Offer of rapid testing and alternative biological samples as practical tools to implement HIV screening programs.

Implementation of HIV testing has the objective to increase screening, identify and counsel persons with infection, link them to clinical services and reduce transmission. Rapid tests and/or alternative biological samples (like oral fluid) give the option for a better general consent in approaching screening, immediate referral of HIV positives to medical treatment and partner notification. We tested the performance characteristics of an oral fluid-based rapid HIV test (Rapidtest HIV lateral flow-Healthchem diag. LLC) in comparison with routinely utilized methods in a selected population of known positive (N = 121) or negative (N = 754) subjects. The sensitivity of the rapid test was 99.1% (one false negative sample) and the specificity 98.8%. Five negatives showed a faint reactivity, 3 of these were reactive also in the reference test, one with a p24 only reaction in Western blot. If these 3 samples were excluded from the analysis the specificity increases to 99.2%. Results from our study confirm that, although a continuous improvement of the test performance is still needed to minimize false negative and positive results, rapid test and alternative biological samples may contribute to HIV prevention strategies by reaching a larger population particularly when and where regular screening procedures are difficult to obtain.

Rapid and accurate quantification of different HCV genotypes by LightCycler Real Time PCR and direct sequencing of HCV amplicons.

Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.

Determination of human papillomavirus (HPV) load and type in high-grade cervical lesions surgically resected from HIV-infected women during follow-up of HPV infection.

BACKGROUND: The role of human papillomavirus (HPV) load and the importance of multiple-strain HPV infections as biomarkers for the development of cervical disease were evaluated in human immunodeficiency virus (HIV)-positive women. METHODS: A total of 108 samples were analyzed, 64 of which were obtained from 16 HIV-positive women who underwent surgical resection of the cervical cone for treatment of a histologically confirmed high-grade cervical intraepithelial neoplasm (cases) and 44 of which were obtained from 22 HIV-positive women who had high-risk HPV but a negative colposcopy result (controls). Each patient underwent periodic examinations at 6-12-month intervals that included colposcopy, Papanicolaou testing, biopsy (if indicated), and cervical brushing for HPV testing. Viral typing was performed by reverse dot-blot hybridization and quantification of viral load by in-house real-time PCR and commercial assays. RESULTS: Analysis of the cervical-brush samples collected when high-grade squamous intraepithelial lesions were diagnosed revealed that all cases had HPV loads that were significantly higher than those of controls (P=.0004 and P=.0003, by PCR and the Hybrid Capture 2 index [Digene], respectively). Decreasing concentrations of HPV load were observed when comparing samples obtained before and after treatment (P<.0001). The number and type of HPV strains that were detected were not statistically different between cases and controls. CONCLUSIONS: The significantly higher HPV load detected in women with high-grade cervical dysplasia, as well as the dramatic decrease in the load after surgical removal of the lesion, suggest that HPV load is a possible prognostic marker of high-grade SIL.

Human papillomavirus infection and its role in the genesis of dysplastic and neoplastic lesions of the squamous epithelia.

Extensive laboratory and epidemiological evidence demonstrate that human papillomavirus (HPV) is the major cause of squamous cervical carcinoma (SCC), its precursor lesions (cervical intraepithelial neoplasia - CIN) and several other benign and malign clinical manifestations including genital warts, condylomata acuminata, Bowenoid papulosis, vaginal, vulvar and anal intraepithelial neoplasia (VIN and AIN) and carcinoma, penile carcinoma and other squamous neoplasias of the head and neck districts. In addition, mother-to-child transmission is probably responsible for recurrent laryngeal and pulmonary papillomatosis in infants. The relevance and high level of scientific interest surrounding HPVs are related to the oncogenic potential of some viral types belonging to this family and the possibility to influence the incidence of various tumour forms likecervical carcinoma, improving the efficacy of specific screening programs or defining preventive strategies like vaccination.

Few modifications of the Cobas Amplicor HIV Monitor 1.5 test allow reliable quantitation of HIV-1 proviral load in peripheral blood mononuclear cells.

Recent studies have suggested that monitoring the amount of HIV provirus in peripheral blood mononuclear cells (PBMCs) may be a useful end point for HAART where, in combination with plasma viral load, it provides additional information as to the possibility of virus eradication. In the present study, a modified version of the Cobas Amplicor HIV-1 Monitor test (CAHIM), currently used to quantify plasma viremia, have been evaluated to also measure the amount of proviral DNA in PBMCs. The analytical and clinical performance of the modified CAHIM test was assessed by quantifying different amounts of a standard HIV-DNA preparation obtained from the 8E5 cell line and by analysing 165 patients and controls samples. In these experiments, the modified test, that showed a linear dynamic range from 1.7 to 4.7 log10 copies/10(6) cells (r = 0.99) with a maximum CV of 20%, proved able to detect and quantify HIV-DNA in all but one clinical samples, with concentrations varying from 1.3 to 3.8 log10 copies/10(6) cells. During anti-retroviral treatment, the assay revealed different proviral DNA time courses associated with viral load changes and inversely correlated with CD4+ cells count. As expected, HIV-DNA was always detectable even when plasma viremia fell below the CAHIM cut-off. The modified CAHIM test specificity was confirmed by testing 20 HIV-negative samples in triplicates. Taken together, the data showed that the modified CAHIM test can be used to monitor HIV proviral DNA changes during HAART and can help in investigating further the clinical use of this marker.

Human papillomavirus viral load: a possible marker for cervical disease in HIV-infected women.

Laboratory markers of human papillomavirus infection have been recognized as relevant tools in programmes designed to reduce the burden of cervical cancer. The ongoing experience with these laboratory markers serves to confirm not only their negative predictive value (close to 100%) but also their positive association with developing or developed lesions. This aspect is particularly relevant in HIV-infected subjects who show an increased prevalence, incidence and severity of infections and lesions even in the era of efficacious control of their immunosuppression. Among the possible virus-related parameters proposed as relevant markers (viral persistence, load, expression, genomic integration capacity) we here analyse the informative value of human papillomavirus viral load measurement as a possible risk marker in this particular clinical setting.