Trends of Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates Between 2012 and 2023: Results From an Open Italian Cohort.

BACKGROUND: Neisseria gonorrhoeae (Ng) is a public health priority because of the rapid evolution of antimicrobial resistance, the emergence of antibiotic resistance, and the absence of a vaccine against Ng. The aim of this study was to investigate trends in the minimum inhibitory concentration and resistance (R) or reduced susceptibility (DS) of Ng cases to ceftriaxone (CRO), azithromycin (AZM), tetracycline (TET), benzylpenicillin (PenG), and ciprofloxacin (CIP) during a 10-year period. METHODS: We conducted a retrospective analysis on an open cohort of Ng cases diagnosed on rectal, urethral, and pharyngeal samples at San Raffaele Scientific Institute, between September 2012 and February 2023. Minimum inhibitory concentrations of antibiotics were determined by gradient-test strips. Bivariate linear regression models were applied on logarithmic minimum inhibitory concentrations values; Cochran-Armitage test was used to determine a linear trend in the proportions of resistant strains. RESULTS: A total of 436 Ng isolates from 352 individuals were analyzed. Minimum inhibitory concentrations of CRO and PenG reduced over time ( P < 0.001, P = 0.030), AZM increased ( P = 0.001), and CIP and TET did not change ( P = 0.473, P = 0.272). The percentages of resistant strains were as follows: PenG, 89.9%; TET, 90.8%; CIP, 48.2%; AZM, and 4.4%. CRO-DS strains were 8.7%, and only 1 case of CRO-R was identified. The proportion of resistant strains increased over time for AZM ( P = 0.007), TET ( P = 0.001), and CIP ( P < 0.001), whereas it decreased for PenG ( P < 0.001) and CRO-DS/R strains ( P < 0.001). CONCLUSIONS: Ng strains showed high susceptibility to CRO, although we identified cases of DS/R and observed high levels of susceptibility to AZM. Overall, the recommended primary regimen for Ng treatment was confirmed to be effective.

Outcome of Darunavir-Cobicistat-Based Regimens in HIV-Infected People Who Have Experienced Virological Failure.

Objective: To evaluate the virological outcome of darunavir-cobicistat (DRVc)-based regimens in adults living with HIV who had experienced virological failure (VF) on any previous drug combination. Methods: This was a retrospective cohort study (CSLHIV Cohort) of adults living with HIV who started a DRVc-based regimen with HIV-RNA >50 copies/mL after VF on any previous drug combination. Data on demographics, antiretroviral treatment since HIV diagnosis, and immunological and metabolic parameters from baseline (start of DRVc) to 48 weeks were analyzed in order to assess the cumulative proportion of those who achieved virological success (VS), defined as at least one instance of HIV-RNA <50 copies/mL within 12 months from baseline. Follow-up lasted from the start of the DRVc-based regimen (baseline) to the first instance of HIV-RNA <50 copies/mL, last available visit, or loss to follow-up or death, whichever occurred first. Univariate and multivariate Cox proportional-hazard regression models were used to identify baseline factors associated with VS. Results: A total of 176 individuals were included, and 120 (68.2%) achieved <50 HIV-RNA copies/mL within 12 months since baseline. On multivariate analysis, baseline HDL cholesterol was independently associated with the occurrence of VS (adjusted HR 1.021, 95% CI 1.004-1.038; p=0.014). Among the 120 subjects with VS, 27 (22.5%) had had VF during a median follow-up of 20.8 months since the first undetectable HIV-RNA. Resistance testing after VF was available in two cases, which harboured the HIV variant-bearing protease inhibitor-resistance mutations D30N, I50V, and N88D. During a median follow-up of 38.4 months, 65 of 176 (36.9%) individuals discontinued DRVc for any reason (37 of 120, 30.8%) and achieved VS vs. 28 of 56 (50%) without VS (p=0.019). Time to discontinuation was longer in people with VS (41.5 vs. 23.0 months, p=0.0007). No statistically significant changes were observed in immunological or lipid profiles during follow-up. Conclusion: Most individuals in this study achieved VS within 12 months from the beginning of a DRVc-based regimen; therefore, this treatment represent a viable option for people who have experienced VF on other regimens.

