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.

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.

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.

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.

Evolution of HIV-1 quasispecies and coreceptor use in cell reservoirs of patients on suppressive antiretroviral therapy.

OBJECTIVES: To track changes in the V3 env region of HIV-1 quasispecies and determine virus coreceptor use in cell reservoirs of patients on long-term suppressive antiretroviral therapy (ART). PATIENTS AND METHODS: Ten patients whose plasma viraemia had been suppressed for a median of 5.5 years were followed for 5 years. The V3 env regions of viruses in peripheral blood mononuclear cells were analysed by ultra-deep sequencing (UDS). HIV-1 tropism was predicted using the geno2pheno 5.75 algorithm and a phenotypic assay. RESULTS: The UDS and phenotypic assay data were concordant for predicting HIV-1 tropism. CXCR4-using viruses detected by UDS accounted for 14.7%-76.5% of the virus populations in samples from five patients at enrolment. Five patients harboured pure R5 virus populations and no X4 viruses emerged during the 5 years. The selection pressures estimated by the dN/dS ratio were acting on the V3 region to produce diversification of the quasispecies in CXCR4-infected patients and purification of the quasispecies in R5-infected patients on effective ART. CONCLUSIONS: UDS showed that the virus quasispecies in cell reservoirs of patients on long-term suppressive ART continued to evolve. CXCR4-using variants became more diversified. Analysis of the selection pressures on the virus quasispecies could provide a clearer picture of virus persistence in patients on effective ART.

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.

Genotypic prediction of HIV-1 CRF01-AE tropism.

HIV-1 subtype CRF01-AE predominates in south Asia and has spread throughout the world. The virus tropism must be determined before using CCR5 antagonists. Genotypic methods could be used, but the prediction algorithms may be inaccurate for non-B subtypes like CRF01-AE and the correlation with the phenotypic approach has not been assessed. We analyzed 61 CRF01-AE V3 clonal sequences of known phenotype from the GenBank database. The sensitivity of the Geno2pheno10 genotypic algorithm was 91%, but its specificity was poor (54%). In contrast, the combined 11/25 and net charge rule was highly specific (98%) but rather insensitive (64%). We thus identified subtype CRF01-AE determinants in the V3 region that are associated with CXCR4 use and developed a new simple rule for optimizing the genotypic prediction of CRF01-AE tropism. The concordance between the predicted CRF01-AE genotype and the phenotype was 95% for the clonal data set. We then validated this algorithm by analyzing the data from 44 patients infected with subtype CRF01-AE, whose tropism was determined using a recombinant phenotypic entry assay and V3-loop bulk sequencing. The CRF01-AE genotypic tool was 70% sensitive and 96% specific for predicting CXCR4 use, and the concordance between genotype and phenotype was 84%, approaching the concordance obtained for predicting the tropism of HIV-1 subtype B. Genotypic predictions that use a subtype CRF01-AE-specific algorithm appear to be preferable for characterizing coreceptor usage both in pathophysiological studies and for ensuring the appropriate use of CCR5 antagonists.

Multicenter assessment of HIV-1 RNA quantitation in semen in the CREAThE network.

Detection of HIV-1 RNA in semen is used commonly to determine the safety of semen processing procedures before assisted reproductive technology (ART). Using two panels of prepared semen samples containing HIV-1 the performances of protocols from 14 centers have been compared. No false-positive results were detected but false-negative results were frequent when the concentration was below 500 HIV-1 RNA copies/ml of seminal plasma. Frequency of HIV-1 RNA detection was higher on seminal cells than on seminal plasma. Assays (or protocols) for quantifying HIV-1 RNA in semen performed less well than standardized blood plasma assays. The HIV load in seminal plasma could be a useful marker of the risk of sexual transmission of the virus. Its use as a marker of global HAART efficiency in the HIV reservoir needs further study. Standardized assays are required for detection and measurement of HIV-1 RNA in semen 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.

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.

No evidence of occult hepatitis C virus (HCV) infection in serum of HCV antibody-positive HCV RNA-negative kidney-transplant patients.

Persistence of hepatitis C virus (HCV) in patients who cleared HCV is still debated. Occult HCV infection is described as the presence of detectable HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) of patients with undetectable plasma HCV-RNA by conventional PCR assays. We have assessed the persistence of HCV in 26 kidney-transplant patients, followed up for 10.5 years (range 2-16), after HCV elimination while on hemodialysis. If HCV really did persist, arising out of the loss of immune control caused by institution of the regimen of immunosuppressive drugs after kidney transplantation, HCV reactivation would have taken place. Their immunosuppression relied on calcineurin inhibitors (100%), and/or steroids (62%), and/or antimetabolites (94%). An induction therapy, given to 22 patients, relied on rabbit antithymocyte globulin (59%) or anti-IL2-receptor blockers (32%). All patients had undetectable HCV RNA as ascertained by several conventional tests. At the last follow-up, no residual HCV RNA was detected in the five liver biopsies, the 26 plasma, and in the 37 nonstimulated and 24 stimulated PBMCs tested with an ultrasensitive RT-PCR assay (detection limit, 2 IU/ml). No biochemical or virologic relapse was seen during follow-up. The absence of HCV relapse in formerly HCV-infected immunocompromised patients suggests the complete eradication of HCV after its elimination while on dialysis.

Determining seminal plasma human immunodeficiency virus type 1 load in the context of efficient highly active antiretroviral therapy.

