Sangeeta Susan Thomas, (2014) A study of the kinetics of cytomegalovirus (CMV) DNA and Immediate early (IE) -mRNA levels in patients with CMV reactivation following bone marrow transplantation. Masters thesis, Christian Medical College, Vellore.
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Abstract
INTRODUCTION: Human cytomegalovirus (HCMV) is one of the members of the Herpesviridae family and is a very common cause of human infection. The seroprevalence for the disease ranges from 50% among adults in United States to 90% among children of 4 years of age in India (1,2). However, the virus remains in equilibrium with the host immune system and disease manifestations are very rare in a healthy individual infected with the virus. Serious morbidity and mortality is seen in individuals with immature immunity such as neonates, and in individuals with compromised immunity such as solid organ and bone marrow transparent recipients. OBJECTIVES: 1) To evaluate the kinetics of CMV DNA levels in BMT patients who reactivate CMV within the first 12 weeks post- transplant. 2) To quantitate the CMV IE-mRNA levels of reactivators using real-time reverse transcriptase PCR (RT-qPCR) METHODS: One hundred and sixty two bone marrow transplant patients were enrolled in the study and 2467 EDTA blood samples were collected from them. Whole blood aliquots of the sample was stored at -20ºC for CMV DNA testing. Peripheral blood mononuclear cells were separated from the samples using Ficoll-Paque and stored in RNA Later solution at -80ºC for CMV IE-mRNA testing. Samples from 25 patients were tested to obtain serial CMV DNA levels in the post transplant period by real time PCR. Samples from 15 patients who reactivated in the post-transplant period, as determined clinically and by CMV DNA levels, were tested for CMV mRNA using RT-qPCR. RESULTS: Of 162 candidates recruited into the study, 95% of them were CMV seropositive before transplantation. The rate of reactivation of CMV in HSCT patients in our study was found to be 21.6%. Among 25 patients who were continuously monitored for CMV DNA levels, 7(28%) showed values which were consistently below 10,000 copies/ml while 18 (72%) had CMV DNA values above 10,000 copies/ml. The CMV IE-mRNA PCR was preliminarily performed for 15 patients and 7(46.6%) patients showed detectable mRNA levels during reactivation. IE-mRNA was detectable in all patients after CMV DNA was detected except for one patient in whom CMV IE-mRNA was detected 3 days before DNA levels were detected. All the patients showed detectable mRNA levels while their CMV DNA levels were increasing in the initial phase of reactivation. Only one patient had detectable CMV IE-mRNA while the CMV DNA level was declining. However this patient also showed an increase in the CMV DNA level one week later. The limit of detection of the assay was determined to be 1198 copies/ml. CONCLUSIONS: CMV IE-mRNA is an indicator of the reactivation of HCMV in bone marrow transplant patients in the post-transplant period. However the sensitivity of the IE-mRNA RT-qPCR needs to be improved so as to detect lower copies of the mRNA. Alternate splicing mechanisms of the IE-mRNA should be carefully evaluated to design primers and probes that ensure detection of all the splice variants by RT-qPCR. The alternate splice variants also need to be tested in the patients who were negative for the 72kDa variant of the IE-mRNA.
| Item Type: | Thesis (Masters) |
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| Uncontrolled Keywords: | Cytomegalovirus (CMV), CMV DNA, CMV Immediate Early (IE)-mRNA, Quantitation, Bone marrow transplant patients, CMV reactivation. |
| Subjects: | MEDICAL > Microbiology |
| Depositing User: | Subramani R |
| Date Deposited: | 23 Mar 2020 16:33 |
| Last Modified: | 17 Aug 2020 07:53 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/12435 |
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