Detection of a characteristic melting profile of a SARS-CoV-2 Kappa variant in Italy using the SARS-CoV-2 Variants ELITe MGB® Kit.

BACKGROUND: Although more than a year has passed since the start of the pandemic, SARS-CoV-2 infection still represents a major challenge for public health all over the world due to viral genome capability of gaining rapid mutations. Whole-genome sequencing (WGS) is the gold standard for variant identification, but it is time consuming and relatively expensive. For this reason, assays targeting multiple regions of the SARS-CoV-2 genome may be useful for a rapid traceability of either known or new variants, anyway, not all the manufacturers are able to sustain the rapid development of variants. OBJECTIVE: We tested forty nasopharyngeal swabs, resulted positive for the presence of SARS-CoV-2 RNA at low cycle threshold (CT < 25), with SARS-CoV-2 Variants ELITe MGB® Kit, which was designed to identify Nigerian variant, possible UK variant and South African or Brazilian variant. RESULTS: During the analysis, we noted an atypical melting curve, different from the other variants recognizable by the kit. The subsequent WGS reported this variant as Kappa, so we assess the possibility of "suspecting" the presence of a Kappa variant using SARS-CoV-2 Variants ELITe MGB® Kit. CONCLUSIONS: Rapid variant screening followed by WGS offers the opportunity to study mutation dynamics and quickly identify possible variants of interest (VOI) and/or variants of concern (VOC), which is crucial in virus spreading control. Furthermore, an accurate analysis of the melting peak could be useful to suspect the presence of new variants.

Quantitative HIV-1 proviral DNA detection: a multicentre analysis.

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.

Prevalence of antiretroviral drug resistance in untreated persons newly diagnosed with HIV-1 infection.

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.

Low avidity antibody: a reliable method to diagnose a recent HIV-1 infection.

Standard serological tests (both EIA and Immunoblotting) have reached high levels of sensitivity and reproducibility, but do not indicate whether infection is recent or longstanding. Since many patients with HIV-1 infection are not usually diagnosed until symptom presentation, the possibility to distinguish between acute and chronic infection has become increasingly important for the purposes of therapeutic decision-making, partner notification and epidemiological surveillance. We evaluated a guanidine-based-antibody-avidity assay in a selected group of recent (within six months from seroconversion) and chronic (more than forty eight months) HIV-1 infections in an attempt to shed more light on the significance of the avidity index in establishing the time of infection. Sera from newly infected individuals showed a low mean avidity index (ranging from 0.35 to 0.60 with a standard deviation 0.09) at baseline and a clear increasing value at the following times of observation. Our data showed that an avidity index <0.70 might be presumptive of infection occurring within 9 months. Avidity index levels might distinguish between acute and chronic infection. The method is semi-automated, inexpensive and easy to perform, and estimates the time elapsed from seroconversion, thereby identifying a recent infection.

Genotypic resistance in plasma and peripheral blood lymphocytes in a group of naive HIV-1 patients.

BACKGROUND: Since HIV infection cannot be completely eliminated due to the establishment of latently infected CD4+ T cell reservoirs, there is an urgent need for a clearer understanding of comparative resistance profiles between plasma and PBMC in HIV-1 patients. OBJECTIVES: To assess the prevalence of mutations associated with drug resistance and to compare cell free and cell-associated strains. STUDY DESIGN: Genotypic resistance analysis on viral DNA and plasma was performed in 31 therapy naive patients with chronic infection to check reverse transcriptase (RT) and protease resistance associated mutations before beginning antiretroviral therapy. RESULTS: Direct sequencing of DNA provirus disclosed key mutations (such as G190A/S, V106A, K103N and T215F) to RT inhibitors more frequently (7 patients out 31) than in plasma RNA (2 out of 31). In addition, major mutations (D30N, M46I, I50V, I84V) associated with drug resistance in the PR region were only found in PBMCs. CONCLUSIONS: Despite the small number of patients, our results show a different resistance profile between plasma and PBMC compartments and may yield additional information for first-line antiretroviral regimens. Further investigations on larger series followed-up for a longer period of time are required to obtain in-depth information on the meaning of the mutations detectable in plasma and/or in PBMCs.

Human T-lymphotropic virus type 1 (HTLV-1) prevalence and quantitative detection of DNA proviral load in individuals with indeterminate/positive serological results.

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.

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review.

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.

Quantitative DNA proviral detection in HIV-1 patients treated with antiretroviral therapy.

