Analytical Assessment of the Vela Diagnostics NGS Assay for HIV Genotyping and Resistance Testing: The Apulian Experience.

Drug-resistance monitoring is one of the hardest challenges in HIV management. Next-generation sequencing (NGS) technologies speed up the detection of drug resistance, allowing the adjustment of antiretroviral therapy and enhancing the quality of life of people living with HIV. Recently, the NGS Sentosa® SQ HIV Genotyping Assay (Vela Diagnostics) received approval for in vitro diagnostics use. This work is the first Italian evaluation of the performance of the Vela Diagnostics NGS platform, assessed with 420 HIV-1 clinical samples. A comparison with Sanger sequencing performance is also reported, highlighting the advantages and disadvantages of the Sentosa® NGS assay. The precision of the technology was studied with reference specimens, while intra- and inter-assay reproducibility were evaluated for selected clinical samples. Vela Diagnostics' NGS assay reached an 87% success rate through 30 runs of analysis in a real-world clinical context. The concordance with Sanger sequencing outcomes was equal to 97.2%. Several detected mismatches were due to NGS's superior sensitivity to low-frequency variants. A high accuracy was observed in testing reference samples. Repeatability and reproducibility assays highlighted the good performance of the NGS platform. Beyond a few technical issues that call for further optimization, the key improvement will be a better balance between costs and processing speed. Once these issues have been solved, the Sentosa® SQ HIV Genotyping Assay will be the way forward for HIV resistance testing.

Anti-spike S1 receptor-binding domain antibodies against SARS-CoV-2 persist several months after infection regardless of disease severity.

Data regarding the immunological memory and long-time kinetics of immunoglobulin (IgG) against viral nucleoprotein (NP) and spike protein S1 receptor-binding domain (S1RBD) of Severe Acute Respiratory Syndrome-associated Coronavirus 2 (SARS-CoV-2) are lacking. All consecutive COVID-19 patients admitted to our Clinic between March 1, 2020, and May 1, 2020, who were tested at hospital admission for anti-S1RBD and anti-NP IgG were enrolled. Serum samples were tested for anti-SARS-CoV-2 antibodies with the use of two commercially available enzyme-linked immunosorbent assays. Results are expressed as optical density measurements at 450 nm (OD450 ). Overall, 111 patients were included; the median (q1-q3) age was 57 (49-73) years, 59 (53%) males. According to disease severity, 31 (28%), 47 (42%), and 33 (30%) patients were considered affected by mild/moderate, severe, and critical SARS-CoV-2 infection, respectively. During hospitalization, patients with the critical disease showed a higher peak value of both anti-NP (median OD450 : 3.66 vs. 3.06 vs. 3.00 respectively, p = .043) and anti-S1RBD IgG (median OD450 : 2.33 vs. 1.6 vs. 0.91, respectively, p < .001). By testing 48 subjects 6 months or above from discharge, a significant decrease of anti-NP IgG was observed (r: -0.5838; p < .0001), whereas anti-S1RBD IgG showed only a modest reduction (r: -0.1507; p = .0647). Accordingly, 10 (21%) and 2 (4%) patients had a negative serological status for anti-NP and anti-S1RBD IgG, respectively; no association with clinical severity was found. IgGs against SARS-CoV-2 persisted several months after discharge, regardless of disease severity, suggesting that vaccination could be a valid strategy to fight the pandemic.

Gp120 substitutions at positions associated with resistance to fostemsavir in treatment-naive HIV-1-positive individuals.

