Mild coronavirus disease 2019: Results of a multicentric prospective study on severe acute respiratory syndrome coronavirus 2 presence in semen fractions and cells and its effects on semen parameters.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2, the virus responsible for coronavirus disease 2019, affects multiple organs. The virus enters cells through angiotensin-converting enzyme-2 and host factors present in genital organs, leading to concern over virus shedding in semen and reproductive function. OBJECTIVES: To investigate severe acute respiratory syndrome coronavirus 2 in semen from patients with a mild infection, identify the seminal infected cells, and explore the effect of the infection on sex hormones and semen parameters. MATERIALS AND METHODS: Prospective study of 54 men with mild severe acute respiratory syndrome coronavirus 2 infection. Semen was collected at 7, 15, 30, 60, 90, 180, and 365 days after symptom onset, and severe acute respiratory syndrome coronavirus 2 RNA was measured in serum, saliva, urine, and semen. The presence of infectious severe acute respiratory syndrome coronavirus 2 in semen was assessed using Vero cell culture. Infected semen cells were identified using immunofluorescence against severe acute respiratory syndrome coronavirus 2 nucleoprotein antigen and cell markers. Semen characteristics as well as testosterone, inhibin B, luteinizing hormone, and follicle-stimulating hormone levels were determined. RESULTS: 11% of patients had at least one severe acute respiratory syndrome coronavirus 2 RNA-positive semen. One patient had viral semen shedding up to day 90 after infection onset, with replication-competent virus isolated from semen and 40% cell fraction at day 7. After sperm preparation, 90% fraction was severe acute respiratory syndrome coronavirus 2 RNA-positive at days 7 and 15. The swim-up fraction was positive only on day 7. In semen, nucleoprotein antigen was detected mainly in exfoliated epithelial cells and less frequently in Sertoli cells. Sperm count and motile sperm count were lower at day 30 than at day 7. Round cells in semen were increased during the acute phase. At days 7 and 15, sperm count and motile sperm count were lower in severe acute respiratory syndrome coronavirus 2 RNA-positive semen compared with negative semen, while semen volume and follicle-stimulating hormone levels were increased. Long-term follow-up shows no evidence of a detrimental effect on hormonal or semen characteristics. DISCUSSION AND CONCLUSION: 11% of patients with mild coronavirus disease 2019 who were not hospitalized had severe acute respiratory syndrome coronavirus 2 excretions in semen, which persisted for up to 90 days in one patient. No germ cells appeared infected by the virus, but the detection of nucleoprotein antigen-positive epithelial semen cells and Sertoli cells suggests genital tract infection. Albeit infrequent, semen may contain the replication-competent virus during the acute phase with potential risk of severe acute respiratory syndrome coronavirus 2 transmissions during sexual contact and assisted reproduction procedures. The effect of mild coronavirus disease 2019 on spermatogenesis and reproductive hormones was moderate and reversible.

HIV-1 genotypic resistance testing using single molecule real-time sequencing.

BACKGROUND: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories. OBJECTIVES: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping. STUDY DESIGN: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches. RESULTS: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001). CONCLUSIONS: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.

Salvage Therapy Including Foscarnet and Ibalizumab for Multidrug-Resistant Human Immunodeficiency Virus Type 2 Infection.

We evaluated Ibalizumab (IBA)-containing standardized optimized salvage regimen (with or without a 4-week foscarnet induction) in individuals harboring multidrug-resistant human immunodeficiency virus type 2 (HIV-2). Nine were included; 2 achieved virological suppression after foscarnet induction with a sustained suppression at Week 24 after IBA initiation, and an additional individual at Week 24 after Ibalizumab initiation.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT- mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy.

BACKGROUND: The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. METHODS: We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. FINDINGS: Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. INTERPRETATION: Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. FUNDING: This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.