MetaShot: an accurate workflow for taxon classification of host-associated microbiome from shotgun metagenomic data.

SUMMARY: Shotgun metagenomics by high-throughput sequencing may allow deep and accurate characterization of host-associated total microbiomes, including bacteria, viruses, protists and fungi. However, the analysis of such sequencing data is still extremely challenging in terms of both overall accuracy and computational efficiency, and current methodologies show substantial variability in misclassification rate and resolution at lower taxonomic ranks or are limited to specific life domains (e.g. only bacteria). We present here MetaShot, a workflow for assessing the total microbiome composition from host-associated shotgun sequence data, and show its overall optimal accuracy performance by analyzing both simulated and real datasets. AVAILABILITY AND IMPLEMENTATION: https://github.com/bfosso/MetaShot. CONTACT: graziano.pesole@uniba.it. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Efficacy of sofosbuvir and ledipasvir in an HCV+ gastro-resected patient.

WHAT IS KNOWN AND OBJECTIVE: The second-generation direct-acting antivirals represented the first major turning point for the eradication of HCV infection in almost all settings of patients. However, no data were available on use in gastro-resected patients. CASE DESCRIPTION: We report on a gastrectomized patient with chronic hepatitis C infection. She was treated with sofosbuvir and ledipasvir (SOF/LDV) for 12 weeks, with measurement of blood levels of the drugs. She obtained sustained virological response at week 12 and 24 without dose adjustment. WHAT IS NEW AND CONCLUSION: This case report can provide information useful for clinical practice in this set of patients and can open new perspectives in evaluating actual SOF/LDV bioavailability.

Hepatitis C diagnosis and treatment in people who use drugs: mind the gap in the linkage to care.

OBJECTIVE: The objective of this study is to identify a simplified rapid screening and linkage-to-care model for HCV among PWUD. PATIENTS AND METHODS: The study stems from a collaborative project bringing together two local Italian Centers for Drug Addiction and the Hepatology-Infectious Diseases Department of Lazzaro Spallanzani. A research physician analyzed the available medical records seeking to identify HCV and HIV infected patients in care in the addiction centers. Between March 2018 and January 2020 subjects were selected from among a cohort of 720 PWUD in the two Centers' care. The study comprises three steps: first, screening for HCVAb; second, the linkage to care; third, clinical assessment to treatment. The research physician recruited patients for the first two steps directly in their local addiction center. The third step was conducted in the Spallanzani. The characteristics of those subjects who adhered to the three-step study program were then compared to those of the non-adhering PWUD. RESULTS: 194 were known HCVAb positive patients. Of the 505 PWUD in the care of the two Centers eligible for screening, 364 were enrolled in the study. 144 resulted HCVAb positive. 269 were tested for HCVRNA. 101 underwent a full assessment. 96 patients started antiviral therapy with DAA. Patients who refused first step screening were older patients and mainly heroin users; in the second step, almost all the HIV/HCV co-infected patients agreed to a viremia test; in the third step all the HIV/HCV co-infected patients refused HCV treatment. CONCLUSIONS: The study suggests an on-site specialist approach conducted directly in the addiction centers themselves starting from screening; it can bring the goal of HCV PWUD microelimination closer.

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte.

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.

Acute HEV hepatitis: clinical and laboratory diagnosis.

OBJECTIVE: Hepatitis E Virus (HEV) is probably the most common cause of acute hepatitis worldwide. It has been regarded for a long time as a disease limited to developing countries. Recently, the refinement of diagnostic techniques, on the one hand, and migratory flows, on the other hand, have also led to the identification of an increased number of HEV infections in industrialized countries. Four HEV genotypes have been identified across the world, with different epidemiological burdens and a wide range of clinical presentations. Here, we report a case series of acute HEV hepatitis observed in the last three years in our hospital. PATIENTS AND METHODS: We performed a search for HEV IgM and IgG in all subjects admitted for acute hepatitis without evidence of other possible infectious, toxic or metabolic causes of liver damage. In subjects with HEV IgM positivity, the search for HEV-RNA was performed. RESULTS: We diagnosed eight acute HEV infections: 2 epidemic and 6 sporadic forms. HEV-RNA was detected in serum in 2 cases. CONCLUSIONS: HEV infection appears to be a cause of acute hepatitis that we must keep in mind even in developed countries.

