Cervical Swabs Research Articles

Prevalence, genotyping and phylogenetic analysis of HPV infecting Palestinian women.

Human Papillomavirus (HPV) is a significant global public health concern due to its association with cervical, other anogenital, and oropharyngeal cancers. This study aimed to evaluate the prevalence and the phylogenetic relationships of HPV among Palestinian in order to inform public health strategies. A cross-sectional study was conducted between September 2023 and April 2024 involving 379 Palestinian women over the age of 18 from 11 governorates in the West Bank. Cervical swabs were collected and analyzed using nested PCR and Sanger sequencing methods to detect and genotype HPV. The study also included phylogenetic analysis to understand the genetic relationships between HPV strains. The overall HPV prevalence was 14.5%. The highest prevalence was observed in the 20-29 age group (19.6%), the Middle region of the West Bank (19.0%), and lower educational attainment. Genotyping revealed a diverse distribution of HPV types, with HPV 11 and HPV 6 being the most common low-risk types, while HPV 16 was the most common high-risk type. About 21.8% of the detected strains were high-risk strains. Phylogenetic analysis indicated significant regional clustering of HPV strains. The study highlights the need for targeted public health interventions, including vaccination and regular screening, particularly for younger women and those with lower educational attainment. Continued surveillance and research are essential to reduce the burden of HPV-related diseases in the West Bank, Palestine.

Influence of COVID-19 pandemic on prevalence and genotype distribution of HPV in cervical cancer screening population

BackgroundHuman Papillomavirus (HPV) DNA screening was a crucial element in the fight against cervical cancer and had been adopted in many countries, including China. However, the onset of the COVID-19 pandemic in March 2020 disrupted this program significantly.MethodsThe aim of this study is to investigate the prevalence and distribution of HPV genotypes among the population undergoing cervical cancer screening during the pandemic period. From January 2017 to December 2022, Peking Union Medical College Hospital gathered 45,496 cervical swabs from individuals undergoing cervical cancer screening. These samples were analyzed to detect fifteen high-risk HPV (HR-HPV) DNA types and a combination of two low-risk HPV (LR-HPV) types.ResultsThe study revealed an overall infection rate of 11.24% (5,114/45,496), with 11.06% (5,032/45,496) of individuals infected with HR-HPV. The number of HPV screening patients and the infection rates of HPV, HR-HPV, LR-HPV, multiple genotype HPV (M-HPV), and single genotype HPV (S-HPV) during the pandemic were lower than those observed before the pandemic. Moreover, the age group with the highest percentage of infected individuals was under 45–49 years, with HPV52, HPV58, HPV16, and HPV51 being the most prevalent genotypes. Notably, HPV66 emerged as the fifth most commonly detected genotype during the pandemic. Additionally, among the eleven age groups examined, women under 25 exhibited the highest detection rate, with HPV52 and HPV16 infection rates exceeding those observed in the pre-pandemic period.ConclusionsThe findings of this study offer significant insights for shaping HPV prevention strategies and enhancing cervical cancer screening initiatives in China following the epidemic.

The role of co-infections on cervical intraepithelial neoplasia prevalence in western Kenya.

Background Screening for co-infections with HIV, HSV-2 and Chlamydia trachomatis (CT ) among high-risk human papilloma virus (hr-HPV) positive women remains essential in alleviating high morbidity of cervical cancer (CC). The aim of this study was to determine the prevalence of cervical intraepithelial neoplasia (CIN) among women referred for CC screening at a referral hospital in Kisumu County, Kenya; and to establish the role of co-infection on CIN. Method In a cross-sectional study, we collected HPV, HIV, HSV-2 and CT data, cervical cytology results, and demographic information from 517 referrals. Blood samples were obtained for HIV and HSV-2 tests; urine for CT test and cervical swabs for hr-HPV test. Results The overall prevalence of CIN was 18.4% (95/517) with CIN1 observed in 56(29.6%), CIN2 in 27(`14.3%), CIN3 + in 12(6.3%) and normal biopsy in 94(49.7%) of the patients out of which high grade CIN2 and above (CIN2+) was 7.54% (39/517) equivalent to 32.5 per 100,000 women per year. HPV/HIV co-infection (infected vs. uninfected: OR 2.79; 95% CI 1.56-5.10, p < 0.001); HPV/HSV-2 co-infection (infected vs. uninfected: OR 2.41, 95% CI: 1.12-5.46, p < 0.024); HPV/CT co-infection (infected vs. uninfected: OR 3.83; 95% CI 1.84-8.51, p < 0.001) were found to be significantly associated with CIN. Conclusion Overall prevalence of CIN was high in the region although high-grade CIN2 + remained relatively lower as reported earlier. Age factor, widowhood and co-infections with HIV, HSV-2 or Chlamydia trachomatis were associated with increased risk of testing positive for CIN.

