|Year : 2015 | Volume
| Issue : 3 | Page : 447-452
Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! - Tissue PCR the saviour?
A Kaul1, D Bhadauria1, V Agarwal2, V Ruhela1, A Kumar1, S Mohendra3, S Barai4, N Prasad1, A Gupta1, RK Sharma1
1 Department of Nephrology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
3 Department of Gastro Medicine, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Department of Nuclear Medicine, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||06-Feb-2014|
|Date of Acceptance||15-Jan-2015|
|Date of Web Publication||12-Jun-2015|
Department of Nephrology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Seronegative Invasive Gastro-intestinal cytomegalovirus disease in renal allograft recipients Background -CMV as oppurtunistic infection affecting the gastrointerstinal tract is the most common cause for tissue invasive CMV disease occuring in 10-30% of organ transplant recepients. Gastrointerstinal CMV disease can be diagnosed in presence of clinical suspecion along with histopathological findings (CMV inclusions) and presence of mucosal lesion(s) on endoscopic examination with collaborative evidences via molecular technique. Aims-Few cases of CMV infection affecting the gastrointerstinal tract show no evidences of dissemintion despite use of highly sensitive molecular techniques. We encountered 6 cases where in despite strong clinical suspecion of Gastrointerstinal CMV disease there were seronegative and endoscopic negative evidences for CMV, blind tissue biopsy yeilded positive results for CMV disease with excellent improvement with antiviral therapy. Conclusions-Blind biopsy specimen for tissue PCR could serve as saviour in an immunocompromised individiual who has a strong clinical symptomatology for GI-CMV disease in absence of viremia, normal endoscopy and histopathology, so that the early therapeutic interventions could help in excellent patient and graft survival.
Keywords: Cytomegalovirus disease invasive, gastrointerstinal, gastrointerstinal disaese, renal transplant
|How to cite this article:|
Kaul A, Bhadauria D, Agarwal V, Ruhela V, Kumar A, Mohendra S, Barai S, Prasad N, Gupta A, Sharma R K. Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! - Tissue PCR the saviour?. Indian J Med Microbiol 2015;33:447-52
|How to cite this URL:|
Kaul A, Bhadauria D, Agarwal V, Ruhela V, Kumar A, Mohendra S, Barai S, Prasad N, Gupta A, Sharma R K. Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! - Tissue PCR the saviour?. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jan 24];33:447-52. Available from: http://www.ijmm.org/text.asp?2015/33/3/447/158596
| ~ Introduction|| |
Newer potent immunosuppressants has been found to be associated with excellent organ survival rates albeit at expense of rising incidence of post-transplant opportunistic infections such as cytomegalovirus (CMV).  CMV infection occurs either as a primary infection or as a result of reactivation of a latent virus in the transplant population usually within 1-4 months of transplantation and causes invasive disease in organs like gastro intestine, lungs and brain etc.  Invasive gastrointestinal tract disease due to CMV disease (GI-CMV) occurs in about 10% of organ recipients. ,, Most of the manifestations are usually secondary to discrete erosions or ulcerations. Life-threatening complications include GI perforation and massive haemorrhage and are associated with significant morbidity and mortality among organ recipients.
GI-CMV disease are often suspected, based on clinical symptoms like abdominal pain and post prandial fullness, nausea and vomiting, chronic diarrhoea and bleeding from upper or lower gastro-intestinal tract (GIT) and significant weight loss; confirmed by presence of viremia, mucosal lesion (s) on endoscopic examination, viral cytopathologic findings on histopathology (CMV inclusions) and of course diagnosis underscored by positive response to therapy. However above mentioned classical clinical situation to confirm GI-CMV disease is not always a case and sometime it is difficult to diagnose, in a patient who has clinical symptoms suggestive of GI-CMV disease but has no viremia despite use of highly sensitive molecular techniques, , along with varied combination of normal or abnormal endoscopy finding and presence or absence of viral cytopathologic changes on histopathology. 
Hence we report 5 difficult to diagnose cases of GI-CMV disease, in absence of viremia, in which we had strong clinical suspicion of this disease but 4 of these patient became even more difficult to diagnose as they also had normal endoscopy and normal histopathology, blind tissue biopsy was done showed positivity for CMV. The improvement in symptomatology with early therapeutics intervention with anti CMV treatment helped in confirmation of diagnosis and excellent patient outcome.
Technique of specimen collection
During colonoscopy examination, multiple biopsies were obtained in buffered formalin and normal saline for histological examination of inflammatory activity and CMV inclusion body and extraction of DNA for PCR respectively. The colonoscopic biopsies collected in normal saline were stored at -80°C until further processing for CMV DNA.
