Type-specific Assays Research Articles

Betapapillomavirus natural history and co-detection with alphapapillomavirus in cervical samples of adult women.

Human papillomaviruses (HPV) of the genus Betapapillomavirus can infect both cutaneous and mucosal sites, but research on their natural history at mucosal sites remains scarce. We examined the risk factors and co-detection patterns of HPVs of the Betapapillomavirus and Alphapapillomavirus genera in cervical samples of the Ludwig-McGill cohort study. We assessed a subset of 505 women from the Ludwig-McGill cohort study from São Paulo, Brazil. Cervical samples over the first year of follow-up were tested for DNA of over 40 alphapapillomavirus types and 43 betapapillomavirus types using a type-specific multiplex genotyping polymerase chain reaction assay. We assessed the risk factors for prevalent and incident betapapillomavirus type detection, and whether types were detected more frequently together than expected assuming independence using permutation tests, logistic regression, and Cox regression. We observed significant within-genus clustering but not cross-genus clustering. Multiple betapapillomavirus types were co-detected in the same sample 2.24 (95% confidence interval [CI]: 1.65-3.29) times more frequently than expected. Conversely, co-detections of alphapapillomavirus and betapapillomavirus types in the same sample occurred only 0.64 (95% CI: 0.51-0.83) times as often as expected under independence. In prospective analyses, positivity to one HPV genus was associated with a nonsignificant lower incidence of detection of types in the other genus. Lifetime number of sex partners and new sex partner acquisition were associated with lower risks of prevalent and incident betapapillomavirus detection. Betapapillomaviruses are commonly found in the cervicovaginal tract. Results suggest potentially different mechanisms of transmission for betapapillomavirus genital infections other than vaginal sex.

Prevalence of Candida albicans in High-Risk Human Papillomavirus-Positive Women: A Study in Diyarbakır Province, Turkey.

The human papillomavirus (HPV) is a significant public health concern due to its association with the development of cervical cancer. Although inflammation caused by Candida spp. has been shown to facilitate oncogenesis, the interactions between HPV and Candida spp. remain unclear. This study aimed to determine the prevalence and genotype distribution of HR-HPV infection HR-HPV-positiveCandida albicans in HR-HPV-positive individuals in Diyarbakır province in Turkey. Cervical samples were taken from 350 participants aged 20-69 years who applied to Diyarbakır Gazi Yaşargil Training and Research Hospital, Gynecology and Obstetrics Clinic. For detection of HPV presence and HR-HPV genotyping, PCR/direct cycle sequencing was used. E6/E7 mRNA expression of HPV-16, -18, -31, -33, and -45 was determined by type-specific real-time NASBA assay (NucliSENS EasyQ(®)HPV v1.1). The presence of Candida albicans in cervical specimens of HR-HPV-positive women was investigated by RAPD-PCR and culture methods. Results. Of the 350 women who participated in the study, 24% were HPV positive and 10.5% were found to be HR-HPV positive. HR-HPV positivity was most frequently detected in the age range of 40-49 years. Among HR-HPV-positive women, C. albicans was found in 59.4%. Conclusion. The most frequent HR-HPV genotype was HPV16, followed by HPV31. Of women who tested positive for HR-HPV, C. albicans was discovered in 59.4%. C. albicans infection may occur when the immune system is weakened or the balance of the vaginal flora is disturbed, increasing tissue damage in the vaginal area and the risk of carcinogenesis of HR-HPV. Therefore, knowing the presence of Candida infection in HR-HPV-positive women is essential to plan the treatment and prevent the risk of secondary disease.

Prevalence of Alpha, Beta, and Gamma Human Papillomaviruses in Patients With Head and Neck Cancer and Noncancer Controls and Relation to Behavioral Factors.

