Absence of detectable SARS-CoV-2 replication in ex vivo cultured cornea and cornea-derived epithelial cells.

PURPOSE: To study the possibility of SARS-CoV-2 to infect human corneal cells and tissues under standard corneal culture conditions using explants of COVID-19 donors and primary cornea-derived epithelial cells. METHODS: Cornea isolated from deceased COVID-19 donors was cultured for 4 weeks, and SARS-CoV-2 replication was monitored by qRT-PCR. Furthermore, primary corneal epithelial cells from healthy donors were cultured ex vivo and infected with SARS-CoV-2 and human cytomegalovirus (HCMV) as a control. Infection status was assessed by western blotting and reporter gene expression using green fluorescent protein-expressing viral strains. ACE2 and TMPRSS2 receptor expression levels in cornea and epithelial cells were assessed by qRT-PCR. RESULTS: We did not detect SARS-CoV-2 replication in 10 corneas isolated from deceased COVID-19 patients and cultured for 4 weeks, indicating absence of infection under natural conditions. Furthermore, high-titer SARS-CoV-2 infection of ex vivo cultured cornea-derived epithelial cells did not result in productive virus replication. In contrast, the same cells were highly permissive for HCMV. This phenotype could potentially be explained by low ACE2 and TMPRSS2 transcriptional activity in cornea and cornea-derived epithelial cells. CONCLUSIONS: Our data suggest that cornea and limbal epithelial cells are refractory to productive SARS-CoV-2 infection. This could be due to the absence of robust receptor expression levels necessary for viral entry. This study adds further evidence to support the very low possibility of transmission of SARS-CoV-2 from an infected corneal transplant donor to a recipient in corneal organ cultures.

Significant decline of HPV 6 infection and genital warts despite low HPV vaccination coverage in young women in Germany: a long-term prospective, cohort data analysis.

BACKGROUND: The introduction of human papillomavirus (HPV) vaccination has resulted in a remarkable decline of genital warts in women and men, but in Germany historical rates of vaccination are relatively low. We report long-term surveillance data on changes in HPV 6 and HPV 11 infection and the prevalence of genital warts in young women in the Wolfsburg HPV epidemiological study (WOLVES). METHODS: Women born in 1983/84, 1988/89, and 1993/94 participated in four cohorts between 2009/10 and 2014/15. Quadrivalent vaccination coverage and prevalence of HPV 6/11 infection and genital warts are reported for participants aged 19-22 years and 24-27 years at the time of sample collection. Statistical analyses were done to compare similarly aged participants using 2 × 2 contingency tables (Röhmel-Mansmann unconditional exact test; two-side alpha of 0.05). RESULTS: A total of 2456 women were recruited. Between 2010 and 2015, there was a statistically significant decrease in the prevalence of HPV 6 infection among women aged 24-27 years (2.1% versus 0.0%; P < 0.0001) and women aged 19-22 years (2.0% versus 0.0%; P = 0.0056). There was no significant decline in HPV 11 infection. In total, 52 of 2341 participants were diagnosed with genital warts. There was a statistically significant drop in the risk of developing genital warts in women aged 24-27 years between 2010 and 2015 (4.7% versus 1.7%, respectively; P = 0.0018). The overall risk of developing genital warts in women aged 19-27 years decreased from 3.1% in 2010 to 1.2% in 2015 (P = 0.0022). CONCLUSIONS: An increase in vaccination coverage was associated with a decreased prevalence of genital warts in young women. A protective effect greater than herd immunity alone was seen despite low vaccination rates. Quadrivalent vaccine had a protective effect on genital HPV 6 infection and an almost fully protective effect on the development of genital warts in the youngest population.

Longitudinal Clinical Performance of the RNA-Based Aptima Human Papillomavirus (AHPV) Assay in Comparison to the DNA-Based Hybrid Capture 2 HPV Test in Two Consecutive Screening Rounds with a 6-Year Interval in Germany.

