Chlamydia trachomatis and Neisseria gonorrhoeae rectal infections: Interplay between rectal microbiome, HPV infection and Torquetenovirus.

Men having sex with men (MSM) represent a key population, in which sexually transmitted rectal infections (STIs) caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and high-risk HPV (HR-HPV) are very common and linked to significant morbidity. Investigating the anorectal microbiome associated with rectal STIs holds potential for deeper insights into the pathogenesis of these infections and the development of innovative control strategies. In this study, we explored the interplay at the rectal site between C. trachomatis, N. gonorrhoeae, HR-HPV infection, and the anorectal microbiome in a cohort of 92 MSM (47 infected by CT and/or NG vs 45 controls). Moreover, we assessed the presence of Torquetenovirus (TTV), a non-pathogenic endogenous virus, considered as a possible predictor of immune system activation. We found a high prevalence of HR-HPV rectal infections (61%), especially in subjects with a concurrent CT/NG rectal infection (70.2%) and in people living with HIV (84%). In addition, we observed that TTV was more prevalent in subjects with CT/NG rectal infections than in non-infected ones (70.2% vs 46.7%, respectively). The anorectal microbiome of patients infected by CT and/or NG exhibited a reduction in Escherichia, while the presence of TTV was significantly associated with higher levels of Bacteroides. We observed a positive correlation of HR-HPV types with Escherichia and Corynebacterium, and a negative correlation with the Firmicutes phylum, and with Prevotella, Oscillospira, Sutterella. Our findings shed light on some of the dynamics occurring within the rectal environment involving chlamydial/gonococcal infections, HPV, TTV, and the anorectal microbiome. These data could open new perspectives for the control and prevention of STIs in MSM.

The Auditory Pathway in Congenitally Cytomegalovirus-Infected Human Fetuses.

Congenital cytomegalovirus (CMV) infection is the main cause of non-hereditary sensorineural hearing loss (SNHL). In order to shed light on SNHL pathophysiology, we examined the auditory pathway in CMV-infected fetuses; the temporal lobe, in particular the auditory cortex, and the inner ear. We investigated both inner ears and temporal lobes of 20 human CMV-infected fetuses at 21 weeks of gestation. As a negative group, five fetuses from spontaneous miscarriages without CMV infection were studied. Inner ears and temporal lobes were histologically examined, immunohistochemistry for CMV and CMV-PCR were performed. On the auditory cortex, we evaluated the local microglial reaction to the infection. CMV-positive cells were found in 14/20 brains and the damage was classified as severe, moderate, or mild, according to histological features. Fetuses with severe brain damage had a statistically higher temporal lobe viral load and a higher number of activated microglial cells in the auditory cortex compared to fetuses with mild brain damage (p: 0.01; p: 0.01). In the inner ears, the marginal cells of the stria vascularis were the most CMV positive. In our study, CMV affected the auditory pathway, suggesting a tropism for this route. In addition, in the auditory cortex, microglial activation may favor further tissue damage contributing to hearing loss.

Inherited Chromosomally Integrated Human Herpesvirus 6: Laboratory and Clinical Features.

Inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the chromosomes of the host germ cell and is vertically transmitted. The aims of this study were to identify iciHHV-6 prevalence in hospitalized patients and clinical features in individuals carrying this integration. HHV-6 PCR on hair follicles was used to confirm iciHHV-6 status when the blood viral load was more than 5 Log10 copies/mL. From January 2012 to June 2022, HHV-6 DNAemia was investigated in 2019 patients. In particular, 49 had a viral load higher than 6 Log10 copies/mL and HHV-6 DNA in hair follicles was positive. A viral load between 5.0 and 5.9 Log10 copies/mL was observed in 10 patients: 6 infants with acute HHV-6 infection and 4 patients with leukopenia and HHV-6 integration. Therefore, the iciHHV-6 prevalence in our population was 2.6% (53/2019). Adult patients with integration presented hematological (24%), autoimmune (11%), autoimmune neurological (19%), not-autoimmune neurological (22%), and other diseases (19%), whereas 5% had no clinically relevant disease. Although in our study population a high percentage of iciHHV-6 adult hospitalized patients presented a specific pathology, it is still unknown whether the integration is responsible for, or contributes to, the disease development.

Recent Advances in the Evaluation of Serological Assays for the Diagnosis of SARS-CoV-2 Infection and COVID-19.

