HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Human papillomavirus testing using HPV APTIMA® assay and an external cellularity control in self-collected samples.

In cervical cancer screening programs, the detection of high-risk human papillomavirus (HR-HPV) is now widely implemented on physician-collected samples and has expanded to include self-collected samples. The use of a cellularity control (CC) is needed to reduce false-negative HPV results. An external mRNA CC for the HPV APTIMA® assay was assessed for its analytical performance and the results were compared with both cervix cytobrush samples taken by physicians and self-collected vaginal samples from 148 women. The performance of the CC was adjusted to control for the presence of cellular mRNA in the ThinPrep® and Multitest® transport media. This CC is user-friendly but implies to perform two independent assays on PANTHER® automate. Self-collected vaginal sampling gives a lower median CC results (13.2 vs. 16.9 min) but a higher risk of negative CC results (3.3 vs. 0%). The usefulness of the CC for the HR-HPV assay may be optimized by the definition of a threshold for a minimum cell number to be tested to increase confidence in HPV-negative results. The systematic use of an RNA CC increases confidence for HPV RNA assays on self-collected vaginal samples.

A severe monkeypox infection in a patient with an advanced HIV infection treated with tecovirimat: clinical and virological outcome.

A patient aged 28 years who is immunocompromised and living with HIV/AIDS became infected with the monkeypox virus (MPXV). His clinical condition deteriorated for 37 days, with fever, skin lesions, and diarrhea before going to the infectious diseases department, where his severe, protracted infection was treated with tecovirimat for 14 days. His condition rapidly improved, and the skin lesions decreased, as did the MPXV loads, with no adverse events. This case indicates that tecovirimat might be effective for treating patients who are immunocompromised and are infected with MPXV.

Effects of Acute Dengue Infection on Sperm and Virus Clearance in Body Fluids of Men.

We investigated the effects of dengue virus (DENV) on semen using samples collected 7, 15, 30, 60, and 90 days after symptom onset from 10 infected volunteers on Réunion Island. We assessed characteristics of semen and reproductive hormones and isolated motile spermatozoa from semen. We assayed semen for DENV using reverse transcription PCR and searched for DENV RNA by virus isolation in Vero E6 cell cultures. Four volunteers had >1 DENV RNA-positive semen samples; 2 volunteers had DENV RNA-positive semen at day 15 and 1 at day 30. No motile sperm were DENV positive. After exposure to positive semen, few Vero E6 cells stained positive for DENV antigens, indicating low levels of replicative virus. We found DENV had shorter duration in semen than in blood. These findings support the possibilities that DENV is sexually transmissible for a short period after acute dengue illness and that acute dengue induces reversible alterations in sperm.

Severe placental lesions due to maternal SARS-CoV-2 infection associated to intrauterine fetal death.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause severe placental lesions leading rapidly to intrauterine fetal death (IUFD). From August 2020 to September 2021, in the pathology department of Toulouse Oncopole, we analyzed 50 placentas from COVID-19-positive unvaccinated mothers. The purpose of our study is to describe the clinicopathological characteristics of these placental damages and to understand the pathophysiology. Ten of them (20%) showed placental lesions with positive immunohistochemistry for SARS-CoV-2 in villous trophoblasts. In five cases (10%), we observed massive placental damage associating trophoblastic necrosis, fibrinous deposits, intervillositis, as well as extensive hemorrhagic changes due to SARS-CoV-2 infection probably responsible of IUFD by functional placental insufficiency. In five other cases, we found similar placental lesions but with a focal distribution that did not lead to IUFD but live birth. These lesions are independent of maternal clinical severity of COVID-19 infection because they occur despite mild maternal symptoms and are therefore difficult to predict. In our cases, they occurred 1-3 weeks after positive SARS-CoV-2 maternal real-time polymerase chain reaction testing and were observed in the 2nd and 3rd trimesters of pregnancies. When these lesions are focal, they do not lead to IUFD and can be involved in intrauterine growth restriction. Our findings, together with recent observations, suggest that future pregnancy guidance should include stricter pandemic precautions such as screening for a wider array of COVID-19 symptoms, enhanced ultrasound monitoring, as well as newborn medical surveillance.

Peripheral Plasma and Semen Cytokine Response to Zika Virus in Humans.

We assessed Zika virus RNA and select cytokine levels in semen, blood, and plasma samples from an infected patient in South America. Viral RNA was detected in semen >2 months after viremia clearance; cytokine profiles differed in semen and plasma. After viremia, Zika virus appears to become compartmentalized in the male reproductive tract.

Detection of Zika, dengue and chikungunya viruses using single-reaction multiplex real-time RT-PCR.

