COVID-Vaccines in Pregnancy: Maternal and Neonatal Response over the First 9 Months after Delivery.

Vaccination against SARS-CoV-2 has been demonstrated to be safe during gestation. Nevertheless, there are no robust data investigating the entity of maternal antibodies' transmission through the placenta to the newborn and the persistence of the antibodies in babies' serum. The objective of this study is to assess the maternal antibody transmission and kinetics among newborns in the first months of life. Women having received one or two doses of anti-SARS-CoV-2 mRNA-vaccines during pregnancy at any gestational age, and their newborns, were recruited and followed-up over 9 months. Ninety-eight women and 103 babies were included. At birth, we observed a significant positive correlation between maternal and neonatal serum anti-SARS-CoV-2 antibody levels and a significant negative correlation between the time since last dose and antibody levels in mothers with two doses. Over the follow-up, the birth antibody level significantly decreased in time according to the received doses number at 3, 6, and 9 months. During the follow-up, we registered 34 dyad SARS-CoV-2 infection cases. The decreasing trend was slower in the SARS-CoV-2 infection group and among breastfed non-infected babies. Antibodies from maternal anti-SARS-CoV-2 vaccination are efficiently transferred via the placenta and potentially even through breast milk. Among newborns, antibodies show relevant durability in the first months of life.

A novel KPC-166 in ceftazidime/avibactam resistant ST307 Klebsiella pneumoniae causing an outbreak in intensive care COVID Unit, Italy.

INTRODUCTION: Aim of the study was the molecular characterization of 21 ceftazidime/avibactam resistant (CZA-R) Klebsiella pneumoniae strains, collected in the period October 2021-March 2022 from an Intensive Care COVID Unit in a Northern Italian Hospital. METHODS: After growth on selective/chromogenic culture media and susceptibility tests assessment, resistance genes content was ascertained for all the isolates by the HybriSpot 12 multiplexing, PCR and Whole-Genome Sequencing (WGS). Clonality was assessed by PFGE and MLST according to the Pasteur scheme. A SNPs-based phylogenetic tree was obtained comparing representative isolates and global genomes. The blaKPC gene horizontal transmission was evaluated by conjugation experiments. blaKPC-166 was cloned in a pCR2.1 vector and transformed in chemically competent TOP10 cells. RESULTS: Sixteen inpatients resulted positive for colonization and/or infection by KPC-producing K. pneumoniae (KPC-Kp) strains. The 21 CZA-R KPC-Kp isolates obtained showed MDR phenotype; susceptibility to meropenem was always retained. All the CZA-R KPC-Kp presented a novel blaKPC variant, named blaKPC-166, showing a single nucleotide substitution (T811C) compared to the blaKPC-94; but related to blaKPC-2. TWO DIFFERENT PULSOTYPES WERE DETECTED: A in 18/21 and B in 1/21 cases, two strains from the same patient being untypable by PFGE. Interestingly, the outbreak was sustained by the high-risk clone ST307, although the ST22, ST6342, ST6418 and ST6811 have also been identified and associated to KPC-166. Worryingly, blaKPC-166 could be transferred horizontally and, after cloning, it conferred resistance to CZA. DISCUSSION: This novel variant confers CZA-resistance and carbapenems susceptibility restoration. As KPC-166 was found expressed by multiple Kp clones, greater efforts should be made to prevent the further dissemination of such strains in Italian clinical settings.

Human Campylobacter spp. infections in Italy.

PURPOSE: Campylobacter is a frequent cause of enteric infections with common antimicrobial resistance issues. The most recent reports of campylobacteriosis in Italy include data from 2013 to 2016. We aimed to provide national epidemiological and microbiological data on human Campylobacter infections in Italy during the period 2017-2021. METHODS: Data was collected from 19 Hospitals in 13 Italian Regions. Bacterial identification was performed by mass spectrometry. Antibiograms were determined with Etest or Kirby-Bauer (EUCAST criteria). RESULTS: In total, 5419 isolations of Campylobacter spp. were performed. The most common species were C. jejuni (n = 4535, 83.7%), followed by C. coli (n = 732, 13.5%) and C. fetus (n = 34, 0.6%). The mean age of patients was 34.61 years and 57.1% were males. Outpatients accounted for 54% of the cases detected. Campylobacter were isolated from faeces in 97.3% of cases and in 2.7% from blood. C. fetus was mostly isolated from blood (88.2% of cases). We tested for antimicrobial susceptibility 4627 isolates (85.4%). Resistance to ciprofloxacin and tetracyclines was 75.5% and 54.8%, respectively; resistance to erythromycin was 4.8%; clarithromycin 2% and azithromycin 2%. 50% of C. jejuni and C. coli were resistant to ≥ 2 antibiotics. Over the study period, resistance to ciprofloxacin and tetracyclines significantly decreased (p < 0.005), while resistance to macrolides remained stable. CONCLUSION: Campylobacter resistance to fluoroquinolones and tetracyclines in Italy is decreasing but is still high, while macrolides retain good activity.

