Vladimira Koudelakova

: The prevalence of high-risk human papillomavirus (hrHPV) types varies across countries, making it essential to estimate prevalence using nationwide samples. Data on hrHPV prevalence in the Czech Republic are very limited. This study aimed to determine the prevalence of various hrHPV types in an unselected screening population of Czech women aged 30–65 years, using paired clinician-obtained cervical swab (CS) and self-collected cervicovaginal swabs (CVS). A total of 1026 eligible women were recruited into two study arms. In arm A, the digene® HC2 DNA Collection Device was used for both CS and CVS. In arm B, the Evalyn Brush was used for CVS, while the Cervex Brush was used for CS. All samples were tested for hrHPV using the digene® HC2 High-Risk HPV DNA Test and genotyped with the PapilloCheck® HPV-Screening assay. The overall hrHPV prevalence was 14.8%, based on positive results from either CVS or CS samples. hrHPV positivity was detected in 10.8% of clinician-obtained CSs and 11.8% of self-collected CVSs. A combined analysis of CS and CVS samples identified the five most prevalent hrHPV genotypes: HPV16, HPV31, HPV39, HPV56, and HPV68. The comparison of hrHPV detection in paired CS and CVS samples showed an overall concordance of 93%. These findings highlight the importance of detecting hrHPV genotypes alongside conventional Pap testing in national cervical screening programs. Furthermore, the results confirm that self-sampling kits represent a suitable alternative to clinician-collected samples. : Clinical trials registration ClinicalTrials.gov Identifier (NCT04133610)

Testing of pooled samples is an effective strategy for increasing testing capacity while saving resources and time. This study aimed to validate pooled testing and gather real-life data on its use for Covid-19 surveillance with a gargle lavage (GL) self-sampling strategy. Two-stage pooled testing with pools of 6 and 12 samples was used for preventive testing of an asymptomatic population and Covid-19 surveillance in Czech schools. Both GL and nasopharyngeal swabs were used for sampling. In total, 61,111 samples were tested. The use of pooled testing for large-scale Covid-19 surveillance reduced consumable costs by almost 75% and increased testing capacity up to 3.8-fold compared to standard methods. RT-PCR experiments revealed a minimal loss of sensitivity (0-2.2%) when using pooled samples, enabling the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes with Ct values >35. The minor loss of sensitivity was counterbalanced by a significantly increased throughput and the ability to substantially increase testing frequencies. Pooled testing is considerably more cost-effective and less time-consuming than standard testing for large-scale Covid-19 surveillance even when the prevalence of SARS-CoV-2 is fluctuating. Gargle lavage self-sampling is a non-invasive technique suitable for sample collection without a healthcare worker's assistance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable disruption worldwide. For efficient SARS-CoV-2 detection, new methods of rapid, non-invasive sampling are needed. This study aimed to investigate the stability of SARS-CoV-2 in a novel medium for gargle-lavage (GL) self-sampling and to compare the performance of SARS-CoV-2 detection in paired self-collected GL and clinician-obtained nasopharyngeal swab (NPS) samples. The stability study for SARS-CoV-2 preservation in a novel medium was performed over 14 days (4 °C, 24–27 °C, and 37 °C). In total, 494 paired GL and NPS samples were obtained at the University Hospital in Olomouc in April 2021. SARS-CoV-2 detection in paired samples was performed with a SARS-CoV-2 Nucleic Acid Detection Kit (Zybio, Chongqing Municipality, Chongqing, China), an Elecsys^® SARS-CoV-2 Antigen assay (Roche Diagnostics, Mannheim, Germany), and a SARS-CoV-2 Antigen ELISA (EUROIMMUN, Lübeck, Germany). The stability study demonstrated excellent SARS-CoV-2 preservation in the novel medium for 14 days. SARS-CoV-2 was detected in 55.7% of NPS samples and 55.7% of GL samples using rRT-PCR, with an overall agreement of 91.9%. The positive percent agreement (PPA) of the rRT-PCR in the GL samples was 92.7%, and the negative percent agreement (NPA) was 90.9%, compared with the NPS samples. The PPA of the rRT-PCR in the NPS and GL samples was 93.2% when all positive tests were used as the reference standard. Both antigen detection assays showed poor sensitivity compared to rRT-PCR (33.2% and 36.0%). rRT-PCR SARS-CoV-2 detection in self-collected GL samples had a similar PPA and NPA to that of NPSs. GL self-sampling offers a suitable and more comfortable alternative for SARS-CoV-2 detection.

Background: Human papillomavirus (HPV) has been shown to adversely affect human reproduction. We aimed to evaluate the prevalence of human papillomavirus (HPV) infection in men and its correlation with semen parameters and reproductive outcomes. Methods: Semen samples and penile swabs were collected from potential sperm donors (SD, n = 97) and male partners of infertile couples (IM, n = 328). The presence of HPV DNA in semen samples and penile swabs was analyzed. Associations between hrHPV positive status and fertility outcomes as well as socio-behavioral and health characteristics were evaluated using the R software package. Results: High-risk HPV (hrHPV) genotypes were detected in 28.9% of SD and 35.1% of IM (P = 0.312). Penile swabs were more frequently positive for hrHPV genotypes than semen samples in both IM (32.3% vs. 11.9%, P < 0.001) and SD (26.8% vs. 6.2%, P = 0.006). Men with hrHPV positive semen samples had lower semen volume (median volume 2.5 ml vs. 3 ml, P = 0.009), sperm concentration (median concentration 16 × 10⁶/ml vs. 31 × 10⁶/ml, P = 0.009) and total sperm count (median count 46 × 10⁶ vs. 82 × 10⁶, P = 0.009) than men with hrHPV negative samples. No association was identified between penile hrHPV status and semen parameters. Conclusions: Our findings indicate that penile HPV infection is common in both potential sperm donors and men from infertile couples. Although HPV positivity is higher in penile swabs, only HPV infection in semen samples affects sperm parameters. However, there was no association between hrHPV positivity in semen and fertility outcomes including abortion rate.

