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Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.
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Since May 2022, approximately 2,500 mpox cases have been reported in Los Angeles County (LAC), California. Beginning in May 2023, the LAC Department of Public Health observed a consistent increase in mpox cases after a prolonged period of low incidence. A total of 56 cases were identified during May 4-August 17, 2023. A minority of mpox patients were fully vaccinated (29%). One patient was hospitalized; no deaths were reported. Two cases of reinfection occurred, both of which were associated with mild illness. The increasing number of cases during this period was significant, as few other health departments in the United States reported an increase in mpox cases during the same period. The outbreak spread similarly to the 2022 U.S. mpox outbreak, mainly through sexual contact among gay, bisexual, and other men who have sex with men. Vaccination against mpox became available in June 2022 and has been shown to be effective at preventing mpox disease. This outbreak was substantially smaller than the 2022 mpox outbreak in LAC (2,280 cases); possible explanations for the lower case count include increased immunity provided from vaccination against mpox and population immunity from previous infections. Nonetheless, mpox continues to spread within LAC, and preventive measures, such as receipt of JYNNEOS vaccination, are recommended for persons at risk of Monkeypox virus exposure.
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Mpox , Minorías Sexuales y de Género , Masculino , Humanos , Homosexualidad Masculina , Los Angeles/epidemiología , Brotes de EnfermedadesRESUMEN
The Los Angeles County Department of Public Health established a surveillance system to identify complicated (advanced human immunodeficiency virus [HIV] or hospitalized) mpox cases. From 1 August to 30 November 2022, we identified 1581 mpox cases, of which 134 (8.5%) were complicated. A subset of 8 cases did not recover after either initiating or completing a course of oral tecovirimat. All 8 patients were HIV positive and had advanced HIV (CD4 count <200 cells/µL). We identified 8 distinct mutations previously associated with tecovirimat resistance in specimens collected from 6 patients. Ongoing surveillance of viral evolution requires close coordination between health departments and frontline providers.
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Seropositividad para VIH , Mpox , Humanos , Los Angeles , Benzamidas , IsoindolesRESUMEN
Remdesivir is the first FDA-approved drug for treating severe SARS-CoV-2 infection and targets RNA-dependent RNA polymerase (RdRp) that is required for viral replication. To monitor for the development of mutations that may result in remdesivir resistance during prolonged treatment, we sequenced SARS-CoV-2 specimens collected at different treatment time points in two transplant patients with severe COVID-19. In the first patient, an allogeneic hematopoietic stem cell transplant recipient, a transient RdRp catalytic subunit mutation (nsp12:A449V) was observed that has not previously been associated with remdesivir resistance. As no in vitro study had been conducted to elucidate the phenotypic effect of nsp12:A449V, its clinical significance is unclear. In the second patient, two other transient RdRp mutations were detected: one in the catalytic subunit (nsp12:V166A) and the other in an accessory subunit important for processivity (nsp7:D67N). This is the first case report for a potential link between the nsp12:V166A mutation and remdesivir resistance in vivo, which had only been previously described by in vitro studies. The nsp7:D67N mutation has not previously been associated with remdesivir resistance, and whether it has a phenotypic effect is unknown. Our study revealed SARS-CoV-2 genetic dynamics during remdesivir treatment in transplant recipients that involved mutations in the RdRp complex (nsp7 and nsp12), which may be the result of selective pressure. These results suggest that close monitoring for potential resistance during the course of remdesivir treatment in highly vulnerable patient populations may be beneficial. Development and utilization of diagnostic RdRp genotyping tests may be a future direction for improving the management of chronic COVID-19.
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The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.
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COVID-19 , Mpox , Infección por el Virus Zika , Virus Zika , Humanos , COVID-19/epidemiología , Pandemias , SARS-CoV-2/genética , GenómicaRESUMEN
The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.
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As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.
