Factors Associated with SARS-CoV-2 Infection Evaluated by Antibody Response in a Sample of Workers from the Emilia-Romagna Region, Northern Italy.

Factors associated with SARS-CoV-2 infection risk are still debated. This case-control study aims to investigate the possible relationship between SARS-CoV-2 infection, evaluated through antibody response, and the main sociodemographic, occupational, clinical-anamnestic, and biochemical factors in a population of Modena province (Northern Italy), mainly workers. Both workers who voluntarily joined the screening campaign proposed by companies and self-referred individuals who underwent serological testing were enrolled. Subjects with antibody positivity were recruited as cases (n = 166) and subjects tested negative (n = 239) as controls. A questionnaire on sociodemographic, occupational, and clinical data was administered through telephone interviews. Serum zinc/iron/copper/chromium/nickel, vitamins D/B12, folates, triglycerides, and LDL/HDL/total cholesterol were measured. Cases lived more often in urban areas (61.8% vs. 57%). Cases and controls did not differ significantly by working macrocategories, but the percentage of workers in the ceramic sector was higher among cases. Low adherence to preventive measures in the workplace was more frequent among seropositives. Folate concentration was significantly lower among cases. Therefore, adequate folate levels, living in rural areas, and good adherence to preventive strategies seem protective against infection. Workers in the ceramic sector seem to be at greater risk; specific factors involved are not defined, but preventive interventions are needed.

Performance of a Novel Real-Time PCR-Based Assay for Rapid Monkeypox Virus Detection in Human Samples.

The ongoing epidemic of mpox, namely human monkeypox virus (MPXV) infection, requires rapid and reliable laboratory diagnosis. We report on the QIAstat-Dx viral vesicular panel PCR assay that allows the detection of (within 75 min) six vesicular disease-causing viruses, including MPXV. We analyzed 168 clinical samples, known to be positive (51 samples) or negative (117 samples) for MPXV clade II, obtained from patients at their mpox diagnosis or follow-up. QIAstat assay results were compared to those of a MPXV-specific reference PCR assay. The QIAstat assay detected MPXV (clade II) in 51 (100%) of 51 samples and did not detect MPXV in 117 (100%) of 117 samples, resulting in a positive or negative agreement of 100% (95% CI, 93.0-100) and 100% (95% CI, 96.8-100), respectively. Of the 20 patients diagnosed with mpox, 18 (90.0%) had at least a vesicular swab and 1 (5.0%) had only an oropharyngeal swab positive for MPXV. At mpox follow-ups, 2 (10.0%) of 20 patients had first-time positive whole blood samples. Thirteen MPXV-negative samples were positive for mpox-mimicking viruses. Our findings show the excellent performance of the QIAstat-Dx assay for MPXV detection in clinical samples. Further studies are needed before considering a large-scale application of the QIAstat-Dx assay.

Human papillomavirus independent status on pathologic response and outcomes in locally advanced cervical cancer managed with chemoradiotherapy followed by surgery.

OBJECTIVE: While human papillomavirus (HPV) has been shown to play a significant role in cervical cancer carcinogenesis (HPV associated cases), a considerable percentage of cervical cancers occur independently of HPV status (HPV independent). METHODS: In this retrospective study of 254 locally advanced cervical cancer patients treated with chemoradiotherapy and radical surgery, HPV genotypes were determined using the Anyplex II HPV28 kit that uses multiplex, real time polymerase chain reaction technology. The primary endpoints of this study were to evaluate the complete response to chemoradiotherapy (pathologic complete response), the presence of microscopic (<3 mm, pathologic micro partial response, group 1) and macroscopic (>3 mm, pathologic macro partial response, group 2) residual carcinoma in the cervix, and the persistence of metastatic lymph nodes (group 3) in HPV independent cervical cancers. Secondary endpoints were evaluation of disease-free survival and overall survival. RESULTS: Of 254 patients studied, 21 cases (8.3%) of cervical cancer were determined to be HPV independent. The percentage of pathologic complete response was found to be higher in the HPV associated group compared with the HPV independent group (p<0.001). In the HPV associated cervical cancer group, 5 year disease free survival was found to be 80.8% versus 59.9% in the HPV independent group (p=0.014). Overall survival was also higher in the HPV associated group (87.9%) compared with the HPV independent patients (69.4%) (p=0.023). In the multivariate analysis, the International Federation of Gynecology and Obstetrics (FIGO) stage and HPV genotypes maintained their relevant impact on pathologic complete response to chemoradiotherapy: FIGO stages IIIC1 and IIIC2 were associated with a 13-fold increased risk for the presence of metastatic lymph nodes compared with group 1 (p<0.001). HPV independent cervical cancers showed the highest risk for the development of macroscopic/stable disease (p=0.007), and persistence of metastatic lymph nodes (p=0.004) versus group 1, respectively. CONCLUSIONS: This study showed that HPV status at diagnosis could be a relevant factor for clinical outcomes in locally advanced cervical cancer patients.