A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping.

Chronic HCV infection is one of the leading causes of liver-related death and in many countries it is a primary reason for having a liver transplant. HCV genotype identification has long been used in the clinical practice, since different genotypes have different response rates and required different doses and durations of IFN/RBV treatment; moreover both the frequency and the pattern of resistance to different Direct-Acting Antivirals (DAAs) classes are subtype specific. Hence the necessity to make an accurate HCV subtyping becomes a fundamental tool to optimize current and future clinical management of HCV infected subjects. In the present study the performance of a next generation sequencing (NGS: based on the Ion Torrent Platform-Vela Sentosa SQ 301 sequencer) HCV genotyping assay has been evaluated. The current method targets a region of the NS5B gene and it is the unique NGS based market CE-IVD assay. As a comparative method a commercial method based on the detection via reverse hybridization of 5'UTR and core regions (Versant HCV Genotype 2.0 Assay, LiPA, Siemens) was selected. A total 207 plasma samples from HCV infected individuals were used. No selection was made for these samples that were submitted for routine HCV genotyping. The results show Vela NGS assay assigns major number of HCV subtypes with respect LiPA. Concerning genotype 1 and 3, the discrepancy of assigned subtypes for LiPA with respect to Vela NGS assay is not relevant (1.8% and 2%, respectively); in contrast, the difference of assigned subtypes for genotypes 2 and 4 is very high (96.6% and 100%, respectively). The resistance mutations data, except for 1a and 1b subtypes, remain scarce; the future relevant challenge will be to identify subtypes-specific drug resistance mutations, which are essential to create highly personalized therapeutic pathways.

Comparison of Versant HBV DNA 3.0 and COBAS AmpliPrep-COBAS TaqMan assays for hepatitis B DNA quantitation: Possible clinical implications.

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p<0.001), and 24.4% of samples showed differences in viral load values >0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p<0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

TT virus infection: role of interferons, interleukin-28 and 29, cytokines and antiviral proteins.

In 1997 a novel virus in the serum of a patient with acute post-transfusion hepatitis of non A-G etiology was identified. This agent was designed TT virus (TTV). It produces persistent viremia and no disease, but the mechanism of its persistence is poorly understood. In the present study mRNA expression of antiviral proteins as MxA, 2' 5' OAS, anti-apopotic protein, cytokines IL- 28, IL- 29 and IFN are examined in a subject affected by B lymphoma and positive for TTV DNA and RNA in this cellular subset, and in BJAB and Dohh2 cell lines.

In vitro activation of HIV RNA expression in peripheral blood lymphocytes as a marker to predict the stability of non-progressive status in long-term survivors.

OBJECTIVES: We investigated a selected group of 11 non-progressor, HIV-infected individuals 20 months prior to this study and found that they all had undetectable levels of viral RNA expression in their peripheral blood lymphocytes (PBL). Phorbol 12-myristate 13-acetate (PMA) and phytohaemagglutinin (PHA) stimulation of PBL produced easily detectable amounts of HIV RNA in only two out of five of these patients. Here we report the results of the virological and clinical follow-up of nine non-progressors from this group. We verified the stability of their non-progressive status and attempted to correlate it to specific virological markers. METHODS: Proviral DNA in lymphocytes was tested by semi-quantitative polymerase chain reaction (PCR). Detection of unspliced (US) and multiple spliced (MS) HIV RNA species in unstimulated and stimulated lymphocytes was performed by reverse transcriptase-PCR (RT-PCR). The amount of p24 antigen released into the media of lymphocyte cultures was measured using a standard procedure. Lymphocyte populations were depleted of CD8 cells by immunomagnetic purging. RESULTS: Follow-up of nine of these subjects showed that the patients who previously showed viral RNA activation following lymphocyte stimulation in vitro, developed a clinical and immunological progression characterized by CD4 count decline and lymphadenopathy. In contrast, all the other subjects maintained progression-free status throughout the follow-up period, with no detectable levels of HIV RNA in the PBL. Notably, this group of subjects showed no activation of viral RNA expression following stimulation of either undepleted or CD8-depleted lymphocytes in vitro. CONCLUSION: The group of non-progressors studied was found to be heterogeneous regarding the stability of the non-progressive status during the follow-up period. Our results suggest that the activation of HIV RNA expression following PMA-PHA treatment of lymphocytes in vitro is an early marker for future progression of the disease.

