ABSTRACT
BACKGROUND AND OBJECTIVE: The SARS-CoV-2 Omicron variant displays increased infectiveness as well as mutations resulting in reduced neutralizing activity of antibodies acquired after vaccination or infection involving earlier strains. To assess the ability of vaccinated COVID-19 convalescent plasma (CCP-V) collected before November 2021 to seroneutralize Omicron, we compared neutralizing antibody (nAb) titres of 63 samples against Omicron and earlier B.1 (D614G) strains. METHODS AND FINDINGS: Relationship between anti-Omicron titres and IgG anti-S1 levels (binding arbitrary unit: BAU/ml) was studied. Although correlated, anti-Omicron titres were significantly lower than anti-B.1 titres (median = 80 [10-1280] vs. 1280 [160-10,240], p < 0.0001). Omicron nAb titres and IgG anti-S1 levels were correlated (Spearman's rank correlation coefficient = 0.67). Anti-S1 IgG threshold at 7000 BAU/ml may allow to discard CCP-V without anti-Omicron activity (nAb titre <40). Conversely, only those with highest titres (≥160) had systematically anti-S1 IgG levels >7000 BAU/ml. CONCLUSION: A fraction of CCP-V collected before November 2021 retains anti-Omicron seroneutralizing activity that may be selected by quantitative anti-IgG assays, but such assays do not easily allow the identification of 'high-titre' CCP-V. However, collecting plasma from vaccinated donors recently infected with Omicron may be the best option to provide optimal CCP-V for immunocompromised patients infected with this variant.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , Humans , Immunization, Passive , COVID-19 SerotherapyABSTRACT
The circulation of Zika virus (ZIKV) in Mali has not been clearly characterized. Therefore, we conducted a serologic survey of 793 asymptomatic volunteers >15 years of age (2016), and 637 blood donors (2013) to assess the seroprevalence of ZIKV infection in 2 ecoclimatic regions of Mali, tropical savannah and warm semiarid region, using ELISA and seroneutralization assays. The overall seroprevalence was ≈12% and increased with age, with no statistical difference between male and female participants. In the warm semiarid study sites we detected immunological markers of an outbreak that occurred in the late 1990s in 18% (95% CI 13%-23%) of participants. In tropical savannah sites, we estimated a low rate of endemic transmission, with 2.5% (95% CI 2.0%-3.1%) of population infected by ZIKV annually. These data demonstrate the circulation of ZIKV in Mali and provide evidence of a previously unidentified outbreak that occurred in the late 1990s.
Subject(s)
Zika Virus Infection , Zika Virus , Blood Donors , Female , Humans , Male , Mali/epidemiology , Seroepidemiologic Studies , Zika Virus Infection/epidemiologyABSTRACT
In the original publication of this article [1], Table 1 has some errors.
ABSTRACT
Here we propose a strategy allowing implementing efficient and practicable large-scale seroepidemiological studies for Zika Virus (ZIKV). It combines screening by a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (CPE-based VNT). In post-epidemic samples from Martinique Island blood donors (a population with a dengue seroprevalence above 90%), this strategy allowed reaching specificity and sensitivity values over 98%. The CPE-based VNT consists of recording CPE directly under the optical microscope, which is easy to identify with ZIKV strain H/PF/2013 at day 5 pi. Overall, considered that CPE-based VNT is cost effective and widely automatable, the NS1 protein-based Zika IgG ELISA+CPE-based VNT combination strategy represents a convenient tool to expedite ZIKV seroprevalence studies.