Distribution of HPV genotypes in Mashhad, Iran: insights from a 2022–2023 study

BackgroundHuman papillomavirus (HPV), is one of the main causes of cervical cancer and also one of the most common sexually transmitted infections (STIs). HPV is responsible for almost all cases of cervical cancer and plays a principal role in causing other cancers including oropharynx, penis, larynx, oral cavity, anus, vulva, and vagina. The study aims to investigate the prevalence and distribution of HPV genotypes among patients referred to private laboratories in Mashhad, located in the northeast of Iran.Methods and materials428 samples including 382 females (89.3%) and 46 males (10.7%) between January 10, 2022, and February 11, 2023, in Mashhad, Iran were evaluated to detect HPV and determine its genotypes. Cervical swabs and urine samples were collected from females and males, respectively. Viral DNA was extracted by using a CedExtra purification kit (cedbio, Iran) and viral genotypes were identified with a High + Low Papillomastrip kit (Operon, Spain). Mann Whitney U test and Chi-square test were accomplished for statistical analysis.ResultFrom the total of 428 participants analyzed, the HPV test result was positive for 129 patients (30.1%) and negative for 299 people (69.9%). Among the participants, 115 female (30.1%) and 14 male (30.4%) were positive for HPV infection. The prevalence of HPV infection among the referring people was about 30%. The most common genotype identified was HPV-6 (10.3%), followed by HPV-16 (8.7%) and HPV-51 (7.7%), the second and third most common genotypes, respectively. Additionally, HPV-39 was detected at a frequency of 6.70%. HPV-11, HPV-61, HPV-91, and HPV-44 with a frequency of 1% were among the least genotypes identified among the patients.ConclusionIn line with the results of this study, the prevalence of HPV genotypes in both genders is 30%. The results likely reflect differences in the prevalence of high-risk HPV genotypes, that are less common. Also, HPV-6 and HPV-16 genotypes that are covered by the vaccine had a significant prevalence. On the other hand, with the prevalence of HPV-51 and HPV-39 genotypes in infected people who are not covered by the Gardasil (quadrivalent) vaccine, there is a risk of related cancers in the future.

Viral whole genome sequencing reveals high variations in APOBEC3 editing between HPV risk categories.

High-risk human papillomavirus (HPV) infections are responsible for cervical cancer. However, little is known about the differences between HPV types and risk categories regarding their genetic diversity and particularly APOBEC3-induced mutations - which contribute to the innate immune response to HPV. Using a capture-based next-generation sequencing, 156 HPV whole genome sequences covering 43 HPV types were generated from paired cervical and anal swabs of 30 Togolese female sex workers (FSWs) sampled in 2017. Genetic diversity and APOBEC3-induced mutations were assessed at the viral whole genome and gene levels. Thirty-four pairwise sequence comparisons covering 24 HPV types in cervical and anal swabs revealed identical infections in the two anatomical sites. Differences in genetic diversity among HPV types was observed between patients. The E6 gene was significantly less conserved in low-risk HPVs (lrHPVs) compared to high-risk HPVs (hrHPVs) (p = 0.009). APOBEC3-induced mutations were found to be more common in lrHPVs than in hrHPVs (p = 0.005), supported by our data and by using large HPV sequence collections from the GenBank database. Focusing on the most common lrHPVs 6 and 11 and hrHPVs 16 and 18, APOBEC3-induced mutations were predominantly found in the E4 and E6 genes in lrHPVs, but were almost absent in these genes in hrHPVs. The variable APOBEC3 mutational signatures could contribute to the different oncogenic potentials between HPVs. Further studies are needed to conclusively determine whether APOBEC3 editing levels are associated to the carcinogenic potential of HPVs at the type and sublineage scales.