Durability of doravirine with dolutegravir dual regimen compared with other dolutegravir-based dual combinations.

OBJECTIVES: The availability of doravirine (DOR) allowed clinicians to prescribe a dolutegravir (DTG)-based two-drug regimen (2DR) in individuals not eligible to receive lamivudine (3TC) or rilpivirine (RPV). The aims of this study were to describe the durability of DTG + DOR compared with DTG/3TC and DTG/RPV and the rate of virological failure and target not-detected maintenance over time. METHODS: This retrospective, monocentric analysis included all subjects who started a DTG-based 2DR from 2018 to 2022 as a simplification. Descriptive statistics and non-parametric tests to describe and compare the groups were applied. Kaplan-Meier probability curves and Cox regression models for regimens durability were used. RESULTS: The study enrolled 710 individuals: 499 treated with DTG/3TC, 140 with DTG/RPV, and 71 with DTG + DOR. A 2DR with DOR was prescribed to older subjects who had a longer infection, greater exposure to different antiretroviral regimens, a higher proportion of resistance-associated mutations, and a worse immune-virologic status. Over a cumulative follow-up of 68 152 weeks, 42 discontinuations were registered (5.9%). DTG + DOR had a risk of treatment interruption of 7.8% at 48 weeks and 9.8% at 96 weeks, significantly higher than the other 2DRs. In the multivariate Cox model, DTG + DOR and DTG/RPV were significantly associated with discontinuation. The maintenance of target not detected during follow-up was similar among groups. The rate of virological failure was higher for DTG + DOR through different event definitions. CONCLUSIONS: DTG + DOR durability was high over a long follow-up albeit lower than for other 2DRs. This combination might be an effective option in people with HIV that has proven difficult to treat.

An overview of SARS-CoV-2 variants circulating in the 2020-2022 period in Lombardy.

Since the beginning of the pandemic, SARS-CoV-2 has shown genetic variability. All the variants that have sustained pandemic waves have shown several mutations, especially in the Spike protein that could affect viral pathogenesis. A total of 15,729 respiratory samples, collected between December 2020 and August 2022, have been included in this study. We report the circulation of SARS-CoV-2 variants in the Lombardy region, Italy, in a 2-year study period. Alpha, Delta, and Omicron variants became predominant causing the majority of cases whereas Beta or Gamma variants mostly caused local outbreaks. Next-generation sequencing revealed several mutations and few deletions in all of the main variants. For example, 147 mutations were observed in the Spike protein of Omicron sublineages; 20% of these mutations occurred in the receptor-binding domain region.

Quantitative SARS-CoV-2 subgenomic RNA as a surrogate marker for viral infectivity: Comparison between culture isolation and direct sgRNA quantification.

Detection of subgenomic (sg) SARS-CoV-2 RNAs are frequently used as a correlate of viral infectiousness, but few data about correlation between sg load and viable virus are available. Here, we defined concordance between culture isolation and E and N sgRNA quantification by ddPCR assays in 51 nasopharyngeal swabs collected from SARS-CoV-2 positive hospitalized patients. Among the 51 samples, 14 were SARS-CoV-2 culture-positive and 37 were negative. According to culture results, the sensitivity and specificity of E and N sgRNA assays were 100% and 100%, and 84% and 86%, respectively. ROC analysis showed that the best E and N cut-offs to predict positive culture isolation were 32 and 161 copies/mL respectively, with an AUC (95% CI) of 0.96 (0.91-1.00) and 0.96 (0.92-1.00), and a diagnostic accuracy of 88% and 92%, respectively. Even if no significant correlations were observed between sgRNA amount and clinical presentation, a higher number of moderate/severe cases and lower number of days from symptoms onset characterized patients with sgRNA equal to or higher than sgRNA cut-offs. Overall, this study suggests that SARS-CoV-2 sgRNA quantification could be helpful to estimate the replicative activity of SARS-CoV-2 and can represent a valid surrogate marker to efficiently recognize patients with active infection. The inclusion of this assay in available SARS-CoV-2 diagnostics procedure might help in optimizing fragile patients monitoring and management.