Subject(s)
Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay/methods , Mass Screening/methods , Neutralization Tests/methods , Serologic Tests/methods , Zika Virus Infection/diagnosis , Zika Virus/immunology , Antibodies, Neutralizing/blood , Cytopathogenic Effect, Viral , Humans , Immunoglobulin G/blood , Martinique/epidemiology , Microscopy , Sensitivity and Specificity , Seroepidemiologic Studies , Zika Virus Infection/epidemiologySubject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/therapy , COVID-19 Serotherapy , Plasma , Antibodies, Viral , Antibodies, Neutralizing , Neutralization TestsABSTRACT
Serosurveys to monitor immunity toward COVID-19 in the population are primarily performed using an ELISA to screen samples for SARS-CoV-2 antibodies, followed by confirmation by a virus neutralization test, which is considered the Gold Standard. However, virus neutralization test may not be feasible for some laboratories because of the requirement for specific facilities and trained personnel. In an attempt to address this limitation, we evaluated three cell-free methods as potential alternatives for assessing SARS-CoV-2 seroprevalence in human population from plasma. We report the establishment of two inhibition ELISAs designed to detect anti-Spike RBD IgG antibodies and a microsphere quantitative suspension array technology assay, based on the Luminex xMAP platform, to measure the presence of antibodies against various SARS-CoV-2 antigens, including anti-RBD. These methods were also compared to a commercial chemiluminescent immunoassay designed for anti-RBD antibodies detection and to the combined ELISA + virus neutralization test strategy. These cell-free assays performed equally to estimate the percentage of positive and negative samples and could be used to determine the prevalence of SARS-CoV-2 antibodies in human population, at least in cohort with high-expected prevalence, without the use of seroneutralization assay.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , COVID-19/epidemiology , SARS-CoV-2 , Seroepidemiologic Studies , Antibodies, Viral , Antigens, Viral , Antibodies, NeutralizingABSTRACT
Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with a mortality rate of up to 30%. First identified in China in 2009, it was later reported in other Asian countries, including Thailand in 2020. SFTSV has been detected in several tick species, including Rhipicephalus sanguineus, known for infesting dogs. We conducted a seroprevalence study of SFTSV in Bangkok and Nong Khai, Thailand, by analyzing 1162 human samples collected between 2019 and 2023. The testing method relied on IgG detection using ELISA and confirmed though a virus seroneutralization test. The results indicated that out of the participants, 12 (1.1%) tested positive for anti-SFTSV IgG antibodies; however, none exhibited positive results in the seroneutralization assay. Additionally, molecular detection of SFTSV, Crimean-Congo hemorrhagic fever (CCHF), Coxiella spp., Bartonella spp., and Rickettsia spp. was performed on 433 Rh. sanguineus ticks collected from 49 dogs in 2023 in Chachoengsao Province, Thailand. No evidence of these pathogens was found in ticks. These findings highlight the importance of exploring viral cross-reactivity. Furthermore, it is important to conduct additional studies to isolate SFTSV from animals and ticks in order to identify the potential transmission routes contributing to human and animal infections in Thailand.
Subject(s)
Phlebovirus , Rhipicephalus sanguineus , Severe Fever with Thrombocytopenia Syndrome , Animals , Thailand/epidemiology , Seroepidemiologic Studies , Rhipicephalus sanguineus/virology , Humans , Phlebovirus/genetics , Phlebovirus/immunology , Phlebovirus/isolation & purification , Middle Aged , Female , Male , Adult , Severe Fever with Thrombocytopenia Syndrome/epidemiology , Severe Fever with Thrombocytopenia Syndrome/virology , Severe Fever with Thrombocytopenia Syndrome/veterinary , Severe Fever with Thrombocytopenia Syndrome/diagnosis , Dogs , Aged , Adolescent , Antibodies, Viral/blood , Young Adult , Child , Child, Preschool , Aged, 80 and over , Infant , Immunoglobulin G/bloodABSTRACT
We conducted a cross-sectional study for SARS-CoV-2 anti-S1 IgG prevalence in French blood donors (n = 32605), from March-2020 to January-2021. A mathematical model combined seroprevalence with a daily number of hospital admissions to estimate the probability of hospitalization upon infection and determine the number of infections while correcting for antibody decay. There was an overall seroprevalence increase over the study period and we estimate that â¼15% of the French population had been infected by SARS-CoV-2 by January-2021. The infection/hospitalization ratio increased with age, from 0.31% (18-30yo) to 4.5% (61-70yo). Half of the IgG-S1 positive individuals had no detectable antibodies 4 to 5 months after infection. The seroprevalence in group O donors (7.43%) was lower (p = 0.003) than in A, B, and AB donors (8.90%). We conclude, based on seroprevalence data and mathematical modeling, that a large proportion of the French population was unprotected against severe disease prior to the vaccination campaign.