OBJECTIVES: Fostemsavir, a novel attachment inhibitor targeting the HIV-1 gp120, has demonstrated wide in vitro activity. However, the high rate of HIV gp120 substitutions could jeopardize its efficacy. We investigated envelope (env) substitutions at positions associated with resistance to fostemsavir in patients with a new HIV-1 diagnosis according to HIV subtype and tropism. METHODS: Gp120 sequences from 409 subjects were retrospectively analysed and the presence of the L116P, A204D, S375H/M/T, M426L, M434I and M475I mutations was evaluated. Other amino acid changes at the same positions were also recorded. The variability at each amino acid position was evaluated using Shannon entropy. RESULTS: The frequency of mutations was: S375T (13.2%); M426L (6.8%); M434I (2.9%); M475I (2.7%); S375H (1.0%)/M (0.8%) and L116P (0.31%). Statistically significant differences were found at positions 375 (R5/non-R5 strains and B/non-B subtypes) and 426 (B/non-B subtypes); post hoc analysis revealed that significance for position 375 was steered by S375T while for position 426 significance was governed by unusual substitutions, in particular M426R (B/non-B, P < 0.00001). The variability of env constant domains appeared to be more relevant in the non-B virus population. CONCLUSIONS: In conclusion, gp120 substitutions were detected in different subtypes and in both R5 and non-R5 variants. Despite the great variability of gp120, the frequency of mutations was low overall and the predominant substitution was S375T, the role of which in reducing fostemsavir efficacy is less substantial.

Influence of HIV-1 V2 sequence variability on bacterial translocation in antiretroviral naïve HIV-1 infected patients.

HIV-1 V2 domain binds α4ß7, which assists lymphocyte homing to gut-associated lymphoid tissue. This triggers bacterial translocation, thus contributing to immune activation. We investigated whether variability of V2 179-181 binding site could influence plasma levels of lipopolysaccharide (LPS) and soluble cluster of differentiation 14 (sCD14), markers of microbial translocation/immune activation. HIV gp120 sequences from antiretroviral naïve patients were analyzed for V2 tripeptide composition, length, net charge, and potential N-linked-glycosylation sites. LPS and sCD14 plasma levels were quantified. Clinical/immuno-virologic data were retrieved. Overall, 174 subjects were enrolled, 8% with acute infection, 71% harboring a subtype B. LDV179-181 was detected in 41% and LDI in 27%. No difference was observed between levels of LPS or sCD14 according to different mimotopes or according to other sequence characteristics. By multivariable analysis, only acute infection was significantly associated with higher sCD14 levels. In conclusion, no association was observed between V2 tripeptide composition and extent of bacterial translocation/immune activation.

X4 viruses are frequently archived in patients with long-term HIV infection but do not seem to influence the "inflamm-aging" process.

BACKGROUND: Co-receptor tropism (CRT) in patients with a long history of HIV-1 infection and antiretroviral treatment has been rarely investigated to date. The aim of this study was to determine the prevalence of X4 and R5 strains in patients with a >15-year follow-up and to investigate the demographical, viral, immunological, clinical and therapeutic determinants of CRT in this population. The possible influence of CRT on the inflammation state related to chronic HIV infection was also examined. METHODS: A total of 118 HIV-1 infected patients with an initial HIV-1-positive test before 1997, and still on follow-up, were enrolled and consecutively submitted to blood sampling. Of these, 111 were on antiretroviral therapy and 89/111 (80.2%) had a plasma viral load (pVL) <25 copies/ml at testing. HIV-1 DNA was extracted and amplified from PBMCs for env gp120 sequencing. CRT was assigned by using geno2pheno and isolates were classified as X4 (FPR ≤20%) or R5 (FPR >20%). Level of serological inflammation biomarkers including IL-6, hsPCR, and D-dimers were measured. RESULTS: An X4 virus was evidenced in HIV-1 proviral DNA of 50 patients (42%) while the remaining 68 patients were classified as R5. The median follow-up was 19 years (range 15-25). No association was observed between CRT and sex, age, nationality, subtype, HIV risk factor, HBV/HCV co-infection, baseline CD4+ cell count and pVL, overall duration of antiretroviral therapy, past exposure to mono-or dual therapies, and duration of NNRTI or PI-based therapy. The presence of an X4 strain was associated with CD4 nadir (p = 0.005), CD4 absolute count over time (p < 0.001), and cumulative positive (copy/years) viremia (p <0.001) during the whole patient history. No differences were found between R5 and X4 patients regarding inflammation marker levels including Il-6, hsPCR and D-dimers. CONCLUSIONS: An archived X4 virus was demonstrated in 42% of patients with a >15-year-history of HIV infection. This presence was clearly associated with a greater exposure to positive viremia and a poorer CD4 trend over time compared to R5, independent of type and duration of antiretroviral treatment. CRT does not seem to influence the inflammation rate of patients aging with HIV.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy.

SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April-December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.

Impact of mutations outside the V3 region on coreceptor tropism phenotypically assessed in patients infected with HIV-1 subtype B.

HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early- and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.

Co-receptor switch during HAART is independent of virological success.

The influence of antiretroviral therapy on co-receptor tropism remains controversial. To verify if co-receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non-responder patients. Baseline blood samples were collected from 36 patients infected with HIV-1 subtype-B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV-1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5-to-X4 and four from X4-to-R5. The lack of reaching virological suppression was not associated with an X4-to-R5 (P = 0.25) or R5-to-X4 (P = 0.14) shift; time-to-viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance-associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 +/- 30.6 in R5-to-X4 shifting patients vs. 161.6 +/- 150.6 in non-shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5-to-X4 viral shift.

Variability OF HIV-1 V2 env domain for integrin binding: Clinical correlates.

The HIV V2179-181 (HXB2 numbering) tripeptide mediates binding to α4ß7 integrin, which is responsible for GALT homing. Our study aimed to assess V2 variability in naive HIV-1 infected patients and its association with clinical and viro-immunological features. Gp120 sequences were obtained from 322 subjects; length, potential N-linked glycosylation sites (PNGs), net-charge (NC) and 179-181tripeptide α4ß7-binding-motif of V2 were evaluated. At multivariate analysis, lower V2 length and higher NC correlated with low CD4 cells; no association was found with PNGs. A greater variability pertained positions 162-163, 164-167, 169, 175-179, 187, 194 and 195 in B sequences, and 163 and 177 in X4 tropic viruses. LDV was the most common tripeptide. Asp180 was highly conserved; Leu179 was more frequently observed in non-B and in recent infections compared to others, while Val181 was found in recent infections and in MSM. Further studies to deeply explore the clinical significance of these associations are warranted.

HIV-1 coreceptor tropism: A syllogistic connection with The Veterans Aging Cohort Study Index and the CD4/CD8 ratio.

BACKGROUND: The association between X4 virus and an increased risk of non-AIDS-events has been reported. Morbidity/mortality due to non-AIDS events, which are properly predicted by the CD4/CD8 ratio and VACS index, have become particularly remarkable in HIV-infected patients receiving effective combined antiretroviral therapy (cART). METHODS: We verified the validity of the syllogism: as HIV-tropism (CRT) contributes to the onset of non-AIDS events which are successfully predicted by the CD4/CD8 ratio and VACS index, then CRT correlates with these two variables. The CD4/CD8 ratio and VACS index at baseline and overtime were analyzed according to CRT tested before the first successful cART regimen in newly-diagnosed patients. RESULTS: Patients with R5 variants had a significantly lower baseline VACS percentage risk [mean (95%CI):18.2%(16.1-20.3) vs 24.3%(18.2-22.5), p = 0.002] and higher baseline CD4/CD8 ratio [mean (95%CI):0.43 (0.38-0.47) vs 0.28 (0.19-0.36), p = 0.002] than non-R5 patients. After an initial drop, VACS increased again in R5 and non-R5 patients and the two trend curves almost overlapped. The CD4/CD8 ratio had an increasing trend in both R5 and non-R5 patients; however, even though non-R5 patients had a greater gain of CD4+, they maintained a lower CD4/CD8 ratio at any time point. CONCLUSION: Our study confirms an association between pre-therapy CRT, CD4/CD8 ratio and VACS. A successful cART regimen positively affects the CD4/CD8 ratio; however, the disadvantage conferred by a non-R5 CRT is maintained overtime. The restoration of VACS in all patients could be directly due to variables included in the VACS calculation and to factors that adversely influence these variables.

