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Total cell-associated HIV-1 DNA is a surrogate marker of the HIV-1 reservoir, however, certified systems for its quantification are not available. The Italian HIV DNA Network was launched to validate HIV-1 DNA quantification methods in use at University and Hospital labs. A quality control panel including HIV-1 DNA standards, reconstructed blood samples (RBSs) and DNA from different HIV-1 subtypes was blindly tested by 12 participating labs by quantitative real-time PCR (n = 6), droplet digital PCR (n = 3) or both (n = 3). The median 95% hit rate was 4.6 (3.7–5.5) copies per test and linearity in the tested range was excellent (R2 = 1.000 [1.000–1.000]). The median values obtained across labs were 3,370 (2,287–4,245), 445 (299–498), 59 (40–81) and 7 (6–11) HIV-1 DNA copies, for the 3,584, 448, 56 and 7-copy standards, respectively. With RBSs, measured values were within twofold with respect to the median in two thirds of cases. HIV-1 subtypes were missed (CRF01_AE by 3 labs) or underestimated by > 1 log (subtypes A, C, D, F by one lab; CRF01_AE by one lab; CRF02_AG by one lab). The overall performance was excellent with HIV-1 DNA standards, however detection of different HIV-1 subtypes must be improved. [ABSTRACT FROM AUTHOR]

Background: Since the end of February 2020, the Coronavirus Disease 2019 (COVID-19) outbreak rapidly spread throughout Italy and other European countries, but limited information has been available about its characteristics in HIV-infected patients. Methods: We have described a case series of patients with HIV infection and COVID-19 diagnosed at the S.Orsola Hospital (Bologna, Italy) during March and April, 2020. Results: We reported a case series of 26 HIV-infected patients with COVID-19. Nineteen subjects were men, the median age was 54 years, 73% of patients had one or more comorbidities. Only 5 patients with interstitial pneumonia were hospitalized, but there were no admissions to intensive care unit and no deaths. Conclusions: In our experience, COVID-19 associated with HIV infection had a clinical presentation comparable to the general population and was frequently associated with chronic comorbidities. [ABSTRACT FROM AUTHOR]

Background: HTLV-1 infection is currently restricted to endemic areas. To define the prevalence of HTLV-1 infection in patients living in Italy, we first carried out a retrospective serological analysis in a group of people originating from African countries referred to our hospital from January 2003 to February 2005. We subsequently applied a real time PCR on peripheral blood mononuclear cells from subjects with positive or indeterminate serological results. Methods: All the sera were first analysed by serological methods (ELISA and/or Western Blotting) and then the peripheral blood mononuclear cells from subjects with positive or inconclusive serological results were analyzed for the presence of proviral DNA by a sensitive SYBR Green real time PCR. In addition, twenty HTLV-I ELISA negative samples were assayed by real time PCR approach as negative controls. Results: Serological results disclosed serum reactivity by ELISA (absorbance values equal or greater than the cut-off value) in 9 out of 3408 individuals attending the Sexually Transmitted Diseases Clinic and/or Oncology Department, and 2 out 534 blood donors enrolled as a control population. Irrespective of positive or inconclusive serological results, all these subjects were analyzed for the presence of proviral DNA in peripheral blood mononuclear cells by SYBR real time PCR. A clear-cut positive result for the presence of HTLV-1 DNA was obtained in two subjects from endemic areas. Conclusion: SYBR real time PCR cut short inconclusive serological results. This rapid and inexpensive assay showed an excellent linear dynamic range, specificity and reproducibility readily revealing and quantifying the presence of virus in PBMCs. Our results highlight the need to monitor the presence of HTLV-1 in countries which have seen a large influx of immigrants in recent years. Epidemiological surveillance and correct diagnosis are recommended to verify the prevalence and incidence of a new undesirable phenomenon. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting., Methods: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm., Results: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools., Conclusion: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

Standard serological tests have reached high levels of sensitivity and reproducibility but do not indicate whether infection is recent or long-standing. Among the 59960 sera analyzed for HIV positivity at the Retrovirus Laboratory, Operative Unit of Microbiology, Bologna, Italy, from January 2010 to July 2011, 134 samples showed an initial positive result. Application of the avidity test, able to distinguish between recent or long-standing HIV infection, classified 59 subjects as recently infected and 75 as chronically infected. Besides all the public health implications, the distinction between acute and chronic infection might serve to establish the time of infection and therefore reach any potential partners who might have been infected in a specific period of time. Although our results are limited to subjects referred to our laboratory and hence represent only a limited part of the problem, the routine application of methods able to distinguish recent from long-lasting infection could help monitor disease incidence, identify high-risk groups, and enhance epidemiological conclusions.

The human immunodeficiency virus type 1 (HIV-1) pandemic is currently in its third decade and approximately 35 million people are infected worldwide. HIV-1 genetic variability results in 9 phylogenetic subtypes and several circulating recombinant forms (CRFs). In Italy, the first phase of the HIV epidemic was mainly confined to the intravenous drug users (IDU) risk group, moreover most studies have focused on different aspects of the non B subtype such as drugs and therapeutic protocols, laboratory methodologies (heteroduplex mobility assay, PCR screening methods) for the identification of phenotypic variants. These studies were mostly locally conducted. In this context, the Red Queen Hypothesis might be suggestive. In the first original expression that comes from Chapter 2, Through the Looking Glass. To improve our knowledge, in the near future, we will need to investigate the demographic and spatiotemporal history of different HIV-1 subtypes circulating in Italy in a large data set of sequences, involving a sample size comparable with the Italian population. To monitor the genetic evolution of the HIV-1 in a large data-set represent an essential strategy to control the local as well as the global HIV-1 epidemic and to develop efficient preventive and therapeutic strategies, with a great impact in clinical practice.

Current knowledge of HIV-primary resistance indicates that the prevalence of transmitted resistant strains has increased to substantial levels over the past few years, with a wide variation depending upon a number of factors. New infections with a virus strain already resistant to antiretroviral drugs, namely non-nucleoside reverse transcriptase inhibitor (NNRTI), have a negative impact on initial treatment response and also shorten the time to first virologic failure. The aim of this study was to determine the prevalence of antiretroviral drug resistance by a genotypic test in a population with newly diagnosed HIV-1 infection at a clinical centre in Bologna between June 2006 and September 2007.