ABSTRACT
This study aimed to estimate the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection within the staff and student populations of the University of Corsica (France) during the second wave of the epidemic. METHODS: A cross-sectional survey was conducted from 23 November 2020 to 31 January 2021. The participants underwent blood sampling using a fingerstick procedure and completed an anonymized questionnaire. Sera were tested for the presence of anti-SARS-CoV-2 IgG (ELISA-S) and, if positive, with an in-house virus neutralization test (VNT). RESULTS: A total of 418 persons were included in the study. The overall seroprevalence was 12.8% (95% confidence interval (CI), 9.8-16.6%). A total of 15 (31%) of the 49 individuals who had a positive ELISA-S also had a positive VNT. Seropositivity was associated with living at the city campus during the week and on weekends (OR = 3.74 [1.40-12.00]), using public transportation/carpooling (OR = 2.00 [1.01-4.02]), and being in contact with a person who tested positive for SARS-CoV-2 (OR = 2.32 [1.20-4.40]). The main symptoms associated with seropositivity were "having had an acute respiratory infection" (OR = 3.05 [1.43-6.43]) and "experiencing loss of smell" (OR = 16.4 [5.87-50.7]). CONCLUSION: These results could be useful for SARS-CoV-2 prevention and control on university campuses.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Cross-Sectional Studies , Humans , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Uncertainties remain regarding the nature and durability of the humoral immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). AIM: This study investigated immunoglobulin G response and neutralizing activity to evaluate the mean antibody concentrations and response duration induced by each vaccination regimen in a French adult population. METHODS: A study including blood sampling and questionnaires was carried out from November 2020 to July 2021 with three separate follow-up phases. Spike proteins and neutralizing antibodies were quantified using ELISA and a virus-neutralization test. RESULTS: Overall, 295 participants were included. Seroprevalences were 11.5% (n = 34), 10.5% (n = 31), and 68.1% (n = 201) in phases 1, 2, and 3, respectively. Importantly, 5.8% (n = 17) of participants lost their natural antibodies. Antibody response of participants with only a prior infection was 88.2 BAU/mL, significantly lower than those vaccinated, which was 1909.3 BAU/mL (p = 0.04). Moreover, the antibody response of vaccinated participants with a prior infection was higher (3593.8 BAU/mL) than those vaccinated without prior infection (3402.9 BAU/mL) (p = 0.78). Vaccinated participants with or without prior infection had a higher seroneutralization rate (91.0%) than those unvaccinated with prior infection (65.0%). CONCLUSION: These results demonstrated that single infection does not confer effective protection against SARS-CoV-2.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Adult , Humans , Follow-Up Studies , SARS-CoV-2 , COVID-19/epidemiology , Antibodies, ViralABSTRACT
Healthcare workers (HCWs) are at increased risk of SARS-CoV-2 infection. The aim of the study was to estimate the SARS-CoV-2 seroprevalence among HCWs in Cochabamba, Bolivia and to determine the potential risk factors. In January 2021, a cross-sectional SARS-CoV-2 seroprevalence study was conducted in 783 volunteer clinical and non-clinical HCWs in tertiary care facilities. It was based on IgG detection using ELISA, chemiluminiscence, and seroneutralisation tests from dried blood spots. Analysis revealed a high seroprevalence (43.4%) of SARS-CoV-2 IgG antibodies. The combination of anosmia and ageusia (OR: 68.11; 95%-CI 24.83-186.80) was predictive of seropositivity. Belonging to the cleaning staff (OR: 1.94; 95%-CI 1.09-3.45), having more than two children in the same house (OR: 1.74; 95%-CI 1.12-2.71), and having been in contact with a close relative with COVID-19 (OR: 3.53; 95%-CI 2.24-5.58) were identified as risk factors for seropositivity in a multivariate analysis. A total of 47.5% of participants had received medication for COVID-19 treatment or prevention, and only ~50% of symptomatic subjects accessed PCR or antigenic testing. This study confirms a massive SARS-CoV-2 attack rate among HCWs in Cochabamba by the end of January 2021. The main risk factors identified are having a low-skilled job, living with children, and having been in contact with an infected relative in the household.
