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The declining cost of performing bacterial whole-genome sequencing (WGS) coupled with the availability of large libraries of sequence data for well-characterized isolates have enabled the application of machine-learning (ML) methods to the development of nonlinear sequence-based predictive models. We tested the ML-based model developed by Next Gen Diagnostics for prediction of cefepime phenotypic susceptibility results in Escherichia coli. A cohort of 100 isolates of E. coli recovered from urine (n = 77) and blood (n = 23) cultures were used. The cefepime MIC was determined in triplicate by reference broth microdilution and classified as susceptible (MIC of ≤2 µg/mL) or not susceptible (MIC of ≥4 µg/mL) using the 2022 Clinical and Laboratory Standards Institute breakpoints. Five isolates generated both susceptible and not susceptible MIC results, yielding categorical agreement of 95% for the reference method to itself. Categorical agreement of ML to MIC interpretations was 97%, with 2 very major (false, susceptible) and 1 major (false, not susceptible) errors. One very major error occurred for an isolate with blaCTX-M-27 (MIC mode, ≥32 µg/mL) and one for an isolate with blaTEM-34 for which the MIC cefepime mode was 4 µg/mL. One major error was for an isolate with blaCTX-M-27 but with a MIC mode of 2 µg/mL. These preliminary data demonstrated performance of ML for a clinically important antimicrobial-species pair at a caliber similar to phenotypic methods, encouraging wider development of sequence-based susceptibility prediction and its validation and use in clinical practice.
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Antibacterianos , Escherichia coli , Humanos , Cefepima/farmacología , Antibacterianos/farmacología , Escherichia coli/genética , Cefalosporinas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Influenza seasons typically begin in October and peak between December and February (1); however, the 2022-23 influenza season in Tennessee began in late September and was characterized by high pediatric hospitalization rates during November. This report describes a field investigation conducted in Tennessee during November 2022, following reports of increasing influenza hospitalizations. Data from surveillance networks, patient surveys, and whole genome sequencing of influenza virus specimens were analyzed to assess influenza activity and secondary illness risk. Influenza activity increased earlier than usual among all age groups, and rates of influenza-associated hospitalization among children were high in November, reaching 12.6 per 100,000 in children aged <5 years, comparable to peak levels typically seen in high-severity seasons. Circulating influenza viruses were genetically similar to vaccine components. Among persons who received testing for influenza at outpatient clinics, children were twice as likely to receive a positive influenza test result as were adults. Among household contacts exposed to someone with influenza, children were more than twice as likely to become ill compared with adults. As the influenza season continues, it is important for all persons, especially those at higher risk for severe disease, to protect themselves from influenza. To prevent influenza and severe influenza complications, all persons aged ≥6 months should get vaccinated, avoid contact with ill persons, and take influenza antivirals if recommended and prescribed.
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Vacunas contra la Influenza , Gripe Humana , Adulto , Niño , Humanos , Lactante , Gripe Humana/prevención & control , Estaciones del Año , Tennessee/epidemiología , Virus de la Influenza B/genética , VacunaciónRESUMEN
Importance: Influenza virus infections declined globally during the COVID-19 pandemic. Loss of natural immunity from lower rates of influenza infection and documented antigenic changes in circulating viruses may have resulted in increased susceptibility to influenza virus infection during the 2021-2022 influenza season. Objective: To compare the risk of influenza virus infection among household contacts of patients with influenza during the 2021-2022 influenza season with risk of influenza virus infection among household contacts during influenza seasons before the COVID-19 pandemic in the US. Design, Setting, and Participants: This prospective study of influenza transmission enrolled households in 2 states before the COVID-19 pandemic (2017-2020) and in 4 US states during the 2021-2022 influenza season. Primary cases were individuals with the earliest laboratory-confirmed influenza A(H3N2) virus infection in a household. Household contacts were people living with the primary cases who self-collected nasal swabs daily for influenza molecular testing and completed symptom diaries daily for 5 to 10 days after enrollment. Exposures: Household contacts living with a primary case. Main Outcomes and Measures: Relative risk of laboratory-confirmed influenza A(H3N2) virus infection in household contacts during the 2021-2022 season compared with prepandemic seasons. Risk estimates were adjusted for age, vaccination status, frequency of interaction with the primary case, and household density. Subgroup analyses by age, vaccination status, and frequency of interaction with the primary case were also conducted. Results: During the prepandemic seasons, 152 primary cases (median age, 13 years; 3.9% Black; 52.0% female) and 353 household contacts (median age, 33 years; 2.8% Black; 54.1% female) were included and during the 2021-2022 influenza season, 84 primary cases (median age, 10 years; 13.1% Black; 52.4% female) and 186 household contacts (median age, 28.5 years; 14.0% Black; 63.4% female) were included in the analysis. During the prepandemic influenza seasons, 20.1% (71/353) of household contacts were infected with influenza A(H3N2) viruses compared with 50.0% (93/186) of household contacts in 2021-2022. The adjusted relative risk of A(H3N2) virus infection in 2021-2022 was 2.31 (95% CI, 1.86-2.86) compared with prepandemic seasons. Conclusions and Relevance: Among cohorts in 5 US states, there was a significantly increased risk of household transmission of influenza A(H3N2) in 2021-2022 compared with prepandemic seasons. Additional research is needed to understand reasons for this association.