Artificial intelligence for differentiating COVID-19 from other viral pneumonias on CT: comparative analysis of different models based on quantitative and radiomic approaches.

BACKGROUND: To develop a pipeline for automatic extraction of quantitative metrics and radiomic features from lung computed tomography (CT) and develop artificial intelligence (AI) models supporting differential diagnosis between coronavirus disease 2019 (COVID-19) and other viral pneumonia (non-COVID-19). METHODS: Chest CT of 1,031 patients (811 for model building; 220 as independent validation set (IVS) with positive swab for severe acute respiratory syndrome coronavirus-2 (647 COVID-19) or other respiratory viruses (384 non-COVID-19) were segmented automatically. A Gaussian model, based on the HU histogram distribution describing well-aerated and ill portions, was optimised to calculate quantitative metrics (QM, n = 20) in both lungs (2L) and four geometrical subdivisions (GS) (upper front, lower front, upper dorsal, lower dorsal; n = 80). Radiomic features (RF) of first (RF1, n = 18) and second (RF2, n = 120) order were extracted from 2L using PyRadiomics tool. Extracted metrics were used to develop four multilayer-perceptron classifiers, built with different combinations of QM and RF: Model1 (RF1-2L); Model2 (QM-2L, QM-GS); Model3 (RF1-2L, RF2-2L); Model4 (RF1-2L, QM-2L, GS-2L, RF2-2L). RESULTS: The classifiers showed accuracy from 0.71 to 0.80 and area under the receiving operating characteristic curve (AUC) from 0.77 to 0.87 in differentiating COVID-19 versus non-COVID-19 pneumonia. Best results were associated with Model3 (AUC 0.867 ± 0.008) and Model4 (AUC 0.870 ± 0.011. For the IVS, the AUC values were 0.834 ± 0.008 for Model3 and 0.828 ± 0.011 for Model4. CONCLUSIONS: Four AI-based models for classifying patients as COVID-19 or non-COVID-19 viral pneumonia showed good diagnostic performances that could support clinical decisions.

CMV seroprevalence and coronary CMV-DNA detection in immunocompetent patients with heart diseases.

BACKGROUND: Acute coronary syndromes (ACS) are a major cause of morbidity and mortality. As cytomegalovirus (CMV) may contribute to cardio-vascular (CV) manifestations, we sought to provide a proof-of-concept for the involvement of coronary and/or systemic CMV-reactivation as a possible ACS trigger. METHODS: We prospectively enrolled consecutive patients undergoing a coronary angiography for ACS (acute-cases, N.=136), or non-ACS reasons (chronic-cases, N.=57). Matched coronary and peripheral blood-samples were processed for quantification of CMV-DNAemia (RT-PCR), CMV-IgG/IgM, and CMV-IgG avidity (ELISA). Peripheral-blood samples from 17 healthy subjects were included as controls. RESULTS: Out of the 193 cases included, 18.1% were aged ≤55 years, 92.5% were Central-European, and 100% immunocompetent. CMV-IgG seroprevalence was 91.7% (95%CI: 87.8-95.6), significantly higher than in healthy-controls (52.9% [95%CI: 29.2-76.5]; P<0.001), yet consistent across age-groups (P=0.602), male/females (P=0.765), and acute/chronic-cases (P=0.157). Median (IQR) IgG titers were 110 (84-163) AU/mL, with 0.62 (0.52-0.72) avidity, supporting a long history of infection. No acute CMV infections were found. In 22.6% (n/N.=40/177) of the IgG-positive cases low-level coronary and/or systemic CMV-DNAemia (always <40 copies/mL) was detected. While no differences in peripheral CMV-DNAemia prevalence were observed nor among cases nor controls, coronary CMV-DNAemia was more frequent in acute-cases without modifiable CV risk-factors (n/N.=4/10; 40.0%), than in chronic-cases (n/N.=6/55, 10.9%; P=0.029), or acute-cases with risk-factors (n/N.=16/112, 14.3%; P=0.058). CONCLUSIONS: CMV-IgG seroprevalence was high in patients with heart diseases. CMV-DNAemia can be found, although uncommonly, in coronary circulation during an ACS, with increased prevalence in older subjects and in absence of CV risk-factors, identifying possible areas for novel interventions.

