A multi-country comparative study of two treponemal tests for the serodiagnosis of syphilis amongst men who have sex with men (MSM): Chemo-luminescent assay vs Treponema pallidum particle agglutination assay.

INTRODUCTION: International guidelines recommend routine screening for syphilis (aetiological agent: Treponema pallidum subspecies pallidum) amongst key populations and vulnerable populations using tests detecting treponemal and non-treponemal antibodies. Whilst treponemal tests have high sensitivities and specificities, they differ regarding subjective or objective interpretation, throughput and workload. Chemiluminescence immunoassays (CLIAs) are cost- and time-effective automated methods for detecting treponemal antibodies. The Treponema pallidum particle agglutination assay (TPPA) has been considered the "gold standard" treponemal assay, however, this includes a highly manual procedure, low throughput and subjective interpretation. The present multi-country study evaluated the ADVIA Centaur® Syphilis CLIA (Siemens Healthcare) assay compared to the reference SERODIA-TP·PA® (Fujirebio Diagnostics) for the serodiagnosis of syphilis amongst men who have sex with men (MSM). METHOD: 1,485 MSM were enrolled in Brighton (UK), Malta, and Verona (Italy) as part of a larger WHO multi-country and multi-site ProSPeRo study. Ethical approval was obtained. Serum was tested with the ADVIA Centaur® Syphilis CLIA assay and SERODIA-TP·PA®, in accordance with the manufacturers' instructions, for a first round of validation. A second round of validation was carried out for discrepant results that were additionally tested with both Western Blot (Westernblot EUROIMMUN®) and an Immunoblot (INNO-LIA, Fujirebio Diagnostics). Sensitivity, specificity, positive and negative predictive value (PPV and NPV), likelihood ratios (positive/negative), and the Diagnostic Odds Ratio (DOR)/pre-post-test probability (Fagan's nomogram) were calculated. RESULTS: Out of 1,485 eligible samples analysed in the first phase, the SERODIA-TP·PA® identified 360 positive and 1,125 negative cases. The ADVIA Centaur® Syphilis CLIA assay (Siemens) identified 366 positives, missclassifying one TPPA-positive sample. In the second phase, the ADVIA Centaur® Syphilis CLIA resulted in 1 false negative and 4 false positive results. Considering the syphilis study prevalence of 24% (95% CI: 22-26.7), The sensitivity of the ADVIA Centaur® Syphilis CLIA assay was 99.7% (95% CI: 98.5-100), and the specificity was 99.4% (95% CI: 98.7-99.7). The ROC area values were 0.996 (95% CI: 0.992-0.999), and both the PPV and NPV values were above 98% (PPV 98.1%, 95% CI: 96.1-99.2; NPV 99.9%, 95% CI: 99.5-100). CONCLUSIONS: The ADVIA Centaur® Syphilis CLIA assay showed similar performance compared to the SERODIA-TP·PA®. Considering the study is based on QUADAS principles and with a homogeneous population, results are also likely to be generalisable to MSM population but potentially not applicable to lower prevalence populations routinely screened for syphilis. The automated CLIA treponemal assay confirmed to be accurate and appropriate for routine initial syphilis screening, i.e. when the reverse testing algorithm is applied.

Quality control and external quality assessment for the independent clinic-based evaluation of point-of-care testing to detect Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in eight countries.