Human papillomaviruses (HPVs) cause head and neck cancer (HNC), which is increasing in incidence in developed countries. We investigated the prevalence of alpha (α), beta (β), and gamma (γ) HPVs among HNC cases and controls, and their relationship with sociodemographic, behavioral, and oral health factors. We obtained oral rinse and brush samples from incident HNC cases (n = 369) and hospital-based controls (n = 439) and tumor samples for a subsample of cases (n = 121). We genotyped samples using polymerase chain reaction with PGMY09-PGMY11 primers and linear array for α-HPV and type-specific multiplex genotyping assay for β-HPV and γ-HPV. Sociodemographic and behavioral data were obtained from interviews. The prevalence of α-, β-, and γ-HPV among controls was 14%, 56%, and 24%, respectively, whereas prevalence among cases was 42%, 50%, and 33%, respectively. Prevalence of α- and γ-HPV, but not β-HPV, increased with increase in sexual activity, smoking, and drinking habits. No HPV genus was associated with oral health. Tumor samples included HPV genotypes exclusively from the α-genus, mostly HPV-16, in 80% of cases. The distribution of α- and γ-HPV, but not β-HPV, seems to vary based on sociodemographic and behavioral characteristics. We did not observe the presence of cutaneous HPV in tumor tissues.

Altered Vaginal Microbiota Composition Correlates With Human Papillomavirus and Mucosal Immune Responses in Women With Symptomatic Cervical Ectopy.

Cervical ectopy is a benign condition of the lower genital tract that is frequently detected in women of reproductive age. Although cervical ectopy is regarded as a physiological condition, some women experience symptoms such as leucorrhoea, persistent bleeding and recurrent vaginal infections that require medical intervention. Cervical ectopy has not been linked to cervical cancer, but it is thought to facilitate the acquisition of sexually transmitted diseases (STDs), like Human Papillomavirus (HPV) infection, as it provides a favorable microenvironment for virus infection and dissemination. We and others have described the presence of oncogenic HPV types in women with symptomatic cervical ectopy. The relevance of this finding and the impact of symptomatic cervical ectopy on the cervicovaginal microenvironment (vaginal microbiota, immune and inflammatory responses) are currently unknown. To shed some light into the interplay between HPV, the vaginal microbiota and mucosal immune and inflammatory responses in the context of this condition, we enrolled 156 women with symptomatic cervical ectopy and determined the presence of HPV using a type-specific multiplex genotyping assay. Overall, HPV was detected in 54.48% women, oncogenic HPV types were found in more than 90% of HPV-positive cases. The most prevalent HPV types were HPV16 (29.4%), HPV31 (21.17%) and HPV18 (15.29%). Next, we evaluated the vaginal microbial composition and diversity by 16S rDNA sequencing, and quantified levels of cytokines and chemokines by flow cytometry using bead-based multiplex assays in a sub-cohort of 63 women. IL-21 and CXCL9 were significantly upregulated in HPV-positive women (p=0.0002 and p=0.013, respectively). Women with symptomatic cervical ectopy and HPV infection had increased diversity (p<0.001), and their vaginal microbiota was enriched in bacterial vaginosis-associated anaerobes (Sneathia, Shuttleworthia, Prevotella, and Atopobium) and depleted in Lactobacillus spp. Furthermore, the vaginal microbiota of women with symptomatic cervical ectopy and HPV infection correlated with vaginal inflammation (IL-1β, rho=0.56, p=0.0004) and increased mucosal homeostatic response (IL-22, rho=0.60, p=0.0001). Taken together, our results suggest that HPV infection and dysbiotic vaginal communities could favor a vaginal microenvironment that might delay the recovery of the cervical epithelium in women with symptomatic cervical ectopy and favor STDs acquisition.

Epigenetic immunophenotyping in monitoring of SARS-CoV2 vaccine response and COVID-19 disease course

Abstract The objective was to explore whether epigenetic immune cell counting can advance efficiency and quality of diagnostic and immune monitoring related to COVID-19. Application areas were monitoring disease course, therapeutic clinical development, and measurements of SARS-CoV2 vaccine responses. Immune cell type specific epigenetic assays have been developed over the last decade. They are primarily used in therapeutic clinical research in oncology and autoimmune disease. Due to the high sample stability and low amount requirements for epigenetic measurements and available assay portfolio for key immune cell populations relevant in COVID-19, the method promised to be useful and practical in the pandemic setting. Epigenetic immunophenotyping using whole blood of hospitalized Covid19 patients was applied and CD3, CD4, CD8 and regulatory T cell populations, NK cells, naïve and memory B cells were quantified, and measurement results show to predict mild or severe COVID-19 disease courses. Furthermore, nasopharyngeal swab and saliva samples were applied demonstrating that epigenetic immune monitoring can measure immune cell content in such non-invasive sample types. Due to the low sample volume and handling requirements and availability of 35 assays for relevant immune cell populations, epigenetic immune monitoring is suitable for therapeutic COVID-19 clinical trials. Another possibility for sample collection is single drops of capillary blood deposited on filter paper (dried blood spots), which have been collected pre and post SARS-CoV2 booster vaccinations in healthy subjects. Such measurements revealed vaccine response for example in changes of cell populations epigenetically active in the markers CCR7 and TIGIT.

