Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia.

INTRODUCTION: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. METHODS: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. RESULTS: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. DISCUSSION: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients.

Clinical validation of full HR-HPV genotyping HPV Selfy assay according to the international guidelines for HPV test requirements for cervical cancer screening on clinician-collected and self-collected samples.

BACKGROUND: According to international guidelines, Human Papillomavirus (HPV) DNA tests represent a valid alternative to Pap Test for primary cervical cancer screening, provided that they guarantee balanced clinical sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or more (CIN2+) lesions. The study aimed to assess whether HPV Selfy (Ulisse BioMed - Trieste, Italy), a full-genotyping HPV DNA test that detects and differentiates 14 high-risk HPV (HR-HPV) types, meets the criteria for primary cervical cancer screening described in the international guidelines, on clinician-collected as well as on self-collected samples. METHODS: For each participant woman, consecutively referring to Azienda Sanitaria Universitaria Giuliano Isontina (Trieste, Italy) and CRO-National Cancer Institute (Aviano, Italy) for the cervical cancer screening program, the following samples were tested: (a) a clinician-collected cervical specimen, analyzed with the reference test (Hybrid Capture®2 test, HC2) and HPV Selfy; and (b) a self-collected vaginal sample, analyzed with HPV Selfy. Enrolled women were also asked to fulfill a questionnaire about self-sampling acceptability. As required by guidelines, a non-inferiority test was conducted to compare the clinical performance of the test under evaluation with its reference test. RESULTS: HPV Selfy clinical sensitivity and specificity resulted non-inferior to those of HC2. By analysis of a total of 889 cervical liquid-based cytology samples from a screening population, of which 98 were from women with CIN2+, HPV Selfy showed relative sensitivity and specificity for CIN2+ of 0.98 and 1.00 respectively (non-inferiority score test: P = 0.01747 and P = 0.00414, respectively); the test reached adequate intra- and inter-laboratory reproducibility. Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen (0.92 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population. CONCLUSIONS: HPV Selfy fulfills all the requirements of the international Meijer's guidelines and has been clinically validated for primary cervical cancer screening purposes. Moreover, HPV Selfy has also been validated for self-sampling according to VALHUDES guidelines. Therefore, at date, HPV Selfy is the only full-genotyping test validated both for screening purposes and for self-sampling. Trial registration ASUGI Trieste n. 16008/2018; CRO Aviano n.17149/2018.

Noninvasive Determination of CMV Serostatus From Dried Buccal Swab Samples: Assay Development, Validation, and Application to 1.2 Million Samples.

BACKGROUND: Buccal swab sampling constitutes an attractive noninvasive alternative to blood drawings for antibody serostatus assays. Here we describe a method to determine the cytomegalovirus immunoglobulin G (CMV IgG) serostatus from dried buccal swab samples. METHODS: Upon solubilization, CMV IgG is determined by an ELISA assay specifically adapted to cope with low IgG concentrations. The derived CMV titer is normalized against the total protein concentration to adjust for incorrectly or less efficiently sampled buccal swabs. Assay parameters were optimized on a set of 713 samples. RESULTS: Validation with 1784 samples revealed distinct results for > 80% of samples with 98.6% specificity and 99.1% sensitivity. Based on the analysis of 1.2 million samples we derived age- and sex-stratified CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile. To confirm accuracy of the assay in routine operation, the CMV status of 6518 donors was reassessed by independent laboratories based on conventional blood samples revealing 96.9% specificity and 97.4% sensitivity. CONCLUSIONS: The assay accurately delivers the CMV IgG serostatus from dried buccal swab samples for > 80% of the participants. Thereby it provides a noninvasive alternative to plasma-based CMV monitoring for nondiagnostic purposes such as hematopoietic stem cell transplantation donor screening or population studies.

Evaluation of self-sampling-based cervical cancer screening strategy using HPV Selfy CE-IVD test coupled with home-collection kit: a clinical study in Italy.