Longitudinal data on the E6/E7 mRNA-based Aptima human papillomavirus (AHPV) assay exceeding three years in comparison to the gold standard Digene Hybrid Capture 2 (HC2) test are not available. We previously reported the cross-sectional data of the German AHPV Screening Trial (GAST) in which 10,040 women were recruited and tested by liquid-based cytology, the HC2 assay, and the AHPV assay. Four hundred eleven test-positive women were followed for up to six years. In addition, 3,295 triple-negative women were screened after a median time of six years. Overall, 28 high-grade cervical intraepithelial neoplasia (CIN3) cases were detected. The absolute risk of developing high-risk HPV-positive CIN3+ over six years among those women that tested negative at baseline was 2.2 (95% confidence interval [95% CI], 1.0 to 4.9) and 3.1 (95% CI, 1.7 to 5.7) per 1,000 women screened by the HC2 and the AHPV tests; the additional risk for those with AHPV-negative compared with HC2-negative results was 0.9 (95% CI, -0.2 to 2.1) per 1,000. In comparison, the absolute risk following a negative LBC test was 9.3 (95% CI, 2.9 to 30.2). The relative sensitivity of AHPV compared to HC2 was 91.5% for CIN3+, and the negative predictive values were 99.8% (95% CI, 99.5 to 99.9%) for HC2 and 99.7% (95% CI, 99.4 to 99.8%) for AHPV. Our data show that the longitudinal performance of the AHPV test over six years is comparable to the performance of the HC2 test and that the absolute risk of CIN3+ over six years following a negative AHPV result in a screening population is low. (This study is registered at ClinicalTrials.gov under registration number NCT02634190.).

Modifiable Risk-factors for Keratinocyte Cancers in Australia: A Case-control Study.

Keratinocyte cancer is the most common malignancy in Caucasians. The aim of this study was to investigate risk-factors responsible for development of keratinocyte cancer in Australia. A case-control study was conducted, including 112 cases of squamous cell carcinoma (SCC), 95 cases of basal cell carcinoma (BCC) and 122 controls. Freckling during adolescence (SCC: odds ratio (OR) 1.04, p < 0.01; BCC: OR 1.05, p < 0.01), propensity to sunburn (SCC: OR 2.75, p = 0.01, BCC: OR 2.68 p = 0.01) and high cumulative sun-exposure (SCC: OR 2.43, p = 0.04; BCC: OR 2.36 p = 0.04) were independent risk-factors for both SCC and BCC. This study provides further evidence that a sun-sensitive phenotype and excessive sun-exposure during adulthood contribute to the risk of developing keratinocyte cancer. Wearing a hat, long-sleeved shirts, and sunscreen did not significantly reduce the risk of keratinocyte cancer in this study.

Study-based evaluation of the Abbott RealTime High Risk HPV test in comparison to the HC2 HR HPV test in women aged ≥30 years using residual LBC ThinPrep specimens.

BACKGROUND: High-risk human papillomavirus (HR HPV) testing is already part of cervical cancer screening programs in a number of countries. New tests need to be validated not only in clinical studies but also in routine screening settings with regard to their clinical performance. METHODS: The Abbott RealTime High Risk HPV Test (RT hrHPV test) was evaluated in a random sample of 1,456 patients from a German routine screening population of 13,372 women ≥30 years of age screened primarily by liquid-based cytology (LBC) that was complemented by 48 CIN3+ cases. Clinical sensitivities, relative specificities and positive predictive values (PPV) for both HPV tests were determined based on histologically confirmed high-grade cervical disease (CIN3+) as clinical outcome. RESULTS: HR HPV prevalence in residual LBC samples was found to be 5.4 % by the RT hrHPV test and 5.6 % by the HR HC2 test, respectively. The Kappa-value for overall agreement between the RT hrHPV test and the HC2 assay for detection of HR HPV was 0.87. Relative sensitivities for detection of CIN3+ in patients with abnormal cytology was 93.8 % for the RT hrHPV assay and 97.9 % for HC2 (p-value = 0.5). Relative specificities and PPVs were comparable for both tests. The highest PPV was calculated for the specific detection of HPV16 by the RT hrHPV test (84.2 %). The RT hrHPV test showed a reduced sensitivity for detection of HVP31-positive CIN3 + . CONCLUSION: The RT hrHPV assay is as sensitive and specific in detecting severe cervical lesions in women with abnormal cytology as the HC2 HR HPV test.