Otitis in patients with Community-Acquired Bacterial Meningitis: A Nationwide Prospective Cohort Study.

BACKGROUND: Otitis is commonly associated with community-acquired bacterial meningitis but role of ear surgery as treatment is debated. In this study, we investigated the impact of otitis and ear surgery on outcome of adults with community-acquired bacterial meningitis. METHODS: We analyzed episodes of adults with community-acquired bacterial meningitis from a nationwide prospective cohort study in the Netherlands, between March 2006 to July 2021. RESULTS: A total of 2,548 episodes of community-acquired bacterial meningitis were evaluated. Otitis was present in 696 episodes (27%). In these patients the primary causative pathogen was Streptococcus pneumoniae (615 of 696 [88%]), followed by Streptococcus pyogenes (5%) and Haemophilus influenzae (4%). In 519 of 632 otitis episodes (82%) an ear-nose-throat specialist was consulted, and surgery was performed in 287 of 519 (55%). The types of surgery performed were myringotomy with ventilation tube insertion in 110 of 287 episodes (38%), mastoidectomy in 103 of 287 (36%) and myringotomy alone in 74 of 287 (26%). Unfavorable outcome occurred in 210 of 696 episodes (30%) and in 65 of 696 episodes was fatal (9%). Otitis was associated with a favorable outcome in a multivariable analysis (odds ratio 0.74; 95% CI 0.59-0.92; p =0.008). There was no association between outcome and ear surgery. CONCLUSIONS: Otitis is a common focus of infection in community-acquired bacterial meningitis in adults, with S. pneumoniae being the most common causative pathogen. Presence of otitis is associated with a favorable outcome. Ear surgery's impact on the outcome of otogenic meningitis patients remains uncertain.

Factors Associated with Low-Level Viremia in People Living with HIV in the Italian Antiviral Response Cohort Analysis Cohort: A Case-Control Study.

Despite effective antiretroviral therapies (ARTs), a subset of people living with HIV (PLWH) still experience low-level viremia (LLV, i.e., 50-1,000 copies/mL). The present study compared PLWH experiencing LLV with those maintaining virological suppression (VS) and explored the potential impact of preexisting drug resistance and other factors on LLV. We conducted a retrospective, 1:1 matched case-control study within a cohort of drug-experienced VS subjects from the Italian Antiviral Response Cohort Analysis database, followed in the period 2009-2019. Cases were individuals experiencing LLV, while controls were those who maintained VS. Matching was for calendar year of first ART regimen. Preexisting drug resistance was calculated as cumulative genotypic susceptibility score (GSS) according to regimen administered at the observational period start. To explore the effect of cumulative GSS, treated as a binary variable (≥2 and <2) and other factors on LLV, we performed a logistic regression analysis. Within a main population of 3,455 PLWH, 337 cases were selected. Cases were comparable to the controls for both gender and age. However, cases showed that they had experienced a longer time since HIV diagnosis, a higher number of drugs previously administered, lower baseline CD4+ T cell count and a higher zenith viral load (VL). By multivariate analysis, we found that higher zenith VL [adjusted odds ratio (aOR) (95% confidence interval [CI]) 1.30 (1.14-1.48)], a cumulative usage of both PI [aOR (95% CI): 2.03 (1.19-3.48)] and InSTI [aOR (95% CI): 2.23 (1.47-3.38)] and a cumulative GSS <2 [aOR (95% CI) 0.67 (0.46-0.98)], were associated with a higher risk in developing LLV. In current high-efficacy ART era, in drug-experienced PLWH, the predictors of increased risk of LLV were the presence of preexisting drug resistance, higher zenith VL, and previous PI, and InSTI exposure.

Bictegravir/Emtricitabine/Tenofovir Alafenamide Treatment: Efficacy and Tolerability in Clinical Practice.