Zika (ZIKV), Dengue (DENV) and Chikungunya viruses (CHIKV) co-circulate in the same geographical areas during the same seasonal period through the same biting arthropods. Therefore a rapid sensitive and specific molecular assay for these viruses would be a considerable help in the disease management and the epidemiological survey. We developed a one-step multiplex real-time PCR for the simultaneous detection of these viruses. Intra and inter-reproducibilities varied from 0.41% to 3.29% and from 1.13% to 4.93% for each virus respectively. The specificity was 100%. Whole blood, plasma and urines were used for comparison with commercially available monoplex assays (RealStar® kits, Altona Diagnostics GmbH, Hamburg, Germany). The concordance was 96%, 92.9% and 95.7% for ZIKV, DENV and CHIKV respectively. No cross reaction and no PCR inhibition were observed for any of the clinical samples. This test can thus be used as a rapid molecular assay for ZIKV, DENV1-4 and CHIKV infections.

High-frequency, high-intensity electromagnetic field effects on Saccharomyces cerevisiae conversion yields and growth rates in a reverberant environment.

Studies of the effects of electromagnetic waves on Saccharomyces cerevisiae emphasize the need to develop instrumented experimental systems ensuring a characterization of the exposition level to enable unambiguous assessment of their potential effects on living organisms. A bioreactor constituted with two separate compartments has been designed. The main element (75% of total volume) supporting all measurement and control systems (temperature, pH, agitation, and aeration) is placed outside the exposure room whereas the secondary element is exposed to irradiation. Measurements of the medium dielectric properties allow the determination of the electromagnetic field at any point inside the irradiated part of the reactor and are consistent with numerical simulations. In these conditions, the growth rate of Saccharomyces cerevisiae and the ethanol yield in aerobic conditions are not significantly modified when submitted to an electromagnetic field of 900 and 2400 MHz with an average exposition of 6.11 V.m-1 and 3.44 V.m-1 respectively.

Kinetics of anti-ZIKV antibodies after Zika infection using two commercial enzyme-linked immunoassays.

High performance assays are essential for the serological diagnosis of recent and past Zika virus (ZIKV) infections but few are presently available. We used two commercially available NS1 antigen-based enzyme-linked immunoassays to study the kinetics of anti-ZIKV IgM and IgG in 15 ZIKV-infected patients for up to 180days after clinical onset. The Diapro assay detected anti-ZIKV IgM reactivity more frequently (100%) and for longer (median 53days) than did the Euroimmun assay (60%; 13days, P<0.005). Both assays detected anti-ZIKV IgG reactivity 11days after clinical onset in all subjects. ZIKV IgG reactivity decreased in 3 subjects, suggesting long-term false-negative results with the Euroimmun assay. Existing anti-Dengue antibodies seem to modify the detection of ZIKV IgG but the specificity of the immunoassays was not assessed. These enzyme-linked immunoassays were user-friendly and provided results rapidly in our hands but they need further assessment before being widely used for diagnosis or public health surveys.

Patterns of residual HIV-1 RNA shedding in the seminal plasma of patients on effective antiretroviral therapy.

BACKGROUND: More and more HIV-1-infected men on effective antiretroviral treatment (ART) have unprotected sex in order to procreate. The main factor influencing transmission is seminal HIV shedding. While the risk of HIV transmission is very low, it is difficult to assess in individuals. Nevertheless, it should be quantified. RESULTS: We retrospectively analysed seminal plasma HIV-1 shedding by 362 treated HIV-infected men attending a medically assisted reproduction centre (1998-2013) in order to determine its frequency, the impact of the antiretroviral regimen on HIV shedding, and to identify shedding patterns. The HIV-1 virus loads in 1396 synchronized blood and semen samples were measured, and antiretroviral treatment, biological and epidemiological data were recorded. We detected isolated HIV-1 shedding into the seminal plasma in 5.3% of patients on efficient antiretroviral treatment, but there was no association with the HIV antiretroviral drug regimen or the CD4 cell count. These men had undergone more regimen changes since treatment initiation and had been on the ongoing drug regimen longer than the non-shedding men. The patterns of HIV seminal shedding among patients with undetectable HIV blood virus load varied greatly. HIV seminal shedding can occur as long as 5 years after starting antiretroviral treatment. CONCLUSIONS: The seminal HIV load was used to monitor risk for infertile HIV-infected patients on an assisted reproductive technology program. This can still be recommended for patients who recently (6 months) started ART, or those with a poor history of adherence to ART but may also be usefull for some patients during counselling. Residual HIV seminal shedding is probably linked to breaks in adherence to antiretroviral treatment but local genital factors cannot be ruled out.