Long-term follow-up of the risk for cervical intraepithelial neoplasia grade 2 or worse in HPV-negative women after conization.

Little research has been conducted on the long-term value of human papillomavirus (HPV) testing after conization. We investigated whether cytology adds to the value of a negative HPV test for long-term prediction of cervical intraepithelial neoplasia grade 2 or worse (CIN2+). In addition, we compared risk of CIN2+ following a negative HPV test in women after conization with that in women from the general population. During 2002-2005, 667 women treated for CIN2+ were tested for HPV and cytology 46 months after conization. Only HPV-negative women were included. Women participating in routine screening were age-matched with post-conization HPV-negative women, leaving 13,230 and 477 women, respectively, for analysis. By linkage to the Pathology Data Bank, we identified all cases of CIN2+ by December 2013. The 3-, 5-, 8- and 10-year risks for CIN2+ were 0.7, 0.9, 2.8 and 5.7% after a negative HPV test and 0.5, 0.8, 2.9 and 6.1% in HPV and cytology-negative women. HPV-negative women in the general population had similar 3-year and 5-year risks of 0.4 and 1.0%; thereafter, they had lower risks of 1.9% at 8 years and 2.7% at 10 years. Our results indicate that HPV testing may be used as a test of cure after conization. In the first 5 years after testing, the risk for CIN2+ of women who were HPV-negative at 34 months after conization was similar to that of HPV-negative women in the general population. After 67 years, however, women who have undergone conization may be at higher risk for CIN2+.

Head-to-Head Comparison of the RNA-Based Aptima Human Papillomavirus (HPV) Assay and the DNA-Based Hybrid Capture 2 HPV Test in a Routine Screening Population of Women Aged 30 to 60 Years in Germany.

Testing for E6/E7 mRNA in cells infected with high-risk (HR) human papillomavirus (HPV) might improve the specificity of HPV testing for the identification of cervical precancerous lesions. Here we compared the RNA-based Aptima HPV (AHPV) assay (Hologic) and the DNA-based Hybrid Capture 2 (HC2) HPV test (Qiagen) to liquid-based cytology (LBC) for women undergoing routine cervical screening. A total of 10,040 women, 30 to 60 years of age, were invited to participate in the study, 9,451 of whom were included in the analysis. Specimens were tested centrally by LBC, the AHPV test, and the HC2 test, and women who tested positive on any test were referred for colposcopy. Genotyping was performed on all HR-HPV-positive samples. Test characteristics were calculated based on histological review. As a result, we identified 90 women with cervical intraepithelial neoplasia grade 2+ (CIN2+), including 43 women with CIN3+. Sensitivity differences between the AHPV test and the HC2 test in detecting CIN2+ (P = 0.180) or CIN3+ (P = 0.0625) lesions were statistically nonsignificant. Of three CIN3 cases that were missed with the AHPV test, two cases presented lesion-free cones and one had a non-HR HPV67 infection. The specificity (

Role of human papillomavirus testing and cytology in follow-up after conization.