Introduction: Few data on the diagnostic performance of serological tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are currently available. We evaluated sensitivity and specificity of five different widely used commercial serological assays for the detection of SARS-CoV-2-specific IgG, IgM, and IgA antibodies using reverse transcriptase-PCR assay in nasopharyngeal swab as reference standard test. Methods: A total of 337 plasma samples collected in the period April-June 2020 from SARS-CoV-2 RT-PCR positive (n = 207) and negative (n = 130) subjects were investigated by one point-of-care lateral flow immunochromatographic assay (LFIA IgG and IgM, Technogenetics) and four fully automated assays: two chemiluminescence immunoassays (CLIA-iFlash IgG and IgM, Shenzhen YHLO Biotech and CLIA-LIAISON® XL IgG, DiaSorin), one electrochemiluminescence immunoassay (ECLIA-Elecsys® total predominant IgG, Roche), and one enzyme-linked immunosorbent assay (ELISA IgA, Euroimmune). Results: The overall sensitivity of all IgG serological assays was >80% and the specificity was >97%. The sensitivity of IgG assays was lower within 2 weeks from the onset of symptoms ranging from 70.8 to 80%. The LFIA and CLIA-iFlash IgM showed an overall low sensitivity of 47.6 and 54.6%, while the specificity was 98.5 and 96.2%, respectively. The ELISA IgA yielded a sensitivity of 84.3% and specificity of 81.7%. However, the ELISA IgA result was indeterminate in 11.7% of cases. Conclusions: IgG serological assays seem to be a reliable tool for the retrospective diagnosis of SARS-CoV-2 infection. IgM assays seem to have a low sensitivity and IgA assay is limited by a substantial rate of indeterminate results.

Time to viral clearance after successful conservative treatment for high-risk HPV-infected high-grade cervical intraepithelial neoplasia and early invasive squamous cervical carcinoma.

Two-thirds of 152 patients treated for high-grade cervical disease, free of persistence/recurrence, and followed-up both with human papillomavirus (HPV) DNA testing and HPV genotyping cleared their high-risk HPV infection within 1year. Viral clearance continued at diminishing rates during the second and the third year, at the end of which it was virtually complete.

Improving Laboratory Efficiency by Automation of Preanalytic Processing of ThinPrep Specimens for Real-Time PCR High-Risk HPV Testing.

Cervical specimens collected in liquid-based cytology (LBC) media are the most common sample type used for high-risk human papillomavirus (HPV) testing. Since preanalytic steps such as vortexing and decapping vials, liquid transfer to a sample input tube with matching unique identifier, and recapping the original vials are required for processing LBC samples prior to running the Abbott RealTime High Risk HPV assay (Abbott, Wiesbaden, Germany), a full manual execution can be complicated, especially in high-throughput diagnostic contexts. Here, a custom-configured worktable setup for the Tecan Freedom EVO (Tecan, Männedorf, Switzerland) designed to automate and control preanalytic steps for ThinPrep (Hologic, Marlborough, MA) samples was used to evaluate the impact of automated versus manual preanalytics. Archival results for manual processing of 226 samples were compared with those obtained with the Tecan protocol, observing a very good overall concordance for final assay interpretation (95.6%). High overall agreement (100%) resulted also from retesting 99 samples by both the preanalytical protocols. High reproducibility was observed analyzing 23 randomly selected samples by automated preprocessing in triplicate. Hence, the new configuration of the Tecan platform translates the manual steps required to process ThinPrep specimens into automated operations, controls sample identification, and allows for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it suitable for screening settings.

The predictive value of human papillomavirus testing for the outcome of patients conservatively treated for stage IA squamous cell cervical carcinoma.

BACKGROUND: Although it is hypothesised that human papillomavirus (HPV) testing may have a role in surveillance of patients conservatively treated for stage IA squamous cell cervical carcinoma, research on this topic has been minimal. OBJECTIVES: To determine: (1) the changes in HPV test result from treatment onward; (2) the time to viral clearance; and (3) the negative predictive value (NPV) and positive predictive value (PPV) of HPV test result for the detection of CIN2 or worse (CIN2+) during follow-up. STUDY DESIGN: In a multicentre retrospective follow-up study of a consecutive series (1997-2009) of 91 patients, longitudinal outcome measures were estimated as cumulative probabilities using the Kaplan-Meier method. RESULTS: For patients testing HPV-positive at the first follow-up visit (n=44), the probability of change to negative rose from 0 to 0.78 between 7 and 21 months after treatment. For HPV-negative patients (n=47), the probability of change to positive rose to 0.13 between 9 and 26 months. After a median follow-up of 50 months (range, 2-80), the NPV for CIN2+ was 1.00. The PPV was 0.60 (95% confidence interval, 0.43-0.77) after 26 months. The median time to detection was 5 months. CONCLUSIONS: If adequately confirmed, these findings would indicate that HPV testing is capable to identify the patients who have had their lesions fully removed, and would make it possible to focus follow-up efforts on a subset of patients at high risk of residual or progressive disease.