The debate on the presence of HIV-1 in human gametes.

The debate about the presence of HIV-1 particles in human gametes and recent experimental results are reported in detail. Using immunocytochemistry, in situ hybridization at electron microscopy level, polymerase chain reaction and in vitro fertilization, it has been demonstrated that human spermatozoa can incorporate HIV-1 using special receptors, different from the usual CD4, and that they remain active and able to vehicle the viral particles into the oocyte, which is regularly fertilized. Moreover, by transmission electron microscopy (TEM), immunocytochemistry and PCR, we demonstrated that cell-free HIV-1 is not able to bind and penetrate the human oocyte in vitro. We attribute this behaviour to the fact that the oocyte and cumulus cells are devoid both of GalAAG and of CD4 receptors. PCR analysis indicated that mRNAs specific for CD4, CXCR4 and CCR5 proteins were absent, too.

PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.

Detection of TT virus in lymph node biopsies of B-cell lymphoma and Hodgkin's disease, and its association with EBV infection.

Human TT virus (TTV) recently isolated from the serum of a patient with post-transfusion hepatitis does seem to have only hepatopathic effect. The virus can also infect the serum, peripheral blood mononuclear cells (PBMC) and bone marrow cells (BMC ). Additional evidence has indicated that TTV is also present in the serum of people with hematopoietic malignancies. A significant increase in the incidence of lymphoma has recently been observed worldwide. We have investigated the presence of TTV DNA in lymph node biopsies of Italian patients affected with the most common lymphoma types in Western Countries: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and nodular sclerosis Hodgkin's disease (NS-HD). The possible role of a co-infection with Epstein-Barr virus (EBV) has also been investigated. DNA was extracted from 73 paraffin-embedded and 38 snap-frozen tissue specimens. From these, only 67 samples (29 paraffin-embedded and 38 snap-frozen tissues) from a total of 56 patients, were suitable for PCR analysis. TTV and EBV were detected by PCR using primers from two different conserved region in TTV and EBV genomes respectively. TTV DNA was detected in 30.0-50.0% of FL, 30.8% of DLBCL and 30.0-50.0% of NS-HD cases, depending on the primers used. All cases of non-specific reactive lymphoid hyperplasia (RLH), used as a putative control, were negative. The two major TTV genotypes circulating in Italy (G1 and G2) were detected in the analysed lymphoid neoplasms. EBV DNA was detected in 40.0% of FL, in 72.7%of DLBCL, in 80.0% of SN-HD and in 40.0% of RLH cases. EBV co-infection was found in 90% of TTV positive cases. The in situ hybridization assay was performed in TTV positive frozen samples. The significant prevalence of TTV DNA in lymphocytes circulating in the lymph nodes of both B-cell lymphomas and HD reported herewith suggests an implication of TTV infection in the development of these lymphoproliferative disorders.

Comparative analysis of total and integrated HIV-1 DNA in peripheral CD4 lymphocytes and monocytes after long treatment with HAART.

OBJECTIVE: To determine the level of total and integrated HIV-1 DNA load in CD4 lymphocytes and monocytes of patients undergoing HAART treatment for at least 2 years. METHODS: CD4 lymphocytes were isolated by subjecting monocyte-depleted blood samples to immune-purging carried out with M-450 Dynabeads. Monocytes were separated by blood through a combined procedure of cell adherence to dishes and complement induced immune lysis with anti-CD3 Mab. HIV DNA in CD4 lymphocytes and monocytes was quantified by polymerase chain reaction (PCR) based limit dilution assay with two PCR protocols, specific for total (LTR PCR) and integrated (Alu PCR) forms of HIV DNA. The replication competence of the provirus harboured in monocyte-depleted peripheral blood mononuclear cells (PBMC) and adherent monocytes was assayed by measuring HIV-1 p24 antigen produced by in-vitro cultures established with these cells. RESULTS: The CD4 lymphocytes of all patients contained a consistent number of HIV DNA copies. Most patients were also positive for HIV DNA in monocytes. The Alu PCR analysis detected, integrated provirus in CD4 lymphocytes of 9 patients and in the monocytes of only three. Four patients had replication-competence virus in their PBL. The monocytes of all patients did not produce virus in vitro. CONCLUSION: The HIV infection of CD4 lymphocytes and monocytes is maintained even after HAART related, apparent, and durable suppression of viral replication. We suggest that the viral persistent infection of monocytes may play a role in maintaining the residual HIV activity found in patients undergoing HAART.