OBJECTIVE: Adequate follow-up of women who have undergone conization for high-grade cervical lesions is crucial in cervical cancer screening programs. We evaluated the performance of testing for high-risk human papillomavirus (HPV) types, cytology alone, and combined testing in predicting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) after conization. DESIGN: Prospective cohort study. SETTING: Denmark. POPULATION: 667 women attending for conization. METHODS: Cervical specimens were collected during 2002-2006 at first visit after conization for cytological examination and Hybrid Capture 2 detection of high-risk HPV. The women were passively followed until 2 years after first follow-up visit by linkage to the nationwide Pathology Data Bank. RESULTS: At first visit after conization (median time, 3.4 months), 20.4% were HPV-positive and 17.2% had atypical squamous intraepithelial lesions or more severe cytology (ASCUS+). The 2-year incidence of CIN2+ after conization was 3.6%. Sensitivity for detection of CIN2+ after conization was 81.0% [95% confidence interval (CI) 58.1-94.6] for positive cytology (ASCUS+ threshold) and 95.2% (95% CI 76.2-99.9) for HPV testing and for combined testing. Specificity of ASCUS+ cytology (85.2%; 95% CI 82.0-88.0) was higher than that of HPV testing (82.4%; 95% CI 79.0-85.4) and markedly higher than that of combined testing (73.2%; 95% CI 69.3-76.8). The margin status had no significant added value. CONCLUSIONS: Testing for high-risk HPV three to four months after conization is more sensitive than ASCUS+ cytology for identifying women at risk for relapse of CIN2+ within 2 years. Further studies are needed to evaluate whether HPV testing could be a stand-alone test in follow up after conization.

A case of recurrent respiratory papillomatosis with malignant transformation, HPV11 DNAemia, high L1 antibody titre and a fatal papillary endocardial lesion.

BACKGROUND: Recurrent respiratory papillomatosis (RRP) is a rare disease, which is characterised by the growth of papillomavirus-induced papillomas within the respiratory tract. Malignant transformation occurs in less than 1% of the cases. CASE PRESENTATION: We report a case of human papillomavirus (HPV) type 11-associated juvenile-onset RRP (JORRP) initially diagnosed at the age of two years. Remarkably high copy numbers of HPV11 DNA and antibody titres targeting the capsid protein L1 were detected in the patient's serum. The patient developed squamous cell carcinomas in both lungs and extraordinarily an HPV11 DNA-positive papillary endocardial lesion in the left atrium of the heart, which caused thromboembolic events leading to the patient's death at 19 years old. CONCLUSION: We here report a severe case of JORRP hallmarked by HPV11 DNAemia and very high antibody titres directed against the major viral capsid protein L1. Furthermore, the extent of malignant transformation and the discovery of a very rare fatal endocardial lesion highlight the unpredictability of JORRP and the complexity of its clinical management.

A population-based observational study comparing Cervista and Hybrid Capture 2 methods: improved relative specificity of the Cervista assay by increasing its cut-off.

BACKGROUND: High-risk human papillomavirus (HR HPV) testing has been shown to be a valuable tool in cervical cancer screening for the detection of cervical pre-cancer and cancer. METHODS: We report a purely observational study evaluating HR HPV prevalences in residual liquid-based cytology (LBC) samples using both the Cervista™ HPV HR Test and the Digene Hybrid Capture 2 High-Risk HPV DNA Test (HC2) in a sample of 1,741 women aged ≥30 years of a German routine screening population of 13,372 women. Test characteristics were calculated and a novel method for measuring test performances was applied by calculating ratios of sensitivity or specificity. RESULTS: The overall agreement of both tests for detection of HR HPV was excellent (κ = 0.8). Relative sensitivities for the detection of histologically confirmed severe cervical intraepithelial dysplasia (CIN3+) were similar for both HPV-tests, which was confirmed by the ratio analysis. However, discrepancy analysis between the Cervista HPV HR test and HC2 revealed a high false positive rate of the Cervista HPV HR test in the cytology normal category. CONCLUSIONS: Performance of the Cervista HPV test in cervical specimens with abnormal cytology is comparable to HC2 as both tests were highly sensitive and specific for the detection of high grade cervical disease. We also demonstrate evidence that modification of the cut-off values drastically reduces the false positive rate in the cytology normal category without affecting the detection of CIN3+, which ultimately improved specificity of the Cervista HPV HR assay.

Persistence and reappearance of high-risk human papillomavirus after conization.