Male and female human papilloma virus infection and assisted reproductive technology outcomes: A comprehensive assessment from prevalence in semen to obstetric outcomes.

Infertility, affecting approximately 16% of the global population, has led to increased reliance on reproductive medicine. The impact of human papillomavirus (HPV) infection in one or both partners on outcomes of Assisted Reproduction Technologies (ART) remains unclear. This prospective cohort study aimed to evaluate prevalence and effects of HPV infection in subjects and couples candidates to ART. A total of n = 510 men and n = 246 women were included and n = 145 couples (n = 290 individuals) had both partners enrolled in the study. The HPV semen infection rate was 17% (95% CI: 14-20) with HPV-42, HPV-16, HPV-53 and HPV-51 as the most frequently detected genotypes. In women, 26% (95% CI: 21-32) tested HPV-positive in cervical swabs. In 6% (95% CI: 3-11) of the couples, both partners were positive but only three couples shared the same genotypes (HPV-16; HPV-39, HPV-51, and HPV-42; HPV-31). Follicular fluids were positive in 20% (95% CI: 11-33) of samples, showing genotype discrepancies with cervical tests. Semen treatment could not completely eliminate the virus in positive samples but reduced the positivity to one-third. No significant differences in semen and embryological variables, clinical pregnancy and live birth rates, neonatal and obstetrics outcomes were observed in subjects with positivity in semen or cervix compared to respective negative groups. Cumulative live birth rates per oocyte retrieval in couples where both partners were negative or both were positive did not differ, being 37% (95% CI: 28%-47%) and 44% (95% CI: 19-73), respectively. In conclusion, HPV testing should not be considered a prerequisite for accessing ART treatments. Robust inferences for natural fertility cannot be made using our findings, as the ART setting does not fully reflect natural conditions.

Viability of Chlamydia trachomatis in different anatomical sites - a systematic review & meta-analysis.

Modern assays for the detection of Chlamydia trachomatis (CT) rely on nucleic acid amplification testing (NAAT) of DNA or ribosomal RNA. However, it is also known that both viable ("living") & non-viable ("dead") CT can be detected by NAAT. Multiple laboratory techniques to measure CT viability have emerged. We searched PubMed, EMBASE, Scopus and Dimensions as well as conference abstracts for entries between January 2000 to May 2023. We included any studies that measured CT viability among NAAT-positive samples. Viability assays include enhanced cell culture, direct fluorescent antibody (DFA), messenger RNA (mRNA) detection via digital droplet PCR (ddPCR), viability PCR (V-PCR) & real-time PCR measuring RNA-to-DNA ratio (RDR) (e.g. InSignia®). A meta-analysis was performed on the proportions of non-viable CT by anatomical site. We screened 31,342 records and included 16 studies in the analysis. The pooled proportions of non-viable CT by site were: 33% (95%CI 19-47%) in rectal swabs (eight studies), 17% (95%CI 7-27%) in cervical swabs (six studies), 15% (95%CI 6-25%) in vaginal swabs (six studies) and 11% (95%CI 9-17%) in urine/urethral swabs (two studies). All included studies found that a proportion of NAAT-detected CT is non-viable. The findings have far-reaching implications for screening programs and studies evaluating new STI tests and antimicrobial regimens.

Prevalence of sexually transmitted infections and human papillomavirus in cervical samples from incarcerated women in São Paulo, Brazil: a retrospective single-center study.

