Performance evaluation of a self-administered point-of-care test for anal HPV screening in PrEP users: data from a community-based PrEP service.

OBJECTIVES: In this study, we compared the performance of a self-administered point-of-care test (POCT) for anal human papillomavirus (HPV) screening with laboratory gold-standard test in pre-exposure prophylaxis (PrEP) users and evaluated its feasibility. METHODS: We enrolled PrEP users from a local community-based PrEP service. Each participant self-collected an anal swab to test anal HPV with a PCR POCT capable of detecting 14 high-risk HPV genotypes. Anonymous questionnaires on self-sampling feasibility were completed. Participants were then referred to local clinics to undergo standard viral genotyping. Concordance between POCT and gold-standard test was measured with absolute agreement and Cohen's kappa. Receiver operating characteristic (ROC) curves were used to calculate POCT sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: 179 subjects got a valid POCT result, most of them men (98.3%) and men who have sex with men (90.4%). 68.2% tested positive for at least one high-risk HPV genotype on POCT. 150 feasibility questionnaires were collected: 92.7% of compilers found the self-swab easy to perform. For 178 subjects, a gold-standard test valid result was also available: 77% tested positive for at least one high-risk HPV genotype. The median time elapsed between the two tests was 9.8 months, due to COVID-19-related service interruptions. Agreement between POCT and gold-standard test was 79.3% (Cohen's kappa=0.49). POCT showed a sensitivity of 81.0%, a specificity of 73.8%, a PPV of 91.0% and an NPV of 54.4%. CONCLUSIONS: POCT showed a moderate agreement with gold-standard test and a discrete sensitivity and specificity, suggesting that it could be a useful and feasible additional tool for HPV screening, especially in low-resource and community-based settings.

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.

Impact of SARS-CoV-2 Omicron and Delta variants in patients requiring intensive care unit (ICU) admission for COVID-19, Northern Italy, December 2021 to January 2022.

This multicenter observational study included 171 COVID-19 adult patients hospitalized in the ICUs of nine hospitals in Lombardy (Northern Italy) from December, 1st 2021, to February, 9th 2022. During the study period, the Delta/Omicron variant ratio of cases decreased with a delay of two weeks in ICU patients compared to that in the community; a higher proportion of COVID-19 unvaccinated patients was infected by Delta than by Omicron whereas a higher rate of COVID-19 boosted patients was Omicron-infected. A higher number of comorbidities and a higher comorbidity score in ICU critically COVID-19 inpatients was positively associated with the Omicron infection as well in vaccinated individuals. Although people infected by Omicron have a lower risk of severe disease than those infected by Delta variant, the outcome, including the risk of ICU admission and the need for mechanical ventilation due to infection by Omicron versus Delta, remains uncertain. The continuous monitoring of the circulating SARS-CoV-2 variants remains a milestone to counteract this pandemic.

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.

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.

Effectiveness of infection-containment measures on SARS-CoV-2 seroprevalence and circulation from May to July 2020, in Milan, Italy.

OBJECTIVE: Through a hospital-based SARS-CoV-2 molecular and serological screening, we evaluated the effectiveness of two months of lockdown and two of surveillance, in Milan, Lombardy, the first to be overwhelmed by COVID-19 pandemics during March-April 2020. METHODS: All subjects presenting at the major hospital of Milan from May-11 to July-5, 2020, underwent a serological screening by chemiluminescent assays. Those admitted were further tested by RT-PCR. RESULTS: The cumulative anti-N IgG seroprevalence in the 2753 subjects analyzed was of 5.1% (95%CI = 4.3%-6.0%), with a peak of 8.4% (6.1%-11.4%) 60-63 days since the peak of diagnoses (March-20). 31/106 (29.2%) anti-N reactive subjects had anti-S1/S2 titers >80 AU/mL. Being tested from May-18 to June-5, or residing in the provinces with higher SARS-CoV-2 circulation, were positively and independently associated with anti-N IgG reactivity (OR [95%CI]: 2.179[1.455-3.264] and 3.127[1.18-8.29], respectively). In the 18 RT-PCR positive, symptomatic subjects, anti-N seroprevalence was 33.3% (95% CI: 14.8%-56.3%). CONCLUSION: SARS-CoV-2 seroprevalence in Milan is low, and in a downward trend after only 60-63 days since the peak of diagnoses. Italian confinement measures were effective, but the risk of contagion remains concrete. In hospital-settings, the performance of molecular and serological screenings upon admission remains highly advisable.