Effect of Lockdowns on Hospital Staff in a COVID Center: A Retrospective Observational Study.

At the onset of the SARS-CoV-2 pandemic, individual and social measures were strengthened through restrictive non-pharmaceutical interventions, labelled with the term "lockdown". In Italy, there were two lockdowns (9 March 2020−3 May 2020 and 3 November 2020−27 March 2021). As part of preventive measures, healthcare workers and the administrative staff population of Policlinico A. Gemelli underwent nasopharyngeal swab tests from 1 March 2020 to 9 February 2022, a long time interval that includes the two aforementioned lockdowns. The population included 8958 people from 1 March 2020 to 31 December 2020; 8981 people from 1 January 2021 to 31 December 2021; and 8981 people from 1 January 2022 to 9 February 2022. We then analysed pseudo-anonymized data, using a retrospective observational approach to evaluate the impact of the lockdown on the incidence of SARS-CoV-2 infections within the population. Given the 14 day contagious period, the swab positivity rate (SPR) among the staff decreased significantly at the end of the first lockdown, every day prior to 18 May 2020, by 0.093 (p < 0.0001, CI = (−0.138−−0.047)). After the fourteenth day post the end of the first lockdown (18 May 2020), the SPR increased daily at a rate of 0.024 (p < 0.0001, 95% CI = (0.013−0.034)). In addition, the SPR appeared to increase significantly every day prior to 17 November 2020 by 0.024 (p < 0.0001, CI = (0.013−0.034)). After the fourteenth day post the start of the second lockdown (17 November 2020), the SPR decreased daily at a rate of 0.039 (p < 0.0001, 95% CI = (−0.050−−0.027)). These data demonstrate that, in our Institution, the lockdowns helped to both protect healthcare workers and maintain adequate standards of care for COVID and non-COVID patients for the duration of the state of emergency in Italy.

The Oncobiome in Gastroenteric and Genitourinary Cancers.

Early evidence suggests a strong association of microorganisms with several human cancers, and great efforts have been made to understand the pathophysiology underlying microbial carcinogenesis. Bacterial dysbiosis causes epithelial barrier failure, immune dysregulation and/or genotoxicity and, consequently, creates a tumor-permissive microenvironment. The majority of the bacteria in our body reside in the gastrointestinal tract, known as gut microbiota, which represents a complex and delicate ecosystem. Gut microbes can reach the pancreas, stomach and colon via the bloodstream. Oral bacterial translocations can also occur. In the stomach, pancreas and colon, low microbial diversity is associated with cancer, in particular with a bad prognosis. The urogenital tract also harbors unique microbiota, distinct from the gut microbiota, which might have a role in the urinary and female/male reproductive cancers' pathogenesis. In healthy women, the majority of bacteria reside in the vagina and cervix and unlike other mucosal sites, the vaginal microbiota exhibits low microbial diversity. Genital dysbiosis might have an active role in the development and/or progression of gynecological malignancies through mechanisms including modulation of oestrogen metabolism. Urinary dysbiosis may influence the pathogenesis of bladder cancer and prostate cancer in males. Modulation of the microbiome via pre, pro and postbiotics, fecal or vaginal microbiota transplantation and engineering bacteria might prove useful in improving cancer treatment response and quality of life. Elucidating the complex host-microbiome interactions will result in prevention and therapeutic efficacy interventions.

Investigation of Possible Factors Influencing the Neutralizing Anti-SARS-CoV-2 Antibody Titer after Six Months from the Second Vaccination Dose in a Sample of Italian Nursing Home Personnel.