Sexually transmitted infections (STIs) cause considerable morbidity worldwide and, depending on the specific pathogen, may lead to serious complications in the female reproductive tract. Incarcerated women are particularly vulnerable to health problems with a disproportionate high rate of STIs, including infections with human papillomavirus (HPV). Here, cervical swab samples collected from 299 women (18 to 64 years) living in one of the women's prisons of São Paulo, Brazil were submitted for liquid-based cytology to determine the prevalence of precancerous lesions. Furthermore, direct detection of 30 genital HPV genotypes (18 high-risk and 12 low-risk types) and 11 additional STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, Herpes simplex virus 1 and 2, Haemophilus ducreyi, Mycoplasma genitalium and hominis, Treponema pallidum, Trichomonas vaginalis, Ureaplasma parvum and urealyticum) were performed by molecular typing using two PCR-based DNA microarray systems, i.e., EUROArray HPV and EUROArray STI (EUROIMMUN), respectively. The overall prevalence of cytological abnormalities was 5.8%, including five women with low-grade and five women with high-grade squamous intraepithelial lesions. The overall prevalence of HPV was 62.2, and 87.1% of the HPV-positive women were infected with oncogenic high-risk (HR) HPV types. HPV types 16 (24.1%), 33 and 52 (both 10.4%) were the most frequently detected. The prevalence of the other STIs was 72.8%. Up to four different pathogens were found in the infected women, the most frequent being Ureaplasma parvum (45.3%), Mycoplasma hominis (36.2%) and Trichomonas vaginalis (24.8%). The high number of HR-HPV infections and other STIs described here highlights the fact that the Brazilian female prison population requires more attention in the country's health policies. The implementation of screening programs and treatment measures might contribute to a decrease in the incidence of STIs and cervical cancer in this vulnerable population. However, for such measures to be effective, further studies are needed to investigate the best practice to get more women to engage in in-prison prevention programs, e.g., through offering further sexual health education and self-sampling.

Rationale and design of the Self-TI Study protocol: a cross-sectional human papillomavirus self-testing pilot study among transgender adults in England

IntroductionPersistent infection with high-risk human papillomavirus (HPV) is the causal agent of several cancers including cervical, anal and oropharyngeal cancer. Transgender men and transmasculine non-binary (TMNB) people with a cervix...

P-333 Combining deep shotgun metagenomic sequencing and culturomics for in-depth characterization of a potential endometrial microbiome

Abstract Study question Does the endometrium host a microbiome, and if so, are these taxa distinct from the vaginal/cervical microbiome? Summary answer Multifaceted analysis of endometrial tissue reveals no distinct microbial taxa compared to the vaginal/cervical samples, questioning whether detected microbiota are endometrium-related or result from cross-contamination. What is known already Recent studies suggest a role for the endometrial microbiota in reproductive health, yet the existence of an endometrial microbiome and its specific bacterial taxa remain enigmatic. The female reproductive tract, with a bacterial abundance gradient, is predominantly Lactobacillus-dominated in the lower region. Inconsistent findings on Lactobacillus dominance in endometrial samples are often ascribed to cross-contamination and methodological constraints, highlighting the need for precise methodologies in studying the low-biomass endometrial environment. Study design, size, duration Fifteen women, undergoing a diagnostic hysteroscopy and endometrial biopsy, as part of the subfertility work-up, were included in this study. Microbiota profiles of endometrial biopsies were compared to paired vaginal and cervical swabs from nine patients using 16S amplicon sequencing. Furthermore, the microbial composition of six additional endometrium biopsies was characterized using both a culturomics-based approach and deep metagenomic sequencing. Participants/materials, setting, methods Endometrial biopsies (n = 15) were taken with a sterile Pipelle de Cornier and snap-frozen in liquid nitrogen, followed by -80 °C storage and 16S amplicon sequencing (n = 9) targeting the V4-region or deep shotgun metagenomic sequencing (n = 6) (Prebiomics, Italy). Simultaneously, in nine women, paired cervical and vaginal swabs (Copan Diagnostics, Italy) were taken and preserved in eNAT medium. In six participants, an additional endometrial biopsy was collected and stored in Amies medium for immediate culturomics. Main results and the role of chance We observed a high similarity in the microbial composition of endometrial biopsies, cervical, and vaginal samples using 16 amplicon sequencing, but also some taxa were only found in the endometrial samples. Notably, the presence of Gemmobacter, a waterborne genus, was only found in endometrial biopsies and could be a source of contamination from water used during hysteroscopy to flush the uterus. Most endometrial microbiome profiles were Lactobacillus dominated, with exceptions observed in three patients. No significant differences for alpha diversity measures (richness and evenness) of the microbiota composition were observed between the different niches. For the first time we applied both culturomics and deep shotgun metagenomic sequencing for the characterization of the microbial composition in the same endometrial biopsies. Commonly identified genera using both techniques include Lactobacillus, Prevotella, Bifidobacterium, Anaerococcus, and Cuticubacterium. Remarkably, at the species level, the two approaches revealed almost no overlapping species within each biopsy, underscoring the importance of combining multiple methodologies in examining low-biomass niches like the endometrium. Limitations, reasons for caution A limitation of this study is the low-biomass nature of endometrial biopsies which are prone to cross-contamination with the lower female reproductive tract. This challenges downstream analysis, as it becomes difficult to distinguish between a relevant microbiological signal and contaminating noise. Wider implications of the findings This study emphasizes the need to consider a multifaceted microbiome approach when exploring low-biomass environments, like the endometrium. The critical consideration of potential cross-contamination by the lower female reproductive tract and its implications for downstream analysis is paramount in clinical microbiology research and demands meticulous handling. Trial registration number not applicable