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COVID-19 , Subtipo H3N2 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , COVID-19/epidemiología , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/diagnóstico , Gripe Humana/epidemiología , Gripe Humana/prevención & control , Gripe Humana/transmisión , Pandemias/prevención & control , Pandemias/estadística & datos numéricos , Estudios Prospectivos , Estaciones del Año , Composición Familiar , Estados Unidos/epidemiología , Trazado de Contacto/estadística & datos numéricos , AutoevaluaciónRESUMEN
Nearly 40 years have elapsed since the invention of the PCR, with its extremely sensitive and specific ability to detect nucleic acids via in vitro enzyme-mediated amplification. In turn, more than 2 years have passed since the onset of the coronavirus disease 2019 (COVID-19) pandemic, during which time molecular diagnostics for infectious diseases have assumed a larger global role than ever before. In this context, we review broadly the progression of molecular techniques in clinical microbiology, to their current prominence. Notably, these methods now entail both the detection and quantification of microbial nucleic acids, along with their sequence-based characterization. Overall, we seek to provide a combined perspective on the techniques themselves, as well as how they have come to shape health care at the intersection of technologic innovation, pathophysiologic knowledge, clinical/laboratory logistics, and even financial/regulatory factors.
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COVID-19 , Enfermedades Transmisibles , Ácidos Nucleicos , Humanos , Patología Molecular , COVID-19/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedades Transmisibles/diagnóstico , Técnicas de Diagnóstico Molecular/métodosRESUMEN
There is concern that in-person schooling during the coronavirus disease 2019 (COVID-19) pandemic will facilitate disease transmission. Through asymptomatic surveillance and contact tracing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we found low rates of asymptomatic SARS-CoV-2 infection and little in-school transmission of COVID-19 when physical distancing and masking strategies were enforced despite a high community prevalence of COVID-19.
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Infecciones Asintomáticas/epidemiología , COVID-19/transmisión , Instituciones Académicas/organización & administración , Adolescente , COVID-19/diagnóstico , COVID-19/epidemiología , Prueba de COVID-19 , Niño , Preescolar , Trazado de Contacto/métodos , Femenino , Humanos , Masculino , Pandemias , Prevalencia , Estudios Prospectivos , SARS-CoV-2 , Instituciones Académicas/estadística & datos numéricos , Tennessee/epidemiologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher Ct values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.