Objective: Analysis of bictegravir/emtricitabine/tenofovir alafenamide (BFTAF) efficacy and safety in virologically suppressed people living with HIV (PLWH) in clinical practice. Patients and methods: The retrospective cohort study, which included adult treatment-experienced and virologically suppressed PLWH, switched to BFTAF from June 2019 to June 2021. Efficacy and safety were evaluated as virological failure (VF=2 consecutive HIV-RNA>50 copies/mL or a single HIV-RNA>400 copies/mL) and treatment failure (TF=VF or discontinuation for any reason) until data freezing (August 2022). Results: Of the 1040 PLWH included, 67.8% switched from elvitegravir/cobicistat/FTAF. VF occurred in 4.2% (n=44), with incidence rate of 1.63 per 1000 person-months of follow-up (PMFU) and probability at 24-30 months of 3.8%-4.0%, respectively. Out of the 44 VF, in 75% virological re-suppression was achieved while maintaining BFTAF. Discontinuation occurred in 15% after a median time of 13.5 months of follow-up, with an incidence rate of 5.67 per 1000 PMFU, and a probability at 24-30 months of 11.9%-15.3%, respectively. Main discontinuation reasons were simplification (51.3%) and toxicity (21.8%, involving CNS in half of cases). TF occurred in 18.6% with an incidence rate of 7.01 per 1000 PMFU after a median time of 13.6 observation months; probability at 24-30 months was 14.8%-18.4%, respectively. Conclusion: BFTAF has proven effective and well tolerated in clinical practice.

Neisseria gonorrhoeae Antimicrobial Resistance: The Future of Antibiotic Therapy.

The growing threat of antibiotic-resistant Neisseria gonorrhoeae, which causes gonorrhea, presents a current public health challenge. Over the years, the pathogen has developed resistance to different antibiotics, leaving few effective treatment options. High-level resistance to key drugs, including ceftriaxone, has become a concerning reality. This article primarily focuses on the treatment of gonorrhea and the current clinical trials aimed at providing new antibiotic treatment options. We explore ongoing efforts to assess new antibiotics, including zoliflodacin, and gepotidacin. These drugs offer new effective treatment options, but their rapid availability remains uncertain. We delve into two ongoing clinical trials: one evaluating the efficacy and safety of gepotidacin compared to the standard ceftriaxone-azithromycin combination and the other assessing the non-inferiority of zoliflodacin versus the combination therapy of ceftriaxone-azithromycin. These trials represent crucial steps in the search for alternative treatments for uncomplicated gonorrhea. Notably, gonorrhea has been included in the "WHO Priority Pathogens List for Research and Development of New Antibiotics". In conclusion, the urgent need for innovative treatment strategies is underscored by the rising threat of antibiotic resistance in N. gonorrhoeae; collaboration among researchers, industries, and healthcare authorities is therefore essential.

Risk of mortality in people with chronic liver diseases hospitalized for Coronavirus disease of 2019 (COVID-19) in a tertiary hospital in Lombardy, Italy.

The impact of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection on patients with pre-existing chronic liver diseases (CLD) remains elusive. The aim of this study was to investigate the in-hospital mortality in patients hospitalized for Coronavirus disease of 2019 (COVID-19) with CLD (CLD group) compared to those without CLD (non-CLD group). We performed a retrospective cohort study including patients with confirmed SARS-CoV-2 infection, hospitalized at San Raffaele Hospital (Milan), stratified according to the presence or absence of CLD. A propensity score was estimated and used to match the two groups by age, gender, body mass index, type 2 diabetes mellitus, and hypertension. Predictors of mortality were assessed using univariate and multivariate logistic regression model. Among 1210 patients with COVID-19, 41 (3.4%) were included in the CLD group and 1169 (96.6%) in the non-CLD group. Using a propensity score, we matched 41 patients in the CLD group with 123 in the non-CLD group. At admission, patients in the CLD group had worse liver function, lower platelets count, and lower c-reactive protein levels. By multivariate analysis, the CLD group showed a higher risk of death: OR 4.04 (95% CI 1.29-12.70; p= 0.017). Our study showed that COVID-19 with chronic liver diseases has a higher risk of mortality during hospitalization.

Meningococcus B Vaccination Effectiveness Against Neisseria gonorrhoeae Infection in People Living With HIV: A Case-Control Study.