O-279 The lower reproductive tract microbiota composition alters upon ovarian stimulation and is associated with, but not accurately predictive of reproductive outcomes following ART

Abstract Study question Are ovarian stimulation (OS) and ART success associated with the microbiota of the lower reproductive tract (LRT), i.e. vagina and cervix? Summary answer Estrogen rise during OS has a pivotal role in the LRT microbiota variation. The LRT microbiota pre-OS, although associated, may not accurately predict ART success. What is known already OS is a key component to enhance success rates in ART. OS triggers a significant rise in serum estrogen levels, which are known to influence the vaginal microbiota. The role of the female LRT microbiota in ART success is increasingly recognized, yet the impact of OS, particularly the estrogen rise, on these microbial communities and how the change may influence ART success remains largely unknown. Additionally, finding putative biomarkers within LRT microbiota could aid in predicting ART success. Understanding this relationship could refine and personalize fertility treatments, but robust evidence is needed to guide microbiota-based clinical decisions in ART. Study design, size, duration Vaginal and cervical swabs were collected longitudinally from patients undergoing ART and this at baseline (pre-OS) and at oocyte retrieval (post-OS). We gathered a final dataset comprising 275 vaginal samples at baseline and 240 samples at oocyte retrieval. Additionally, we defined a prospective cohort of n = 228x2 samples corresponding to patients for which both pre- and post-OS samples were available. The main results as detected within the vaginal samples were confirmed in the cervical samples. Participants/materials, setting, methods Bacterial DNA from −80 °C preserved swabs was extracted in a Tecan Nucleic Acid Extraction Platform, using the QIAamp DNA-Stool-Mini-Kit. Samples for paired-end Illumina MiSeq were constructed using a two-step PCR amplicon approach targeting the V4 region of the 16S rRNA. Then, LotuS, DADA2, the RDP classifier trainset 16 and variance stabilisation (poscounts, DESeq2) were used for read de-multiplexing, pre-processing, taxonomy assignment and transformation, respectively, as recommended for zero-inflated compositional data. Main results and the role of chance First, a confounder analysis revealed that both the menstrual phase and intercourse correlated with the LRT’s baseline microbiota composition (multivariate distance-based redundancy analysis [dbRDA], AdjP&amp;lt;0.05, N = 275). Next, in the paired cohort, we observed that the OS-associated rise in estrogen was correlated with confounder-independent compositional (confounder-adjusted dbRDA, AdjP=0.002, R2=1.14%, N = 228x2) and microbial-feature changes (Wilcoxon test, AdjP&amp;lt;0.05, Ndelta=228) in the LRT. Interestingly, post-OS we noted a decrease in Lactobacillus proportions compared to baseline (in participants sampled at either the follicular or luteal phase of the menstrual cycle). Additionally, post-OS microbiota composition was not associated with the live birth rate (LBR, following the first embryo transfer of the cycle) or the cumulative LBR (CLBR, following all embryo transfers of the cycle), nor with the total number of high-quality (HQ) embryos available (dbRDA, AdjP&amp;gt;0.05, N = 240). Notably, pre-OS microbiota communities and LRT Anaerococcus and Peptoniphilus proportions were associated with (C)LBR (confounder-adjusted dbRDA, AdjP&amp;lt;0.05; Logistic regression, odds-ratio&amp;lt;1, AdjP&amp;lt;0.05, N = 275). Machine learning models determined that vaginal Peptoniphilus and Anaerococcusproportions alone did not have good accuracy/ROC for predicting (C)LBR (test-set;&amp;lt;70%). Notably, the number of HQ embryos showed good accuracy/ROC for CLBR (test-set; 80%/84.5%). Including vaginal Anaerococcus proportions only slightly improved the model’s accuracy/ROC (test-set; 82.5%/85.6%). Limitations, reasons for caution The data’s proportional nature restricted assessing whether the estrogen rise decreased Lactobacillus proportions or increased other bacteria. Luteal phase sampling was avoided given the intravaginal progesterone support. Larger datasets may further improve predictive models. Wider implications of the findings Our study highlights the confounding impact of baseline variables and the influence of the OS-associated estrogen rise on LRT microbiota. It questions the clinical relevance of the LRT microbiota’s impact on reproductive outcomes following ART, warning for caution in microbiota-based decisions in ART. Trial registration number NCT03105453