Factors predicting the persistence of genital human papillomavirus infections and PAP smear abnormality in HIV-positive and HIV-negative women during prospective follow-up.

As part of an extensive multi-institutional DIANAIDS study focused on assessing the risk factors, natural history, diagnosis and follow-up of genital human papillomavirus (HPV) infections in HIV-infected women, the present communication reports a sub-cohort of 142 women (89 HIV+ and 48 HIV-), followed-up for a mean of 14.07 (+/-10.84) months to analyse the factors predicting the persistence and clearance of HPV infections (polymerase chain reaction [PCR] and sequencing) and cervical Papanicolaou (PAP) smear abnormalities, using both univariate (Kaplan-Meier) and multivariate (Cox) survival analysis. The appearance of new HPV infections during the follow-up was significantly more frequent in HIV-positive than in HIV-negative women, odds ratio (OR) 8.800 (95% confidence interval [CI]: 1.199-64.611), and also the clearance rate was significantly less frequent in HIV-positive than in HIV-negative women, 69.2% vs 22.8%, respectively (OR 0.330; 95% CI: 0.163-0.670). These two groups were also markedly different with respect to the clinical course of the cervical lesions, in the frequency of progressive disease (determined by PAP smear) was higher in HIV-positive group (12/89) than in HIV-negative women (2/52) (OR 3.506; 95% CI 0.816-15.055) (P = 0.055), in whom the disease regressed more frequently than in HIV-positive women (13.5% vs 7.9%) (OR 0.584; 95% CI 0.217-1.573). Using (1) HPV-positivity, (2) oncogenic HPV-type and (3) significant PAP smear abnormality at the end of follow-up as outcome measures, (1) was significantly (P < 0.001) predicted by the following variables in univariate analysis: age, mode of contraception, CD4 count, and HIV-positivity. The significant predictors of (2) were age and mode of contraception. The outcome measure (3) was significantly predicted by CD4 count, PAP smear abnormality and PCR status at entry. In the multivariate analysis, the significant independent predictive factors for HPV-positivity proved to be only the HIV status (P < 0.001), and PCR status at entry, p53 polymorphism at aa-72, oncogenic HPV type and significant PAP smear at entry remained independent predictors, with the significance level of P < 0.05. None of the significant predictors of oncogenic HPV type in univariate analysis retained their independent value in multivariate analysis. Oncogenic HPV type at entry proved to be an independent predictor of significant PAP smear (P < 0.05). The present results indicate that HIV-infected women, even on highly active antiretroviral therapy, demonstrate a more aggressive clinical course of cervical HPV infections, and fail to eradicate the disease more frequently than HIV-negative women. This persistence of HPV-positivity, oncogenic HPV type and significant PAP smear abnormality can be predicted by the results of PAP test and HPV typing in univariate analyses, and partly retain their independent predictive value also in multivariate analysis. Clearly, in addition to regular monitoring by PAP smear, HPV testing for the oncogenic HPV types seems to provide additional prognostic information in the management of cervical lesions in HIV-infected women.

Dynamics of viral load in plasma and HIV DNA in lymphocytes during highly active antiretroviral therapy (HAART): high viral burden in macrophages after 1 year of treatment.