BACKGROUND: We assessed the vaccination effectiveness (VE) of multicomponent meningococcal serogroup B (4CMenB) vaccine against gonorrhea among people living with HIV (PLWH) with a previous diagnosis of sexually transmitted infection. METHODS: Unmatched case-control study on men who have sex with men living with HIV, in care at San Raffaele Scientific Institute, Milan, Italy, with gonorrhea, syphilis, chlamydia, or anal human papillomavirus between July 2016 (beginning of 4CMenB vaccination) and February 2021 (date of freezing). For the analysis, cases were people with ≥1 gonorrhea infection since July 2016, and controls were people with ≥1 syphilis, chlamydia, or anal human papillomavirus infection since July 2016. Logistic regression was used to provide the estimate of 4CMenB VE against gonorrhea. RESULTS: Included people living with HIV were 1051 (103 cases, 948 controls); 349 of 1051 (33%) received 2 doses of 4CMenB vaccination. The median follow-up was 3.8 years (2.1-4.3 years). The unadjusted estimate for VE against gonorrhea was 42% (95% confidence interval, 6%-64%; P = 0.027). Logistic regression showed that VE against gonorrhea remained significant (44%; 95% confidence interval, 9%-65%; P = 0.020) after adjusting for some factors that might have a potential influence on VE or those with significant unbalanced distributions between cases and controls at univariable analysis. CONCLUSIONS: 4CMenB vaccination is associated with a lower risk of gonorrhea in the setting of men who have sex with men living with HIV with a previous sexually transmitted infection.

Switching from a Non-Protease inhibitor-Based Regimen To the Fixed Dose Combination of Darunavir/Cobicistat/Emtricitabine/Tenofovir Alafenamide in Clinical Practice.

Background: The primary objective of this study was to estimate the proportion of people living with HIV (PLWH) who switched from a non-protease inhibitor (PI)-based regimen [integrase strand transfer inhibitor (InSTI)-based or non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen] to darunavir, cobicistat, emtricitabine, tenofovir alafenamide (D/C/F/TAF). Methods: This was a retrospective study on PLWH treated with a non-PI regimen in January 2017, who switched to D/C/F/TAF or to another antiretroviral therapy (ART) within November 2019. Follow-up was from the start date of D/C/F/TAF until the last available visit or discontinuation for any reason of this regimen. Virological failure (VF) was defined as 2 consecutive HIV-RNA values >50 copies/mL. Characteristics were reported as median (interquartile range) or frequency (%). A univariate Poisson regression model was used to measure the incidence rate of switch to D/C/F/TAF. Changes in laboratory parameters during D/C/F/TAF were assessed by univariate mixed linear models. Results: Overall, 3076 PLWH were included; 83% were male, median age at ART switch was 50 (42-56) years and median time on ART was 5.2 (0.3-13.0) years. PLWH had a median follow-up of 4.76 (3.70-6.38) years; during 17,099 person-years of follow-up (PYFU), 423/3076 (14%) participants discontinued the non-PI-based regimen and 106/423 (25%) switched to D/C/F/TAF, with an overall incidence rate of switch to D/C/F/TAF of 6.2 per 1000-PYFU (95% CI: 5.0-7.4). Among PLWH who switched to D/C/F/TAF, the ongoing regimen was based on NNRTIs in 37 (35%) and on InSTIs in 69 (65%). Main reasons leading to switch to D/C/F/TAF included neuropsychiatric adverse events (37%), VF (26%) and Kaposi sarcoma progression (5%). Conclusion: In the last years, a non-negligible proportion of patients on an NNRTI- or an InSTI-based regimen switched to D/C/F/TAF.

Levels of Alpha-Fetoprotein and Association with Mortality in Hepatocellular Carcinoma of HIV-1-Infected Patients.

METHODS: This is a retrospective cohort study on patients living with HIV-1 infection (PLWH) followed at the Division of Infectious Diseases of the San Raffaele Hospital, with cirrhosis and HCC diagnosed between 1999 and 2018 and with an available AFP value at HCC diagnosis. The area under the receiver operating characteristic curve (AUC) was used to estimate the accuracy of baseline AFP in predicting death. Factors associated with the risk of death were identified using multivariable Cox proportional-hazards regression models. RESULTS: Overall, 53 PLWH were evaluated: 18 patients received a curative treatment (9 liver transplantation, 5 liver resections and 4 radiofrequency ablation) and 35 a noncurative treatment (17 chemo or radio embolization, 10 sorafenib and 8 best supportive care). Baseline AFP was predictive of death [AUC 0.71, 95% confidence interval (CI) 0.54-0.83], and the optimal cut-off was 28.8 ng/mL. At multivariable analysis, BL AFP ≥28.8 ng/mL was associated with death [adjusted hazard ratio (aHR) 7.05, 95% CI 1.94-25.71 P = 0.003]. Other factors were HBV infection (aHR 8.57, 95% CI 1.47-50.08, P = 0.017) and treatment allocation (curative vs. noncurative, aHR 0.08, 95% CI 0.02-0.40, P = 0.0004). CONCLUSIONS: Our findings suggest that in PLWH AFP serum levels ≥28.8 ng/mL, HBV coinfection and treatment allocation represent predictive markers for death at the time of HCC diagnosis.