Colposcopically Directed Biopsy Before Ablative Treatment Versus Direct Ablative Treatment in Patients With Cervical Oncogenic HPV.

In the past, the standard of care for women with abnormal cervical cytology has been the performance of colposcopically guided biopsy, followed by conization or large loop excision of the transition zone (LLETZ) where biopsy revealed pre-cancerous or cancerous areas. More straightforward protocols are emerging which advocate performing LLETZ in all women with highly suspicious cytology, suspicious colposcopic impression, or the presence of high-risk oncogenic human papilloma virus (HPV) strains in their cervical swabs. This, theoretically, would reduce the rate of false-negative diagnoses, but at the price of overtreating a significant number of healthy women. We retrospectively analyzed cervical cancer screening protocols in two large cohorts of women with high-risk HPV. The study compared outcomes between patients undergoing a colposcopically directed biopsy before LLETZ (n=683) and those proceeding directly to LLETZ without a biopsy (n=136). The primary focus was to assess whether intervening biopsies would reduce unnecessary ablative procedures without compromising the detection of high-grade lesions. The biopsy group had a high false-negative rate, with several high-grade lesions (CIN3) and a case of invasive cancer initially underdiagnosed. Conversely, the direct-to-LLETZ approach, while ensuring no high-grade lesions were missed, led to overtreatment of lower grade lesions. These findings raise concern about the reliance on biopsy results for treatment decisions. Neither protocol was entirely satisfactory, although the more aggressive one avoided the potentially life-threatening consequence of false-negative results. Further research is mandatory to accurately diagnose all cases requiring aggressive treatment, without subjecting healthy women to ablative treatments they do not need.

Comparison of HPV DNA Screening using menstrual blood collected in sanitary pads versus cervical swabs in women with both single and multiple sexual partners

Background: Persistent high-risk HPV infection is the main cause of cervical cancer. Sexually active women are at risk of HPV infection, especially if they have multiple sexual partners. To detect HPV, the HPV DNA test can be conducted using menstrual blood samples as an alternative to cervical cancer screening. Objective: This study aims to analyze the comparison of results between HPV DNA screening with menstrual blood in sanitary pads and cervical swabs in women with single and multi-partner sexual. Methods: This study was an analytical observational study with a cross-sectional design. The sample used was 44 women (21 single partners and 23 multiple sexual partner) aged &gt;18 years, sexually active, experiencing regular menstruation. Menstrual blood samples in sanitary pads and cervical swabs were examined PCR method. Statistical tests used Mann Whitney with a significance level of p≤0.05, diagnostic test and ROC description. Results: HPV DNA was found in 47.6% (10/21) in menstrual blood and 47.8% (11/21) in cervical swabs with a significance value of p=1,000 in women with single sexual partners. HPV DNA was detected in 82.6% (19/23) in menstrual blood and 87% (20/23) in cervical swabs with a significance value of p=1,000 in women with multiple sexual partners. Menstrual blood in sanitary pads has a sensitivity value of 90.32%, specificity 92.31%, positive predictive value 96.5%, negative predictive value 80% and accuracy of 91.32% to detect HPV DNA. Conclusion: The conclusion from this analysis shows that there is concordance between HPV DNA screening with menstrual blood in sanitary pads and cervical swabs with high accuracy values

Portable Device with Nicking Enzyme Enhanced Special RCA on μPADs toward Sensitive Detection of High-Risk HPV Infection.