Diagnostic Performance in Differentiating COVID-19 from Other Viral Pneumonias on CT Imaging: Multi-Reader Analysis Compared with an Artificial Intelligence-Based Model.

Growing evidence suggests that artificial intelligence tools could help radiologists in differentiating COVID-19 pneumonia from other types of viral (non-COVID-19) pneumonia. To test this hypothesis, an R-AI classifier capable of discriminating between COVID-19 and non-COVID-19 pneumonia was developed using CT chest scans of 1031 patients with positive swab for SARS-CoV-2 (n = 647) and other respiratory viruses (n = 384). The model was trained with 811 CT scans, while 220 CT scans (n = 151 COVID-19; n = 69 non-COVID-19) were used for independent validation. Four readers were enrolled to blindly evaluate the validation dataset using the CO-RADS score. A pandemic-like high suspicion scenario (CO-RADS 3 considered as COVID-19) and a low suspicion scenario (CO-RADS 3 considered as non-COVID-19) were simulated. Inter-reader agreement and performance metrics were calculated for human readers and R-AI classifier. The readers showed good agreement in assigning CO-RADS score (Gwet's AC2 = 0.71, p < 0.001). Considering human performance, accuracy = 78% and accuracy = 74% were obtained in the high and low suspicion scenarios, respectively, while the AI classifier achieved accuracy = 79% in distinguishing COVID-19 from non-COVID-19 pneumonia on the independent validation dataset. The R-AI classifier performance was equivalent or superior to human readers in all comparisons. Therefore, a R-AI classifier may support human readers in the difficult task of distinguishing COVID-19 from other types of viral pneumonia on CT imaging.

High prevalence of anal papillomavirus infection in men who have sex with men PrEP users.

OBJECTIVES: Human papillomavirus (HPV) is the most common STI and is associated with a wide range of diseases from anogenital warts to malignancies. Anal HPV infection is considerably more common in men who have sex with men (MSM) living with HIV. Aims of the present study are to (i) describe the prevalence of anal HPV infection in MSM who started pre-exposure prophylaxis (PrEP) and (ii) analyse factors associated with anal infection from genotypes that would be covered by nonavalent vaccination. METHODS: This monocentric, cross-sectional study included all subjects who started PrEP from May 2018 to November 2021. PrEP candidates underwent full behavioural and clinical evaluation, including digital anal rectal examination and swabbing for HPV determination. Descriptive statistics, Mann-Whitney U test for continuous and χ2 tests for categorical variables were adopted. Unadjusted and adjusted regression analyses were performed to assess factors associated with positive anal swabs and to the presence of genotypes covered by the nonavalent vaccination. RESULTS: The analysis included 288 subjects: anal swabs tested positive in 87.2% of cases, 79.2% of the subjects had a high-risk genotype (mainly 16), whereas 67.4% had a genotype covered by nonavalent vaccine. Sexual role was the only factor associated with anal HPV infection. Use of recreational drugs and a diagnosis of ≥2 STIs correlated with the presence of genotypes that would have been covered by vaccine, while previous vaccination had a protective role. CONCLUSIONS: PrEP candidates showed a high prevalence of anal HPV infection, especially due to high-risk genotypes, comparable to what has been reported in MSM living with HIV.

Antibody Response to COVID-19 Booster Vaccination in Healthcare Workers.

