Sowmya, P (2008) Nucleic acid based molecular biological methods for quantitation and characterization of human cytomegalovirus in the clinical specimens from immunocompromised patients. Doctoral thesis, The Tamilnadu Dr.M.G.R. Medical University, Chennai.
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Abstract
The main objective of the study was to develop and establish the usefulness of nucleic acid based molecular methods for detection and quantitation of HCMV in various clinical specimens from patients with clinically suspected HCMV infections. In addition the HCMV strains encountered in these patients were characterized based on five different glycoprotein genes viz. glycoprotein B belonging to gCI complex; glycoprotein H, glycoprotein O and glycoprotein L belonging to gCIII complex and glycoprotein N belonging to gCII complex. ♦ pp65 antigenemia assay, a rapid, sensitive and semi-quantitative assay based on the detection and quantitation of lower matrix protein pp65 of HCMV for detection of active HCMV infection was standardized. Initially, two different methods namely the Conventional Dextran sedimentation (CDS) and Direct erythrocyte lysis (DEL) were compared for isolation of leucocyte from peripheral blood. Since, a good agreement in the quantitation of pp65 positive cells was found between the two methods, they were alternatively used in the rest of the study. pp65 antigen was not detectable in the peripheral blood of the controls (healthy blood donors seropositive for HCMV) confirming the earlier view that detection of the antigen in a patient is an evidence for active HCMV infection. ♦ Since, nucleic acid based molecular methods showed great promise in detection of several viruses and also reports suggesting that the region of interest can affect the sensitivity of the Polymerase chain reaction, three different targets viz. mtrII, UL-83 and gO gene of HCMV were evaluated against pp65 antigenemia assay as ‘gold standard’. PCR for mtr II was found to be more suitable for qualitative detection of HCMV in Chennai region, India. The other findings from the study include the inability of the qualitative PCRs to detect latent viruses circulating in the seropositive controls and all the three regions becoming positive with high level of antigenemia (>50 cells/ 2x 105 PBLs). ♦ Since, Post renal transplant recipients and HIV patients with HCMV infections remain asymptomatic with lower viral load, quantitation of HCMV in these patient groups become increasingly important. Also, since the results of the previous study suggested a high viral load when all the three regions became positive, multiplexing of the three primers were attempted. The multiplex PCR for the three regions of HCMV proved to be useful in quantitation of HCMV since it generated four different patterns that segregated with different pp65 antigenemia levels. ♦ Taqman probe based Real-time PCR was standardized for the mtrII region of HCMV. The mtr II region was chosen as it provided 100% sensitivity for detection of HCMV DNA in immunocompromised patients. The real-time PCR was sensitive, rapid (results available in 2 hours) and had a high reproducibility. It had an ability to detect the HCMV genome circulating in peripheral blood specimens of healthy controls. The median viral load in the healthy controls was 26.5 copies/ml. ♦ The multiplex PCR was evaluated against pp65 antigenemia assay and Taqman-probe based real time PCR for its efficacy in quantitation of HCMV. Each pattern of the multiplex PCR was assigned a viral load based on the results of Taqman-probe based real time PCR. The median antigenemia levels and viral loads in clinical specimens showing Pattern I and Pattern II of the multiplex PCR was significantly higher than Pattern III and Pattern IV. A moderate positive correlation ((rs) = 0. 7778) was seen between the results of pp65 antigenemia assay and Taqman – probe based real time PCR in the study. The multiplex PCR was less expensive and had the ability to distinguish clinical specimens with high and low viral load which may be effective in predicting patients who might develop HCMV disease and to monitor effectiveness of antiviral therapy. ♦ Since, the genotypic difference of HCMV affects pathogenesis and tropism of HCMV, the genotypic methods for determining the distribution of HCMV genotypes in the study population was developed and evaluated. ♦ Initially, a nested PCR based RFLP for gB gene was attempted however the method was unable to identify genotype gB3 in the study population. The study indicated that the sequence variations in the gB genome of HCMV may complicate genotyping of HCMV. Confirmation of primer specificity by the BLAST program may not suffice for the genotyping studies. Complete alignment of the prototype sequences of all the genotypes that exist for the gene of interest by a multiple alignment program with the primers