BACKGROUND: Sexually transmitted infections caused by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) remain significant global health problems. The World Health Organization (WHO) has recently conducted a multi-faceted, multi-country validation study (ProSPeRo), which included an evaluation of the Xpert CT/NG and Xpert TV assays on the GeneXpert system (Cepheid, Sunnyvale, Ca., USA) in clinic-based settings across eight countries. To support the study, a training and quality management system was implemented and evaluated. METHODS: A comprehensive training program for the study was developed. Quality control (QC) and external quality assessment (EQA) samples were provided by an accredited quality assurance provider. QC testing was conducted at 14 point-of-care testing (POCT) clinics, while EQA samples were tested by the POCT sites and a reference laboratory supporting each clinic. RESULTS: For QC testing, concordance with the expected results for CT and NG was > 99% and rates of unsuccessful tests were < 4%. For TV testing, concordance was similar (97%), but rates of unsuccessful tests were high (18%), particularly in the 'TV negative' sample. For EQA testing initially conducted in 2018, concordance was 100% for CT and NG, and 90% for TV for the reference laboratory group (which used non-GeneXpert systems). Concordance for the POCT group was also high (> 94%) for all analytes, but this cohort (which used GeneXpert systems) exhibited a high rate of unsuccessful TV tests. All but one of these unsuccessful tests was subcategorised as 'invalid'. CONCLUSIONS: The high level of concordance for QC and EQA testing confirm that the trained operators at the POC clinical sites were competent to conduct POC testing and that the training and quality systems implemented for the ProSPeRo study were effective. The quality materials used were satisfactory for CT and NG but exhibited poor performance for TV testing on the GeneXpert system. The WHO should continue to work with industry and EQA providers to provide improved materials that are reliable, stable and cost effective for quality management, as it seeks to rollout molecular-based STI POCT in non-laboratory-based settings. TRIAL REGISTRATION: Ethics approval to conduct the ProSPeRo study was granted by the WHO Ethics Review Committee.

Independent clinic-based evaluation of dual POCTs for screening for HIV and syphilis in men who have sex with men in Italy, Malta, Peru, and the United Kingdom.

INTRODUCTION: Globally, the incidence of HIV and syphilis can be reduced by the use of validated point of care tests (POCTs). As part of the WHO PRoSPeRo Network, we aimed to evaluate the performance, acceptability, and operational characteristics of two dual HIV/syphilis POCTs (Bioline HIV/Syphilis Duo (Abbott) and DPP® HIV-Syphilis assay (Chembio) for the screening of HIV and syphilis amongst men who have sex with men (MSM). METHOD AND ANALYSES: A cross sectional study of 2,577 MSM in Italy, Malta, Peru, and the United Kingdom (UK) presenting to seven clinic sites, were enrolled. Finger prick blood was collected to perform POCTs and results compared with standard laboratory investigations on venepuncture blood. Acceptability and operational characteristics were assessed using questionnaires. Diagnostic meta-analysis was used to combine data from the evaluation sites. RESULTS: Based on laboratory tests, 23.46% (n = 598/2549) of participants were confirmed HIV positive, and 35.88% of participants (n = 901/2511) were positive on treponemal reference testing. Of all participants showing evidence of antibodies to Treponema pallidum, 50.56% (n = 455/900) were Rapid Plasma Reagin (RPR) test reactive. Of HIV positive individuals, 60.62% (n = 354/584) had evidence of antibodies to T. pallidum, and of these 60.45% (n = 214/354) exhibited reactive RPR tests indicating probable (co)infection. For Bioline POCT, pooled sensitivities and specificities for HIV were 98.95% and 99.89% respectively, and for syphilis were 73.79% and 99.57%. For Chembio pooled sensitivities and specificities for HIV were 98.66% and 99.55%, and for syphilis were 78.60% and 99.48%. Both tests can detect greater than 90% of probable active syphilis cases, as defined by reactive RPR and treponemal test results. These dual POCTs were preferred by 74.77% (n = 1,926) of participants, due to their convenience, and the operational characteristics made them acceptable to health care providers (HCPs). CONCLUSIONS: Both the Bioline and the Chembio dual POCT for syphilis and HIV had acceptable performance, acceptability and operational characteristics amongst MSM in the PRoSPeRo network. These dual POCTs could serve as a strategic, more cost effective, patient and healthcare provider (HCP) friendly alternative to conventional testing; in clinical and other field settings, especially those in resource-limited settings.

Clinic-based evaluation of the dual Xpert CT/NG assay on the GeneXpert System for screening for extragenital chlamydial and gonococcal infections amongst men who have sex with men.