The titer of the anti-SARS-CoV-2 antibodies produced after vaccination shows a relevant decay over time, as demonstrated in several studies. However, less is known on the possible factors affecting the entity of this decay. The aim of this study is to analyze a group of individual factors which are possibly associated with anti-SARS-CoV-2 antibody titer decay six months after the second vaccine dose. We report here the results of a follow-up serological analysis and a questionnaire-based evaluation of a sample of workers from an Italian nursing home, vaccinated with two doses of BNT162b2 vaccine in early 2021. The baseline data were collected one month after the vaccine, while in the present analysis we report the data collected six months later. Our data show a relevant decay of the neutralizing antibody titer, even if for all the workers a largely positive response was detected. Moreover, our results demonstrate a possible association between younger age and the absence of previous COVID-19 infection, and a higher decay rate of the anti-SARS-CoV-2 antibodies titer.

Rapid Detection of the Omicron (B.1.1.529) SARS-CoV-2 Variant Using a COVID-19 Diagnostic PCR Assay.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the last variant of concern (VOC) identified to date. Compared to whole-genome or gene-specific sequencing methods, reverse-transcription PCR assays may be a simpler approach to study VOCs. We used a point-of-care COVID-19 diagnostic PCR assay to detect the Omicron SARS-CoV-2 variant in the respiratory tract samples of COVID-19 patients who had tested positive for SARS-CoV-2 RNA between April 2021 and January 2022. Sequencing analyses had shown that 87 samples were positive for the Omicron variant and 43 samples were positive for a non-Omicron variant (Delta, 18 samples; Alpha, 13 samples; Gamma, 10 samples; Beta, 1 sample; or Epsilon, 1 sample). According to results by the PCR assay, whose primers anneal a nucleocapsid (N) gene region that comprises the E31/R32/S33 deletion (also termed the del31/33 mutation), we found that N gene target failure/dropout (i.e., a negative/low result) occurred in 86 (98.8%) of 87 Omicron variant-positive samples tested. These results were assessed in relation to those of the spike (S) gene, which expectedly, was detected in all (100%) 130 samples. A total of 43 (100%) of 43 Delta, Alpha, Gamma, Beta, or Epsilon variant-positive samples had a positive result with the N gene. Importantly, in 86 of 87 Omicron variant-positive samples, the del31/33 mutation was detected together with a P13L mutation, which was, instead, detected alone in the Omicron variant-positive sample that had a positive N-gene result. IMPORTANCE Rapid detection of the Omicron SARS-CoV-2 variant in patients' respiratory tract samples may influence therapeutic choices, because this variant is known to escape from certain monoclonal antibodies. Our findings strengthen the importance of manufacturers' efforts to improve the existing COVID-19 diagnostic PCR assays and/or to develop novel variant-specific PCR assays. Furthermore, our findings show that only a small fraction of SARS-CoV-2-positive samples may require whole-genome sequencing analysis, which is still crucial to validate PCR assay results. We acknowledge that the emergence of novel variants containing mutations outside the PCR assay target region could, however, allow an assay to work as per specifications without being able to identify a SARS-CoV-2-positive sample as a variant. Future work and more experience in this topic will help to reduce the risk of misidentification of SARS-CoV-2 variants that is unavoidable when using the current PCR assays.

SARS-CoV-2 Antigen Test Results to Infer Active or Non-Active Virus Replication Status in COVID-19 Patients.

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2-7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16-30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2.

Haemophilus parasuis (Glaesserella parasuis) as a Potential Driver of Molecular Mimicry and Inflammation in Rheumatoid Arthritis.

Background: Haemophilus parasuis (Hps; now Glaesserella parasuis) is an infectious agent that causes severe arthritis in swines and shares sequence similarity with residues 261-273 of collagen type 2 (Coll261-273), a possible autoantigen in rheumatoid arthritis (RA). Objectives/methods: We tested the presence of Hps sequencing 16S ribosomal RNA in crevicular fluid, synovial fluids, and tissues in patients with arthritis (RA and other peripheral arthritides) and in healthy controls. Moreover, we examined the cross-recognition of Hps by Coll261-273-specific T cells in HLA-DRB1*04pos RA patients, by T-cell receptor (TCR) beta chain spectratyping and T-cell phenotyping. Results: Hps DNA was present in 57.4% of the tooth crevicular fluids of RA patients and in 31.6% of controls. Anti-Hps IgM and IgG titers were detectable and correlated with disease duration and the age of the patients. Peripheral blood mononuclear cells (PBMCs) were stimulated with Hps virulence-associated trimeric autotransporter peptide (VtaA10755-766), homologous to human Coll261-273 or co-cultured with live Hps. In both conditions, the expanded TCR repertoire overlapped with Coll261-273 and led to the production of IL-17. Discussion: We show that the DNA of an infectious agent (Hps), not previously described as pathogen in humans, is present in most patients with RA and that an Hps peptide is able to activate T cells specific for Coll261-273, likely inducing or maintaining a molecular mimicry mechanism. Conclusion: The cross-reactivity between VtaA10755-766 of a non-human infectious agent and human Coll261-273 suggests an involvement in the pathogenesis of RA. This mechanism appears emphasized in predisposed individuals, such as patients with shared epitope.