Subject(s)
Antibodies, Viral/blood , COVID-19/epidemiology , COVID-19/immunology , Health Personnel/statistics & numerical data , Adolescent , Adult , Antibodies, Viral/immunology , Bolivia/epidemiology , Cross-Sectional Studies , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Prevalence , Risk Factors , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Seroepidemiologic Studies , Tertiary Healthcare/statistics & numerical data , Young AdultABSTRACT
Soluble ACE2 (sACE2) decoys are promising agents to inhibit SARS-CoV-2, as their efficiency is unlikely to be affected by escape mutations. However, their success is limited by their relatively poor potency. To address this challenge, multimeric sACE2 consisting of SunTag or MoonTag systems is developed. These systems are extremely effective in neutralizing SARS-CoV-2 in pseudoviral systems and in clinical isolates, perform better than the dimeric or trimeric sACE2, and exhibit greater than 100-fold neutralization efficiency, compared to monomeric sACE2. SunTag or MoonTag fused to a more potent sACE2 (v1) achieves a sub-nanomolar IC50 , comparable with clinical monoclonal antibodies. Pseudoviruses bearing mutations for variants of concern, including delta and omicron, are also neutralized efficiently with multimeric sACE2. Finally, therapeutic treatment of sACE2(v1)-MoonTag provides protection against SARS-CoV-2 infection in an in vivo mouse model. Therefore, highly potent multimeric sACE2 may offer a promising treatment approach against SARS-CoV-2 infections.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 Drug Treatment , Animals , Antibodies, Monoclonal/therapeutic use , Mice , SARS-CoV-2ABSTRACT
PURPOSE: The COVID-19 vaccination campaign began in December 2020, in France, and primarily targeted the oldest people. Our study aimed to determine the level of acceptance of vaccination in a population of older patients with cancer. METHODS: From January 2021, we offered vaccination with the BNT162b2 COVID-19 vaccine to all patients 70 years and older referred to our geriatric oncology center in Marseille University Hospital (AP-HM) for geriatric assessment before initiation of an oncological treatment. Objectives were to evaluate acceptance rate of COVID-19 vaccination and to assess vaccine safety, reactogenicity, and efficacy two months after the first dose. RESULTS: Between January 18, 2021 and May 7, 2021, 150 older patients with cancer were offered vaccination after a geriatric assessment. The majority were men (61.3%), with a mean age of 81 years. The two most frequent primary tumors were digestive (29.4%) and thoracic (18%). The vaccine acceptance rate was 82.6% and the complete vaccination rate (2 doses) reached 75.3%. Among the vaccinated patients, 15.9% reported mild side effects after the first dose and 23.4% after the second dose, mostly arm pain and fatigue. COVID-19 cases were observed in 5.1% of vaccinated patients compared with 16.7% in unvaccinated patients. Of the 22 vaccinated patients who agreed to have their serum tested, 15 had antibodies against the spike protein at day 21 after the first dose. CONCLUSION: Our study showed a high acceptance rate of COVID-19 vaccination, with good tolerance in this frail population. These results highlight the benefits of organizing vaccination campaigns at the very beginning of oncological management in older patients. CLINICAL TRIAL REGISTRATION: This study was registered May 23, 2019 in ClinicalTrials.gov (NCT03960593).