BACKGROUND: Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections have increased globally. Asymptomatic infections represent a significant risk of long-term complications. Men who have sex with men (MSM) are disproportionally affected, underscoring the need to offer screening programmes to this population. CT/NG Point of Care Testing (POCT) constitutes a strategic tool to improve the continuum of STI care, however extensive real-life evaluations amongst at risk populations are lacking. The aim of this study is to estimate the GeneXpert CT/NG assay performance and usability for CT and NG at genital and extragenital sites for screening amongst MSM. METHODS: This study was a multi-site sexual health clinic-based evaluation (Italy, Malta and Peru) with consecutive enrolment. A first void urine sample (divided in two aliquots), two oropharyngeal and two anorectal swabs were collected for each study participant. One specimen set (one for each anatomical site) was tested with the dual index test (Cepheid) at the clinics by the healthcare staff, the other set with FDA/CE approved Nucleic Acid Amplification Tests (NAATs) at the laboratory. Clinical sites and reference laboratories participated in an internal and external quality control programme. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values for each anatomical site were estimated using a meta-analytic approach. RESULTS: One thousand seven hundred two MSM were recruited across all clinical sites for a total of 5049 biological specimens. NG and CT were respectively detected in 274 and 287 of samples. Overall, the NG POCT sensitivity and specificity was 91.43% and 99.75% in urine (LR + 372.80, LR- 0.09), 89.68% and 99.55% in rectal specimens (LR + 197.30, LR- 0.10) and 75.87% and 98.77% at the pharynx respectively (LR + 61.94, LR- 0.24). The CT component of the POCT sensitivity was 84.82% and specificity 99.63% in urine (LR + 228.68, LR- 0.15), 78.07% and 99.19% respectively on rectal site (LR + 96.23, LR-0.22), 67.79% and 99.88% respectively at pharyngeal site (LR + 554.89, LR- 0.32). 95.95% of MSM reported to be willing to wait for POCT results and no provider reported difficulties in terms of performance or interpretation of the results of the Xpert CT/NG. CONCLUSION: Rapid turnaround time, ease of use and high acceptability make the Xpert CT/NG testing system a strategic tool for increasing testing frequency, reaching those not yet tested and offering the possibility of immediate treatment if needed. The assay showed good negative likelihood ratios and confirms its use to rule out CT/NG infections. Sensitivity varied across sites and pathogens. Periodic staff training at the testing sites should be mandatory.

Evaluation of a ddPCR Commercial Assay for the Absolute Quantification of the Monkeypox Virus West Africa in Clinical Samples.

BACKGROUND: Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the orthopoxvirus genus in the family Poxviridae. Distinct clades are identified: the clade I belonging to the Central African (or Congo Basin) clade and the subclades IIa and IIb belonging to the West African clade. Here, a commercial droplet digital PCR (ddPCR) assay was evaluated for the quantification of the MPXV West Africa clade in clinical samples. METHODS: The ddPCR reaction was assessed as a duplex assay using RPP30 as an internal amplification control. A total of 60 clinical specimens were tested, 40 positives (skin lesions, n=10; rectal swabs, n = 10; pharyngeal swabs, n = 10; and whole blood, n = 10), and 20 negatives (n = 5 for each biological matrix) were found at the routine molecular diagnostics (orthopoxvirus qPCR followed by confirmation with Sanger sequencing). To evaluate the analytical sensitivity, the ddPCR reaction was first analyzed on serial dilutions of synthetic DNA spiked in water and in negative biological matrices, achieving a limit of detection of 3.5 copy/µL. RESULTS: Regarding the clinical samples, compared to routine molecular diagnostics, the ddPCR duplex assay showed 100% of specificity for all biological matrices and 100% sensitivity (10/10) for lesions, 100% (10/10) for rectal swabs, 90% (9/10) for pharyngeal swabs, and 60% (6/10) for whole blood. CONCLUSION: Overall, our data showed that the commercial ddPCR assay allowed the DNA detection of MPXV in 87.5% (35/40) of our cohort, highlighting useful technical indications for the different specimens with a potential greatest performance for skin lesions and rectal swabs.

Mycoplasma genitalium antibiotic resistance-associated mutations in genital and extragenital samples from men-who-have-sex-with-men attending a STI clinic in Verona, Italy.