OBJECTIVE: Women with early cervical cancer or intraepithelial neoplasia grades 2 and 3 (CIN2+) are treated by conization; however, they still have a higher risk for subsequent CIN2+ than the general female population. Persistence of high-risk (HR) human papillomavirus (HPV) is a key factor in the development of CIN2+. We investigated persistence and reappearance of type-specific HR HPV infection after conization and evaluated possible co-factors. METHODS: During 2002-2006, cervical swabs from 604 women were collected before conization, at 4-6 months and at 8-12 months after conization. HPV was detected by HC2 and genotyped by LiPAv2. Information on co-factors was collected through a questionnaire. Associations were assessed by multivariate logistic regression analysis. RESULTS: HR HPV persistence rate was 9.5%. The α5/6 species were more likely to persist than α9 species (OR, 2.28; 95% CI, 1.11-4.70). For single infections, a doubling in viral load at enrolment increased the risk for persistence by 36% (95% CI, 1.13-1.63). In addition, margin status was associated with risk of persistence. Smoking, oral contraceptive use and severity of the cervical lesion did not significantly affect persistence. Among the HPV infections that had cleared, 2.2% reappeared. CONCLUSION: Our study indicates that viral load is important in predicting HPV persistence. The α5/6 species were most likely to persist. However, most of these HPV types have a lower carcinogenic potential than the α7/α9 species and may be by-standers. Further studies are needed to assess whether pre-conization viral load can also predict subsequent CIN2+.

Prevalence of high-risk HPV types and associated genital diseases in women born in 1988/89 or 1983/84--results of WOLVES, a population-based epidemiological study in Wolfsburg, Germany.

BACKGROUND: High-risk human papilloma virus (HR-HPV) infection is associated with the development of cervical cancer. HPV vaccination reduces the risk of developing malignant lesions and is expected to change the dynamics of HPV transmission. Data from non-vaccinated women may provide an important benchmark to allow the impact of HPV vaccination programs to be assessed.This study was designed to prospectively determine the changing dynamics of HR-HPV infection and associated genital diseases in young women, most of whom were non-vaccinated. METHODS: Data from a population-based cohort study, comprising women of two predefined birth cohorts (women born in 1983/84 or 1988/89), were analyzed between 19 October 2009 and 31 December 2010 to determine risk factors for high-risk HPV infection and the association between specific HR-HPV types and atypical Pap smear test results. HPV status was determined by Hybrid Capture 2 (HC2) assay and genotyping. RESULTS: The prevalence of HR-HPV was 22.8% in the 1983/84 cohort (150/659) and 23.7% in the 1988/99 cohort (142/599). Only the number of sexual partners was a significant risk factor for HPV infection (odds ratios 22.687 and 6.124 for more than five versus one partner 84 cohort,/84 and 1988/89 cohorts, respectively) in multivariate analysis. HPV16 positive-women were significantly more likely to have abnormal Pap smears of any degree than HPV16-negative women (22.0% versus 3.61%, p < 0.0001 for the 1983/84 cohort and 9.09% versus 2.52%, p = 0.0482 for the 1988/89 cohort). CIN3 was diagnosed in six women 84 cohort,/84 cohort and two in the 1988/89 cohort. All women with CIN3 tested positive for HC2-HR and all six CIN3 cases 84 cohort,/84 cohort tested positive for HPV16. In the 1988/89 cohort, the rate of HPV16 infection was significantly lower in vaccinated than non-vaccinated women (1.59% versus 8.88%; p = 0.003). CONCLUSIONS: HR-HPV infection was highly prevalent in both cohorts and associated with an increased risk of abnormal Pap smears and biopsy proven CIN2+. HPV16 infection was associated with a high risk of clinically relevant lesions. HPV vaccination significantly decreased the risk of HPV16 infection.

Prevalence of low-risk HPV types and genital warts in women born 1988/89 or 1983/84 -results of WOLVES, a population-based epidemiological study in Wolfsburg, Germany.