Neutralizing monoclonal antibodies for the treatment and prophylaxis of SARS-CoV-2 infection.

Neutralizing monoclonal antibody therapies against the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) play a significant role both in the prevention and treatment of the coronavirus disease 2019 (COVID-19). In this review we discuss the monoclonal antibody therapies that have received preliminary authorization for use in COVID-19 patients by the U.S. Food and Drug Administration or the European Medicines Agency. We review here their mechanisms of action, their efficacy in prophylaxis and treatment, their indications for use, and the impact of SARS-CoV-2 variants of concern on their activity.

Symptoms and signs of conjunctivitis as predictors of disease course in COVID-19 syndrome.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can induce conjunctivitis signs and symptoms. However, limited information is available on their impact on COVID-19 disease phenotype. Quantification of ocular signs/symptoms can provide a rapid, non-invasive proxy for predicting clinical phenotype. Moreover, the existence and entity of conjunctival viral shedding is still debated. This has relevant implications to manage disease spread. The purpose of this study was to investigate conjunctivitis signs and symptoms and their correlation with clinical parameters, conjunctival viral shedding in patients with COVID-19. METHODS: Fifty-three patients hospitalized between February 25th and September 16th, 2020 at the San Raffaele Hospital, in Milan, Lombardy, Italy with a confirmed diagnosis of SARS-CoV-2 were evaluated. Presence of interstitial pneumonia was confirmed with computed tomography scan imaging. Ocular signs and symptoms, anosmia/ageusia, clinical/laboratory parameters, and reverse transcriptase-polymerase chain reaction (RT-PCR) from nasopharyngeal and conjunctival swabs for COVID-19 virus were analyzed. RESULTS: Forty-six out of 53 patients showed a positive nasopharyngeal swab for SARS-CoV-2 infection at the time of conjunctival evaluation. All the conjunctival swabs were negative. Conjunctivitis symptoms were present in 37% of patients. Physician-assessed ocular signs were detected in 28% of patients. Patients with ocular symptoms or signs tended to be older: 76.8 years (62.4-83.3) vs 57.2 years (48.1-74.0), p = 0.062 and had a longer hospitalization: 38 days (18-49) vs. 14 days (11-21), p = 0.005. Plasma levels of Interleukin-6 were higher in patients with signs or symptoms in comparison with those without them: 43.5 pg/ml (19.7-49.4) vs. 8 pg/ml (3.6-20.7), p = 0.02. Red cell distribution width was also significantly higher: 15 (14.3-16.7) vs 13.2 (12.4-14.4), p = 0.001. CONCLUSIONS: We found that over a third of the patients had ocular signs or symptoms. These had higher prevalence in patients with a more severe infection. No viral shedding was detected in the conjunctiva. Our results suggest that prompt detection of conjunctivitis signs/symptoms can serve as a helpful proxy to predict COVID-19 clinical phenotype.

Serum IgG1 and IgG4 could contribute to partial control of viral rebound in chronically HIV-1-infected patients.