Background: Mycoplasma genitalium (MG) is one of the most warning emerging sexually transmitted pathogens also due to its ability in developing resistance to antibiotics. MG causes different conditions ranging from asymptomatic infections to acute mucous inflammation. Resistance-guided therapy has demonstrated the best cure rates and macrolide resistance testing is recommended in many international guidelines. However, diagnostic and resistance testing can only be based on molecular methods, and the gap between genotypic resistance and microbiological clearance has not been fully evaluated yet. This study aims at finding mutations associated with MG antibiotic resistance and investigating the relationship with microbiological clearance amongst MSM. Methods: From 2017 to 2021, genital (urine) and extragenital (pharyngeal and anorectal swabs) biological specimens were provided by men-who-have-sex-with-men (MSM) attending the STI clinic of the Infectious Disease Unit at the Verona University Hospital, Verona, Italy. A total of 1040 MSM were evaluated and 107 samples from 96 subjects resulted positive for MG. Among the MG-positive samples, all those available for further analysis (n=47) were considered for detection of mutations known to be associated with macrolide and quinolone resistance. 23S rRNA, gyrA and parC genes were analyzed by Sanger sequencing and Allplex™ MG and AziR Assay (Seegene). Results: A total of 96/1040 (9.2%) subjects tested positive for MG in at least one anatomical site. MG was detected in 107 specimens: 33 urine samples, 72 rectal swabs and 2 pharyngeal swabs. Among them, 47 samples from 42 MSM were available for investigating the presence of mutations associated with macrolide and quinolone resistance: 30/47 (63.8%) showed mutations in 23S rRNA while 10/47 (21.3%) in parC or gyrA genes. All patients with positive Test of Cure (ToC) after first-line treatment with azithromycin (n=15) were infected with 23S rRNA-mutated MG strains. All patients undergoing second-line moxifloxacin treatment (n=13) resulted negative at ToC, even those carrying MG strains with mutations in parC gene (n=6). Conclusion: Our observations confirm that mutations in 23S rRNA gene are associated with azithromycin treatment failure and that mutations in parC gene alone are not always associated with phenotypic resistance to moxifloxacin. This reinforces the importance of macrolide resistance testing to guide the treatment and reduce antibiotic pressure on MG strains.

COVID-19 seroprevalence amongst healthcare workers: potential biases in estimating infection prevalence.

SARS-CoV-2 serological tests are used to assess the infection seroprevalence within a population. This study aims at assessing potential biases in estimating infection prevalence amongst healthcare workers (HCWs) when different diagnostic criteria are considered. A multi-site cross-sectional study was carried out in April-September 2020 amongst 1.367 Italian HCWs. SARS-CoV-2 prevalence was assessed using three diagnostic criteria: RT-PCR on nasopharyngeal swab, point-of-care fingerprick serological test (POCT) result and COVID-19 clinical pathognomonic presentation. A logistic regression model was used to estimate the probability of POCT-positive result in relation to the time since infection (RT-PCR positivity). Among 1.367 HCWs, 69.2% were working in COVID-19 units. Statistically significant differences in age, role and gender were observed between COVID-19/non-COVID-19 units. Prevalence of SARS-CoV-2 infection varied according to the criterion considered: 6.7% for POCT, 8.1% for RT-PCR, 10.0% for either POCT or RT-PCR, 9.6% for infection pathognomonic clinical presentation and 17.6% when at least one of the previous criteria was present. The probability of POCT-positive result decreased by 1.1% every 10 days from the infection. This study highlights potential biases in estimating SARS-CoV-2 point-prevalence data according to the criteria used. Although informative on infection susceptibility and herd immunity level, POCT serological tests are not the best predictors of previous COVID-19 infections for public health monitoring programmes.

Populations size estimations using SS-PSE among MSM in four European cities: how many MSM are living with HIV?

BACKGROUND: Although men who have sex with men (MSM) are considered at high risk for transmission of sexually transmitted infections, including HIV, there are few studies estimating the population size of MSM in Europe. We used network data from a survey of MSM in four cities to perform successive sampling-population size estimations (SS-PSE) to estimate MSM population sizes. METHODS: Data were collected in 2013-14 in Bratislava, Bucharest, Verona and Vilnius using respondent-driven sampling (RDS). SS-PSE uses a Bayesian framework to approximate the RDS sampling structure via a successive sampling model and uses the selection order of the sample to provide information about the distribution of network sizes over the population members of MSM. RESULTS: We estimate roughly 4600 MSM in Bratislava, 25 300 MSM in Bucharest, 7200 in Verona and 2900 in Vilnius. This represents 2.9% of the estimated adult male population in Bratislava, 2.3% in Bucharest, 2.7% in Verona and 1.5% in Vilnius. The number of MSM living with HIV would roughly be 200 in Bratislava, 4554 in Bucharest, 690 in Verona and 100 in Vilnius. CONCLUSIONS: Benefits of this method are that no additional information from an RDS survey needs to be collected, that the sizes can be calculated ex post facto a survey and that there is a software programme that can run the SS-PSE models. However, this method relies on having reliable priors. Although many countries are estimating the sizes of their vulnerable populations, European countries have yet to incorporate similar and novel methods.