RÉSUMÉ: De plus en plus d'hommes sous traitement antirétroviral (ART) ont des rapports sexuels non protégés à des fins de procréation. Le principal déterminant de la transmission sexuelle est. l'excrétion séminale du VIH. Malgré un risque de transmission très faible, il reste difficile à évaluer au niveau individuel. Dans ce contexte, l'étude de l'excrétion séminale du VIH, notamment chez des hommes sous traitement antirétroviral, est. d'un grand intérêt. RÉSULTATS: Nous avons analysé rétrospectivement l'excrétion séminale du HIV chez 362 hommes sous traitement antirétroviral consultant un centre d'assistance médicale à la procréation (1998­2013) pour déterminer sa fréquence, l'impact des antirétroviraux sur l'excrétion du HIV et identifier les profils d'excrétion. Les charges virales HIV-1 ont été mesurées dans 1396 échantillons de sang et de sperme prélevés concomitamment et les traitements, les données biologiques et épidémiologiques recueillis. Nous avons détecté une excrétion dans le plasma séminal isolée chez 5,3% des patients sous traitement antirétroviral efficace mais nous n'avons pas trouvé d'association avec la composition du traitement antirétroviral ou le taux de lymphocytes T CD4+. Ces hommes avaient eu plus de changements thérapeutiques et leur traitement avait été instauré depuis plus longtemps que pour les hommes non excréteurs. Les profils d'excrétion séminale du HIV parmi les patients avec une charge virale indétectable dans le sang étaient très variables. L'excrétion séminale du HIV peut survenir jusqu'à 5 ans après l'instauration du premier traitement antirétroviral. CONCLUSIONS: La charge virale séminale du HIV était l'outil classique d'évaluation du risque viral de transmission pour les patients infertiles infectés par HIV et inclus dans les programmes d'assistance médicale à la procréation. Ceci peut continuer à être recommandé chez les patients ayant débuté un traitement antirétroviral dans les 6 mois précédent ou chez ceux avec des antécédents de mauvaise adhérence au traitement mais peut aussi être utile pour le conseil de certains patients. Le risque résiduel d'excrétion séminale du HIV est. probablement lié à des défauts d'adhérence au traitement antirétroviral mais des facteurs génitaux ne peuvent pas être éliminés.

Effect of acute Zika virus infection on sperm and virus clearance in body fluids: a prospective observational study.

BACKGROUND: Evidence of human sexual transmission during Zika virus emergence is a matter of concern, particularly in procreation, but to date, kinetics of seminal shedding and the effects of infection on human reproductive function have not been described. To investigate the effects of Zika virus infection on semen and clearance of Zika virus from semen and body fluids, we aimed to study a cohort of Zika virus-infected men. METHODS: This prospective observational study recruited men presenting with acute Zika virus infection at Pointe-à-Pitre University Hospital in Guadeloupe, French Caribbean, where a Zika virus outbreak occurred between April and November, 2016. Blood, urine, and semen were collected at days 7, 11, 20, 30, 60, 90, and 120 after symptom onset, and semen characteristics, such as total sperm count, sperm motility, vitality, and morphology, and reproductive hormone concentrations, such as testosterone, inhibin, follicle-stimulating hormone, and luteinising hormone, were assessed. At days 7, 11, and 20, semen was processed to isolate motile spermatozoa. Zika virus RNA was detected by RT-PCR using whole blood, serum, urine, seminal plasma, semen cells, and motile spermatozoa fractions. Zika virus was isolated from different sperm fractions on Vero E6 cultures. FINDINGS: 15 male volunteers (mean age 35 years [SD 5; range 25-44) with acute Zika virus infection and positive Zika virus RNA detection in blood or urine were enrolled. Total sperm count was decreased from median 119 × 106 spermatozoa (IQR 22-234) at day 7 to 45·2 × 106 (16·5-89·6) at day 30 and 70 × 106 (28·5-81·4) at day 60, respectively, after Zika virus infection. Inhibin values increased from 93·5 pg/mL (IQR 55-162) at day 7 to 150 pg/mL (78-209) at day 120 when total sperm count recovered. In motile spermatozoa obtained after density gradient separation, Zika virus RNA was found in three of 14 patients at day 7, four of 15 at day 11, and four of 15 at day 20, and replication-competent virus was found in the tested patient. Seminal shedding kinetics seemed heterogeneous among patients. Whole blood was the fluid most frequently positive for Zika virus RNA (62 of 92 samples) and three patients remained positive at day 120. INTERPRETATION: Semen alterations early after acute Zika virus infection might affect fertility and could be explained by virus effects on the testis and epididymis. Frequency of shedding and high viral load in semen, together with the presence of replicative virus in a motile spermatozoa fraction, can lead to Zika virus transmission during sexual contact and assisted reproduction procedures. Whole blood seems to be the best specimen for Zika virus RNA detection, diagnosis, and follow-up. FUNDING: Agence de la Biomédecine/Agence Régionale de Santé de la Guadeloupe/Inserm-REACTing.