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Prueba de COVID-19/economía , Prueba de COVID-19/métodos , COVID-19/economía , COVID-19/epidemiología , Países en Desarrollo/economía , SARS-CoV-2 , HumanosRESUMEN
BACKGROUND: The spectrum of illness and predictors of severity among children with SARS-CoV-2 infection are incompletely understood. METHODS: Active surveillance was performed for SARS-CoV-2 by polymerase chain reaction among symptomatic pediatric patients in a quaternary care academic hospital laboratory beginning March 12, 2020. We obtained sociodemographic and clinical data 5 (+/-3) and 30 days after diagnosis via phone follow-up and medical record review. Logistic regression was used to assess predictors of hospitalization. RESULTS: The first 1000 symptomatic pediatric patients were diagnosed in our institution between March 13, 2020 and September 28, 2020. Cough (52 %), headache (43 %), and sore throat (36 %) were the most common symptoms. Forty-one (4 %) were hospitalized; 8 required ICU admission, and 2 required mechanical ventilation (< 1 %). One patient developed multisystem inflammatory syndrome in children; one death was possibly associated with SARS-CoV-2 infection. Symptom resolution occurred by follow-up day 5 in 398/892 (45 %) patients and by day 30 in 443/471 (94 %) patients. Pre-existing medical condition (OR 7.7; 95 % CI 3.9-16.0), dyspnea (OR 6.8; 95 % CI 3.2-14.1), Black race or Hispanic ethnicity (OR 2.7; 95 % CI 1.3-5.5), and vomiting (OR 5.4; 95 % CI 1.2-20.6) were the strongest predictors of hospitalization. The model displayed excellent discriminative ability (AUC = 0.82, 95 % CI 0.76-0.88, Brier score = 0.03). CONCLUSIONS: In 1000 pediatric patients with systematic follow-up, most SARS-CoV-2 infections were mild, brief, and rarely required hospitalization. Pediatric predictors of hospitalization included comorbid conditions, Black race, Hispanic ethnicity, dyspnea and vomiting and were distinct from those reported among adults.
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COVID-19 , Prestación Integrada de Atención de Salud , Adulto , Niño , Hospitalización , Humanos , Estudios Prospectivos , SARS-CoV-2 , Síndrome de Respuesta Inflamatoria SistémicaRESUMEN
The COVID-19 outbreak has had a major impact on clinical microbiology laboratories in the past several months. This commentary covers current issues and challenges for the laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the preanalytical stage, collecting the proper respiratory tract specimen at the right time from the right anatomic site is essential for a prompt and accurate molecular diagnosis of COVID-19. Appropriate measures are required to keep laboratory staff safe while producing reliable test results. In the analytic stage, real-time reverse transcription-PCR (RT-PCR) assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 infection while antibody-based techniques are being introduced as supplemental tools. In the postanalytical stage, testing results should be carefully interpreted using both molecular and serological findings. Finally, random-access, integrated devices available at the point of care with scalable capacities will facilitate the rapid and accurate diagnosis and monitoring of SARS-CoV-2 infections and greatly assist in the control of this outbreak.
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Betacoronavirus , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Betacoronavirus/genética , Betacoronavirus/inmunología , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Vacunas contra la COVID-19 , Humanos , Pandemias , Reacción en Cadena de la Polimerasa , SARS-CoV-2RESUMEN
PURPOSE: Recent research shows an increasing recognition that organisms not traditionally considered infectious in nature contribute to disease processes. Propionibacterium acnes (P. acnes) is a gram-positive, aerotolerant anaerobe prevalent in the sebaceous gland-rich areas of the human skin. A ubiquitous slow-growing organism with the capacity to form biofilm, P. acnes, recognized for its role in acne vulgaris and medical device-related infections, is now also linked to a number of other human diseases. While bacterial culture and molecular techniques are used to investigate the involvement of P. acnes in such diseases, definitive demonstration of P. acnes infection requires a technique (or techniques) sensitive to the presence of biofilms and insensitive to the presence of potential contamination. Fortunately, there are imaging techniques meeting these criteria, in particular, fluorescence in situ hybridization and immunofluorescence coupled with confocal laser scanning microscopy, as well as immunohistochemistry. METHODS: Our literature review considers a range of microscopy-based studies that provides definitive evidence of P. acnes colonization within tissue from a number of human diseases (acne vulgaris, degenerative disc and prostate disease and atherosclerosis), some of which are currently not considered to have an infectious etiology. RESULTS/CONCLUSION: We conclude that P. acnes is an opportunistic pathogen with a likely underestimated role in the development of various human diseases associated with significant morbidity and, in some cases, mortality. As such, these findings offer the potential for new studies aimed at understanding the pathological mechanisms driving the observed disease associations, as well as novel diagnostic strategies and treatment strategies, particularly for degenerative disc disease. These slides can be retrieved under Electronic Supplementary Material.