Subject(s)
COVID-19 , Neoplasms , Vaccines , Aged , Aged, 80 and over , BNT162 Vaccine , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Female , Humans , Male , Neoplasms/therapy , VaccinationABSTRACT
Zika virus (ZIKV) is an arthropod-borne RNA virus (arbovirus), belonging to the Spondweni serogroup. ZIKV was first described in Africa in 1947 and remained sporadic until Micronesia outbreak in 2007, which was followed by outbreaks in the Pacific Islands, Latin America, and the Caribbean. Subsequent to the epidemics, ZIKV revealed its severity as virus was sexually transmissible, and it was associated with serious fetal and neurological complications. ZIKV originated from Africa; however, little is known about the epidemiology of the virus in African populations. Following a recent study in Cameroon that evidenced low ZIKV epidemiology associated with a presumptive (peri-)sylvatic transmission, we performed a seroepidemiological study in Republic of the Congo, neighbor of Cameroon. To accomplish this, 386 serum specimens from volunteer blood donors collected in 2011 from rural and urban areas of Republic of the Congo were tested with ZIKV-specific methodology; primary screening with anti-NS1 ZIKV IgG ELISA followed by confirmation with cytopathic effect (CPE)-based virus neutralization test (VNT). ZIKV seropositivity was determined as low as 1.8%, varying slightly between urban and rural areas (1.7% and 3.6%). These results demonstrate that the majority of the population of Republic of the Congo is immunologically naïve against ZIKV with a presumptive (peri-)sylvatic transmission cycle, which emphasizes the importance of surveillance studies in Africa.
Subject(s)
Antibodies, Viral/blood , Zika Virus Infection/blood , Zika Virus Infection/epidemiology , Zika Virus/immunology , Blood Donors , Congo/epidemiology , Female , History, 16th Century , Humans , Male , Seroepidemiologic Studies , Young AdultABSTRACT
Objectives: We aimed to describe the mechanisms of colistin resistance in Acinetobacter baumannii. Materials and Methods: Twenty-nine patients diagnosed with colistin-resistant A. baumannii infection were included to the study. The mutations in pmrCAB, lpxA, lpxC, and lpxD genes, expression of pmrCAB, carbapenemases, and mcr-1 positivity were studied. Results: Twenty-seven (93%) of the patients received IV colistin therapy during their stay, and the case fatality rate was 45%. All mutations in pmrC and pmrB were found to be accompanied with a mutation in lpxD. The most common mutations were I42V and L150F in pmrC (65%), E117K in lpxD (65%), and A138T in pmrB (58.6%). The colistin minimum inhibitory concentrations (MICs) of the isolates having any of these four mutations were higher than the isolates with no mutations (p < 0.001). The two most common mutations in pmrC (I42V and L150F) were found to be associated with higher expressions of pmrA and pmrC and higher colistin MIC values (p = 0.010 and 0.031). All isolates were blaOXA-23 positive. Conclusion: Coexistence of the lpxD mutation along with mutations in pmrCAB indicates synergistic function of these genes in development of colistin resistance in A. baumannii.
Subject(s)
Acinetobacter Infections/microbiology , Acinetobacter baumannii/genetics , Colistin/pharmacology , Drug Resistance, Bacterial/genetics , Promoter Regions, Genetic/genetics , Acinetobacter Infections/drug therapy , Acinetobacter baumannii/drug effects , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial/drug effects , Female , Humans , Male , Microbial Sensitivity Tests/methods , Middle Aged , Mutation/genetics , Transcription Factors/genetics , beta-Lactamases/geneticsABSTRACT
BACKGROUND: Zika virus (ZIKV), was widely reported in Latin America and has been associated with neuropathologies, as microcephaly, but only few seroprevalence studies have been published to date. Our objective was to determine the seroprevalence amongst Bolivian blood donors and estimate the future potential circulation of the virus. METHODOLOGY: A ZIKV seroprevalence study was conducted between December 2016 and April 2017 in 814 asymptomatic Bolivian volunteer blood donors residing in various eco-environments corresponding to contrasting entomological activities. It was based on detection of IgG to ZIKV using NS1 ELISA screening, followed by a seroneutralisation test in case of positive or equivocal ELISA result. CONCLUSIONS/SIGNIFICANCE: Analysis revealed that ZIKV circulation occurred in tropical areas (Beni: 39%; Santa Cruz de la Sierra: 21.5%) but not in highlands (~0% in Cochabamba, La Paz, Tarija). It was modulated by Aedes aegypti activity and the virus spread was not limited by previous immunity to dengue. Cases were geo-localised in a wide range of urban areas in Santa Cruz and Trinidad. No differences in seroprevalence related to gender or age-groups could be identified. It is concluded that ZIKV has been intensely circulating in the Beni region and has still a significant potential for propagating in the area of Santa Cruz.