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Técnicas y Procedimientos Diagnósticos , Brotes de Enfermedades , Mpox , Brotes de Enfermedades/prevención & control , Humanos , Laboratorios , Mpox/diagnóstico , Mpox/epidemiología , Orthopoxvirus , Estados Unidos/epidemiología , Virus de la ViruelaRESUMEN
OBJECTIVES: This study aimed to assess whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Epsilon variant (B.1.429/427) is more virulent, leading to more hospitalization and more severe disease requiring intensive care unit (ICU) admission. METHODS: SARS-CoV-2 genomic surveillance was performed on respiratory samples from 231 unique patients, collected at a single large health system in Southern California between November 2020 and March 2021 during the winter surge. RESULTS: The frequencies of the Epsilon variant among outpatients, hospitalized patients, and ICU patients were indifferent. CONCLUSIONS: Our study suggests that the Epsilon variant is not associated with increased hospitalization and ICU admission.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , California/epidemiología , Genómica , Hospitalización , Humanos , Unidades de Cuidados Intensivos , SARS-CoV-2/genéticaRESUMEN
We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.
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Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/transmisión , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Mutación/genética , Secuenciación Completa del Genoma/métodosRESUMEN
A suspected outbreak of influenza A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at a long-term care facility in Los Angeles County was, months later, determined to not involve influenza. To prevent inadvertent transmission of infections, facilities should use highly specific influenza diagnostics and follow Centers for Disease Control and Prevention (CDC) guidelines that specifically address infection control challenges.
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COVID-19 , Gripe Humana , Brotes de Enfermedades , Humanos , Gripe Humana/diagnóstico , Gripe Humana/epidemiología , Cuidados a Largo Plazo , SARS-CoV-2RESUMEN
We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.
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On June 22, 2017, the Los Angeles County Department of Public Health (LAC DPH) was notified of seven patients who were seen at an eye care clinic on June 8, 2017, and later developed symptoms of epidemic keratoconjunctivitis (EKC). EKC is a contagious, severe form of viral conjunctivitis that can cause pain and blurred vision for up to 4 weeks (1). LAC DPH conducted an investigation, which identified 17 patients with EKC, including 15 who had visited the optometry clinic and two who were household contacts of clinic patients. Observations in the clinic found deficiencies in disinfection of tonometers (an instrument connected to a slit lamp and used to test for glaucoma by measuring intraocular pressure) and multiuse eye drop administration. Staff member education and revision of disinfection practices interrupted further transmission. Patient specimens tested positive for human adenovirus (HAdV) type D53 (HAdV-53). As the first documented EKC outbreak associated with HAdV-D53 in the United States, this outbreak highlights the need for rigorous implementation of recommended infection prevention practices in eye care settings.
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Adenoviridae/aislamiento & purificación , Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Queratoconjuntivitis/epidemiología , Optometría , Adulto , Anciano , Análisis por Conglomerados , Infección Hospitalaria/transmisión , Femenino , Humanos , Los Angeles/epidemiología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: More laboratories are screening for syphilis with automated treponemal immunoassays. We compared direct costs and downstream consequences when a local public health laboratory switches from a traditional algorithm (nontreponemal screening) to a reverse algorithm (treponemal screening). METHODS: We created a decision analysis model based on laboratory and surveillance data to estimate the cost-effectiveness of a reverse syphilis-screening algorithm from the perspectives of the Los Angeles County Public Health Laboratory and the Los Angeles County Department of Public Health (laboratory + STD Program costs) in 2015 US dollars. RESULTS: The estimated total costs for the Department (Public Health Laboratories) were $2,153,225 ($367,119) for the traditional algorithm and $2,197,478 ($239,855) for the reverse algorithm. Reverse algorithm screening was estimated to detect an additional 626 cases of syphilis, 9.7% more than the traditional algorithm. The incremental cost-effectiveness ratio for the reverse algorithm from the Public Health Department's perspective was $39 per additional syphilis case detected. Cost of follow-up, screening test costs, positivity rates, and frequency of repeat infections most affected the cost-effectiveness of reverse algorithm. Costs were significantly higher for the reverse algorithm when the enzyme Immunoassay/chemiluminescence immunoassay screening test cost was the same as the published Centers for Medicaid Services treponemal test cost. CONCLUSIONS: Using the reverse algorithm would have been slightly more expensive for the Los Angeles County Department of Public Health, but would have identified more syphilis cases and would have resulted in lower laboratory costs.