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Biopelículas , Infecciones por Bacterias Grampositivas , Degeneración del Disco Intervertebral , Microscopía , Propionibacterium acnes , Acné Vulgar/diagnóstico por imagen , Acné Vulgar/microbiología , Infecciones por Bacterias Grampositivas/diagnóstico por imagen , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Degeneración del Disco Intervertebral/diagnóstico por imagen , Degeneración del Disco Intervertebral/microbiologíaRESUMEN
PURPOSE: The presence of Propionibacterium acnes in a substantial component of resected disc specimens obtained from patients undergoing discectomy or microdiscectomy has led to the suggestion that this prominent human skin and oral commensal may exacerbate the pathology of degenerative disc disease. This hypothesis, therefore, raises the exciting possibility that antibiotics could play an important role in treating this debilitating condition. To date, however, little information about antibiotic penetration into the intervertebral disc is available. METHODS: Intervertebral disc tissue obtained from 54 microdiscectomy patients given prophylactic cefazolin (n = 25), clindamycin (n = 17) or vancomycin (n = 12) was assayed by high-performance liquid chromatography, with cefaclor as an internal standard, to determine the concentration of antibiotic penetrating into the disc tissue. RESULTS: Intervertebral disc tissues from patients receiving the positively charged antibiotic clindamycin contained a significantly greater percentage of the antibacterial dose than the tissue from patients receiving negatively charged cefazolin (P < 0.0001) and vancomycin, which has a slight positive charge (P < 0.0001). CONCLUSION: Positively charged antibiotics appear more appropriate for future studies investigating potential options for the treatment of low-virulence disc infections. These slides can be retrieved under Electronic Supplementary Material.
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Antibacterianos/farmacocinética , Cefazolina/farmacocinética , Clindamicina/farmacocinética , Infecciones por Bacterias Grampositivas/prevención & control , Degeneración del Disco Intervertebral/cirugía , Desplazamiento del Disco Intervertebral/cirugía , Disco Intervertebral/metabolismo , Propionibacterium acnes , Vancomicina/farmacocinética , Adulto , Antibacterianos/uso terapéutico , Cefazolina/uso terapéutico , Clindamicina/uso terapéutico , Humanos , Vancomicina/uso terapéuticoRESUMEN
Unfortunately, the complete conflict of interest statement was missed out in the original publication. The same is given below.
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BACKGROUND: Biofilms represent a complex milieu of matrix-enclosed microorganisms, which can significantly contribute to the pathology of chronic wounds. In this study, we compare the activity of 3 commercial antimicrobial wound care solutions, Vashe (HOCl based), PhaseOne (HOCl based), and Sulfamylon (mafenide acetate), for their in vitro activity against bacterial and fungal biofilms. METHODS: Reference and clinical isolates of 6 Gram-negative bacterial species (36 total strains), 3 Gram-positive bacteria (21 strains), and 3 Candida species (9 strains) were used to create biofilms. Various working concentrations of the 3 antiseptic agents were incubated with the biofilms in microwell plates; they were monitored from 1 minute to 24 hours to compare bacterial and fungal viability through colony forming unit analysis. RESULTS: Vashe and PhaseOne displayed excellent bactericidal and fungicidal activity, whereas Sulfamylon demonstrated minimal activity against the biofilms tested. With the exception of Candida albicans, all biofilms were eliminated at either 1 or 10 minutes using Vashe and PhaseOne solutions. In most cases, mafenide was unable to eliminate both bacterial and fungal biofilms, even with 24 hours of treatment. CONCLUSIONS: Biofilms represent a major clinical challenge, with no clear consensus for treatment of chronic wounds or prosthetic devices. Our results suggest that hypochlorous acid-based wound solutions such as Vashe and PhaseOne are more efficacious than mafenide in eliminating bacterial and fungal biofilms. Further studies are necessary to investigate and compare the in vivo efficacy of these products in clinical care.
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Antiinfecciosos/administración & dosificación , Antifúngicos/administración & dosificación , Biopelículas/efectos de los fármacos , Soluciones/administración & dosificación , Heridas y Lesiones/microbiología , Administración Tópica , Enfermedad Crónica , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Sensibilidad y Especificidad , Heridas y Lesiones/tratamiento farmacológicoRESUMEN
One of the most common urologic problems afflicting millions of people worldwide is urinary tract infection (UTI). The severity of UTIs ranges from asymptomatic bacteriuria to acute cystitis, and in severe cases, pyelonephritis and urosepsis. The primary cause of UTIs is uropathogenic Escherichia coli (UPEC), for which current antibiotic therapies often fail. UPEC forms multicellular communities known as biofilms on urinary catheters, as well as on and within bladder epithelial cells. Biofilm formation protects UPEC from environmental conditions, antimicrobial therapy, and the host immune system. Previous studies have investigated UPEC biofilm formation in aerobic conditions (21% oxygen); however, urine oxygen tension is reduced (4-6%), and urine contains molecules that can be used by UPEC as alternative terminal electron acceptors (ATEAs) for respiration. This study was designed to determine whether these different terminal electron acceptors utilized by E. coli influence biofilm formation. A panel of 50 urine-associated E. coli isolates was tested for the ability to form biofilm under anaerobic conditions and in the presence of ATEAs. Biofilm production was reduced under all tested sub-atmospheric levels of oxygen, with the notable exception of 4% oxygen, the reported concentration of oxygen within the bladder.