Comparison of Cobas® HPV and Anyplex™ II HPV28 assays for detecting and genotyping human papillomavirus.

Persistent infection with high-risk human papillomavirus (HPV-HR) is a recognized cause of cervical cancer. The aim of this study was to compare analytical and clinical performances of the Cobas® HPV and Anyplex™ II HPV28 assays for HPV detection and genotyping. A total of 94 cervical samples were tested. For HPV-HR, the results agreed very well (94.68%), with 100% agreement when detecting CIN2+. The Anyplex™ II HPV28 assay detected more genotypes than the Cobas® HPV Test, but their clinical performances were similar.

Zika Virus Infection and Prolonged Viremia in Whole-Blood Specimens.

We tested whole-blood and plasma samples from immunocompetent patients who had had benign Zika virus infections and found that Zika virus RNA persisted in whole blood substantially longer than in plasma. This finding may have implications for diagnosis of acute symptomatic and asymptomatic infections and for testing of blood donations.

Brief Report: HIV-1 Tropism During Primary Infections in France: 1996-2014.

HIV-1 was mainly CCR5 tropic in recent seroconverters. We analyzed the coreceptor use in 239 primary HIV-1 infections (PHIs) between 1996 and 2014 using a validated recombinant virus phenotypic entry assay. CXCR4-using viruses were detected in 8.3%, 3.8%, and 6.1% of PHIs from 1996 to 2004, 2005 to 2009, and 2010 to 2014, respectively. The presence of CXCR4-using viruses was associated with the virological failure of antiretroviral treatment initiated during PHI (odds ratio, 7.9; 95% confidence interval, 1.1 to 56.5). The phenotypic tropism assay data show that the prevalence of X4 tropic transmitted viruses was stable in this French cohort of PHIs between 1996 and 2014.

Comparison of the effect of semen from HIV-infected and uninfected men on CD4+ T-cell infection.

OBJECTIVES: Semen composition is influenced by HIV-1 infection, yet the impact of semen components on HIV infection of primary target cells has only been studied in samples from HIV-uninfected donors. DESIGN: We compared the effect of seminal plasma (SP) from chronically HIV-infected (SP+) versus uninfected donors (SP-) on HIV-1 infection of peripheral blood mononuclear cells (PBMCs) and CD4 T cells. METHODS: Primary cells were infected with HIV-1 in the presence of SP+ or SP- and analyzed for infection level, metabolic activity, HIV receptor expression, proliferation and activation. SP+ and SP- were compared for infection-enhancing peptides, cytokines and prostaglandin E2 levels. RESULTS: SP- efficiently enhanced HIV-1 R5 infection of CD4 T cells, whereas SP+ enhancing activity was significantly reduced. RANTES (CCL5) concentrations were elevated in SP+ relative to SP-, whereas the concentrations of infectivity-enhancing peptides [semen-derived enhancer of viral infection (SEVI), SEM1, SEM2] were similar. CCR5 membrane expression levels were reduced on CD4 T cells shortly postexposure to SP+ compared with SP- and correlated to R5-tropic HIV-1 infection levels, and CCR5 ligands' concentrations in semen. SP+ and SP- displayed similar enhancing activity on PBMC infection by X4-tropic HIV-1. Addition/depletion of RANTES (regulated on activation, normal T-cell expressed and secreted) from SPs modulated their effect on PBMC infection by R5-tropic HIV-1. CONCLUSION: Semen from HIV-infected donors exhibits a significantly reduced enhancing potential on CD4 T-cell infection by R5-tropic HIV-1 when compared with semen from uninfected donors. Our data indicate that elevated seminal concentrations of RANTES in HIV-infected men can influence the ability of semen to enhance infection.

Modulation of gene expression in CD4+ T lymphocytes following in vitro HIV infection: a comparison between human and chimpanzee.

Chimpanzees are susceptible to experimental infection by human deficiency virus (HIV)-1, but unlike humans, they exceptionally develop an immunodeficiency syndrome after HIV-1 inoculation. To explore the difference between human and chimpanzee, we analyzed the expression of 1547 genes of various functions in human or chimpanzee CD4+ lymphoblasts inoculated in vitro with HIV-1. We observed that, 1 day after HIV inoculation, fifty-eight genes were up-regulated in lymphoblasts of the three humans while their expression remained unchanged in lymphoblasts of the three chimpanzees. One gene is involved in adhesion of HIV (catenin-alpha), three in the immune response (semaphorin 4D, placental growth factor, IL-6), three in apoptosis (deleted in colorectal carcinoma, caspase 9 and FOXO1A). No difference between species was revealed for the expression of 373 genes related to glycosylation pathways. The in vitro human/chimpanzee comparison reveals new candidate genes up-regulated after inoculation with HIV-1 only in human lymphoblasts and which could be related to the higher sensitivity of human to HIV-induced AIDS.