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Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

ABSTRACT: AIMS: Viral uveitis and retinitis, usually caused by herpesviruses, are common in immunosuppressed patients. The diagnosis of viral anterior uveitis and retinitis is usually clinical. The polymerase chain reaction (PCR) has been used for the diagnosis of some viral infections, especially those caused by herpesviruses. This paper reports the use of PCR in the diagnosis of viral retinitis in vitreous samples from Brazilian patients. METHODS: PCR was used for the diagnosis of necrotising retinitis in vitreous samples from patients from the Hospital São Geraldo, Universidade Federal de Minas Gerais, Brazil. The vitreous samples were collected by paracentesis and stored until analysis. Samples were analysed by PCR using specific primers designed to amplify herpes simplex virus 1 (HSV-1), varicella zoster virus (VZV), or human cytomegalovirus (HCMV). In a case of anterior uveitis, PCR was performed with a sample from the anterior chamber. RESULTS: Herpesvirus DNA was amplified in 11 of 17 samples. HCVM DNA was detected in nine samples but DNA from HSV-1 and VZV were detected only once each. CONCLUSION: These results strongly suggest that PCR could be used for a rapid complementary diagnosis of viral uveitis and retinitis. A prospective study to evaluate the PCR results, clinical evolution, and treatment is imperative to corroborate the real value of PCR in diagnosis and how it could help the clinicians' approach.

SUBMITTER: Nogueira ML

PROVIDER: S-EPMC1731349 | biostudies-other | 2001 Feb

REPOSITORIES: biostudies-other

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Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

Nogueira M L ML Siqueira R C RC Freitas N N Amorim J B JB Bonjardim C A CA Ferreira P C PC Oréfice F F Kroon E G EG

Journal of clinical pathology 20010201 2

<h4>Aims</h4>Viral uveitis and retinitis, usually caused by herpesviruses, are common in immunosuppressed patients. The diagnosis of viral anterior uveitis and retinitis is usually clinical. The polymerase chain reaction (PCR) has been used for the diagnosis of some viral infections, especially those caused by herpesviruses. This paper reports the use of PCR in the diagnosis of viral retinitis in vitreous samples from Brazilian patients.<h4>Methods</h4>PCR was used for the diagnosis of necrotisi ...[more]

PMID: 11215276

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Project description:BACKGROUND: Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24?months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8. METHODS: A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood. RESULTS: The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (102 to 1010 copies/?L TE Buffer) with a limit of detection of 4.85x103, 5.61x102, and 2.59x102 copies/?L TE Buffer and a limit of quantification of 4.85x103, 3.01x102, and 1.38x102 copies/?L TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x105 HHV-8 copies/?L DNA extract (range: 4.37x103 to 1.47x106 copies/?L DNA extract): When normalised these equate to an average viral load of 2.44x104 HHV-8 copies/103 cells (range: 2.20x102 to 7.38x105 HHV-8 copies/103 cells). CONCLUSIONS: These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now available for research, for clinical diagnosis of HHV-8 infection, and for monitoring treatment efficacy.

Project description:Since its discovery in 1994, KSHV (also called human herpesvirus-8 or HHV8) has been implicated in a variety of disorders. Although the association of KSHV with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease has been well established, its presence in some other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, the conflicting data may be attributable to variations in the methods, primer sets, and target sequences selected. To establish an efficient and reliable PCR approach for KSHV detection we designed eight sets of primers to six regions (ORFK1, ORFK2, ORFK9, ORK26, ORF72, and ORF74) of the KSHV genome using appropriate database and software. The detection sensitivity of these primers was carefully assessed and their reliability was strictly validated in a series of positive (15 KS and PEL samples) and negative (16 lymphoid tissues) controls. We found that primer sets to the ORFK9 region showed the highest sensitivity, whereas primer sets to ORFK1 and ORF74 showed the lowest sensitivity. Primer sets to ORFK9, ORF26 and ORF72 regions detected all of the positive cases, whereas other primer sets showed varying detection rates or nonspecific bands. All 16 negative controls were negative with all primer sets. However, six of 16 negative controls became positive when we used nested PCR targeting ORF26. Therefore, multiple target KSHV sequences increase the detection efficiency, while nested PCR protocols are likely to introduce false positivity. Using ORFK9, ORF26 and ORF72 primer sets, we screened bone marrow biopsies from 18 cases of multiple myeloma, and failed to detect any KSHV sequences. This finding supports the conclusion that KSHV is not associated with multiple myeloma. Indeed, our results further confirm that although KSHV is universally present in Kaposi's sarcoma and primary effusion lymphoma, it is not ubiquitious.

Dataset Information

Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

Publications

Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis.

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