The serological prevalence of SARS-CoV-2 infection in patients with chronic myeloid leukemia is similar to that in the general population.

BACKGROUND: Patients with hematological malignancies are at an increased risk of SARS-CoV-2 disease (COVID-19) and adverse outcome. However, a low mortality rate has been reported in patients with chronic myeloid leukemia (CML). Preclinical evidence suggests that tyrosine kinase inhibitors (TKIs) may have a protective role against severe COVID-19. METHODS: We conducted a cross-sectional study of 564 consecutive patients with CML who were tested for anti-SARS-CoV-2 IgG/IgM antibodies at their first outpatient visit between May and early November 2020 in five hematologic centers representative of three Italian regions. RESULTS: The estimated serological prevalence of SARS-CoV-2 infection in patients with CML after the first pandemic wave was similar to that in the general population (about 2%), both at national and regional levels. CML patients with positive anti-SARS-CoV-2 serology were more frequently male (p = 0.027) and active workers (p = 0.012), while there was no significant association with TKI treatment type. Only 3 out of 11 IgG-positive patients had previously received a molecular diagnosis of COVID-19, while the remainders were asymptomatic or with mild symptoms. CONCLUSIONS: Our data confirm that the course of SARS-CoV-2 infection in patients with CML is generally mild and reassure about the safety of continuing TKIs during the COVID-19 pandemic. Furthermore, we suggest that patients with CML succeed to mount an antibody response after exposure to SARS-CoV-2, similar to the general population.

A Review of Vaccinations in Adult Patients with Secondary Immunodeficiency.

Vaccine-preventable diseases and their related complications are associated with increased morbidity and mortality in patients with altered immunocompetence. Optimised immunisation in this patient population is challenging because of limited data from vaccine trials, suboptimal vaccine efficacy and safety concerns. Reliable efficacy data are lacking among patients with altered immunocompetence, and existing recommendations are mainly based on expert consensus and may vary geographically. Inactivated vaccines can be generally used without risks in this group, but their efficacy may be reduced, and immunisation schedules vary according to local guidelines, age, and type and stage of the underlying disease. Live vaccines, if indicated, should be administered with care because of the risk of vaccine-associated disease. We have reviewed the current evidence on vaccination principles and recommendations in adult patients with secondary immunodeficiencies, including asplenia, HIV infection, stem cell and solid organ transplant, haematological malignancies, inflammatory bowel disease and other chronic disorders.

Estimating the percentage of European MSM eligible for PrEP: insights from a bio-behavioural survey in thirteen cities.

OBJECTIVES: This paper aims to estimate the percentage of European men who have sex with men (MSM) who may benefit from pre-exposure prophylaxis (PrEP), applying the three most widely used HIV risk indices for MSM (MSM Risk Index, Menza score, San Diego Early Test (SDET) score) and drawing on a large-scale multisite bio-behavioural survey (Sialon II). METHODS: The Sialon II study was a bio-behavioural survey among MSM implemented in 13 European cities using either time-location sampling or respondent-driven sampling. Biological and behavioural data from 4901 MSM were collected. Only behavioural data of HIV-negative individuals were considered. Three widely used risk indices to assess HIV acquisition risk among MSM were used to estimate individual HIV risk scores and PrEP eligibility criteria. RESULTS: 4219 HIV-negative MSM were considered. Regardless the HIV risk score used and the city, percentages of MSM eligible for PrEP were found to range between 5.19% and 73.84%. Overall, the MSM Risk Index and the Menza score yielded broadly similar percentages, whereas the SDET Index provided estimates constantly lower across all cities. Although all the three scores correlated positively (r>0.6), their concordance was highly variable (0.01

Nocardia infection over 5 years (2011-2015) in an Italian tertiary care hospital.