In the Czech Republic, the current pandemic led to over 1.67 million SARS-CoV-2- positive cases since the recording of the first case on 1 March 2020. SARS-CoV-2 genome analysis is an important tool for effective real-time quantitative PCR (RT-qPCR) diagnostics, epidemiology monitoring, as well as vaccination strategy. To date, there is no comprehensive report on the distribution of SARS-CoV-2 genome variants in either the Czech Republic, including Central and Eastern Europe in general, during the first year of pandemic. In this study, we have analysed a representative cohort of SARS-CoV-2 genomes from 229 nasopharyngeal swabs of COVID-19 positive patients collected between March 2020 and February 2021 using validated reference-based sequencing workflow. We document the changing frequency of dominant variants of SARS-CoV-2 (from B.1 -> B.1.1.266 -> B.1.258 -> B.1.1.7) throughout the first year of the pandemic and list specific variants that could impact the diagnostic efficiency RT-qPCR assays. Moreover, our reference-based workflow provided evidence of superinfection in several samples, which may have contributed to one of the highest per capita numbers of COVID-19 cases and deaths during the first year of the pandemic in the Czech Republic.

Background: Data about the genotype-specific human papillomavirus (HPV) prevalence in the Czech Republic is limited. We aimed to evaluate the prevalence and concordance of genotype-specific HPV infection detected in semen samples, penile swabs and cervical swabs from non-vaccinated heterosexual couples without HPV-associated disease. Methods: Semen samples and penile swabs were collected from male partners and cervical swabs were collected from female partners of heterosexual couples treated for infertility (n = 195). Presence of HPV DNA in semen samples and cervical swabs was analyzed using the cobas^® HPV Test and PapilloCheck^®. Only the PapilloCheck^® test was used to detect HPV in penile swabs. The genotype-specific prevalence and concordance of HPV infection not targeted by vaccine were evaluated using Fisher exact test. Results: Both partners were infected with any HPV type in 13.8% (27/195) of couples and, of these couples, 55.6% (15/27) harbored at least one mutual genotype. High-risk HPV (hrHPV) genotypes were detected in 12.3% (24/195) of semen samples, 31.3% (61/195) of penile swabs, and 19.5% (38/195) of cervical swabs (P < 0.001). The most prevalent hrHPV genotype were HPV53 (2.56%; 5/195) in semen samples, HPV16 (6.67%, 13/195) in penile swabs and HPV39 (3.59%, 7/195) in cervical swabs. Low-risk (lrHPV) genotypes were detected in 5.13% (10/195) of semen samples, 15.9% (31/195) of penile swabs, and 4.10% (8/195) of cervical swabs (P < 0.001). Male sexual partners of HPV-positive women were more likely to be infected with at least one of the same HPV types than female sexual partners of HPV-positive men (34.9% vs. 17.9%, P = 0.055). Conclusions: This study showed that the detection of HPV infection differ by anatomic site and gender. Regardless the anatomic site, high prevalence of HPV genital infection was found in both Czech men and women.

The purpose of our study was to examine the presence of human papillomavirus (HPV) DNA in Czech patients with non-small cell lung cancer (NSCLC). A highly sensitive quantitative polymerase chain reaction (qPCR) detecting the E6 gene of HPV16, 18, 31, and 56 was designed. The limit of detection was assessed using serial dilutions of HPV-positive plasmids. The qPCR was validated on a set of 402 cervical swabs where the qPCR, Cobas, and PapilloCheck methods were tested in parallel. Finally, qPCR was used for HPV detection in a set of 80 patients with primary NSCLC, both from formalin-fixed paraffin-embedded (FFPE) and fresh frozen (FF) tissue samples. The qPCR method was able to reliably detect at least 4 copies of the E6 gene per reaction in HPV16, 18, and 31, and 40 copies per reaction in HPV56. The sensitivity and specificity of the qPCR were 75.6-99.3% and 63.9-100% respectively, depending on the HPV genotype and reference method used. HPV DNA was not detected in the FFPE and FF samples from the set of 80 NSCLC patients. No hrHPV DNA was found in primary NSCLC tumors from a Czech population.

Human papillomavirus 68 (HPV68) is a probable carcinogenic HPV genotype which is included in almost all HPV screening assays and exists as two genetically variable subtypes (HPV68a and HPV68b). Routine HPV sample testing has shown that the cobas 4800 HPV Test (Roche) provides higher false-negative rates for HPV68 status than PapilloCheck HPV-Screening (Greiner Bio-One). The aim of our study was to evaluate the efficacy of cobas 4800 in HPV68 detection. A total of 2,145 cervical/cervicovaginal samples from women aged 17–88 were tested for HPV68 status using the cobas 4800 and PapilloCheck HPV tests. Viral load was assessed by quantitative PCR in all of the HPV68-positive cases. HPV68a/b subtyping was performed with real-time PCR followed by high resolution melting curve analysis, and was subsequently confirmed by Sanger sequencing. Cobas 4800 detected HPV positivity in only 13/33 HPV68 single-genotype infection cases. Viral load was comparable across both tested subgroups. HRM analysis and Sanger sequencing identified the HPV68a subtype in all of the 20 instances of cobas 4800 false negatives. HPV68a and HPV68b were detected in 3/13 and 10/13 cases identified as other HPV-positive by cobas 4800. The HPV68a subtype was missed by cobas 4800 in more than 85% of all HPV68a-positive cases. Therefore, commercially available assays may underestimate HPV68 prevalence.