Extraction of DNA from colonic tissues for the detection of CMV DNA by PCR
The DNA from biopsy specimens were extracted by QIAmp DNA Mini Kit (QIAGEN).
The tissue specimens were immersed and ground in liquid nitrogen. The powder derived was transferred to a 1.5 ml microcentrifuge tube and 180 μl of ATL buffer was added. 20 μl of Proteinase K was added to homogenate and mixed by vortexing and incubated at 56°C in a water bath for 3 hrs until the tissue is completely lysed. The microcentrifuge tube is briefly centrifuged to remove drops from inside of the lid. 200 μl of AL buffer is added and mixed by pulse-vortexing for 15 sec and incubated at 70°C for 10 minutes and centrifuged to remove drops from the lid. 200 μl of ethanol (96-100%) is added to the sample and mixed by pulse-vortexing for 15 sec. The mixture obtained is carefully applied to the QIAmp Spin Column (2 ml collection tube) without wetting the rim. It is centrifuged at 8000 rpm for 1 minute. The QIAmp spin column is placed in a clean 2 ml collection tube. 500 μl of AW1 buffer is added and centrifuged at 8000 rpm for 1 minute and the filtrate is collected in a clean 2 ml collection tube. 500 μl of AW2 buffer is added to this filtrate and centrifuged at full speed at 14000 rpm for 3 minutes. The fluid in the spin column is collected in a clean 1.5 ml centrifuge tube and 200 of AE buffer is added and incubated at room temperature for 1 minute and then centrifuged at 8000 rpm for 1 minute. This elution step is repeated to increase the DNA yield. The AE buffer eluted DNA can be stored at − 20°C until further use.
Amplification of DNA by PCR
PCR was performed using the above eluted DNA from the biopsies. Sequence of the forward and reverse primers (CMV5′ CTGTCGGTGATGGTCTCTTC-3′, CMV5′ CCCGACACGCGGAAAAGAAA-3′) used in PCR for the detection of symptomatic CMV infection; and was performed in a reaction mixture containing 5 μl target DNA, 1 μl of 100 pM each forward and reverse primer, 0.7 μl of 3U Taq polymerase, 4 μl of 10 mM each dNTPs, 5 μl 10 × reaction buffer (Bangalore Genei), 1 μl of 10% Bovine Serum Albumin (BSA) and 32.3 μl nuclease-free distilled water to a total volume of 50 μl. PCR tubes were subjected to 95°C for 5 min for one cycle and 35 cycles of amplification (94°C for 1 min, 55°C for 2 min and 72°C for 3 min with final extension at 72°C for 10 min) in a DNA thermal cycler (Gene Amp PCR System 9700). Positive and negative controls were run for each PCR batch. Amplified PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide (EtBr) and visualized with UV light. Presence of a 251 base pair amplicon was considered as CMV DNA.
Criteria for diagnosis of CMV infection
Positive result in one or more of the three tests (IgM antibody, CMV DNA and inclusion body in H and E-stained sections) was considered as evidence of CMV infection.
All the cases were diagnosed at different period following transplant and there was no outbreak [Figure 1].
|Figure 1: PCR amplification of DNA from colonic biopsy for CMV. Lane 6, molecular size markers (Phi x 174 DNA/Hae III Digest); lanes 4 and 5, negative and positive controls (251bp) respectively; lanes 1-3, study specimens (1, 3 tested positive for CMV DNA)|
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A 35-year-old male who was nondiabetic, had received a live related renal transplant in April 2010, mother being donor, was on triple immunosuppression [Tacrolimus, mycophenolate mofetil (MMF) and Prednisolone] presented to us 9 month after transplant surgery with complaints of post prandial fullness, recurrent vomiting, anorexia and weight loss for last more than 1 month. These symptoms were refractory to conventional antiemetic and prokinetics drugs. Gastric emptying study was done which was prolonged to 529 minutes [Figure 2]. Upper GI endoscopy showed only evidence of gastritis with duodenal ulcerations and biopsy was taken from both from gastric and duodenum. Histopathology revealed viral inclusion and tissue PCR positive for CMV despite his blood CMV PCR being negative. He was started on valgancyclovir in treatment doses following which his symptoms were settled. Repeat gastric emptying study became normal after completion of treatment for 3 months.