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Biopelículas/crecimiento & desarrollo , Infecciones por Escherichia coli/metabolismo , Oxígeno/metabolismo , Vejiga Urinaria/microbiología , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena/fisiología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/orina , Humanos , Hipoxia/metabolismo , Hipoxia/microbiología , Hipoxia/orina , Oxígeno/orina , Infecciones Urinarias/microbiología , Infecciones Urinarias/orinaRESUMEN
Helicobacter cinaedi is an emerging opportunistic pathogen associated with infections of diverse anatomic sites. Nevertheless, the species demonstrates fastidious axenic growth; it has been described as requiring a microaerobic atmosphere, along with a strong preference for supplemental H2 gas. In this context, we examined the hypothesis that in vitro growth of H. cinaedi could be enhanced by coculture with human epithelial cells. When inoculated (in Ham's F12 medium) over Caco-2 monolayers, the type strain (ATCC BAA-847) gained the ability to proliferate under H2-free aerobic conditions. Identical results were observed during coculture with several other monolayer types (LS-174T, AGS, and HeLa). Under chemically defined conditions, 40 amino acids and carboxylates were screened for their effect on the organism's atmospheric requirements. Several molecules promoted H2-free aerobic proliferation, although it occurred most prominently with millimolar concentrations of l-lactate. The growth response of H. cinaedi to Caco-2 cells and l-lactate was confirmed with a collection of 12 human-derived clinical strains. mRNA sequencing was next performed on the type strain under various growth conditions. In addition to providing a whole-transcriptome profile of H. cinaedi, this analysis demonstrated strong constitutive expression of the l-lactate utilization locus, as well as differential transcription of terminal respiratory proteins as a function of Caco-2 coculture and l-lactate supplementation. Overall, these findings challenge traditional views of H. cinaedi as an obligate microaerophile. IMPORTANCE: H. cinaedi is an increasingly recognized pathogen in people with compromised immune systems. Atypical among other members of its bacterial class, H. cinaedi has been associated with infections of diverse anatomic sites. Growing H. cineadi in the laboratory is quite difficult, due in large part to the need for a specialized atmosphere. The suboptimal growth of H. cinaedi is an obstacle to clinical diagnosis, and it also limits investigation into the organism's biology. The current work shows that H. cinaedi has more flexible atmospheric requirements in the presence of host cells and a common host-derived molecule. This nutritional interplay raises new questions about how the organism behaves during human infections and provides insights for how to optimize its laboratory cultivation.
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Células Epiteliales/microbiología , Helicobacter/crecimiento & desarrollo , Ácido Láctico/farmacología , Aerobiosis , Células CACO-2 , Técnicas de Cocultivo , Células Epiteliales/efectos de los fármacos , Células HeLa , Helicobacter/efectos de los fármacos , Helicobacter/fisiología , Infecciones por Helicobacter/diagnóstico , Infecciones por Helicobacter/microbiología , Humanos , Hidrógeno , Ácido Láctico/metabolismoRESUMEN
Persistent neutrophilic meningitis presents a diagnostic challenge, because the differential diagnosis is broad and includes atypical infectious causes. We describe a case of persistent neutrophilic meningitis due to Aspergillus fumigatus in an immunocompetent man who had no evidence of sinopulmonary or cutaneous disease. An epidural glucocorticoid injection was identified as a potential route of entry for this organism into the central nervous system, and the case was reported to the state health department.