BACKGROUND: Primary human papillomaviruses (HPV) cervical cancer screening can be strengthened by offering home-collection of biological specimen as a valuable option to increase screening coverage. As recommended by World Health Organization (WHO), screening programs should consider whether the inclusion of HPV self-sampling as a complementary option within their existing screening algorithms could address the gaps in current coverage. However, few HPV screening tests are validated for self-sampling according to international guidelines. This study aimed to test a self-sampling-based screening strategy, complementary to the main screening program based on clinician-collected cervical samples. The study took place in Trieste, Italy, and it aimed to evaluate the feasibility of self-testing at home under an opt-in system during COVID-19 pandemic in order to exploit self-sampling to reduce the screening delay generated by the lockdown. METHODS: 500 women, who should have received the screening call in 2020, were asked, via phone call, to participate in the study. To whom agreed, a home-collection kit, including a vaginal dry swab for specimen collection, was sent. The recipients performed the sample self-collection and sent back the swab through traditional mail using a prepaid envelope. Once received by the hospital, the samples were analyzed with HPV Selfy (Ulisse BioMed, Italy), a CE-IVD HPV screening test specifically validated for self-collection. Results were further compared using cobas® 4800 HPV (Roche, Switzerland). RESULTS: 80% women sent back their swab, showing one of the highest return rate obtained in comparable studies. 34 HPV-positive women were followed up and underwent the Pap test, that revealed 8 low squamous intraepithelial lesions (LSIL) cases, later triaged to colposcopy. HPV Selfy was confirmed to be an adequate test for self-sampling-based screening. CONCLUSIONS: This study further confirmed the feasibility of self-test at home screening strategy based on self-sampling with an opt-in system as a support method to enhance cervical cancer screening coverage in Italy. Enrolled women showed a high appreciation for this approach. HPV Selfy test demonstrated to be a valuable assay for cervical cancer screening based on home self-collection. TRIAL REGISTRATION: ASUGI Trieste n. 16008/2018 and amendment 02-11/09/2020.

Preliminary Results of Feasibility and Acceptability of Self-Collection for Cervical Screening in Italian Women.

BACKGROUND: Given the diagnostic accuracy of HPV-DNA tests in terms of self-collected samples, in order to implement self-sampling in cervical screening programs, the standardization of the pre-analytical phase, including decisions concerning the choice of medium, the volume of elution, and storage conditions, are necessary, in addition to understanding the potential factors involved in acceptability by women. On this basis, we carried out a cross-sectional study to assess (i) the stability of dry vaginal self-collected samples stored at room temperature for up to 4 weeks after elution in 2 mL of eNat® (Copan) medium, and (ii) the acceptability of self-collection in enrolled women. METHODS: 185 women were enrolled in the LILT (Italian League Against Tumors) regional project. A self-sampling kit, including a dry FLOQSwab® (Copan), instructions for use, and a satisfaction questionnaire, were supplied for each woman and sent by mail to the laboratory. The HPV-DNA test was carried out using the Anyplex™ II HPV HR (Seegene) kit. To evaluate the specimen's stability, 185 dry vaginal swabs were eluted in eNat®, a lyses-based molecular medium and tested for HPV detection at two different time points (<6 days and 1 month after elution). The Cohen's Kappa coefficients and McNemar test were used to assess the agreement of HPV-DNA at different times. RESULTS: We found high agreement in terms of HPV-DNA results among the samples tested at two different time points (Cohen K = 0.98; p < 0.0001). Moreover, most of the women found it easy to use self-collection devices and the pictorial instructions clear to understand. Approximately half of the enrolled women declared preferring self-sampling to clinician-collected methods. CONCLUSION: Our results display the high reliability and accuracy of HPV-DNA tests using dry vaginal self-collection FLOQSwabs® devices eluted in 2 mL of molecular medium. The analysis of the questionnaire showed a high acceptability of self-collection among women, although a high percentage preferred standard collection devices. Overall, our preliminary results support the adoption of self-collection in screening programs, even though further analyses should be performed to optimize and standardize protocols for HPV tests on self-samples, and educational campaigns are needed to adequately inform and increase responsiveness in a target population.

Comparison of Different Self-Sampling Devices for Molecular Detection of Human Papillomavirus (HPV) and Other Sexually Transmitted Infections (STIs): A Pilot Study.