Objective: To evaluate the mean increase of anti-S IgG antibody titer between the basal, pre-booster level to the titer assessed 14 days after the booster dose of BNT162b2. Patients and Methods: The RENAISSANCE study is an observational, longitudinal, prospective, population-based study, conducted on healthcare workers of Niguarda Hospital in Milan, Italy who received a BNT162b2 booster dose at least 180 days after their second dose or after positivity for SARS-CoV-2 and accepted to take part in the study. The RENAISSANCE study was conducted from January 1, 2021 through December 28, 2021. Findings: 1,738 subjects were enrolled among healthcare workers registered for the booster administration at our hospital. Overall, 0.4% of subjects were seronegative at the pre-booster evaluation, and 1 subject had a titer equal to 50 AU/ml: none of the evaluated subjects was seronegative after the booster dose. Thus, the efficacy of the booster in our population was universal. Mean increase of pre- to post-booster titer was more significant in subjects who never had SARS-CoV-2 (44 times CI 95% 42-46) compared to those who had it, before (33 times, CI 95% 13-70) or after the first vaccination cycle (12 times, CI 95% 11-14). Differently from sex, age and pre-booster titers affected the post-booster antibody response. Nevertheless, the post-booster titer was very similar in all subgroups, and independent of a prior exposure to SARS-CoV-2, pre-booster titer, sex or age. Conclusion: Our study shows a potent universal antibody response of the booster dose of BNT162b2, regardless of pre-booster vaccine seronegativity.

Anaerobic bloodstream infections in Italy (ITANAEROBY): A 5-year retrospective nationwide survey.

INTRODUCTION: A lack of updated data on the burden and profile of anaerobic bloodstream infections (ABIs) exists. We assessed the incidence of ABIs and trends in antimicrobial resistance in anaerobes isolated from blood in Italy. MATERIAL AND METHODS: We conducted a retrospective study on 17 Italian hospitals (2016-2020). Anaerobes isolated from blood culture and their in vitro susceptibility profiles (EUCAST-interpreted) were registered and analyzed. RESULTS: A total of 1960 ABIs were identified. The mean age of ABIs patients was 68.6 ± 18.5 years, 57.6% were males. The overall incidence rate of ABIs was 1.01 per 10.000 patient-days. Forty-seven% of ABIs occurred in medical wards, 17% in ICUs, 14% in surgical wards, 7% in hemato-oncology, 14% in outpatients. The three most common anti-anaerobic tested drugs were metronidazole (92%), clindamycin (89%) and amoxicillin/clavulanate (83%). The three most common isolated anaerobes were Bacteroides fragilis (n = 529), Cutibacterium acnes (n = 262) and Clostridium perfringens (n = 134). The lowest resistance rate (1.5%) was to carbapenems, whereas the highest rate (51%) was to penicillin. Clindamycin resistance was >20% for Bacteroides spp., Prevotella spp. and Clostridium spp. Metronidazole resistance was 9.2% after excluding C. acnes and Actinomyces spp. Bacteroides spp. showed an increased prevalence of clindamycin resistance through the study period: 19% in 2016, 33% in 2020 (p ≤ 0.001). CONCLUSIONS: Our data provide a comprehensive overview of the epidemiology of ABIs in Italy, filling a gap that has existed since 1995. Caution is needed when clindamycin is used as empirical anti-anaerobic drug.

Polymorphism of the HLA system and weak antibody response to BNT162b2 mRNA vaccine.

The polymorphism of the HLA system has been extensively studied in COVID-19 infection, however there are no data about the role of HLA on vaccine response. We report here the HLA-A, -B, -C, and DRB1 allelic frequencies of n = 111 individuals after BNT162b2 mRNA vaccine, selected on the basis of lower antibody levels (<5% percentile) after the second dose among a total of n = 2569 vaccinees, and compare them with the frequencies of a reference population. We found that differences in the frequencies of the alleles HLA-A*03:01, A*33:03, B*58:01 and at least one haplotype (HLA-A*24:02~C*07:01~B*18:01~DRB1*11:04) are associated with a weaker antibody response after vaccination, together with the age of vaccinees. Our results might suggest a role played by some HLA alleles or haplotypes in antibody production after the BNT162b2 mRNA vaccine, giving insights into the tracking of potentially susceptible individuals across populations. Further studies are needed to better define our exploratory findings and dissect the role of HLA polymorphism on response to anti-COVID-19 vaccines.