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Aspergilosis/diagnóstico , Aspergillus fumigatus/aislamiento & purificación , Encéfalo/patología , Líquido Cefalorraquídeo/parasitología , Contaminación de Medicamentos , Meningitis Fúngica/diagnóstico , Aspergilosis/etiología , Encéfalo/diagnóstico por imagen , Cerebelo/irrigación sanguínea , Infarto Cerebral/etiología , Infarto Cerebral/patología , Diagnóstico Diferencial , Brotes de Enfermedades , Resultado Fatal , Glucocorticoides/administración & dosificación , Cefalea/etiología , Humanos , Inyecciones Epidurales/efectos adversos , Hemorragias Intracraneales/diagnóstico por imagen , Hemorragias Intracraneales/etiología , Dolor de la Región Lumbar/tratamiento farmacológico , Dolor de la Región Lumbar/etiología , Masculino , Meningitis Fúngica/epidemiología , Meningitis Fúngica/etiología , Persona de Mediana Edad , Tomografía Computarizada por Rayos X , Estados UnidosRESUMEN
Introduction: SARS-CoV-2 has demonstrated that, in targeted circumstances, viral quantification within respiratory specimens can valuably inform patient management, as well as research. Nevertheless, the pandemic has illustrated concomitant challenges for obtaining high-quality (and broadly comparable) respiratory viral loads. This includes a critical need for standardization and calibration, even though the necessary resources may not always be available for emergent pathogens and non-bloodstream specimens. Methods: To these ends, we describe a novel strategy for implementing quantitative SARS-CoV-2 testing with International Unit-based calibration. Earlier in the course of the pandemic-when analytic resources were far more limited-select residual SARS-CoV-2 positive specimens from routine care in our diagnostic laboratory were pooled to formulate a clinically realistic secondary standard of high volume and analyte concentration, which was cross-calibrated to the primary SARS-CoV-2 standard of the World Health Organization. Results: The resultant calibrators were integrated into the original CDC RT-qPCR assay for SARS-CoV-2, whose (now broadened) performance characteristics were defined to generate a test appropriate for both clinical and research use. This test allowed for the quantification of virus in respiratory specimens down to a validated lower limit of quantification of 103.4 IU/ml. Conclusions: By self-formulating calibrators from this derivative-of-care secondary standard, we successfully validated respiratory viral loads without the commercial availability (at that time) of quantitative assays or calibrators. As the SARS-CoV-2 pandemic continues to decline-and even beyond this pathogen-this strategy may be applicable for laboratories seeking to implement viral load testing for nontraditional specimen types despite limited resources.
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COVID-19 , Humanos , COVID-19/diagnóstico , Prueba de COVID-19 , Calibración , SARS-CoV-2/genética , Carga ViralRESUMEN
In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.
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ADN , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa/métodos , ADN/química , ADN/análisis , SARS-CoV-2/genética , Cartilla de ADN/química , COVID-19 , HumanosRESUMEN
Biofilm growth facilitates the interaction of uropathogenic Escherichia coli (UPEC) with the host environment. The extracellular polymeric substances (EPS) of UPEC biofilms are composed prominently of curli amyloid fiber and cellulose polysaccharide. When the organism is propagated as a colony biofilm on agar media, these macromolecules can generate pronounced macroscopic structures. Moreover, curli/cellulose associate tightly with Congo red, generating a characteristic pink-to-red staining pattern when the media is supplemented with this dye. Among different clinical isolates of UPEC, changes in the abundance of curli/cellulose can lead to diverse colony biofilm phenotypes on a strain-by-strain basis. Nevertheless, for any given isolate, these phenotypes are classically homogenous throughout the colony biofilm. Here, we report that a subset of clinical UPEC isolates display heterogenous 'peppermint' colony biofilms, with distinct pale and red subpopulations. Through isolation of these subpopulations and whole genome sequencing, we demonstrate various emergent mutations associated with the phenomenon, including within the gene encoding the outer membrane lipoprotein nlpI. Deletion of nlpI within independent strain-backgrounds increased biofilm rugosity, while its overexpression induced the peppermint phenotype. Upregulation of EPS-associated proteins and transcripts was likewise observed in the absence of nlpI. Overall, these results demonstrate that EPS elaboration in UPEC is impacted by nlpI. More broadly, this phenomenon of intra-strain colony biofilm heterogeneity may be leveraged as a tool to identify additional members within the broad collection of genes that regulate or otherwise affect biofilm formation.