BACKGROUND: Cervical cancer is the fourth most common cancer in women, and it is well known that high-risk human papillomavirus (hrHPV) infections are the necessary carcinogenic factors for the development of cervical tumors. Moreover, the interaction between HPV and other sexually transmitted infections (STIs) may increase the risk of cancer progression. Self-sampling has been demonstrated to represent a valid and well-accepted alternative, favoring women's participation in screening programs. This study aimed to investigate the use of FLOQSwabs® (FS) as compared to two other vaginal self-collection devices for the detection of hrHPV and other sexually transmitted infections. METHODS: Cervical and vaginal self-samples were collected, using two different combinations of vaginal self-sampling devices, from 40 women referred to colposcopy for a documented abnormal Pap smear. All samples were tested for hrHPV and seven STI pathogens using two commercial molecular assays. RESULTS: Data on hrHPV detection from the first group of women showed an almost perfect agreement (kappa: 0.89) between cervical vs. FS vaginal self-samples, and a substantial agreement (kappa: 0.79) between cervical and HerSwab™ (HS) samples. In the second group of women, an almost perfect agreement (kappa: 0.90) was demonstrated in the detection of hrHPV between cervical samples vs. FS, and a moderate agreement (kappa: 0.60) for cervical vs. Evalyn®Brush (EB) self-collected samples. STI detections showed a very good agreement (kappa: 0.89 and kappa: 1.00) both among FS vs. HS and FS vs. EB, respectively. There was no statistically significant difference between the different devices used. The most frequently detected hrHPV genotypes in the studied population were HPV 16, 31, 35, 51, and 56; whilst the most frequently identified STI pathogens were Ureaplasma parvum and Mycoplasma hominis. Overall, investigated women did not report any discomfort in using the different vaginal self-collection devices. CONCLUSION: Evaluation of the three different vaginal self-collection devices confirmed their overall good acceptability by the studied population, as well as a similar agreement for hrHPV detection as compared to cervical samples. Our study indicated that the use of self-collected samples offers an alternative strategy to improve women's participation in cervical cancer screening programs, but also underlined the importance of evaluating the concordance in hrHPV detection of collection devices in combination with the molecular hrHPV assay.

Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNat® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs.

Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt® (HOLOGIC®, USA) medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries. This pilot study aimed to evaluate the analytical performance of eNat ® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 (Seegene, Korea), Papilloplex® High Risk HPV (GeneFirst, UK), and HPV OncoPredict (Hiantis, Italy). 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 µL using Microlab Nimbus platform (Seegene, Korea) and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman. hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses.

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery.

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids' stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2-8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2-8 °C was 83-100% and 75-95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Efficacy of Unsupervised Self-Collected Mid-Turbinate FLOQSwabs for the Diagnosis of Coronavirus Disease 2019 (COVID-19).

CONTEXT: The Global Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has resulted in explosive patterns of transmission in most countries. Nasopharyngeal swabs were the specimen's collection tools recommended for the diagnosis of SARS-CoV-2 infection, and for monitoring infection outbreaks in communities. Our objective was to report the quality and efficacy of unsupervised self-collected mid turbinate "dry FLOQSwabs" (MT FLOQSwabs) (56380CS01, Copan). There were 111 specimens collected for the study: 36 by health care personnel, from themselves, to verify the quality and efficacy of mid-turbinate swabs; 75 to compare and assess the diagnostic performance, among health care personnel, of nasopharyngeal swabs and self-collected mid-turbinate FLOQSwabs. A collection of 51 specimens was enrolled to define the efficacy of the Testami program (validation). Our analyses demonstrate that self-collected mid-turbinate dry swabs ensure an accuracy of 97.3%, as compared to the standard nasopharyngeal swabs collected by health care workers. Furthermore, the mid-turbinate FLOQSwabs can be stored without medium for six days at room temperature without affecting the molecular diagnosis of the SARS-CoV-2 virus infection. Self-collection of diagnostic specimens at home could offer an avenue to increase testing availability for SARS-CoV-2 infection without asking people to travel to a clinic or a laboratory, thus reducing people's exposure to infection. Our findings demonstrate that unsupervised self-collection swabs, transported dry, are sensitive, practical and easy-to-use tools and should be considered for diagnosis of SARS-COV-2 and coronavirus disease 2019 (COVID-19) surveillance.

Development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing.

We developed and evaluated flocked nasal midturbinate swabs obtained from 55 asymptomatic and 108 symptomatic volunteers. Self-collected swabs obtained from asymptomatic volunteers yielded numbers of respiratory epithelial cells comparable to those of staff-collected nasal (n = 55) or nasopharyngeal (n = 20) swabs. Specific viruses were detected in swabs self-collected by 42/108 (38.9%) symptomatic volunteers by multiplex PCR.

Non-invasive detection of IgG antibodies from common pathogenic viruses using oral flocked swabs.