SARS-COV-2 screening in allogeneic hematopoietic stem cell donors: Implications for the evaluation process and eligibility

Abstract Introduction Soon after the onset of the SARS-CoV-2 pandemic, viral screening by nasopharyngeal swab became mandatory for allogeneic hematopoietic stem cell (HSC) donor eligibility. Methods We described our monocenter experience with allogeneic HSC donors from February 1 to the October 31, 2020 to verify whether the introduction of SARS-CoV-2 screening altered the donor eligibility and/or entailed a prolongation of the evaluation process. Results A total of 21 allogeneic HSC donors were screened during the above-mentioned period upon request by the local transplant physicians or by the Italian Bone Marrow Donor Registry; among the HSC donors (n = 17) who completed the eligibility process and further received the nasopharyngeal swab, all but one were negative for the presence of SARS-CoV-2. The positive donor remained asymptomatic for the whole duration of the infection, which lasted six weeks. However, he was temporarily excluded from donation. The median duration of the evaluation process was not significantly different, compared to the same period of 2019 (p-value = 0.11). Conclusion The mandatory SARS-CoV-2 screening in allogeneic HSC donors allowed for the detection of 6% positivity in this monocenter series over a 9-month period. Despite the inconvenience of this unexpected non-eligibility, the exclusion of a SARS-CoV-2 positive donor represented an important safety measure for the donor, with respect to a new and still partially unknown virus. The screening did not alter the length of the donor evaluation and thus, did not cause a delay in the eligibility process.

SARS-COV-2 screening in allogeneic hematopoietic stem cell donors: Implications for the evaluation process and eligibility.

INTRODUCTION: Soon after the onset of the SARS-CoV-2 pandemic, viral screening by nasopharyngeal swab became mandatory for allogeneic hematopoietic stem cell (HSC) donor eligibility. METHODS: We described our monocenter experience with allogeneic HSC donors from February 1 to the October 31, 2020 to verify whether the introduction of SARS-CoV-2 screening altered the donor eligibility and/or entailed a prolongation of the evaluation process. RESULTS: A total of 21 allogeneic HSC donors were screened during the above-mentioned period upon request by the local transplant physicians or by the Italian Bone Marrow Donor Registry; among the HSC donors (n = 17) who completed the eligibility process and further received the nasopharyngeal swab, all but one were negative for the presence of SARS-CoV-2. The positive donor remained asymptomatic for the whole duration of the infection, which lasted six weeks. However, he was temporarily excluded from donation. The median duration of the evaluation process was not significantly different, compared to the same period of 2019 (p-value = 0.11). CONCLUSION: The mandatory SARS-CoV-2 screening in allogeneic HSC donors allowed for the detection of 6% positivity in this monocenter series over a 9-month period. Despite the inconvenience of this unexpected non-eligibility, the exclusion of a SARS-CoV-2 positive donor represented an important safety measure for the donor, with respect to a new and still partially unknown virus. The screening did not alter the length of the donor evaluation and thus, did not cause a delay in the eligibility process.

Results of the RENAISSANCE Study: REsponse to BNT162b2 COVID-19 vacciNe-short- And long-term Immune reSponSe evAluatioN in health Care workErs.

OBJECTIVE: To evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike (S) IgG antibody production after vaccination with BNT162b2 and the protection from symptomatic breakthrough infections in health care workers. METHODS: This prospective observational study (RENAISSANCE) had as a primary end point the evaluation of serologic response to BNT162b2 14 days after a second dose. SARS-CoV-2 anti-S IgG antibodies were evaluated with LIAISON SARS-CoV-2 TrimericS IgG assay (DiaSorin S.p.A.), which is able to detect the presence of both binding and neutralizing antibodies for trimeric spike glycoprotein. Participants were recruited from February 1, 2021, to February 22, 2021. Occurrence of vaccine breakthrough infections was assessed by reverse transcription-polymerase chain reaction on symptomatic and contact cases up to June 6, 2021. RESULTS: Of 2569 staff evaluated, only 4 were nonresponders (0.16%; 95% CI, 0.04% to 0.41%). All 4 nonresponders were severely immunosuppressed and receiving treatment with mycophenolate mofetil or mycophenolic acid. At 14 days after the second dose, 67.5% (1733) of staff had anti-S IgG titers of 2000 BAU/mL or higher; 19.2% (494), between 1500 and 2000 BAU/mL; 9.8% (251), between 1000 and 1500 BAU/mL; and 3.4% (87), 1000 BAU/mL or lower. Women had a higher probability of having higher titers than men (64.5% [1044/1618] vs 58.3% [410/703]; P=.005). This was confirmed after adjustment for age group (odds ratio, 1.275; 95% CI, 1.062 to 1.531; P=.009). Four months after the end of the vaccination program, only 13 participants (0.26%) had experienced a breakthrough SARS-CoV-2 infection, including 1 nonresponder. This was the only participant requiring hospitalization for severe COVID-19. CONCLUSION: The vaccination campaign among health care workers at the ASST GOM Niguarda has resulted in a marked serologic response and reduction of incident COVID-19 cases. Yet, the lack of protection should not be overlooked in immunocompromised individuals.