The semen plasma virus load is measured to ensure the safety of sperm processing during medically assisted procreation (MAP) for couples with a human immunodeficiency virus type 1 (HIV-1)-infected man. A practical, automated protocol using the COBAS Ampliprep CAP/CTM kit in the COBAS TaqMan96 system was developed to measure the HIV-1 load in semen plasma samples. HIV-1 was detected in 13.4% of the semen samples processed at our MAP center. Of the eight patients having a detectable semen HIV-1 load, five had no detectable virus in their blood plasma. This highlights the residual risk of HIV-1 transmission during unprotected intercourse and raises the question of the possible consequences of ineffective highly active antiretroviral therapy in the genital tract.

Genotypic prediction of human immunodeficiency virus type 1 CRF02-AG tropism.

We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.

Serum concentrations of ribavirin and pegylated interferon and viral responses in patients infected with HIV and HCV.

The hepatitis C virus (HCV) infects a substantial proportion of patients infected with human immunodeficiency virus (HIV). Patients infected with both HCV and HIV respond poorly to anti-HCV treatment with pegylated interferon alpha and ribavirin. But few data are available on the influence of ribavirin and interferon concentrations on treatment outcome for these patients. This study investigated the relationship between the serum pegylated interferon and ribavirin concentrations 3 and 6 months after treatment initiation, and treatment outcome in 35 HCV-HIV coinfected patients. The pegylated interferon and ribavirin concentrations at months 3 and 6 were similar. The pegylated interferon concentrations at 3 months in responders and nonresponders were similar. However, responders tended to have higher ribavirin concentrations (2,322 ng/ml) than nonresponders (1,833 ng/ml; P = 0.08). Responders infected with HCV genotype 1 or 4 had higher ribavirin concentrations (2,672 ng/ml) than did similarly infected nonresponders (1,758 ng/ml; P = 0.04). ROC curve analysis showed that a ribavirin concentration of 2,300 ng/ml was the best threshold for predicting a nonresponse (ROC area = 0.80 +/- 0.12). Thus ribavirin concentrations influence treatment outcome in HIV patients infected with HCV genotype 1 or 4. Monitoring ribavirin concentrations could help adapt ribavirin concentrations and improve the sustained virological response.

Efficacy and safety of peginterferon alpha-2a/ribavirin in treatment-naive Cameroonian patients with chronic hepatitis C.

Data were examined from a day-to-day clinical practice in Yaounde, Cameroon to evaluate the efficacy and safety of peginterferon alfa-2a and ribavirin in treatment-naive Cameroonian patients with chronic hepatitis C. Ninety adults with chronic hepatitis C (mean age, 53 +/- 8 years; 79% males; 37.8% genotype 1; 23.3% genotype 2; and 38.9% genotype 4) were given at least 12 weeks of combination therapy between February 2003 and August 2007. Of these, 54 completed the treatment and the 24-week follow up. Subsequently, 18 continued treatment and 18 (20%) discontinued the treatment, 6 (6.7%) due to adverse effects. An intention-to-treat analysis showed that 38 (52.8%) had an end-of-treatment virologic response and 34 (47.2%) had a sustained virologic response. Sustained virologic response were significantly higher among patients with hepatitis C virus (HCV) genotype 2 (83.4%) than in those with genotype 1 (31%) or genotype 4 (42.3%) (P < 0.05). Non HCV-2 genotype, pretreatment fibrosis score >2, HCV RNA level >8.0 x 10(5) IU/ml and a non-virologic response at 12 weeks of treatment were associated with poor sustained virologic response (P < 0.05). Thus, HCV can be treated in a Sub-Saharan African country. It indicates that Cameroonian HCV-1 and -4 patients have a poorer sustained virologic response than the published results for Western and Middle-East countries. Virus subtype may influence the treatment outcome, since there is a great genetic diversity within Cameroonian HCV-1 and -4 genotypes.

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.

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.

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.

The hepatitis C virus epidemic in Cameroon: genetic evidence for rapid transmission between 1920 and 1960.

Hepatitis C virus (HCV) infection in Cameroon is characterized by widespread seropositivity and great virus genetic diversity (3 genotypes and over 10 subtypes). A total of 244 HCV NS5B sequences of 382-405 bp long (95 type 1, 58 type 2, and 91 type 4) were phylogenetically analyzed to estimate the history of the HCV epidemic in Cameroon. The newly developed Bayesian coalescent approach was used to infer the history of each HCV type. The estimated dates of the most recent common ancestors (MRCA) for genotypes 1 (1500; 95% confidence interval (95% CI): 1300-1650) and 4 (1500; 95% CI: 1350-1700) were in the same range, while the date for genotype 2 MRCA (1600; 95% CI: 1400-1750) was slightly more recent. The mean genetic distance between HCV genotype 1 sequences was greater than that of HCV type 4 sequences, itself greater than that of HCV type 2 sequences. The initial infected populations of all three genotypes did not grow until recently, when they grew exponentially. The growth rate has now begun to slow, with a less steep exponential growth curve. The period of exponential growth of all the three genotypes was between 1920 and 1960. These results (i) confirm that HCV genotypes 1 and 4 have produced long-term endemics, (ii) suggest that genotype 2 was introduced into Cameroon more recently, and (iii) indicate that the exponential spread of the three genotypes between 1920 and 1960 coincided with the mass campaign against trypanosomiasis and mass vaccinations in Cameroon.