Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7.

Human herpesvirus 7 is a highly seroprevalent beta-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

Reverse transcriptase-polymerase chain reaction to evaluate human cytomegalovirus lytic gene expression.

This paper describes the development of four Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays devised to evaluate lytic (UL123, immediate-early; UL54, early; UL65, late; and UL99, true late) human cytomegalovirus (HCMV) transcripts. Subsequently, the assays have been validated evaluating the HCMV lytic gene expression in 28 samples (peripheral blood leukocytes, PBLs) from 14 renal transplant recipients. Although the assessment of HCMV transcriptional profile could be useful to evaluate viral reactivation state, further studies on dynamics of viral transcripts in different clinical settings are needed to determine the role of transcriptional profiling in virological monitoring and therapeutic management in immunocompromised patients.

Evaluation of two set of primers for detection of immediate early gene UL123 of human cytomegalovirus (HCMV).

Human cytomegalovirus (HCMV) is a widespread agent causing a life-long persistent and generally asymptomatic infection in immunocompetent individuals. In contrast, immunocompromised subjects are the most susceptible group to experience HCMV disease. First genes to be expressed during the replication cycle are the immediate early (IE) genes, the products of which have pleiotropic effects on host cell metabolism. Aim of this study was to compare two set of primers in the optimization and standardization of a RT-PCR assay for qualitative detection of mRNA encoded by the IE gene UL123 (IE1). The RT-PCR assays were then used to evaluate the UL123 gene expression in 29 peripheral blood leukocyte (PBL) samples obtained from 14 renal transplant recipients. In particular, 21/29 (72.4%) were positive with set A of primers vs. 24/29 (82.8%) with set B. Only one sample were negative with set B and positive with set A. Twenty-four of 29 samples (82.8%) were pp65-antigaenemia positive: 21 mRNA-UL123 positive with set A vs. 22 with set B; all viraemia-positive patients were mRNA-UL123 positive with both set A and B. Five of 29 samples were pp65-antigaenemia negative: 1 mRNA-UL 123 positive with set A vs. 2 with set B; all of them were viraemia-negative. These two RT-PCR assays could provide a reliable, rapid and sensitive system enabling the detection and identification of UL123 transcripts and could be usefully employed to study the pathogenesis of HCMV-related diseases.

Development of a multiplex polymerase chain reaction for detection and typing of major human herpesviruses in cerebrospinal fluid.

Infections of the central nervous system (CNS) represent a difficult diagnostic problem for both clinicians and microbiologists. In particular, the Herpesviridae family plays a central etiological role in CNS viral infections. These diseases have acquired growing importance in the past few years owing to the increasing number of immunocompromised patients and the availability of new antiviral drugs. Prompt detection and diagnosis of CNS viral infections are critical because most infections are treatable, while a delayed recognition may lead to life-threatening conditions or severe sequelae. The traditional methods for detection of herpesviruses in CNS infections exhibit several drawbacks, whereas the polymerase chain reaction (PCR) on cerebrospinal fluid has revolutionized the neurovirology and is becoming an essential part of the diagnostic work-up of patients with suspected CNS viral infections. A sensitive multiplex PCR method was developed for the simultaneous detection of 6 human herpesviruses (human cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, Epstein-Barr virus, varicella-zoster virus, and human herpesvirus 6) with the aim of simplifying detection and reducing time and costs. The accuracy, reproducibility, specificity, and sensitivity of these assays were established.

Detection and typing of BKV, JCV, and SV40 by multiplex nested polymerase chain reaction.

A multiplex nested polymerase chain reaction (PCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK, and SV40 in a single tube. In the first amplification step the same set of primers was used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV, and SV40. The second round was carried out using a set of primers designed to obtain products of different size for each related virus. Subsequently, the sensitivity of the multiplex nested PCR was maximized by optimizing parameters such as primer, magnesium, and dNTP concentrations. The sensitivity of the method ranged between 1 and 10 copies of the polyomavirus genome. The assay was then used for detecting polyomavirus DNA in urine, serum, and biopsy specimens from renal transplant recipients. Based on the results obtained, the multiplex nested PCR developed in our study represents a useful tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and to better define the role of polyomaviruses in human pathology.

Human herpes virus 8 infection in kidney transplant patients from an area of northwestern Italy (Piemonte region).

BACKGROUND: Human herpes virus 8 (HHV-8) infection is associated with Kaposi sarcoma (KS) and other neoplastic and non-neoplastic manifestations. A strong association between HHV-8 and KS has been evidenced in transplant recipients, particularly kidney recipients. METHODS: We have investigated the HHV-8 seroprevalence by an immunoenzymatic assay in 408 patients awaiting kidney transplantation and in the corresponding 356 donors; moreover, we have tested for the presence of HHV-8 DNA by nested PCR in available serum samples of the same graft recipients at 6, 12 and >18 months post-transplantation (overall 156 specimens). Associated factors, such as age, sex, area of residence, potential for HHV-8 transmission via organ transplantation and development of KS were also investigated. RESULTS: Twenty (4.9%) of 408 patients and 7 (1.9%) of 356 donors were seropositive. HHV-8 seropositive patients were on average about 6 years older than seronegative individuals. No difference in prevalence by gender was found. Considering the area of residence of seropositive patients, 4/161 (2.48%) were resident in Piemonte vs 16/247 (6.47%) in other areas of Italy (P = n.s.). During the follow-up post-transplantation, HHV-8 DNA was found only in four patients who were seropositive before transplantation, in three cases the corresponding donor was seronegative, in one the corresponding donor was also seropositive and the recipient developed KS. At >18 months post-transplantation, two patients were HHV-8 DNA positive, both were seronegative pre-transplantation and their corresponding donors were seronegative. CONCLUSIONS: HHV-8 seroprevalence in the Piemonte region seems to be low, also in a population of kidney transplant recipients. Based on our data, it does not seem that the immunosuppressive regimen favours the reactivation of HHV-8. Our results do not suggest the possibility of HHV-8 transmission via organ transplantation. Incidence of KS among HHV-8 seropositive patients was very low.