Detection of Circulating HPV16 DNA as a Biomarker for Cervical Cancer by a Bead-Based HPV Genotyping Assay.

ABSTRACTHuman papillomavirus (HPV) circulating tumor DNA (HPV ctDNA) was proposed as a biomarker for the detection and disease monitoring of HPV-related cancers. One hundred eighty plasma samples obtained from women diagnosed with HPV16-positive cervical cancer (CC) (n = 100), HPV16-positive premalignant lesions (cervical intraepithelial neoplasia grade 3 [CIN3]) (n = 20), and HPV DNA-negative controls (n = 60) were randomly selected from the archives for evaluating the performance of a bead-based HPV genotyping assay (E7 type-specific multiplex genotyping assay [E7-MPG]) in detecting HPV16 ctDNA. The performance of the E7-MPG was compared with those of DNA detection by droplet digital PCR (ddPCR) and detection of HPV16 E6 antibodies evaluated in an independent study. Internal controls to assess DNA quality were included in the molecular assays, i.e., beta-globin and ESR1, respectively. The sensitivity and specificity of E7-MPG and/or E6 antibodies to detect HPV16-positive CCs were evaluated. HPV16 ctDNA was detected using the E7-MPG in 42.3% of all plasma samples and in 74.7% of plasma samples from HPV16-positive CC cases. The validation of E7-MPG data by ddPCR showed that the sensitivity of the E7-MPG test for HPV16-positive CC detection was higher than that of ddPCR (74.7% versus 63.1%; P < 0.001). When both HPV16 ctDNA and E6 antibodies were considered, the sensitivity for HPV16-positive CC detection increased from 74.7% to 86.1%, while the specificity was unchanged at 97.8%. The performance of E7-MPG for the detection of HPV16 ctDNA appears to be at least as sensitive as that of ddPCR, offering an additional tool for ctDNA detection of HPV16-positive CC. The use of an additional blood marker of HPV infection, such as E6 antibodies, further improved the detection of CC.IMPORTANCE The validity of HPV ctDNA as a marker of HPV-driven cancers has been previously reported. Herein we validated an alternative to ddPCR for HPV16 ctDNA detection, using a bead-based HPV genotyping assay that offers the potential advantage of reducing the cost of clinical management due to the multiplex capability of the test, thus facilitating its use in clinical settings. In addition, we analyzed HPV ctDNA in the context of E6 antibodies as an additional HPV marker. The HPV16 ctDNA biomarker appeared to be highly specific and, to a lesser extent, sensitive for the detection of CC, mainly indicated for those at an advanced tumor stage. In this proof-of-principle study, E6 antibodies were mainly detected in early tumor stages of CC, while HPV ctDNA was mainly positive at advanced tumor stages.

Prevalence of human papillomavirus types in head and neck cancer sub-sites in the Indian population.

Although a subset of head and neck cancers (HNC) has been associated worldwide with mucosal high-risk human papillomaviruses (HPV), information on the prevalence of HPV-positive HNC in India is limited. In this study, we examined the prevalence of 21 subtypes of HPV in sub-sites of HNC (n = 175) in the western region of India. Type-specific multiplex genotyping assay was conducted at the Centre for Cancer Epidemiology, Tata Memorial Centre, to determine the prevalence of HPV subtypes. The HPV prevalence was observed to be 28.43%, 41.67%, 38.89% and 15.79% in the oral cavity, oropharynx, hypopharynx and larynx tumour tissues, respectively. The HPV 16 genotype was most common in all HNC tumour tissues (30.29%), followed by HPV 58 (0.57%).