|Figure 2: Radionuclide Gastric emptying study using solid meal shows a gross delayed gastric emptying in the pre-therapy stage (a) which normalizes after therapy (b)|
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A 35-year-old male who received a live related renal allograft in August 2004, was on triple immunosuppression (Tacrolimus, MMF and Prednisolone). He presented to us 6 year after post transplant surgery, with complaints of chronic small bowel diarrhoea and significant weight loss for last 6 month. He was treated empirically with antibiotics and supportive management but had no response. All possibilities of causes of diarrhoea like infective, drug induced, allergic and malignant were kept and worked up. Repeated stool microscopy for opportunistic infections and cultures were sent and found to be negative. CMV PCR was negative. His immunosuppression was empirically modified to Tacrolimus, Azathioprine and prednisolone but his symptoms continued to persist. To rule out a possibility of intestinal post transplant lymhoproliferative disorder (PTLD) imaging like CT scan abdomen and repeated colonoscopy were performed but were normal. At last one more colonoscopy was done and rectal tissue was taken blindly for biopsy sent for histopathology [Figure 3]. His histopathological specimen was normal but tissue PCR for CMV was positive despite his blood PCR for CMV being negative. Due to clinical suspicion and tissue PCR positivity for CMV, he was initiated on parentral ganciclovir in treatment dose and his symptoms got settled within 5 days of starting therapy. He was later shifted to oral valgancyclovir and treated for a total duration of 3 months. He stabilized and became symptom free with adequate weight gain following antiviral therapy.
|Figure 3: (a) Gastric mucosa shows cytomegaly and nucleomegaly of capillary endothelial cells with presence of round eosinophilic intranuclear cytomegalovirus (CMV) inclusions and perinuclear halo (left arrow) along with granular cytoplasmic inclusions (right arrow). The lamina propria shows mixed inflammatory cell infiltrate. Gastric glands show flattening and denudation of epithelium and inflammatory infiltrate (star). (Hematoxylin and eosin; ×400). (b) Rectal mucosa shows maintained crypt architecture with presence of mild mononuclear inflammatory cell infiltrate in the lamina propria. (Hematoxylin and eosin; ×200 (c) Antral biopsy revealing erosions of which tissue biopsy revealed positivity for CMV|
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A 35-year-old male who received a live unrelated renal allograft in December 2008, wife being donor, received Basiliximab induction. He was on triple immunosuppression (tacrolimus, MMF and Prednisolone). He got admitted with us, 3 years post transplant, with complaints of recurrent episodes of diarrhoea associated with significant weight loss (10-12 kilograms) for last 5 months with not associated fever. His further work up revealed anemia and leucopoenia but normal graft function and low vitamin B 12 and folate levels, thought to be MMF related. His immunosuppression was modified to tacrolimus, Azathioprine and prednisolone but his symptoms persisted despite modification. Blood PCR for CMV was negative. CT scan abdomen was done to rule out PTLD and was normal. He underwent upper and lower GI endoscopy twice but were normal. However, blind biopsies were taken from D2 duodenum and rectal tissue, during the 2 nd procedure, despite a normal looking tract because of strong clinical suspicion of CMV infection. His rectal tissue PCR for CMV came out to be positive although tissue histopathology was negative. Patient was treated with intravenous ganciclovir in treatment dose followed by maintenance dose for 3 months. He responded well to treatment with improvement in his symptoms and weight gain and also hematologic parameters.
A 49-year-old male who received a live unrelated renal allograft in August 2009, wife being donor, received Basiliximab induction. He was on triple immunosuppression (tacrolimus, MMF and Prednisolone), developed biopsy proven antibody mediated rejection immediate post transplant, treated with antithymocyte globulin and renal function settled to a Serum creatinine of 1.8 mg%. He got admitted with us, 3 years post transplant, with complaints of recurrent episodes of diarrhoea associated with significant weight loss (6 − 7 kilograms) for last 4 months. He had associated pancytopenia, bone marrow was done revealing myeloid hyperplasia and work up for anemia revealed low vitamin B 12 and folate levels, thought to be MMF related. His immunosuppression was modified to tacrolimus, Azathioprine and prednisolone but his symptoms persisted despite modification. Blood PCR for CMV was negative. CT scan abdomen was done to rule out PTLD and was normal. He underwent upper and lower GI endoscopy once in last 4 months but was normal. However, blind biopsies were taken from D2 duodenum and rectal tissue, during the 2 nd procedure, despite a normal looking tract because of strong clinical suspicion of CMV infection. His rectal tissue PCR for CMV came out to be positive although tissue histopathology was negative. Patient was treated with intravenous ganciclovir in treatment dose followed by maintenance dose for 3 months. He responded well to treatment with improvement in his symptoms and weight gain and also settlement in his hematologic parameters.