The amount of proviral DNA in peripheral blood mononuclear cells (PBMCs) from 93 HIV-1 seropositive patients on long-lasting antiretroviral therapy was measured by the SYBR green real-time PCR technique. Variable levels of proviral DNA, ranging from 14 to 1847 copies of HIV-1 DNA per 10(6) PBMC were found, without a significant correlation between proviral load and plasma HIV-1 RNA levels or CD4(+) lymphocyte counts. To investigate the possible role of HIV-1 DNA levels as prognostic markers in clinical practice, the amount of proviral DNA and peripheral blood CD4(+) lymphocyte counts were further evaluated after 5 months of continued therapy in 32 patients who maintained a persistently undetectable viremia throughout the observation period. Interestingly, a clear-cut decrease (> or =0.5 log) in proviral DNA levels was significantly associated with a definite increase in CD4(+) lymphocyte counts. Even though plasma HIV-1 RNA levels remain the basic parameter to monitor both the intensity of viral replication and the efficacy of therapeutic interventions, the results obtained in our study seem to indicate that measuring proviral DNA levels could represent an adjunct prognostic marker, especially useful in patients whose HIV-1 RNA levels drop below detectable limits.

Different patterns of HIV-1 DNA after therapy discontinuation.

BACKGROUND: By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16-24 weeks to study the possible relationship between DNA and RNA viral load. METHODS: The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia (RNA viral load). RESULTS: Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA. CONCLUSION: Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects.

HIV-1 tat protein and cell proliferation and survival: a brief review.

Many studies have demonstrated that HIV-1 Tat plays a pivotal role both in the HIV-1 replication cycle and in the pathogenesis of HIV-1 infection. Indeed, Tat affects the HIV-1 replication cycle regulation increasing the proviral transcription rate several hundred-fold and acting on the elongation of viral transcripts. Moreover, Tat displays several important biological activities committed to uninfected and infected cells by a paracrine/autocrine mechanism due to secretion of Tat from infected cells. In particular, Tat modulates the expression of several cellular genes and triggers the activation of some signal transduction pathways and transcription factors suggesting a complex role in the scenario of HIV-1 infection. This review focuses on some aspects of Tat biological activity with particular regard to effects of Tat on cell proliferation and survival regulation.

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique.

BACKGROUND: The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. OBJECTIVE: We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. STUDY DESIGN: Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. RESULTS/CONCLUSIONS: The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients.

HIV-1 viral load represents a basic marker for evaluation of the rate and severity of HIV-1 related disease and to monitor the effectiveness of treatment. An SYBR green-based real-time RT-PCR (SYBR green real-time RT-PCR) revealed by Light Cycler technology was evaluated for quantitation of HIV-1 RNA viral load in plasma of HIV-1 seropositive patients. The performance of the SYBR green real-time PCR was assessed on 56 HIV-1 seropositive patients under highly active retroviral therapy (HAART) and 25 blood donors. The results demonstrated that this technique detected 50 HIV-1 RNA copies per millilitre of plasma. Moreover, we compared real-time RT-PCR with the b-DNA technique considered widely a reference technique for HIV-1 RNA viral load measurement. The parallel quantitative analysis of HIV-1 positive samples showed a high correlation (r=0.908) between the two methods. Although b-DNA and the real-time-based method gave similar sensitivity, the assay determined quantitatively HIV-1 RNA copies in 4 out of 16 samples shown as undetectable by b-DNA. The SYBR green real-time RT-PCR represents a good alternative to b-DNA assay in HIV-1 viral load determination especially during the monitoring of HAART treatment.

Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

OBJECTIVES: To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. METHODS: Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. RESULTS: At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. CONCLUSIONS: The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection.

HIV-1 DNA load analysis in peripheral blood lymphocytes and monocytes from naïve and HAART-treated individuals.

OBJECTIVE: To evaluate HIV-1 DNA load in PBLs and monocytes from both long-term HAART-treated and antiretroviral naïve HIV-1 infected patients. METHODS: Cross-sectional quantitative analysis of HIV-1 DNA load was performed in PBLs and monocytes, purified from 34 long-term HAART-treated and 34 naïve HIV-1 infected patients, and compared to RNA viral load and CD4+ cell count. RESULTS: HAART-treated patients showed significantly lower levels of viral DNA both in PBLs and monocytes in comparison with naïve individuals. Variable levels of HIV-1 DNA amount in monocytes were detected in all naïve patients but only in 12 of 34 HAART-treated individuals. PBLs HIV-1 DNA load was inversely correlated to CD4+ cell count in naïve and HAART-treated patients whereas no association was detected in monocytes. CONCLUSIONS: Long-term HAART decreased HIV-1 DNA load in PBLs and monocytes demonstrating a valuable inhibitor effect, especially in short-lived reservoirs. In addition, the positive correlation of DNA burden between PBLs and monocytes may suggest a dynamic relation between these reservoirs in the course of disease. HIV-1 DNA load quantitative analysis in PBLs and monocytes may be considered an important approach to study the HIV-1 reservoir and the effectiveness of HAART therapy in HIV-1 seropositive patients.

Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy.

OBJECTIVE: The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population. METHODS: The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995-2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses. RESULTS: 18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.