BACKGROUND: Wolfsburg HPV Epidemiological Study (WOLVES) is a population-based cohort study on HPV infections and associated diseases in the pre-vaccination era in young women in Wolfsburg, Germany. METHODS: Women born 1983/84 or 1988/89 were invited to participate. Participants were recruited in gynecology practices, and completed a questionnaire with socioeconomic, sexual and medical data including vaccination status. Pelvic examination with Pap smear and HPV testing (HC2 = Hybrid Capture 2) was obligatory. HC2-positive and 10% of HC2-negative samples were tested for specific HPV types with SPF-10-PCR, and in inconclusive cases with DNA sequencing. Women with genital warts (GW) and those with atypical Pap smears were transferred for colposcopy. GWs were classified as typical condylomata acuminata (TCA), flat condyloma (FC) and seborrheic wart-like (SWL). RESULTS: In total, 1258 subjects were recruited from the target population of 2850 (44.1%). Overall the prevalence of HC2 low-risk (LR) types was 8.5%. HPV6 was the most frequent LR type (2.1%), followed by HPV42 (1.1%), HPV11 and HPV44 (each 0.4%). LiPA showed a low sensitivity for HPV types 42, 90 and 91, which were detected only by HC2 and HPV sequencing. Nine women (0.7%) were transferred with incident GW: five TCA, two FC and two SWL. All TCA were associated with HPV6 in corresponding cervical swabs and warts. Tissues of SWL contained HPV6 (n = 1) and HPV16 (n = 1). The cumulative life-risk for GW was 1.4% in the 1988/89 and 4.8% in the 1983/84 cohort. Eight of 107 HC2-LR + and five of nine cases of GW had concomitant abnormal Pap smears. All CIN lesions could be linked to high-risk HPV types but borderline and low-grade abnormal smears were explained by vaginal and cervical TCA in four cases. CONCLUSIONS: HC2 was a specific test for the detection of established and potential LR types. In this first WOLVES analysis, HPV6 was the most frequent HPV type and the single LR type linked to disease. The observed GW incidence of 715 per 100,000 fits well with estimates of healthcare providers. Although life risks for GW were lower than in Scandinavian analyses, the societal burden within the WOLVES populations was considerable.

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in Human Corneal Tissues.

PURPOSE: To examine corneal tissue for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) positivity regarding implications for tissue procurement, processing, corneal transplantation, and ocular surgery on healthy patients. We performed quantitative reverse transcription-polymerase chain reaction qRT-PCR-testing for SARS-CoV-2 RNA on corneal stroma and endothelium, bulbar conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium of coronavirus disease 2019 (COVID-19) postmortem donors. METHODS: Included in this study were 10 bulbi of 5 COVID-19 patients who died because of respiratory insufficiency. Informed consent and institutional review board approval was obtained before this study (241/2020BO2). SARS-CoV-2 was detected by using a pharyngeal swab and bronchoalveolar lavage. Tissue procurement and tissue preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for each of the abovementioned tissues and intraocular fluids. RESULTS: The qRT-PCRs yielded no viral RNA in the following ocular tissues and intraocular fluid: corneal stroma and endothelium, bulbar-limbal conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium. CONCLUSIONS: In this study, no SARS-CoV-2-RNA was detected in conjunctiva, anterior chamber fluid, and corneal tissues (endothelium, stroma, and epithelium) of COVID-19 donors. This implicates that the risk for SARS-CoV-2 infection using corneal or conjunctival tissue is very low. However, further studies on a higher number of COVID-19 patients are necessary to confirm these results. This might be of high importance for donor tissue procurement, processing, and corneal transplantation.

Severe acute respiratory Syndrome-Coronavirus-2: Can it be detected in the retina?