A 50-year-old male who received a live unrelated renal allograft in August 2011, wife being donor, received Basiliximab induction. He was on triple immunosuppression (tacrolimus, MMF and Prednisolone), developed biopsy proven antibody mediated rejection 8 months post transplant, treated with antithymocyte globulin and renal function settled to a Serum creatinine of 1.4 mg %. He got admitted with us, 14 months post transplant, with complaints of recurrent episodes of diarrhoea associated with significant weight loss (8-10 kilograms) for last 4 months. He had associated pancytopenia, bone marrow was done revealing no significant abnormality and work up for anaemia revealed low vitamin B 12 and folate levels, thought to be MMF related. His immunosuppression was modified to tacrolimus, Azathioprine and prednisolone but his symptoms persisted despite modification. Blood PCR for CMV was negative. CT scan abdomen was done to rule out PTLD and was normal. He underwent upper and lower GI endoscopy was done was normal, however, blind biopsies were taken from D2 duodenum and rectal tissue, despite a normal looking tract because of strong clinical suspicion of CMV infection. His rectal tissue PCR for CMV came out to be positive although tissue histopathology was negative. Patient was treated with intravenous ganciclovir in treatment dose followed by maintenance dose for 3 months. He responded well to treatment with improvement in his symptoms and weight gain and also settlement in his hematologic parameters [Table 1].
| ~ Discussion|| |
CMV which is responsible for life-threatening opportunistic infection in solid-organ transplant patients and GIT seem to be the main target for end-organ involvement, due to reactivation of latent infection with subsequent systemic dissemination. Anti viral therapy is usually started in patient of GI-CMV disease based on clinical symptomatology along with detectable CMV viremia by plasma PCR. Diagnosis can be further confirmed by endoscopy guided histopathologic evidences of CMV inclusion bodies, positive tissue PCR and response to antiviral therapy.
All our patients had no viremia but positive tissue PCR, along with symptomatology suggestive of CMV disease. The possible explanation for absence of viremia but positive tissue PCR in a patient of GI-CMV disease could be 1. Viral titre below the lower limit of detection by currently employed molecular methods ,,, 2. Different CMV glycoprotein B genotypes may be associated with viral tropism for specific organs influencing CMV dissemination. ,,
Diagnosis was relatively easy in the first patient and antiviral therapy was instituted without delay after confirmation by histopathological finding and tissue PCR positivity, as endoscopic abnormalities guided us to take biopsy from involved site. But therapy was delayed in remaining four patients despite symptomatology suggestive of GI-CMV disease, because of absence of viremia and repeated normal endoscopies. Antiviral therapy was started in these four patients after exclusion of other causes of chronic diarrhoea and PCR positivity for CMV in histopathologically unremarkable specimen or tissue taken from endoscopically normal looking gastrointestinal tract. In situations of strong clinical suspicion despite repeated normal endoscopic and histopathologic evidences for GI-CMV disease, tissue PCR positivity puts the clinician to think whether the CMV disease is a bystander or active disease. Some studies have observed that patients with gastrointestinal CMV disease may have evidences of other disease process accompanying raising the issue that whether CMV is a bystander or the primary pathology in such individuals. However none of our patients had any other associated cause for these symptoms and all of them showed excellent recovery in symptoms with definitive antiviral therapy ruling out the possibility of CMV as bystander. ,
The possible explanation for normal endoscopy despite tissue PCR positivity could be substantiated by study done by S M Graham et al. who looked into evidences of opportunistic infection in transplant among kidney and pancreas transplant and observed evidence of CMV infection in 22% despite a normal endoscopic study,  putting forward a suggestion whether these gut motility disorders could be due to associated abnormalities of the autonomic nervous system which is a cause among 30% of patients with chronic intestinal pseudo-obstruction. Much of this could be explained by a systemic viral infection acting permanently to alter normal peristaltic activity via effects on the myenteric plexus, extrinsic enteric neurones, or directly on enteric muscle. Clinical case reports have observed positive viral serology and tissue localisation of viral inclusions in the myenteric plexus being described for EBV, VZV and CMV. ,,,, Chang et al. concluded that early colonoscopy is needed in transplant recipients with chronic diarrhoea associated with anaemia and that biopsy is necessary to diagnose CMV. 
| ~ Conclusion|| |
biopsy specimen for tissue PCR could serve as a saviour in an immunocompromised individual who has strong clinical symptomatology for GI-CMV disease but has absence of viremia, normal endoscopy and normal histopathology, so that the early therapeutic intervention could help in excellent patient and graft survival and not considering it as a bystander.
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[Figure 1], [Figure 2], [Figure 3]