High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.

A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.

Performance of HPV DNA testing in the follow-up after treatment of high-grade cervical lesions, adenocarcinoma in situ (AIS) and microinvasive carcinoma.

BACKGROUND: Over the last two decades it has become clear that distinct types of human papillomavirus (HPV), the so-called high-risk types (hrHPV), are the major cause of cervical cancer. The hrHPV-DNA testing has shown excellent performance in several clinical applications from screening to the follow-up of conservatively treated patients. METHODS: We conducted a systematic review of the recent literature on the performance of HPV DNA testing in follow-up after treatment of high-grade cervical lesions, adenocarcinoma in situ, and microinvasive carcinoma compared to Pap smear cytology. RESULTS: Observational studies have demonstrated that the high risk hrHPV-DNA test is significantly more sensitive (95%) compared to follow-up cytology(70%) in detecting post-treatment squamous intraepithelial high-grade lesions. Moreover, in patients treated conservatively for cervical adenocarcinoma in situ, the hrHPV-DNA test is the most significant independent predictor of recurrent disease or progression to invasive cancer, and the combination of viral DNA testing and cytology reaches 90% sensitivity in detecting persistent lesions at the first follow-up visit and 100% at the second follow-up visit. The cause of microinvasive squamous cervical carcinoma is increasingly treated with conservative therapies in order to preserve fertility, and an effective strategy allowing early detection of residual or progressive disease has become more and more important in post-treatment follow-up. Primary results seem to indicate that the median time for viral clearance is relatively longer compared with patients treated for CIN and suggest a prolonged surveillance for these patients. However, the potential clinical value of HPV-DNA testing in this clinical setting needs to be confirmed by further observations. CONCLUSIONS: The excellent sensitivity, negative predictive value, and optimal reproducibility of the hrHPV DNA testing, currently is considered a powerful tool in the clinicians' hands to better manage post-treatment follow-up either in cervical squamous lesion or in situ adenocarcinoma.

Virologic and genetic evaluation of vemurafenib-induced skin cancers.

We describe the occurrence of a giant squamous cell carcinoma in a patient receiving vemurafenib for the treatment of late melanoma mestastases. Although the development of keratoacanthomas and squamous cell carcinomas (SCC) has been described during vemurafenib therapy, most of the reported cases are treated with surgical excision. In the present case, SCC regressed after drug withdrawal.

Detection of HPV16 African variants and quantitative analysis of viral DNA methylation in oropharyngeal squamous cell carcinomas.

BACKGROUND: HPV16 is the most prevalent genotype in oropharyngeal squamous cell carcinomas (OPSCCs), but there are few information about intratype variants prevalence and their clinical relevance in these tumors. Moreover, since HPV DNA detection alone is not sufficient to determine a favorable prognosis for OPSCCs, additional diagnostic/prognostic biomarkers are necessary. OBJECTIVES: To assess the prevalence of HPV intratype variants and evaluate the role of HPV16 DNA methylation as prognostic marker in OPSCCs. STUDY DESIGN: We analyzed 81 biopsies of OPSCCs for HPV detection and genotyping with a pyrosequencing-based HPV DNA-test. We also quantified viral DNA methylation frequency in the LCR by pyrosequencing in patients with transcriptionally-active HPV16-infection, analyzing it in function of available clinical/virological data. RESULTS: The overall HPV prevalence was 74.1% and HPV16 was confirmed the most prevalent genotype (51/60, 85.0%). Interestingly, we detected, for the first time, HPV16 African variants in 10/51 cases (19.6%), pointing out their clinical relevance also out of the anogenital district. Regarding viral DNA methylation, the E2BS1 showed a significantly higher mean methylation frequency compared to E2BS3/4, as previously observed in high-grade precancerous lesions of the uterine cervix. On the other hand, patient with a mean methylation frequency in the early promoter >10% showed only a tendency to have a worse prognosis. CONCLUSIONS: Our results highlight both the involvement of HPV16 African variants in OPSCCs development and the necessity of further studies to deepen the role of viral methylation in these tumors, in order to better define similarities/differences compared to cervical cancer.

Detection of high-risk human papillomavirus type 16 and 18 using isothermal helicase-dependent amplification.