Neutralizing Anti-SARS-CoV-2 Antibody Titer and Reported Adverse Effects, in a Sample of Italian Nursing Home Personnel after Two Doses of the BNT162b2 Vaccine Administered Four Weeks Apart.

BACKGROUND: The immunization of healthcare workers (HCWs) plays a recognized key role in prevention in the COVID-19 pandemic: in Italy, the vaccination campaign began at the end of December 2020. A better knowledge of the on-field immune response in HCWs, of adverse effects and of the main factors involved is fundamental. METHODS: We performed a study on workers at a nursing home in Northern Italy, vaccinated in January-February 2021 with two doses of the BNT162b2 vaccine four weeks apart, instead of the three weeks provided for in the original manufacturer protocol. One month after the second dose, the serological titer of IgG-neutralizing anti-RBD antibodies of the subunit S1 of the spike protein of SARS-CoV-2 was determined. The socio-demographic and clinical characteristics of the subjects and adverse effects of vaccination were collected by questionnaire. RESULTS: In all of the workers, high antibody titer, ranging between 20 and 760 times the minimum protective level were observed. Titers were significantly higher in subjects with a previous COVID-19 diagnosis. Adverse effects after the vaccine were more frequent after the second dose, but no severe adverse effects were observed. CONCLUSIONS: The two doses of the BNT162b2 vaccine, even if administered four weeks apart, induced high titers of anti-SARS-CoV-2 neutralizing IgG in all the operators included in the study.

Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups.

OBJECTIVES: Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. METHODS: We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. RESULTS: With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25-<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. CONCLUSIONS: Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Evaluation of three commercial assays for SARS-CoV-2 molecular detection in upper respiratory tract samples.

The increasing COVID-19 widespread has created the necessity to assess the diagnostic accuracy of newly introduced (RT-PCR based) assays for SARS-CoV-2 RNA detection in respiratory tract samples. We compared the results of the Allplex™ 2019-nCoV assay with those of the Simplexa™ COVID-19 Direct assay and the Quanty COVID-19 assay, respectively, all performed on 125 nasal/oropharyngeal swab samples of patients with COVID-19 suspicion. Fifty-four samples were positive, and 71 were negative with the Allplex™ assay, whereas 47 of 54 samples were also positive with the Simplexa™ assay. The Quanty assay detected 55 positive samples, including the 54 positive samples with the Allplex™ assay and 1 sample that was Allplex™ negative but Simplexa™ positive. Using a consensus result criterion as the reference standard allowed to resolve the eight samples with discordant results (one Allplex™ negative and seven Simplexa™ negative) as truly false negative. Interestingly, a Spearman's negative association was found between the viral RNA loads quantified by the Quanty assay and the CT values of RT PCRs performed with either the Allplex™ assay or the Simplexa™ assay. However, the strength of this association was higher for the Allplex™ assay (N gene, ρ = - 0.92; RdRP gene, ρ = - 0.91) than for the Simplexa™ assay (ORF1ab gene, ρ = - 0.65; S gene, ρ = - 0.80). The Allplex™ 2019-nCoV, the Simplexa™ COVID-19 Direct, and the Quanty COVID-19 assays yielded comparable results. However, the role these assays might play in future clinical practice warrants larger comparison studies.

Should face masks be worn to contain the spread of COVID-19 in the postlockdown phase?

BACKGROUND: In East Asia, face masks are commonly worn to reduce viral spread. In Euope and North America, however, their use has been stigmatised for a long time, although this view has radically changed during the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Notwithstanding this, it is still unclear whether face masks worn by COVID-19 carriers may indeed prevent viral transmission and environmental contamination. The objective of this study was to evaluate the effectiveness of surgical face masks in filtering SARS-CoV-2. METHODS: Four male patients with COVID-19 were recruited for the study. Two patients wore a surgical mask for 5 h, while two others did not. The spread of the virus in the environment was evaluated through the approved Allplex 2019-nCoV assay. RESULTS: In the room with the two patients without surgical masks, the swab performed on the headboard and sides of the beds was positive for SARS-CoV-2 contamination. In the other room, where two patients were wearing surgical masks, all of the swabs obtained after 5 h tested negative. CONCLUSIONS: The results of the current study add to the growing body of literature supporting the use of face masks as a measure to contain the spread of SARS-CoV-2 by retaining potentially contagious droplets that can infect other people and/or contaminate surfaces. Based on the current evidence, face masks should therefore be considered a useful and low-cost device in addition to social distancing and hand hygiene during the postlockdown phase.