HIV-1 negatively affects the survival/maturation of cord blood CD34(+) hematopoietic progenitor cells differentiated towards megakaryocytic lineage by HIV-1 gp120/CD4 membrane interaction.

To investigate the mechanisms involved in the human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia (TP), human umbilical cord blood (UCB) CD34(+) hematopoietic progenitor cells (HPCs) were challenged with HIV-1(IIIb) and then differentiated by thrombopoietin (TPO) towards megakaryocytic lineage. This study showed that HIV-1, heat-inactivated HIV-1, and HIV-1 recombinant gp120 (rgp120) activated apoptotic process of megakaryocyte (MK) progenitors/precursors and decreased higher ploidy MK cell fraction. All these inhibitory effects on MK survival/maturation and platelets formation were elicited by the interaction between gp120 and CD4 receptor on the cell membrane in the absence of HIV-1 productive infection. In fact, in our experimental conditions, HPCs were resistant to HIV-1 infection and no detectable productive infection was observed. We also evaluated whether the expression of specific cytokines, such as TGF-beta1 and APRIL, involved in the regulation of HPCs and MKs proliferation, was modulated by HIV-1. The specific protein and mRNA detection analysis, during TPO-induced differentiation, demonstrated that HIV-1 upregulates TGF-beta1 and downregulates APRIL expression through the CD4 engagement by gp120. Altogether, these data suggest that survival/differentiation of HPCs committed to MK lineage is negatively affected by HIV-1 gp120/CD4 interaction. This long-term inhibitory effect is also correlated to specific cytokines regulation and it may represent an additional mechanism to explain the TP occurring in HIV-1 patients.

Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples.

This paper describes the development of a SYBR Green-based multiplex real time RT-PCR for the simultaneous detection of HCV and HIV-1 genomes in plasma samples. Viral genomes were identified in the same sample by their distinctive melting temperature (Tm) which are 81.6 and 86.5 degrees C for HIV-1 gag 142 bp amplicon and HCV 5'-NCR region 226 bp amplicon, respectively. Analysis of known scalar concentrations of reference plasma indicated that the multiplex procedure detects at least 500 copies/ml of both HIV-1 and HCV. In addition, we also assayed HIV-1 and HCV viral load in 30 co-infected patients and in 15 blood donors, confirming the sensitivity and specificity of the assay. This method may represent a useful alternative method for the detection of HIV-1/HCV co-infection, reliable for a rapid and relatively inexpensive screening of blood donors.

HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity.

In this study, we showed the existence of a positive correlation between the amount of human immunodeficiency virus-type 1 (HIV-1) RNA in HIV-1 seropositive subjects and the plasma levels of TRAIL. Since it has been previously demonstrated that HIV-1 Tat protein up-regulates the expression of TRAIL in monocytic cells whereas tat-expressing lymphoid cells are more resistant to TRAIL cytotoxicity, we next investigated the effect of Tat on the expression/activity of both apical caspase-8 and -10, which play a key role in mediating the initial phases of apoptosis by TRAIL, and c-FLIP. Jurkat lymphoblastoid human T cell lines stably transfected with a plasmid expressing wild-type (HIV-1) tat gene showed normal levels of caspase-8 but significantly decreased levels of caspase-10 at both mRNA and protein levels with respect to Jurkat transfected with the control plasmid or with a mutated (cys22) non-functional tat cDNA. A significant decrease of caspase-10 expression/activity was also observed in transient transfection experiments with plasmid carrying tat cDNA. Moreover, c-FLIP(L) and c-FLIP(S) isoforms were up-regulated in tat-expressing cells at both mRNA and protein level in comparison with control cells. Taken together, these results provide a molecular basis to explain the resistance of tat-expressing Jurkat cells to apoptosis induced by TRAIL and, possibly, to other death-inducing ligands.

Serum antibody reactivity to human intracisternal A-type particle retrovirus proteins in systemic sclerosis patients.

Serum antibodies against human intracisternal A-type particle (HIAP) endogenous retrovirus have been found to be associated with various autoimmune pathologies. To evaluate the presence of serum antibody reactivity to HIAP proteins in systemic sclerosis, a Western blot analysis was performed on sera from 42 patients with systemic sclerosis, in comparison with 18 sera from patients with primary biliary cirrhosis and 52 healthy subjects. A positive Western blot was found in 55.5% of serum samples from patients with primary biliary cirrhosis and in 66.0% of patients with systemic sclerosis. None of the 52 healthy subjects showed positive results. Although this difference may be attributable either to an autoimmune response to antigenically related cellular proteins or to a specific antibody response to HIAP proteins expressed as an incidental consequence of attendant pathological processes, the high prevalence of antibodies against HIAP proteins demonstrated in patients with systemic sclerosis may be considered a hallmark of this disease.