Development of an accurate, rapid, and cost-effective portable device is in high demand for point-of-care molecular diagnosis toward disease screening. Here we report a one-pot homogeneous isothermal assay that leverages nicking endonuclease and minimum secondary structured rolling circle amplification (N-MSSRCA) for fast and sensitive quantification of nucleic acids on distance microfluidic paper-based analytical devices (dμPAD) by a portable custom-made fluorescence detector. Human papillomavirus (HPV) oncogenic E7 mRNA as the biomarker for cervical cancer was used as the model analyte. N-MSSRCA integrates ligase for target recognition, the nicking enzyme for primer generation, and the dual function of the Phi29 DNA polymerase for both on- and off-loop amplification. The proposed method was capable of detecting 1 and 10 fM of the analyte using the microplate reader and portable detector with dμPAD, respectively, with ∼1 h assay time. A cohort study of 40 cervical swab samples shows N-MSSRCA reached positive and negative predictive values of 87.5% and 93.5% using the portable detector with dμPAD, compared to 91.67% and 100% using the microplate reader. N-MSSRCA demonstrates potential in early screening of high-risk HPV infection as a generic strategy to detect various nucleic acids in point-of-care scenarios.

Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag–Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag–Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.

The vaginal microbial signatures of preterm birth woman

To explore the differences of vaginal microbes in women with preterm birth (PTB), and to construct prediction model. We searched for articles related to vaginal microbiology in preterm women and obtained four 16S rRNA-sequence datasets. We analyzed that for species diversity and differences, and constructed a random forest model with 20 differential genera. We introduce an independent whole genome-sequencing (WGS) data for validation. In addition, we collected vaginal and cervical swabs from 33 pregnant women who delivered spontaneously full-term and preterm infants, performed WGS in our lab to further validate the model. Compared to term birth (TB) samples, PTB women vagina were characterized by a decrease in Firmicutes, Lactobacillus, and an increase in diversity accompanied by the colonization of pathogenic bacteria such as Gardnerella, Atopobium and Prevotella. Twenty genus markers, including Lactobacillus, Prevotella, Streptococcus, and Gardnerella performed well in predicting PTB, with study-to-study transfer validation and LODO validation, different gestation validation showing good results, and in two independent cohorts (external WGS cohorts and woman samples WGS cohorts) in which the accuracy was maintained. PTB women have unique vaginal microbiota characteristics. A predictive model of PTB was constructed and its value validated from multiple perspectives.

Challenges in the diagnosis and control of female genital schistosomiasis in sub-Saharan Africa: an exemplar case report associated with mixed and putative hybrid schistosome infection in Nsanje District, Southern Malawi

Female genital schistosomiasis (FGS) remains an often overlooked chronic complication of urogenital schistosomiasis in adolescent girls and women. Moreover, the role of zoonotic or hybrid schistosome infection(s) is poorly appreciated, but is increasingly becoming an emerging public health concern in sub-Saharan Africa. In Southern Malawi, during the “Hybridization in UroGenital Schistosomiasis (HUGS)” study visit, we describe the case of a 33-year-old woman with suspected FGS who partook in a detailed external assessment with internal cervical examination using a portable colposcope. She provided several biological samples for analysis with traditional and molecular parasitological methods—urine, cervicovaginal lavage (CVL), cervical swabs, and external mass and cervical biopsies—alongside provision of detailed demographic information after a thorough medical history questionnaire and an in-depth interview. These samples were screened for the presence of Schistosoma ova on microscopy and DNA genotyping using a novel real-time PCR assay in parallel to pre-published probe-based PCR assays capable of identifying and discriminating up to six named Schistosoma species. A further molecular screen of sexually transmitted infections (STIs) including Trichomonas vaginalis, Chlamydia spp., and human papilloma virus (HPV) was conducted on her genital swab and CVL. Overt FGS was diagnosed on clinical colposcopy alongside inspection of the cervical biopsy by microscopy, real-time PCR, and histopathology. The urine filtration, microscopy and real-time PCR of the CVL and swab were negative. This evidences the typical diagnostic challenge, and cases such as this will pose an unmet need in satisfactory patient management. In addition to Schistosoma haematobium, the presence of the zoonotic species Schistosoma mattheei and concurrent STIs raise questions as to the long-term effectiveness of the current control strategies of the National Control Programme to eliminate schistosomiasis as a public health problem. Improved availability of and regular accessibility to praziquantel treatment for women at risk such as this are urgently needed. Furthermore, targeted health education, increased community awareness, and dovetailing of synergistic activities and strategies with other health stakeholders such as those in sexual and reproductive health, as well as HIV/AIDS programs in the Ministry of Health, are needed here and in neighboring countries.