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine

Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/μl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against the putative novel genotypes. RT-qPCR assays targeting BTV segment 9 and 10 detected both strains (SPvvvv/02 and SPvvvv/03) whereas a BTV segment 1 RT-qPCR assay was unable to detect either BTV strain. The work presented here expands upon the current repertoire of RT-qPCR assays for BTV genotype determination.

Kronik Hepatit C Enfeksiyonlu Hastalarda Genotip Dağılımı ve Risk Faktörleri

Objective: Hepatitis C virus (HCV) is a common infection around the world and an important public health problem. Determination of HCV genotype is important epidemiologically and for treatment approaches. In this study, the aim was to assess the genotype distribution and associated risk factors for patients monitored at our center in northern Turkey.&#x0D; Methods: A cross-sectional study was carried out of patients with confirmed HCV infection. Our study retrospectively assessed 175 patients with chronic hepatitis C diagnosis in the Infectious Diseases clinic from 2016-2019 and with antiviral treatment administered. The samples were tested by type specific genotyping assay. The relationship between demographic characteristics and potential risk factors and genotype was investigated.&#x0D; Results: Genotype 1b was identified as the dominant genotype (95%). In 5% of patients, non-1b genotypes were present (genotype 1a, 3 and mixed). Genotype 1b was more common in patients over 50 years of age, while the patients with other genotypes were younger. The most frequent risk factor was identified as surgical intervention history. While young age, transplantation and intravenous drug use were identified as risk factors for development of infection with non-1b genotypes, household HCV contact was significant for genotype 1b.&#x0D; Conclusion: In our study, the dominant genotype was identified as genotype 1b. Among the risk factors in our study, the most frequently identified are surgical interventions and dental treatment. The variation in traditional risk factors will cause an increase in non-1b genotypes. We think it is important to correctly analyze these variations in the global struggle with HCV.

Dengue outbreaks: Report of short heterologous protection in Reunion Island

Background: Reunion Island is facing a dengue outbreak with a first epidemic wave in 2018 and a second one in 2019. During this outbreak, almost 25 000 confirmed cases and more than 75 000 clinically compatible cases were notified. While only DENV2 was isolated among autochthonous cases in 2018, DENV1 locally emerged in 2019. This occurrence of DENV1 from April was most probably from a transmission chain initiated around an imported case that travelled in Southeast Asia and in which DENV1 was isolated. While infection confers lifelong immunity to the same serotype, heterologous protection is shorter. Even though the length of this protection is not precisely known, it is often reported that the complete heterologous protection would last 2 months and that partial protection would extend to 2 years. Case description: Here we report on 11 patients in which 2 positive PCRs for dengue were declared during the 2 epidemic waves. All these patients suffered from symptomatic infections and when signs and symptoms were collected (in 6 of them), back pain, myalgia, loss of appetite and asthenia were the most frequent followed by fever, arthralgia and vomiting. Noticeably, none experienced a severe episode nor required to be hospitalized. At this stage, typing using type-specific RT-PCR assays of the two positive samples was performed in one patient and confirmed the secondary infection. DENV1 was isolated in 3 samples from 2019 from 3 patients also diagnosed in 2018. Because of an IgG positive serology, it strongly suggests the heterologous infection despite no typing in 2018. Due to technical issues, 3 samples could not be typed. Four typing results are pending. The median length between the 2 infections was 13 months [min-max: 4.5–18 months]. Discussion: These preliminary findings showed that two symptomatic dengue infections can occur even if infections are only distanced of 4 months and suggests that heterologous protection is shorter than expected. Conclusion: These results contrast with the 24 months protection usually reported. Of note and as previously described, it seems that the risk of developing a severe form during secondary infection is increasing with the time interval between the 2 infections.

Challenges and possible solutions for decoding extranasal olfactory receptors.