HIV-RNA decay in paired blood and semen samples of subjects receiving their first dolutegravir-based ART regimen.

BACKGROUND AND OBJECTIVES: We aimed to investigate to what extent a first-line DTG-based ART regimen reduces HIV-RNA in semen compared to plasma. STUDY DESIGN: In this prospective, observational study, ART-naïve, HIV-infected males starting their first ART regimen with DTG plus TDF/FTC or ABC/3TC were enrolled. Paired blood (BP) and seminal plasma (SP) samples were collected at baseline (T0) and at week-2/4/12/24 after ART initiation. Sexually transmitted infections (STI) were ruled out before enrolment. RESULTS: Median baseline HIV-RNA levels were lower in SP compared to BP (657 versus 38.200 copies/ml, p < 0.001), three subjects had undetectable semen HIV-RNA. After 12 weeks of treatment, HIV-RNA was below the quantification limit in both BP and SP of 11 pts (61.1%). Discordant results were obtained in 6 subjects (33.3%), showing quantifiable HIV-RNA in blood only (2 cases) and in semen only (4 cases). Finally, one subject had a positive HIV-RNA in SP/BP. At W24, only in 2/16 subjects (12.5%) HIV-RNA was detectable in semen, while in the others it was negative on SP/BP. No concurrent STI was found in subjects with detectable VL in semen. CONCLUSIONS: DTG demonstrated effectiveness in reducing VL with different kinetics in blood and semen, despite seminal viral suppression after 6 months of ART was not obtained in the totality of subjects.

A multidrug-resistant (MDR) HIV type 1 infection in a homosexual man and identified source patient.

We report a case (IC) of multidrug-resistant (MDR) HIV-1 infection in which the identification of the source patient (S) was supported by phylogenetic analysis of the pol gene and by the similarity of env sequences. HIV isolates from IC and S were characterized as non-syncytium viruses: a X4 variant (R(11) E(26)) was identified in both cases according to the V3 loop sequence. The pol mutational profile of IC included multiple protease and reverse-transcriptase inhibitor mutations similar to those in S. The lamivudine/tenofovir/tipranavir/ritonavir/enfuvirtide association was effective for IC but not for S.

Difficulties in classifying a/g recombinants: methodological problems or genetic variability?

Thirty pol gene plasma-derived sequences clustering with the circulating recombinant form (CRF) 02_AG (IbNG) (bootstrap 100%) were evaluated to analyze the genomic composition. Subtype assignment was also phylogenetically confirmed by C2-V3 region analysis for 18/21 sequences evaluated. Thereafter, we compared the genomic recombination of the CRF02_AG/IbNG prototype as predicted by bootscanning and Jumping HMMER software (jpHMM) to that of our strains. With these methods, 27% and 50%, respectively, of our clinical sequences demonstrated the same pol structure as the prototype CRF02_A/G-IbNG. However, in subtrees built for each segment predicted by jpHMM (with a bootstrap value of more than 75%), all fragments clustered with IbNG and were distinct from A and G clades. Overall, our sequences resulted in true members of CRF02_AG-IbNG, which, however, appeared to be a subtype phylogenetically separate from A or G, at least with regard to the pol gene.

V3 sequences and paired HIV isolates from 52 non-subtype B HIV type 1-infected patients.

Viral isolation and V3 sequencing were performed for 52 patients with non-subtype B viruses. The HIV-1 isolation rate was 93%, and 98% of isolates were characterized as NSI. V3 sequences corresponding to NSI isolates were compared to non-subtype B sequences with corresponding SI isolates from the Los Alamos database. The two sequence groups significantly differed in number of sequences with 35 amino acids, net charge, Brigg's coefficient, loss of NGS at positions 6-8, and 11/25 genotype (p < 0.0001). Substantial discrepancies in V3 variability were also observed. Basic amino acids at positions 8, 21, 23, and 24 were more frequent in SI sequences as were uncharged amino acids at positions 5, 6, 7, 8, 12, 13, 25, and 34. When characterizing paired viral isolates and V3 sequences from patients with non-subtype B HIV-1, current V3 sequence-based criteria from subtype B appeared to discriminate well between NSI and SI sequences from non-subtype B patients.