A novel clinical metaproteomics workflow enables bioinformatic analysis of host-microbe dynamics in disease.

Clinical metaproteomics has immense potential to offer functional insights into the microbiome and its contributions to human disease. However, there are numerous challenges in the metaproteomic analysis of clinical samples, including handling of very large protein sequence databases for sensitive and accurate peptide and protein identification from mass spectrometry data, as well as taxonomic and functional annotation of quantified peptides and proteins to enable interpretation of results. To address these challenges, we have developed a novel clinical metaproteomics workflow that provides customized bioinformatic identification, verification, quantification, and taxonomic and functional annotation. This bioinformatic workflow is implemented in the Galaxy ecosystem and has been used to characterize diverse clinical sample types, such as nasopharyngeal swabs and bronchoalveolar lavage fluid. Here, we demonstrate its effectiveness and availability for use by the research community via analysis of residual fluid from cervical swabs.

Special RCA based sensitive point‐of‐care detection of HPV mRNA for cervical cancer screening

AbstractDeveloping the sensitive point‐of‐care testing (POCT) of oncogenic nucleic acids from human papillomavirus (HPV) infection is essential in preventing cervical cancer, especially in resource‐limited settings. Rolling circle amplification (RCA) is attractive in achieving POCT via nucleic acid‐based aggregation under isothermal conditions. However, the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity. Here, a minimum secondary structured RCA technique (MSS‐RCA) is developed by designing a unique circular template, demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions. The amplification efficiency of MSS‐RCA could be kinetically manipulated by controlling the secondary structure of the circular template. Introducing the invertase probe to MSS‐RCA, HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter (PGM) with a sensitivity of 5 fm (50 zmol in 10 µL). This integrated MSS‐RCA‐PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00% and negative predictive values of 96.00% (77.77% to 99.40%, 95% CI). MSS‐RCA‐PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.

Comparison of cervicovaginal fluid extracellular vesicles isolated from paired cervical brushes and vaginal swabs

AbstractEndometriosis is a common gynaecological condition, with a long diagnostic delay. Surgery is required to confirm a diagnosis, highlighting the need for a non‐invasive biomarker. Extracellular vesicles (EVs) may have a role in endometriosis pathogenesis, yet there is limited EV biomarker literature available. This study aimed to investigate the feasibility of isolating cervico‐vaginal fluid EVs sampled using cervical brushes and vaginal swabs and to compare these methods. After providing informed consent, patients undergoing surgery for suspected endometriosis had cervical brush and vaginal swab samples collected under general anaesthetic. Isolated EVs were characterised through negative stain transmission electron microscopy (TEM), Western blotting (TSG101, CD63, Calnexin, ApoB, Albumin), tunable resistive pulse sensing (TRPS), microBCA assays and RT‐qPCR of miRNAs. PCR was performed on samples prior to EV isolation to assess bacteria present in samples. Cervical brush and vaginal swab EVs were intact vesicles with limited co‐isolated contaminants. Cervical brushes had higher concentrations of particles compared to match vaginal swabs, although both samples had low concentrations. Protein and miRNA yield were similar between matched samples. PCR demonstrated only a small amount DNA within samples was bacterial (&gt;0.5%). Cervico‐vaginal fluids EVs were successfully isolated from cervical brushes and vaginal swabs, demonstrating a new method of sampling reproductive EVs. EV yield from both sample types was low. Similar protein and miRNA levels suggest either sampling method may be suitable for biomarker studies.