Salivary antibodies are useful in surveillance and vaccination studies. However, low antibody levels and degradation by endonucleases are problematic. Oral flocked swabs are a potential non-invasive alternative for detecting viral antibodies. Seroprevalence for Cytomegalovirus (CMV), Varicella-Zoster virus (VZV), Epstein-Barr virus (EBV), Measles and Mumps IgG antibodies were determined from 50 matched serum, saliva and swabs samples from healthy volunteers using commercial ELISAs. CMV IgG, VZV IgG, and EBV EBNA-1 IgG, VCA IgG, and Measles IgG swab versus serum sensitivities were 95.8%, 96.0%, 92.1%, 95.5%, 84.5%, respectively, and swabs correlated well with saliva. Sensitivity of Mumps IgG in swabs and saliva was poor at 60.5%, and 68.2%, respectively. Specificities for IgG antibodies were 100% for CMV, EBV and Mumps, but could not be determined for VZV and Measles due to exclusively seropositive volunteers. Except for Mumps IgG, swabs correlate well with serum, are easy to self-collect and are stable at room temperature.

Comparison of the RIDASCREEN norovirus enzyme immunoassay to IDEIA NLV GI/GII by testing stools also assayed by RT-PCR and electron microscopy.

RIDASCREEN norovirus enzyme immunoassay (EIA) detected 80.3% of norovirus-infected feces samples compared to 60.6% by IDEIA NLV GI/GII from 228 patients with no false positives by either assay. RT-PCR and electron microscopy percent sensitivity and specificity were 98.5, 100 and 36.4 and 96.9, respectively.

Diagnosis of Human Prion Disease Using Real-Time Quaking-Induced Conversion Testing of Olfactory Mucosa and Cerebrospinal Fluid Samples.

Importance: Early and accurate in vivo diagnosis of Creutzfeldt-Jakob disease (CJD) is necessary for quickly distinguishing treatable from untreatable rapidly progressive dementias and for future therapeutic trials. This early diagnosis is becoming possible using the real-time quaking-induced conversion (RT-QuIC) seeding assay, which detects minute amounts of the disease-specific pathologic prion protein in cerebrospinal fluid (CSF) or olfactory mucosa (OM) samples. Objective: To develop an algorithm for accurate and early diagnosis of CJD by using the RT-QuIC assay on CSF samples, OM samples, or both. Design, Setting, and Participants: In this case-control study, samples of CSF and OM were collected from 86 patients with a clinical diagnosis of probable (n = 51), possible (n = 24), or suspected (n = 11) CJD and 104 negative control samples (54 CSF and 50 OM). The CSF and OM samples were analyzed using conventional RT-QuIC. The CSF samples underwent further testing using improved RT-QuIC conditions. In addition, the diagnostic performance of a novel, easy-to-use, gentle flocked swab for sampling of OM was evaluated. Data were collected from January 1 to June 30, 2015. Main Outcome and Measures: Correlations between RT-QuIC results and the final diagnosis of recruited patients. Results: Among the 86 patients (37 men [43%] and 49 women [57%]; mean [SD] age, 65.7 [11.5] years) included for analysis, all 61 patients with sporadic CJD had positive RT-QuIC findings using OM or CSF samples or both for an overall RT-QuIC diagnostic sensitivity of 100% (95% CI, 93%-100%). All patients with a final diagnosis of non-prion disease (71 CSF and 67 OM samples) had negative RT-QuIC findings for 100% specificity (95% CI, 94%-100%). Of 8 symptomatic patients with various mutations causing CJD or Gerstmann-Sträussler-Scheinker syndrome, 6 had positive and 2 had negative RT-QuIC findings for a sensitivity of 75% (95% CI, 36%-96%). Conclusions and Relevance: A proposed diagnostic algorithm for sporadic CJD combines CSF and OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease.

Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients.

Significantly more epithelial cells were collected by flocked swabs than by rayon swabs in parallel nasopharyngeal and nasal swabs taken from 16 volunteers. Nasopharyngeal sampling of 61 symptomatic patients also yielded more cells by flocked than rayon swabs, providing better clinical specimens for diagnosis.

Use of flocked swabs and a universal transport medium to enhance molecular detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

The use of new flocked swabs, compared to kit swabs, enhanced the ability of three commercial nucleic acid amplification tests to detect low levels of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids when the organisms were diluted in a universal transport medium as mocked specimens.