Persistent infection with human papillomavirus (HPV) induces cervical cancer. Here, we describe a sensitive, specific, and rapid assay for high-risk HPV16 and 18 detection by isothermal helicase-dependent amplification. This method can be used as cost-effective diagnostic method for low-income countries, where highest incidences worldwide of cervical cancer are registered.

Keeping pace with parvovirus B19 genetic variability: a multiplex genotype-specific quantitative PCR assay.

Three genotypes have been identified within the parvovirus B19 species (B19V), and such genetic diversity may have significant implications for the development of molecular detection assays. In the present study, B19V genetic variability has been examined on a subset of genomic sequences available in the NCBI nucleotide database, and a quantitative PCR (qPCR) assay able to detect, differentiate, and quantify all viral variants has been established. The designed primers and probes have been used for the development of alternative detection formats, based on a combined use of intercalating dye and genotype-specific hydrolysis probes. The qPCR assay analytical performances have been determined on the 1st WHO International Reference Panel for Parvovirus B19 Genotypes. The developed qPCR protocols allow for the detection of genotypes 1 to 3 with equal accuracy, and with a limit of detection (LOD) of 200 IU/ml. A comparison of routine performance was carried out with respect to a previously established assay specifically validated on B19V genotype 1. For 130 clinical samples analyzed, 126 showed concordant results (31 positive and 97 negative), while 4 showed discordant results. Overall, the genotype-specific qPCR assay showed a sensitivity of 93.94% and a specificity of 97.94%, with an agreement rate of 96.92%. The proposed qPCR assay and the alternative protocols developed, each with robust performance, may allow choice with respect to operational systems and diagnostic requirements and might contribute to provide a more reliable diagnostic service and epidemiological surveillance of B19 virus.

Comparison of HPV sign genotyping test with INNO-LiPA HPV genotyping extra assay on histologic and cytologic cervical specimens.

An accurate tool for viral typing is important for management of patients with human papillomavirus (HPV) infection and to monitor HPV vaccine efficacy. This study evaluated the performance of the HPV sign® Genotyping Test by analyzing 87 archival cervical specimens and compared results with historical data by INNO-LiPA HPV Genotyping Extra assay. There was a substantial concordance for HPV detection in clinical samples (k 0.66), with an overall agreement rate of 85.1%. The genotyping overall agreement, considering one by one the HPV infection detected, was 95.7%. The HPV sign test showed, however, lower sensitivity than INNO-LiPA for HPV 31, 53, and 66. On the other hand, The HPV16 sensitivity was higher for HPV sign (90.0%, confidence interval [CI] 0.79-1.01) than for INNO-LiPA (83.3%, CI 0.70-0.97). Furthermore, HPV sign allowed identifying the presence of HPV16 intratype variants. In conclusion, HPV sign is a promising method for HPV genotyping and has revealed advantages in detecting a broad spectrum of HPV types and variants.

Factors predicting the outcome of conservatively treated adenocarcinoma in situ of the uterine cervix: an analysis of 166 cases.

OBJECTIVE: The present study assessed the clinical outcome of patients conservatively treated for cervical adenocarcinoma in situ (AIS) and their predictive factors using univariate and multivariate population averaged (PA) generalized estimating equation (GEE) model in a longitudinal setting. METHODS: A series of 166 consecutive women (mean age 39.8 yrs; range 23-63 yrs) underwent conservative treatment of AIS as the primary treatment and were followed-up (mean 40.9 mo) using colposcopy, PAP-smear, biopsy and HPV-testing with Hybrid Capture 2. RESULTS: Hysterectomy was performed as part of the primary management in 47 patients, who were excluded from the follow-up (FU) analysis. Out of 119 women closely followed-up, additional therapeutic procedures were performed in 69. At study conclusion, 7 patients (5.9%) showed persistent disease, while 8 (6.7%) had progressed to invasive adenocarcinoma (AC). Positive HR-HPV test was the only independent predictor of disease recurrence (adjusted OR=2.72; 95%CI 1.08-6.87), and together with free cone margins (OR=0.20; 95%CI 0.04-0.92), HR-HPV positivity was also the single most powerful predictor of disease progression to AC, with OR=3.74; 95%CI 1.84-7.61 (p=0.0001) in multivariate PA-GEE. CONCLUSIONS: These results suggest that testing HR-HPV positive at any time point during FU is the most significant independent predictor of progressive disease, while showing free margins in cone has a significant protective effect against progression to AC. Furthermore, because 4.3% women with persistent, recurrent or progressive disease experienced a late (5th and 6th FU) diagnosis of HG-CGIN or microinvasive AC, a close surveillance should be scheduled for at least three years in conservatively treated AIS patients.

Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting.

BACKGROUND: The Abbott RealTime High Risk HPV assay (ART) is an automated multiplex real-time PCR test for detection of DNA from 14 high risk (HR) HPV types in cervical specimens and simultaneous distinction of HPV16 and HPV18 from other HR-HPV. OBJECTIVES: To evaluate the performance of the ART assay in specimens referred for HPV testing to our laboratory (referral population) by comparison with historical data from HC2 and INNO-LiPA as well as histological status, if available. STUDY DESIGN: 412 cervical specimens were collected from women between 18 and 70 years of age: 301 previously tested by HC2 without clinical data and 111 previously tested by HC2 and INNO-LiPA with histological diagnosis of CIN3+. RESULTS: Our study demonstrated good overall agreement between ART, HC2 and INNO-LiPA. In the group of the CIN3+ specimens HR-HPV was detected by ART in 93.07% (95% CI: 88.12-98.02), while HR-HPV detection rates with HC2 and INNO-LiPA were 91.09% (95% CI: 85.53-96.65) and 95.05% (95% CI: 90.82-99.28), respectively. The typing capability of ART for HPV16, HPV18 and a pool of twelve other HR-HPV types was investigated by comparison with INNO-LiPA demonstrating high overall assay concordance (89.81%; k 0.87). CONCLUSIONS: The Abbott RealTime assay showed similar clinical performance for detection of CIN3+ compared with HC2. The high level of automation and ability to identify HPV16, HPV18 and other HR-HPV make this assay a very attractive option for HR-HPV testing, potentially improving patient management by risk stratification of cytological abnormal populations.

High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups.

BACKGROUND: Human papillomavirus (HPV) infection is a necessary event in the development of cervical carcinoma. High risk (HR) HPV genotypes, however, may progress differentially from low grade lesions to malignancy. OBJECTIVES: The necessity to genotype and quantify HPV-DNA in cervical screening programs, in the follow up post-surgical treatments and in monitoring the effectiveness of HPV vaccination programs, requires access to economical, high-throughput and flexible molecular technologies. STUDY DESIGN: A high-throughput two-step LNA real time PCR assay was developed consisting of real time PCR reactions with fluorescent Locked Nucleic Acid (LNA) probes. The first step permits classification into three prognostic-risk groups of nine HR HPV genotypes (16, 18, 31, 33, 35, 45, 52, 56 and 58) most frequently found associated with cervical lesions in Europe. The second step allows us to genotype/quantify the HPV-DNA only when clinical, epidemiological or prophylactic aims exist. RESULTS: The specificity, repeatability, detection and quantitation limit, and linearity of the assay were evaluated and appear to be in agreement with guidelines for the validation of analytical procedures. The overall genotype concordance on cervical samples between our assay and INNOLiPA test was 94% (k 0.83) indicating good agreement. CONCLUSIONS: The two-step PCR assay can give much information relative to the predictive value of different HR HPV types and can quantify the genotype-specific viral load. In particular, its ability to detect and quantify nine HR HPV genotypes can help provide more efficient and successful patient care and may be useful for the monitoring of the efficacy of HPV vaccines.

Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion.

The presence of HPV 16 E6*I/E6*II spliced transcripts, in cervical lesions of different grade, was analyzed to characterize the transcription pattern. The presence and amount of spliced transcripts were correlated with DNA viral markers such as E2/E6 ratio and physical state. The detection of HPV 16 E6*I/E6*II mRNAs was set up by an SYBR Green real-time reverse transcriptase PCR assay with an optimal dynamic range and sensitivity. The assay was applied to the analysis of 71 specimens, positive to HPV 16 as a sole infection, from women with abnormal cervical smears, precisely 31 low-grade squamous intraepithelial lesions and 40 high-grade lesions. Samples negative to both transcripts were found only in low-grade cervical lesions. Three different transcription profiles were found in the low- and high-grade lesions analyzed: in low-grade lesions samples positive only to E6*II and in high-grade lesions samples positive only to E6*I were detected. In low- and high-grade lesions, samples positive to both E6*I and E6*II were found. In the samples positive for both transcripts, the E6*I/E6*II ratio was higher than that in the majority of high-grade lesions and lower than that in all the low-grade lesions. Analyzing the transcription pattern in relation to E2/E6 ratio and to the DNA physical state, the presence of high values of E6*I was associated mainly with low values of E2/E6 ratio and of mixed DNA forms. The detection of HPV 16 E6*I/E6*II mRNAs may serve to identify transcription patterns indicative of cervical disease progression and help physicians to decide clinical management.