Olfactory receptors are primarily known to be expressed in the olfactory epithelium of the nasal cavity and therefore assist in odor perception. With the advent of high-throughput omics technologies such as tissue microarray or RNA sequencing, a large number of olfactory receptors have been reported to be expressed in the nonolfactory tissues. Although these technologies uncovered the expression of these olfactory receptors in the nonchemosensory tissues, unfortunately, they failed to reveal the information about their cell type of origin. Accurate characterization of the cell types should be the first step towards devising cell type-specific assays for their functional evaluation. Single-cell RNA-sequencing technology resolved some of these apparent limitations and opened new means to interrogate the expression of these extranasal olfactory receptors at the single-cell resolution. Moreover, the availability of large-scale, multi-organ/species single-cell expression atlases offer ample resources for the systematic reannotation of these receptors in a cell type-specific manner. In this Viewpoint article, we discuss some of the technical limitations that impede the in-depth understanding of these extranasal olfactory receptors, with a special focus on odorant receptors. Moreover, we also propose a list of single cell-based omics technologies that could further promulgate the opportunity to decipher the regulatory network that drives the odorant receptors expression at atypical locations.

Outbreaks of Adenovirus-associated Respiratory Illness on 5 College Campuses in the United States, 2018-2019.

Human adenoviruses (HAdVs) are commonly associated with acute respiratory illness. HAdV outbreaks are well documented in congregate military training settings, but less is known about outbreaks on college campuses. During fall 2018 and spring 2019, 5 United States (US) colleges reported increases in HAdV-associated respiratory illness. Investigations were performed to better understand HAdV epidemiology in this setting. A case was defined as a student at one of the 5 colleges, with acute respiratory illness and laboratory-confirmed HAdV infection during October 2018-December 2018 or March-May 2019. Available respiratory specimens were typed by HAdV type-specific real-time polymerase chain reaction assays, and for a subset, whole genome sequencing was performed. We reviewed available medical records and cases were invited to complete a questionnaire, which included questions on symptom presentation, social history, and absenteeism. We identified 168 HAdV cases. Median age was 19 (range, 17-22) years and 102 cases (61%) were male. Eleven cases were hospitalized, 10 with pneumonia; 2 cases died. Among questionnaire respondents, 80% (75/94) missed ≥ 1 day of class because of their illness. Among those with a type identified (79%), HAdV types 4 and 7 were equally detected, with frequency of each varying by site. Genome types 4a1 and 7d were identified, respectively, by whole genome sequence analysis. HAdV respiratory illness was associated with substantial morbidity and missed class time among young, generally healthy adults on 5 US college campuses. HAdVs should be considered a cause of respiratory illness outbreaks in congregate settings such as college campuses.

Role of human papillomavirus infection in the etiology of vulvar cancer in Italian women

BackgroundVulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract. We aimed to determine the mucosal high-risk human papillomavirus (HPV)-attributable fraction of VSCCs from Italian women using multiple markers of viral infections.MethodsVSCCs and 8 metastatic lymph node samples from 107 Italian women were analyzed by a highly type-specific multiplex genotyping assay for the presence of DNA from 119 different HPVs. Tissues were further analyzed for HPV RNA and for upregulation of the cellular protein p16INK4a.ResultsThe rate of mucosal HPV-related tumors defined by viral DNA and RNA positivity was low (7.8%). HPV16 was the most prevalent, followed by 53, 56, and 58. Only five (4.9%) p16INK4a-positive tumors were also positive for both viral DNA and RNA. One (14.3%) metastatic lymph node sample was positive for all three markers. DNA of cutaneous HPVs was detected in only two VSCCs, i.e. genus beta types 5 and 110.ConclusionA small proportion of Italian VSCCs is putatively HPV-related, i.e. positive for both viral DNA and RNA of the same type, thus reinforcing the importance of HPV vaccination. Moreover, this study suggests that a direct role of HPV from genus beta and gamma in vulvar carcinogenesis is unlikely.

Amino Acid Mutations in NS5B Protein among Treatment-naive Genotype 3A Infected Patients.