Evidence-based of conjunctival COVID-19 positivity: An Italian experience: Gemelli Against COVID Group.

BACKGROUND: The possible transmission of severe acute respiratory coronavirus 2 (SARS-CoV-2) by tears and conjunctiva is still debated. METHODS: Main outcome was to investigate the agreement between nasopharyngeal swab (NPs) and conjunctival swabs (Cs) in patients with SARS-CoV-2 infection. We divided patients into four groups: (1) NPs and Cs both negative (C-NF-), (2) NPs positive and Cs negative (NFs+Cs-), (3) NPs negative and Cs positive (NFs-Cs+), and (4) NPs and Cs both positive (NFs-Cs+). The secondary outcomes were to correlate Cs results with systemic clinical parameters such as: oxygen saturation (SpO2), dyspnea degree (DP), radiologic pulmonary impairment based on chest radiography (XR) or computed tomography (CT), blood chemistry as D-Dimer (D-Dimer), fibrinogen, ferritin, lactate dehydrogenase (LDH), and C-reactive protein (C-RP). RESULTS: A total of 100 conjunctival swabs in 50 patients with SARS-CoV-2 have been enrolled in this interventional clinical trials. Ocular signs (conjunctivitis) were present in five patients (10%). NPs and Cs highlighted a poor level of agreement (0.025; p = 0.404). Median SpO2 levels are the highest in the NF-C- group (98%) and the lowest (90%) in the group NF+C+ (p = 0.001). Pulmonary impairment was statistically significantly different between NFs and Cs groups (p = 0.019). Pulmonary impairment score increased from NFs-Cs- group (3.8 ± 3.9), to NFs+Cs+ group (6.7 ± 4.1). Intensive care unit patients showed higher COVID-19 Cs positivity in conjunctiva (12.5%) against hospitalized ones (5.8%). CONCLUSIONS: In patients hospitalized for SARS-CoV-2 the virus can be detected in conjunctival swab. Intensive care unit patients may reveal a higher COVID-19 presence in the conjunctiva. The most severe pulmonary impairment can be observed in NFs and Cs positivity. TRIAL REGISTRATION: Clinicaltrials.gov registration. ETHICAL COMMITTEE AUTHORIZATION: ID number: 0013008/20.

Peritoneal HPV-DNA test in cervical cancer (PIONEER study): A proof of concept.

The aim of this study was to investigate the prevalence of peritoneal human papillomavirus (HPV) infection in different clinical cervical cancer (CC) settings, and its association with potential clinical and/or histological factors. This is a single-center, prospective, observational study. Consecutive patients with newly diagnosed or recurrent/persistent CC, between March 2019 and April 2020, were included. A group of patients undergoing surgery for benign gynecological conditions was included as control group. All patients underwent HPV-DNA test in the cervix and in the peritoneal cavity simultaneously at time of surgery. Two-hundred seventy-two patients had cervical and peritoneal HPV test analyzed. Cervical and peritoneal HPV positivity (PHP) was found in 235 (88.0%) and 78 (28.7%) patients, respectively; the prevalence of PHP was 17.7% in early stage, 28.8% in locally advanced cervical cancer (LACC) and 46.6% in the metastatic/persistent/recurrent setting (P = .001). No control patient was found to have peritoneal HPV infection. Higher frequency of PHP was documented in patients with larger tumor size (P = .003), presence of cervical HPV 16/18 genotypes (P < .001), higher number of cervical high-risk (HR)-HPV per patient (P = .018) and peritoneal carcinomatosis (P < .001). Multivariate analysis demonstrated that lack of preoperative cervical conization in early stages (P = .030), while higher International Federation of Gynecology and Obstetrics (FIGO) stage (P = .021) and presence of cervical HPV 16/18 (P = .001) in LACC, was associated with PHP. This is a proof-of-concept study. A number of potential clinical implications, including prognosis, could be obtained by further studies.