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients.

Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.

Prolongation of incubation time improves clinical diagnosis of Mycobacterium xenopi infection and allows susceptibility testing of mycobacterial strains against multiple antibiotics.

OBJECTIVES: Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) whose clinical diagnosis and drug susceptibility studies are frequently hampered by poor in vitro growth. Extending the culture incubation time from 42 days (common-standard) to 56 days could improve the likelihood of diagnosis and provide strains for phenotypic drug susceptibility profiling of this poorly studied but clinically relevant mycobacterium. METHODS: Time-to-positivity of mycobacterial cultures incubated for 56 days were analysed and compared. Clinical mycobacteriosis was defined by ATS/IDSA criteria. In vitro susceptibility of M. xenopi isolates was tested by broth microdilution. RESULTS: Of 3852 mycobacteria-positive cultures (26 different mycobacterial species),M. xenopi required by far the longest growth time in culture, exceeding the 42 days commonly used in routine diagnostics in 41.2% of cases versus 4.7% for other NTM and 2.0% for Mycobacterium tuberculosis complex (P<0.001). Prolonging the incubation time to 56 days had a great impact on M. xenopi diagnosis, as 56.3% (27/48) of patients would have not fulfilled the ATS/IDSA criteria at an incubation limited to 42 days. All 40 M. xenopi isolates from patients with clinical mycobacteriosis were fully susceptibility to macrolides and rifamycins in vitro and to moxifloxacin, amikacin and linezolid. CONCLUSION: These results indicate that a significant percentage (56.3%) of positive culture forM. xenopi would have incorrectly been reported as negative to clinicians without prolonging the incubation time to 56 days. Moreover, 56.3% of patients with M. xenopi disease would have missed the diagnosis along with an appropriate germ-based antimycobacterial treatment, otherwise fully effective.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Successful treatment with isavuconazole of subcutaneous phaeohyphomycosis in a kidney transplant recipient.

Phaeohyphomycosis is a diverse group of uncommon mycotic infections caused by dematiaceous fungi which appears to be increasing in incidence, particularly in transplant recipients. Alternaria is the most frequent isolated genus. Subcutaneous, pulmonary and disseminated disease are the most common sites of Alternaria infection in solid organ transplant recipients. We report the first case, to our knowledge, of a kidney transplant recipient with Alternaria alternata subcutaneous infection who was successfully treated with isavuconazole.

Performance of real-time PCR Xpert ®MTB/RIF in diagnosing extrapulmonary tuberculosis.

The real time PCR Xpert ® MTB/RIF is fundamental for rapid diagnosis in paucibacillary respiratory samples and for the detection of multidrug-resistant TB cases. This paper aimed to determine its performance on different extrapulmonary samples. We determined sensitivity, specificity, positive and negative predictive value on respiratory and non-respiratory samples collected from January 2010 to June 2014. The protocol for the Xpert ® MTB/RIF PCR suggested by Cepheid was strictly followed for all specimens. In 12257 respiratory samples we observed a sensitivity of 87.1% and a specificity of 99.9%. There were 2818 extrapulmonary specimens, of which 250 were followed by a positive culture for Mycobacterium tuberculosis complex, whereas 72 samples were culture-negative: tuberculosis was clinically confirmed in 71 of them and was excluded for one sample. The sensitivity of the test on urine, pus and CSF samples was 88.2%, 95.6% and 100% respectively. In contrast, the sensitivity of gastric aspirates and biopsies was 81.8% and 83.6% respectively, whereas results of total cavitary fluids were significantly worse than expected (53.7% sensitivity). Our experience shows that Xpert MTB/RIF assay is an accurate, sensitive, and specific test for the rapid detection of pulmonary and extra-pulmonary TB with the only exception of cavitary fluids.