OBJECTIVES: Few studies have investigated chronically infected individuals after antiretroviral therapy (ART) interruption (ATI, analytical therapy interruption); thus, we investigated the association between some HIV-specific antibodies and viral control. DESIGN: All enrolled patients were previously described in the APACHE study. Briefly, the study was conducted on HIV-1 chronically infected patients, with HIV-RNA less than 50 copies/ml for at least 10 years, CD4+ cell count greater than 500 cells/µl and HIV-DNA less than 100 copies/106 PBMC. The ART regimen in use at the time of ATI was resumed at confirmed viral rebound (CVR, defined as two consecutive HIV-RNA >50 copies/ml). METHODS: Collection of sera and analysis of both binding antibodies (BAbs) and neutralizing antibodies (NAbs) was performed at three different time points: ATI, CVR and time of viral re-suppression after ART resumption. RESULTS: IgG subclasses (IgG1, IgG2, IgG3 and IgG4) from the four patients with highest levels of neutralization were found to block viral infection. All patients had CVR after ATI at a median time of 21 days (14-56). After ART resumption, all the enrolled patients achieved HIV-RNA less than 50 copies/ml in 42 days (21-98). We observed a strong increase of either BAbs and NAbs titers from ATI to viral re-suppression in one patient, who showed the longest period of virus undetectability during ATI. In this patient, BAbs and NAbs specifically belonged to both IgG1 and IgG4 subclasses, directed to env antigen. CONCLUSION: env-specific NAbs and BAbs belonging to IgG1, IgG4 subclasses could be helpful to monitor long-term responses able to control virus replication and eradicate HIV infection.

Virological and Serological Discordant Profiles in COVID-19 Pneumonia: Two Atypical Clinical Cases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is primarily diagnosed through viral RNA positivity in nasopharyngeal swabs, and it is associated with the early detection of specific immunoglobulins to SARS-CoV-2 proteins. We describe two moderate coronavirus disease 2019 (COVID-19) patients with WHO score 4/5 at the time of hospitalization, pneumonia, and oxygen saturation <94% and with a strong discrepancy between viral RNA and antibodies to SARS-CoV-2. One patient was positive for viral RNA but completely negative for binding and neutralizing antibodies, whereas the second patient was negative for viral RNA but with high levels of both neutralizing and binding antibodies. This observation is relevant to better understand the pathogenesis of this novel infection.

SARS-CoV-2 IgG/IgM Rapid Test as a Diagnostic Tool in Hospitalized Patients and Healthcare Workers, at a large Teaching Hospital in northern Italy, during the 2020 COVID-19 Pandemic.

We describe the outcome of a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) IgG/IgM rapid test, and discuss the potential suitability of antibody testing. Retrospective single cohort study on patients with suspected Coronavirus Disease 2019 (COVID-19) and asymptomatic Healthcare Workers, enrolled from March to April 2020. Subjects had quantitative PCR (qPCR) test for detection of SARS-CoV-2 via nasal swab and serological testing using the COVID-19 IgG/ IgM Rapid Test (PRIMA Lab SA) immunochromatographic assay. Some subjects underwent chemiluminescence immunoassay (CLIA) after rapid test. The aim of the study was to analyse the proportion of those who developed a positive IgM/IgG response for SARS-CoV-2. The correspondence between the results from rapid testing and CLIA, when available, was evaluated. 97 subjects underwent qPCR for SARS-CoV-2 through nasal swab, which resulted positive in 40/43 (93.0%) of symptomatic patients, 2/40 (5%) of asymptomatic HCW, in no subjects with suspected COVID- 19 (clinical and radiological findings) then excluded by repeated nasal swabs and alternative diagnosis (COVID-19-negative patients, CNPs), and in 6/6 (100%) of patients with confirmed diagnosis and negative follow-up nasal swabs (COVID-19-recovered patients, CRPs). IgM resulted positive in 8/43 (18.6%) of symptomatic patients and in 1/6 (16.7%) of CRPs. IgG resulted positive in 36/43 (83.7%) of symptomatic patients, 2/40 (5%) of HCW, and in 1/8 (12.5%) and 6/6 (100%) of CNPs and CRPs, respectively. A comparison between an IgG/IgM Rapid Test and a following CLIA test showed consistency in negative results in 25/28 of HCW and 8/8 of CNPs tested. Our preliminary data support the role of IgG/IgM Rapid Test (PRIMA Lab SA) immunochromatographic assay as a point-of-care test that may complement molecular tests in the screening of SARS-CoV-2 carriers. The test may gain particular relevance in shortening the time needed to refer patients to a COVID or non-COVID Hospital area and to achieve diagnosis in patients with persistently negative nasal swabs.