BACKGROUND/OBJECTIVES: The systemic organ involvement of SARS-CoV-2 needs to be thoroughly investigated including the possibility of an ocular reservoir in humans. To examine retinal tissues and vitreous for histopathology and SARS-CoV-2 presence with regard to possible effects on the human retina and/ or vitreous. We performed histopathological analyses and quantitative (q)RT-PCR-testing for SARS-CoV-2 RNA on retinal tissues and vitreous of COVID-19 postmortem donors. SUBJECTS/METHODS: Included in this study were 10 eyes of 5 deceased COVID-19 patients. The diagnosis of SARS-CoV-2 infection was confirmed via pharyngeal swabs and broncho-alveolar fluids. The highest level of personal protective equipment (PPE) and measures was employed during fluid-tissue procurement and preparation. Histopathological examinations and qRT-PCR-testing were carried out for all retinal tissues and vitreous fluids. RESULTS: The histopathological examinations revealed no signs of morphologically identifiable retinal inflammation or vessel occlusions based on hematoxylin and eosin stains. By qRT-PCRs, we detected no significant level of viral RNA in human retina and vitreous. CONCLUSIONS: In this study, no significant level of SARS-CoV-2-RNA was detected in the human retinal and vitreous fluid samples of deceased COVID-19 patients. Histopathological examinations confirmed no morphological sign of damage to retinal vasculature or tissues. Further studies are needed to confirm or refute the results.

Antibody Response against SARS-CoV-2 and Seasonal Coronaviruses in Nonhospitalized COVID-19 Patients.

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2, and two other patients (4%) were positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.

Prevalence of low-risk and high-risk types of human papillomavirus and other risk factors for HPV infection in Germany within different age groups in women up to 30 years of age: an epidemiological observational study.

Human papillomavirus (HPV) infection is frequent in young women and persistent infection may lead to cervical cancer. Therefore, vaccination against HPV is recommended for young women in the age group from 12-17 years in Germany. However, epidemiological data on the prevalence of HPV types and risk factors for infection for younger women in Germany is scarce. To address this, an observational study was performed in Germany including 1,692 women aged 10-30 years. After a routine Pap smear, cervical swabs were tested for high-risk and low-risk HPV, respectively, using the Hybrid Capture 2 (HC2) test, and genotyped using the PCR-based tests SPF(10)/LiPA(25) and PapilloCheck®. In addition, the women were interviewed regarding their medical history and lifestyle factors. Three hundred seventy-seven (22.28%) women had positive HC2 results. The proportion of HPV positive women was highest in the 20-22 age group with 28.3%. Predominant HPV types were HPV 16, 42, 51 and HPV 16, 51, 31 as defined by PapilloCheck® and SPF(10)/LiPA(25), respectively. 95.8% of women did not show signs of any cervical lesion. Adjusted analysis identified number of sexual partners (OR:1.105; 95% CI:[1.069-1.142]), smoking (OR:1.508; [1.155-1.968]), and vaccination against HPV (OR:0.589; [0.398-0.872]) rather than increasing age as risk associated with HPV infection. Comparison of the genotyping assays showed that they correspond well regarding the high-risk HPV types but less well for low-risk HPV types. This epidemiological study shows that high-risk HPV infection is common in young women in Germany. According to our data, vaccination of young women could have a potential impact on the prevention of HPV infection and cervical disease.

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in ocular tissues.

PURPOSE: To evaluate the status of ocular donor tissues of a COVID-19 postmortem donor. METHODS: SARS-CoV-2 was detected via a pharyngeal swab and broncho-alveolar lavage in the COVID-19 suspect. Postmortem tissue procurement and preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for the following ocular tissues and fluids: conjunctival fluid swabs, bulbar conjunctiva, corneal epithelium, corneal stroma, corneal endothelium, anterior chamber fluid, lens, iris, vitreous, retina, uvea, sclera, and optic nerve. Informed consent and Institutional Review Board approval was obtained prior to this study (196/2020BO2; Date of approval: 03/26/2020; Ethics Committee of the University of Tuebingen). RESULTS: In all ocular tissue and fluid samples no SARS-CoV-2 RNA was detected via qRT-PCR of the confirmed COVID-19 postmortem donor. CONCLUSIONS AND IMPORTANCE: Late-stage COVID-19 patients might not harbor an ocular reservoir of SARS-CoV-2. The risk of transmitting SARS-CoV-2 via ocular tissues and fluids might be low. This may bear future implications for patient management in ophthalmological practice, surgery and transplantation.

Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.