used for amplification may be necessary for genotyping of HCMV. ♦ Later, seminested PCR-based RFLP and multiplex PCR were developed for genotyping of gB gene. Multiplex nested PCR for gB gene was more advantageous than the commonly employed PCR based RFLP for gB genotyping since it was rapid and allowed easier detection of mixed infections with multiple gB genotypes in clinical specimens. ♦ In the study determining the distribution of gB genotypes by multiplex PCR a predominance of gB1 genotype followed by gB3 and gB2 irrespective of the patient group or specimen type was found. Absence of gB4 and gB5 in the study population and gB mix in the infant group was observed. Presence of gB2 in the intraocular fluid was statistically significant. No absolute segregation of gB genotype based on gender or patient group was observed. In the HIV patient group with discordant results between the blood and intraocular fluids, the genotype other than gB1 in the blood, was detected in the intraocular fluid. ♦ PCR-based RFLPs were standardized to determine the distribution of genotypes pertaining to the genes of gCIII complex viz. gH, gL and gO. In all the three cases, the existing protocols were modified to achieve the following: direct application on clinical specimen without requirement for isolation, detection of mixed genotypes and visualization of restriction products on agarose gel. ♦ The analysis revealed no significant differences in the distribution of the gCIII variants based on gender. Statistically significant difference in the distribution of gH genotypes and gO genotypes were found in different patient groups. gL1 and gL2 genotypes were not found in the study population and predominance of gL4 genotype irrespective of the patient groups and specimen types were also observed. Fifty percent of HIV patients in the study was shown to harbour HCMV strains with gH1-gO1-gL4 configuration. ♦ An existing uniplex PCR based RFLP was converted to seminested PCR based RFLP to attain optimal sensitivity for genotyping gN gene of HCMV strains from direct clinical specimens without the need for viral isolation. The distribution of gN genotypes in the study population was in accordance to the distribution seen in other geographical areas. There was no statistically significant difference in the distribution of gN genotypes with respect to gender or clinical specimens. The genotype gN4c predominated among the genotypes irrespective of the patient types or specimen though its frequency was little less in PRT than other groups of patients. ♦ Following conclusions were drawn from the studies on cumulative genotyping of the five regions of HCMV: The study confirms that HCMV are highly heterogenous group of viruses and exhibit several genetic combinations. Theoretically 1120 strains may exist based on the assumption of number of genotypes existing for the five glycoprotein genes and the present study showed 48 different strains circulating in the study population. Genetic linkages are limited though some associations of genotypes occur more frequently than others. Hence, variation in one part of the gene does not reflect variations in other parts of HCMV genome. Analysis of single genes may not aid in understanding the tissue tropism or pathogenesis of HCMV. Infection with one HCMV strain does not provide protection against reinfection with a new strain, since a higher number of mixed infections with multiple genotypes were encountered in Post-renal transplant patients and HIV infected individuals. The order of genetic loci suitable for detection of mixed genotype as confirmed by the study is gB>gO>gH=gL>gN. The leucocyte fraction of peripheral blood is recommended for detection of mixed genotypes in PRT and HIV patients. A statistically significant difference was observed in the viral loads as determined by Taqman probe-based real time PCR between the specimens carrying single genotype and multiple genotypes. Hence, detection of mixed genotypes in the hypervariable regions of HCMV genome can be a surrogate marker for increase in viral load. The mixed genotype in any loci was not observed in the infant group in this study. ♦ Phylogenetic analysis of the four isolates and fifteen other HCMV strains revealed that no novel variant existed with respect to the regions of the gene analyzed and all the strains could be accommodated into one or the other genotypes already established.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Uncontrolled Keywords: | Nucleic acid, molecular biological methods, quantitation, characterization, human cytomegalovirus, clinical specimens, immunocompromised patients. |
| Subjects: | MEDICAL > Microbiology |
| Depositing User: | Laksham S |
| Date Deposited: | 22 Jun 2017 10:01 |
| Last Modified: | 26 Jan 2021 01:14 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/249 |
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