This study was conducted reviewing clinical records of 14 patients affected by nocardiosis over 5 years in a tertiary care hospital. Nocardia abscessus was responsible for one third of infections, deviating significantly from the results reported by other epidemiological investigations and highlighting the key role of molecular identification tests. Indeed, a precise identification of species is crucial for the determination of antibiotic sensitivity patterns and, consequently, for the choice of antibiotic treatment. Noteworthy, 40% of isolates of N. abscessus (formerly N. asteroides complex) showed resistance to carbapenems, which are usually recommended for empirical therapy.

The treatment of latent TB infection in HIV-positive people: the Verona experience.

Not available.

Symptomatic congenital Cytomegalovirus deafness: the impact of a six-week course of antiviral treatment on hearing improvement.

Congenital Cytomegalovirus infection is the leading non-genetic cause of neurosensory deafness. We compared the outcomes of a treated group of children to an untreated group. The effect of antiviral therapy on hearing improvement between baseline and 2-year follow-up was statistically significant. These results suggest that the benefit of 6-week therapy is not limited to preventing further hearing deterioration.

Field evaluation of two point-of-care tests for syphilis among men who have sex with men, Verona, Italy.

OBJECTIVES: The incidence of HIV and syphilis among men who have sex with men (MSM) in Europe has recently increased. Rapid point-of-care tests (POCTs) for syphilis can improve access to screening. The purpose of this study was to evaluate the performance of two syphilis POCTs compared with laboratory tests among MSM. METHODS: The study was undertaken in Verona, Italy. Asymptomatic MSM, potentially exposed to syphilis, were enrolled prospectively. The POCTs evaluated were SD Bioline Syphilis 3.0 and Chembio DPP Syphilis Screen & Confirm Assay on both serum and fingerprick blood. The results of the POCTs were read by the naked eye by two independent readers and their concordance assessed. RESULTS: A total of 289 MSM were enrolled in the study. Based on laboratory tests, 35 MSM (12.1%) were TPPA-positive alone and 16 (5.5%) were both Treponema pallidum particle agglutination test (TPPA) and rapid plasma reagin (RPR)-positive. The specificities of both POCTs were above 99% on both serum and fingerstick blood specimens, while sensitivities varied considerably. The sensitivity of the SD Bioline test was lower on fingerprick blood (51.4% and 54.3%, readers 1 and 2, respectively) compared with that on serum (80.0% and 82.9%). In contrast, the Chembio test exhibited similar sensitivity values for serum and fingerprick samples (57.7% and 64.0% on serum vs 65.4% and 69.2% on fingerprick for the treponemal component; 63.6% on both samples by both readers for the non-treponemal component). The positive predictive value ranged between 100% and 93.9% for the treponemal component of both syphilis POCTs, but was lower (76.3%-100%)%) for the non-treponemal component of the Chembio POCT. The negative predictive value surpassed 90% for both tests on both samples. The agreement between readers was very high (>99%). CONCLUSION: The diagnostic performance of the syphilis POCTs was lower than expected; however, considering the prevalence of syphilis among MSM, POCTs should be recommended to improve syphilis detection among MSM.

Disseminated Mycobacterium avium complex disease in a patient with left ventricular assist device (Heart Mate II).

Although disseminated Mycobacterium avium complex disease occurs mainly in immunocompromised hosts, especially HIV-infected patients in the last stage of the disease (AIDS), this condition is still rare in immunocompetent subjects. We report the case of a Caucasian man who received a left ventricular assist device two years before as a bridge to heart transplantation, that began to present signs and symptoms of mycobacterial infection. The diagnostic work-up we performed showed the presence of Mycobacterium intracellulare in lungs and both peripherical and bone marrow blood. Although evaluated, we found no abnormalities in the patient's immune system that can be related to mycobacterial infection. The beginning of a specific therapy made the patient slowly improve and further nuclear medicine assay (PET-TC) showed a good reduction in radio-labelled drug captation.