To determine the frequency of mutations at specific amino acid positions in full length NS5B gene among chronic HCV genotype 3a infected patients of Peshawar, who had not taken any previous treatment. Cross-sectional descriptive study. Institute of Basic Medical Sciences, Khyber Medical University, Peshawar (IBMS, KMU) and Comsats Institute of Information Technology (CIIT), Islamabad from September 2016 to December 2017. HCV genotype determination was carried out among 310 actively infected, treatment-naive patients, using type specific PCR-based genotyping assay. In a total of 162 (52%) HCV genotype 3a isolates, NS5B gene was amplified in 126 (78%) samples using qualitative PCR and sequencing. NS5B gene sequences were analysed for clinically relevant mutations against standard HCV 3a reference sequence (Isolate NZL1, BAA04609) using MEGA 6 software. Analysis of HCV NS5B amino acid sequences (aa.1-591), comprising essential motif A-F revealed four novel mutations: A67V, T131I, R374H and M425L in 27 (21%) viral isolates. Mutation D/N244S and D/N310K were found in 14 (11%) of the pre-treatment isolates. Mutations at positions 282 and 316 (S282T and C316N/Y) were not identified among studied isolates. This study reports mutations based on complete NS5B protein of HCV 3a genome that could help predict treatment response among the chronically infected with HCV genotype 3a patients of Peshawar.

Genotype distribution in relation to viral load in a large cohort of Indian patients with chronic hepatitis C virus infection: A retrospective analysis.

Hepatitis C virus (HCV) displays high genetic diversity, characterized by regional variations in the prevalence of genotype posing challenges to the development of vaccines and definitive treatment. Very few reports exist on the distribution and frequency change of HCV genotypes in India. In the present retrospective study, we aimed to understand the distribution pattern of HCV genotypes and viral load among HCV-infected patients attending the Asian Institute of Gastroenterology, Hyderabad, India, a tertiary care hospital. Patients referred to the Hepatology Department from January 2009 to December 2015 were screened for this study. Eight hundred and sixty-two chronic HCV patients were included in this study. Genotyping was performed using type-specific probe-based hybridization assay and viral load was estimated by real-time polymerase chain reaction. Out of 862 patients, genotype 1 was detected predominantly in 392 (45.5%), followed by genotype 3 in 344 (39.9%) patients; genotypes 4, 6, and 2 were detected in 115 (13.3%), 8 (0.9%), and 3 (0.3%) patients, respectively. The number of patients having genotype 1 increased in frequency while genotype 3 became less from the year 2009 to 2015. Patients having genotype 1 had significantly (p < 0.0001) higher viral load compared with the patients infected with other genotypes. Our study results demonstrate a change in HCV genotypic distribution pattern from genotypes 3 to 1 during the span of 7years in patients referred to our hospital. In the light of the reported difference in the pathogenic potential of various HCV genotypes, detection of HCV genotype appears to be still essential for better patient management.

Effect of the bivalent HPV vaccine on viral load of vaccine and non-vaccine HPV types in incident clearing and persistent infections in young Dutch females.

BackgroundHPV vaccination with the bivalent vaccine is efficacious against HPV16 and 18 infections and cross-protection against non-vaccine HPV types has been demonstrated. Here, we assessed (cross-) protective effects of the bivalent HPV16/18 vaccine on incident and persistent infections and viral load (VL) of fifteen HPV types in an observational cohort study monitoring HPV vaccine effects.MethodsVaginal samples were obtained annually. Type-specific VL assays were developed for HPV6,11,31 33,35,39,45,51,52,56,58,59 and 66 and used in addition to existing HPV16 and 18 assays. Rate differences of incident clearing and persistent infections were correlated with differences in VL and vaccination status.ResultsHPV16/18 vaccination resulted in significantly lower incidence of HPV16/18 infections and significantly lower VL in breakthrough HPV16 (p<0.01) and 18 infections (p<0.01). The effects of vaccination on non-vaccine type VL were ambiguous. Incidence and/or persistence rates of HPV31, 33, 35 and 45 were reduced in the vaccinated group. However, no significant type specific VL effects were found against HPV31, 33, 45, 52 in the vaccinated group. For HPV 6, 59 and 66 no significant reductions in numbers of incident and persistent infections were found, however borderline) VL reductions following vaccination were observed for HPV6 (p = 0.01), 59 (p = 0.10) and 66 (p = 0.03), suggesting a minor effect of the vaccine on the VL level of these HPV types. Overall, vaccination resulted in infections with slightly lower VL, irrespective of HPV type.ConclusionsIn conclusion, vaccination with the bivalent HPV16/18 vaccine results in significantly reduced numbers of HPV16 and 18 incidence rates and reduced VL in breakthrough infections. Significant reductions in incident and/or persistent HPV31, 33, 35 and 45 infections were found, but no significant effect was observed on the VL for infections with these types. For the other non-vaccine HPV types no reduction in incident and/or persistent infections were found, but overall the VL tended to be somewhat lower in vaccinated women.

