fluorescence detection using ELFA. The solid phase receptacle (SPR) serves as the solid as well as the pipetting device for the assay.The instructions of the manufacturer were followed for performance and interpretation of the assays.
 

4.8 CMV IgG avidity assay
Vidas CMV IgG Avidity Kit was used with Vidas. This is a simple technique which enables weak avidity antibodies, produced at the early stage of primary infection, to be differentiated from high avidity antibodies which are characteristic of a former infection. This enables exclusion of a recent primary infection of less than 3 months. The assay also combines a two-step enzyme immunoassay sandwich method using  with a final fluorescent detection using ELFA. The kit makes use of urea buffer for dissociating antigen antibody links.

4.9 Data analysis
In our study, for the purpose of analysis, CMV case (as per case definition) was taken as the objective for detection and the various viral diagnostic tests were assessed for their ability to detect the case. Data was collected using samples from the patients attending the transplant clinic and IPD of the hospital. Data sheets were filled for all patients and finally the data was compiled. Descriptive statistics were performed to summarize the basic characteristics of the sample population. Parameters checked were sensitivity, specificity, positive predictive value, negative predictive value and overall accuracy of the test, to detect the cases. The χ2 test was used to test the significance of association and p-value of <0.05 was taken as significant. Final analysis of data was done using the SPSS statistical package (SPSS version 12.0, SPSS Inc, Chicago, IL.).
 

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