Genotypic analysis of the protease and reverse transcriptase of non-B HIV type 1 clinical isolates from naïve and treated subjects.

One hundred and ninety-two pol sequences of drug-naïve and drug-experienced subjects infected with non-B HIV-1 subtypes were analyzed to identify treatment-related amino acid changes which might be relevant for drug-resistance and possibly not included in the accepted mutation list for the B subtype. The correspondence analysis identified non-B-specific and subtype-specific polymorphisms which should not be mistaken for mutations. Multiple chi(2) were performed to detect the differences between naïve vs treated subjects and between different subtypes. To verify the contribution of each single mutation to the resistance levels as predicted by the Virtual Phenotype-LM, simple univariate linear regression was used with fold resistance as a dependent variable and individual mutations as predictors. Commonly accepted protease (PR) and reverse transcriptase (RT) positions along with mutants at RT positions 118 and 90 were significantly associated with treatment. Two unusual PR (K14R and I66F) and five RT positions (E28K, S68G, H221Y, L228R/H and P294A) were also associated with treatment (p<0.01). Only minimal variations were observed with respect to commonly accepted amino acid changes. All amino acid changes correlated with treatment influenced the resistance levels to each single drug. Our findings demonstrate that there are no substantial differences regarding known resistance-associated mutations and the newly emergent substitutions between non-B and B subtype strains.

HIV infection and protease genetic diversity in a rural area of the Southern Central African Republic.

Thirty-eight of 175 (21.7%) consecutive women of childbearing age from a rural area near Bangassou were tested HIV-positive. Ten protease and two protease/reverse transcriptase sequences (31.5% samples) were obtained. Eight sequences clustered into subtype J, A, G; two sequences were 13_cpx recombinant forms and two were indeterminate. The high proportion of HIV found suggests a recent outbreak of diversified HIV strains.

HIV-1 subtypes and circulating recombinant forms (CRFs) from HIV-infected patients residing in two regions of central and southern Italy.

A total of 347 pol gene sequences from 88 Tuscan and 259 Apulian subjects (including 52 non-Italians and 9 children) were analyzed phylogenetically. Forty-four (12.6%) non-B subtypes were found, including 3.4% C, 1.4% F1, 0.8% G, and 0.3% each for J and A pure subtypes, and 3.7% CRF02_AG, 1.4% CRF01_AE, 0.6% BF, and 0.3% CRF06-cpx recombinant forms. An additional sample close-matched the pol gene of an unique recombinant form (URF AGK 99GR303). The non-B subtypes were from 40 adults and 4 children; 12 of these 44 patients were epidemiologically linked. Thirty-three of the 44 non-B viruses pertained to non-Italian immigrants and 11 to Italians, signifying that 63.4% immigrants and 3.7% Italians harbored non-B subtypes. The overall frequency of non-B subtypes was higher in Tuscany than in Apulia (18.1% vs. 10.8%). Moreover, 6.1% and 3.0% non-B subtypes were found among Italians from Florence and Apulia, respectively, while 52.1% and 72.4% of immigrants living in Tuscany and Apulia harbored non-B subtypes. Women infected by means of sexual contact prevailed among non-Italian adults; the majority of Italians were males and admitted high-risk sexual behavior. Four Italians had a history of extensive travel in countries of high endemicity. Social and epidemiological changes are responsible for an increasing circulation of non-B subtypes in Italy. Although non-B subtypes principally infect non-Italian patients, in Italy they can no longer be considered exclusively restricted to subjects from endemic areas.