Sex differences in electrolyte imbalances caused by SARS-CoV-2: A cross-sectional study.

BACKGROUND: Since SARS-CoV-2 spread, evidence regarding sex differences in progression and prognosis of COVID-19 have emerged. Besides this, studies on patients' clinical characteristics have described electrolyte imbalances as one of the recurrent features of COVID-19. METHODS: We performed a cross-sectional study on all patients admitted to the emergency department (ED) from 1 March to 31 May 2020 who had undergone a blood gas analysis and a nasopharyngeal swab test for SARS-CoV-2 by rtPCR. We defined positive patients as cases (n = 710) and negatives as controls (n = 619), for a total number of patients of 1.329. The study was approved by the local ethics committee Area 3 Milan. Data were automatically extracted from the hospital laboratory SQL-based repository in anonymised form. We considered as outcomes potassium (K+ ), sodium (Na+ ), chlorine (Cl- ) and calcium (Ca++ ) as continuous and as categorical variables, in their relation with age, sex and SARS-CoV-2 infection status. RESULTS: We observed a higher prevalence of hypokalaemia among patients positive for SARS-CoV-2 (13.7% vs 6% of negative subjects). Positive patients had a higher probability to be admitted to the ED with hypokalaemia (OR 2.75, 95% CI 1.8-4.1, P < .0001) and women were twice as likely to be affected than men (OR 2.43, 95% CI 1.67-3.54, P < .001). Odds ratios for positive patients to manifest with an alteration in serum Na+ was (OR 1.6, 95% CI 1.17-2.35, P < .001) and serum chlorine (OR 1.6, 95% CI 1.03-2.69, P < .001). Notably, OR for positive patients to be hypocalcaemic was 7.2 (95% CI 4.8-10.6, P < .0001) with a low probability for women to be hypocalcaemic (OR 0.63, 95% CI 0.4-0.8, P = .005). CONCLUSIONS: SARS-CoV-2 infection is associated with a higher prevalence of hypokalaemia, hypocalcaemia, hypochloraemia and sodium alterations. Hypokalaemia is more frequent among women and hypocalcaemia among men.

Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study.

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available. Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells. Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R 2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550-8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease. Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

Frontline Screening for SARS-CoV-2 Infection at Emergency Department Admission by Third Generation Rapid Antigen Test: Can We Spare RT-qPCR?

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.

SARS-CoV-2 RNA in plasma samples of COVID-19 affected individuals: a cross-sectional proof-of-concept study.

BACKGROUND: Recent studies showed that plasma SARS-CoV-2 RNA seems to be associated with worse COVID-19 outcome. However, whether specific population can be at higher risk of viremia are to date unexplored. METHODS: This cross-sectional proof-of-concept study included 41 SARS-CoV-2-positive adult individuals (six affected by haematological malignancies) hospitalized at two major hospital in Milan, for those demographic, clinical and laboratory data were available. SARS-CoV-2 load was quantified by ddPCR in paired plasma and respiratory samples. To assess significant differences between patients with and patients without viremia, Fisher exact test and Wilcoxon test were used for categorical and continuous variables, respectively. RESULTS: Plasma SARS-CoV-2 RNA was found in 8 patients (19.5%), with a median (IQR) value of 694 (209-1023) copies/mL. Viremic patients were characterized by an higher mortality rate (50.0% vs 9.1%; p = 0.018) respect to patients without viremia. Viremic patients were more frequently affected by haematological malignancies (62.5% vs. 3.0%; p < 0.001), and had higher viral load in respiratory samples (9,404,000 [586,060-10,000,000] vs 1560 [312-25,160] copies/mL; p = 0.002). CONCLUSIONS: Even if based on a small sample population, this proof-of-concept study poses the basis for an early identification of patients at higher risk of SARS-CoV-2 viremia, and therefore likely to develop severe COVID-19, and supports the need of a quantitative viral load determination in blood and respiratory samples of haematologic patients with COVID-19 in order to predict prognosis and consequently to help their further management.