In this study we evaluated the level of HIV RNA in plasma and HIV DNA in peripheral blood cells. Sixteen antiretroviral-experienced HIV patients with severe immune suppression were included in the study. After the first month, 56.2% of the patients showed undetectable levels of HIV RNA, this percentage remaining stable after 1 year (53.3%). At enrollment, 7 patients (43.7%) with a low CD4+ T cell count (mean 22 per mm3 versus 73) showed HIV DNA levels below the limit of detection (5 copies/10(5)) in lymphocytes. They all subsequently had increased HIV DNA that became detectable between the first and the third month of therapy, associated with an increase of the CD4+ T cell count (mean 22 to 95/mm3); in 4 of these patients this increase was transitory, becoming undetectable again after one year. In 7 out of the 8 patients with detectable HIV DNA at enrollment, the HIV DNA level decreased over time. Seven out of 15 patients at 1 year (46.7%) showed both undetectable levels of HIV RNA in plasma and HIV DNA in lymphocytes (p<0.05); these patients had a higher CD4+ T cell count at baseline (mean 75 versus 25/mm3) and a higher increase (306 versus 177/mm3) after 1 year. PCR-based dilution assay carried out at 1 year showed that all patients had a consistent amount of HIV DNA positive- CD4+ T lymphocytes and macrophages, with higher values in these last cells. The data indicate that a durable reservoir of virus is still present in both lymphocytes and monocytes, even after long-lasting HAART treatment.

Using the number of koilocytes to predict HIV serostatus in women with HPV-associated SIL.

OBJECTIVE: To investigate the relationship between specific cytopathologic changes, koilocyte counts and human papillomavirus (HPV) types in HIV-positive and -negative women. STUDY DESIGN: A cohort of 459 women (266 HIV+ and 193 HIV-), were examined in a multicentric study (Early Diagnosis of Neoplasia in AIDS) involving 14 gynecologic centers. Altogether, 97 women had cervical smears consistent with squamous intraepithelial lesions (SIL). Koilocytes were found in 60/97 SIL slides, subjected to quantitative counting in 30 predetermined fields. HPV genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: SIL lesions were four times more frequent (29%) in HIV-positive women than in HIV-negative women (10%) (odds ratio = 3.80). HPV DNA was equally frequent in both groups. There was a strong association between the number of koilocytes and HIV serostatus in both high grade and low grade SIL diagnoses. The presence of eight or more koilocytes had a specificity of 93% and sensitivity of 76% toward the diagnosis of HIV-positive status. No HIV-negative woman had a count > 8 koilocytes. No association was shown between koilocyte count and HPV genotype. CONCLUSION: An elevated number of koilocytes could suggest the possibility of HIV infection. Pap smear examination might give the first clue to HIV positivity in otherwise-unsuspected cases.

Squamous intraepithelial lesions (SILs) and HPV associated changes in HIV infected women or at risk of HIV. DIANAIDS Cooperative Study Group.

OBJECTIVES: The study concerns the prevalence of squamous intraepithelial lesions (SILs) and the specific cervical cytopathological features of a group of HIV-positive and a group of HIV-negative women recruited in a multicentric cohort study. The assessment of HPV-DNA genotypes was carried out in both groups. METHOD: 459 women, 266 HIV-positive and 193 HIV-negative women at risk were examined in an Italian multi-institutional study involving 14 gynaecological centres. RESULTS: In our samples, the risk of SILs was 29.4% for HIV-positive women and 10% for HIV-negative women (O.R. = 3.90, C.I. 95%: 2.20-6.98) while HPV-DNA-PCR genotypes had a high prevalence in both groups of HIV-positive and HIV-negative women. Cytopathological features of SILs in HIV-positive women were: a higher number of koilocytes and a more marked atypia of high grade neoplastic cells. CONCLUSIONS: A higher prevalence of SILs as well as a specific cervical cytopathology might suggest HIV infection.

Acute HIV infection: a misleading presentation.

Detecting acute HIV infection is important, but often the infection is difficult to recognize. We report the case of a 23-year-old man with persistent genital ulceration; all microbiological examinations from the ulcers were negative. HIV-1/2 antibodies were positive, but the HIV-1 Western blot (WB) was indeterminate and HIV-1 p24 antigen was persistently negative, with a low HIV-1 RNA level. One month later, the genital ulcerations disappeared and the WB test showed complete seroconversion; nonetheless HIV p24 antigen remained negative and HIV-RNA was persistently low. HIV-1 proviral DNA was detected by nested polymerase chain reaction (PCR) from the initial ulcers swabs. This case was notable due to genital ulceration being the only sign of primary HIV infection. Furthermore, the serological pattern was unusual, and HIV-RNA was unexpectedly low. This underlines the importance of screening for HIV being infection in the setting of sexually transmitted infections (STIs), and also in cases of atypical STI-like presentations.