Subject(s)
Blood Donors , Mass Screening , Zika Virus Infection/epidemiology , Adolescent , Adult , Aedes/virology , Animals , Bolivia/epidemiology , Chikungunya Fever/epidemiology , Dengue/epidemiology , Female , Humans , Male , Middle Aged , Mosquito Vectors/virology , Seroepidemiologic Studies , Volunteers , Young Adult , Zika VirusABSTRACT
Bloodstream infections caused by Escherichia coli ST131 and ST131 H30-Rx subclones have emerged worldwide. This study was carried out to evaluate the prevalence of the ST131-Rx subclone and characterize the virulence properties of the Rx isolates among the bloodstream E. coli isolates. A total of 297 non-duplicated E. coli bloodstream isolates were studied. Antibiotic susceptibilities were tested using the disc diffusion method. PCR amplification and sequencing was used to identify ST131 and H30-Rx, the virulence gene, the ß-lactamase and virotype. Quinolone resistance among bacteraemic E. coli strains was 51â%, and it was 98â% among E. coli ST131 isolates. The ST131 isolates accounted for 16â% (49) of all isolates and all ST131 isolates belonged to the extraintestinal pathogenic E. coli. The proportion of H30 subclone among the ST131 isolates was 98â%, and 75â% of H30 isolates belonged to the H30-Rx subclone. The prevalence of ST131 increased from 13 to 23â% in 4âyears; however, there was a decrease in the ratio of H30-Rx infections. CTX-M-15 was detected in 85â% of ST131 and all of the H30-Rx isolates. The virulence genes associated with adhesion, cell protection, iron uptake and toxins (papA, iha, kpsMTII, iut and sat) were more common in ST131 than in non-ST131 isolates. Most of the ST131 and H30-Rx isolates were of the C virotype. All papA-positive isolates were in virotype C. The E. coli ST131 clone has increased rapidly among bloodstream isolates. However, a decrease in the proportion of the H30-Rx subclone in the quinolone-resistant population suggests the possibility of dissemination of other virulent and quinolone-resistant subclones in hospital settings.
ABSTRACT
In this study, risk factors for ST131 H30 and H30-Rx subclones among urinary tract infections (UTIs) caused by multidrug-resistant (MDR) Escherichia coli were described. Urine samples were collected from consecutive outpatients registered to the outpatient clinics of Baskent University Hospital (Ankara, Turkey) with complaints of acute cystitis in 2011. A total of 107 MDR E. coli isolates were included in the study. Of the 107 isolates studied, 26 (24.3%) were typed as ST131 clone. Extended-spectrum ß-lactamase (ESBL)-producers accounted for 59 (55.1%) of the 107 isolates. Among the 59 ESBL-positive isolates, 18 (31%) were found to belong to the ST131 clone. Of the 18 ESBL-positive ST131 isolates, 17 (94%) were defined as H30 subclone, among which 16 (94%) represented the H30-Rx subclone. Among the 48 ESBL-negative isolates, 8 (17%) ST131 isolates were detected, 7 (88%) of which belonged to H30 subclone; 5 (71%) of the H30 subclone isolates were classified under H30-Rx subclone. In multivariate analysis, hospitalisation within last year was the only host risk factor associated with MDR E. coli ST131 H30-Rx subclone UTI (OR=3.5, 95% CI 1.04-12.17; P=0.042). CTX-M-15 production was found to be highly associated with the presence of ST131 H30-Rx subclone (OR=4.8, 95% CI 1.54-15.32; P=0.007). In conclusion, urinary MDR E. coli ST131 H30-Rx subclone was found to be important in the dissemination of MDR UTIs in the community. Approximately 20% of the MDR isolates were H30-Rx subclone. Infection with this subclone was found to be healthcare-associated.