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Algoritmos , Tamizaje Masivo/economía , Tamizaje Masivo/métodos , Sífilis/diagnóstico , Sífilis/epidemiología , Análisis Costo-Beneficio , Humanos , Técnicas para Inmunoenzimas , Prevalencia , Sensibilidad y Especificidad , Serodiagnóstico de la Sífilis/métodos , Treponema pallidum/inmunología , Estados Unidos/epidemiología , United States Public Health ServiceRESUMEN
INTRODUCTION: Clinical management and identification of respiratory diseases has become more rapid and increasingly specific due to widespread use of PCR(polymerase chain reaction) multiplex technologies. Although significantly improving clinical diagnosis, multiplexed PCR assays could have a greater impact on local and global disease surveillance. The authors wish to propose methods of evaluating respiratory multiplex assays to maximize diagnostic yields specifically for surveillance efforts. Areas covered: The authors review multiplexed assays and critically assess what barriers have limited these assays for disease surveillance and how these barriers might be addressed. The manuscript focuses specifically on the case study of using multiplexed assays for surveillance of respiratory pathogens. The authors also provide a method of validation of specific surveillance measures. Expert commentary: Current commercially available respiratory multiplex PCR assays are widely used for clinical diagnosis; however, specific barriers have limited their use for surveillance. Key barriers include differences in testing phase requirements and diagnostic performance evaluation. In this work the authors clarify phase testing requirements and introduce unique diagnostic performance measures that simplify the use of these assays on a per target basis for disease surveillance.
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Aprobación de Pruebas de Diagnóstico/normas , Técnicas de Diagnóstico Molecular/normas , Reacción en Cadena de la Polimerasa Multiplex/normas , Infecciones del Sistema Respiratorio/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Estados Unidos , United States Food and Drug AdministrationRESUMEN
We compared the performance and ease of use for three high-throughput treponemal immunoassays: Phoenix Biotech Trep-Sure Total Antibody EIA, Siemens ADVIA® Centaur Syphilis Assay, and DiaSorin LIAISON® Treponema Assay. One thousand serum samples submitted for routine screening were used in this study. Each assay demonstrated comparable sensitivity, specificity, and percent agreement (98-100%) compared with Treponema pallidum particle agglutination (TP-PA). Thus, treponemal immunoassays are an acceptable alternative for syphilis screening or confirmatory testing. Batch sizes and technologist active time varied between each treponemal immunoassay; the chemiluminescence platforms offered significantly greater ability to batch (random access vs. fixed batch sizes) in less time. When we compared the results obtained using a reverse algorithm approach to those obtained using a traditional algorithm, we found that the reverse algorithm identified 38 additional seropositive individuals that were not detected using the traditional algorithm. Clinical evaluation was useful for resolving cases with discordant serology.
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Técnicas para Inmunoenzimas/métodos , Mediciones Luminiscentes/métodos , Pruebas Serológicas/métodos , Sífilis/diagnóstico , Algoritmos , Anticuerpos Antibacterianos/sangre , Técnicas Bacteriológicas , Humanos , Sífilis/microbiologíaRESUMEN
Plague is a disease caused by Yersinia pestis. Septicemic and pneumonic plague have a high mortality rate if untreated. Here we describe the challenges of accurately diagnosing a nonfatal pediatric case of septicemic plague with involvement of multiple organs; to our knowledge, the first documented case of multifocal plague osteomyelitis.