Performance evaluation of four type-specific commercial assays for detection of herpes simplex virus type 1 antibodies in a Middle East and North Africa population

BackgroundThe number of diagnostic assays for the detection of herpes simplex virus type 1 (HSV-1) antibodies has increased over the years. However, their performance characteristics could vary among global populations. ObjectiveTo investigate performance of two commercial ELISA kits, HerpeSelect® 1 ELISA and Euroimmun Anti-HSV-1 (gC1) ELISA (IgG); and two commercial immunoblot (IB)/Western blot (WB) assays, HerpeSelect® 1 and 2 Immunoblot IgG, and Euroimmun Anti-HSV-1/HSV-2 gG2 Euroline-WB (IgG/IgM); in detecting HSV-1 antibodies in a Middle East and North Africa (MENA) population. Study designBlood specimens were collected from blood donors in Doha, Qatar, June 2013–2016. Twenty specimens were randomly selected from 10 MENA nationalities (Egypt, Iran, Jordan, Lebanon, Pakistan, Palestine, Qatar, Sudan, Syria, and Yemen; total = 200), and tested for HSV-1 antibodies. ResultsAcross all six comparisons between assays, positive percent agreement ranged between 95.7% (95% CI: 91.4–98.3%) and 100.0% (95% CI: 97.8–100.0%). Negative percent agreement ranged between 86.2% (95% CI: 68.3-96.1%) and 96.2% (95% CI: 80.4–99.9%). Overall percent agreement ranged between 95.7% (95% CI: 91.7–97.8%) and 99.4% (95% CI: 96.7–99.9%). Cohen’s kappa statistic ranged between 0.84 (95% CI: 0.73–0.95) and 0.98 (95% CI: 0.93–1.00). Compared against IB/WB, HerpeSelect® and Euroimmun had sensitivities and specificities >96% and >86%, respectively. Positive and negative predictive values were >97% and >83%, respectively. ConclusionThe assays showed excellent concordance with one another, and with a high kappa statistic. The ELISA kits demonstrated robust diagnostic performance compared to the IB/WB assays. These findings support the assays’ utility in clinical diagnosis and research in MENA populations.

Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification

BackgroundOropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification.MethodsWe used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification.ResultsOur LAMP assays could detect 105, 103, 104, and 105 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification.ConclusionsThe high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.

Racial/ethnic differences in HPV 16/18 genotypes and integration status among women with a history of cytological abnormalities

ObjectiveHPV genotype distribution varies by race/ethnicity, but is unclear whether there are racial/ethnic variations in HPV 16/18 integration in the host genome. We describe HPV16/18 infection and integration status in a racially/ethnically diverse sample of women with a recent abnormal Pap test. MethodsPatients (n=640) represent a subset of women participating in a clinical trial. Cervical swabs were tested for HPV16/18 DNA using type-specific polymerase chain reaction assays. Viral integration status was assessed using type-specific integration assays and categorized as fully integrated, fully non-integrated, or mixed. Unconditional logistic regression was used to generate unadjusted (OR) and adjusted odds ratios (aOR) to assess the association between self-reported race/ethnicity and risk of these outcomes. ResultsHispanic and non-Hispanic black women had half the odds of prevalent HPV16 compared to non-Hispanic white women (aORs: 0.43 and 0.45, respectively). The prevalence odds of HPV18 was less than half among Hispanic women (aOR: 0.48), but not significantly different between black and white women (aOR: 0.72). Among women with prevalent HPV16, the odds of fully integrated viral DNA were significantly higher among black women (aORs: 2.78) and marginally higher among Hispanic women (aOR: 1.93). No racial/ethnic differences were observed for HPV18 DNA integration. ConclusionsWhile HPV16 and 18 infections were less prevalent among Hispanic and black women compared to whites, their HPV16 DNA was more likely to be present in a fully integrated state. This could potentially contribute to the higher rates of abnormal cytology and cervical dysplasia observed among Hispanic and black women.

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.

The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies.