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Modified two-tiered testing (MTTT) algorithms for Lyme disease (LD), which involve the sequential use of orthogonal enzyme immunoassays (EIAs) without immunoblotting, are acceptable alternatives to standard two-tiered testing (STTT; EIA followed by immunoblots) provided the EIAs have been FDA-cleared for this intended use. We evaluated four Zeus Scientific LD EIAs used in two distinct MTTT algorithms for FDA review. MTTT 1 used a VlsE1/pepC10 polyvalent EIA followed by a whole-cell sonicate (WCS) polyvalent EIA. MTTT 2 used the same first-tier EIA followed by separate IgM and IgG WCS EIAs. In a retrospective phase, we compared each MTTT algorithm to STTT using archived samples from LD patients or control subjects. In a prospective phase, we used the same algorithms to analyze consecutive excess samples submitted for routine LD serology to three clinical laboratories. For the retrospective phase, MTTTs 1 and 2 were more sensitive (56% and 74%) than STTT (41%; P ≤ 0.03) among 61 patients with acute erythema migrans (EM). In LD patients with neuroborreliosis, carditis, or arthritis (n = 75), sensitivity was comparable between algorithms (96 to 100%; P = 1.0). Among 190 control subjects without past LD, all algorithms were highly and comparably specific (≥99%, P = 0.48). For the prospective phase, (n = 2,932), positive percent-agreement (PPA), negative percent-agreement (NPA), and overall agreement of MTTT 1 with STTT were 93%, 97.7% and 97.4% (kappa 0.80). MTTT 2 yielded higher PPA (98%) but lower NPA (96.1%) and overall agreement (96.2%, kappa 0.74; all P < 0.05). Compared with STTT, both MTTT algorithms provided increased sensitivity in EM patients, comparable sensitivity in later disease and non-inferior specificity.
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Borrelia burgdorferi , Enfermedad de Lyme , Algoritmos , Animales , Anticuerpos Antibacterianos , Peces , Humanos , Inmunoglobulina M , Estudios Prospectivos , Estudios Retrospectivos , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
Background: Ceftazidime/avibactam (CAZ-AVI) may improve outcomes among patients with carbapenem-resistant Enterobacteriaceae (CRE) infections compared to conventional therapies. However, CAZ-AVI's cost-effectiveness is unknown.Methods: We used a decision analytic model to estimate the health and economic consequences of CAZ-AVI-based therapy compared to colistin-based therapy (COL) for a hypothetical cohort of patients with CRE pneumonia or bacteremia over a 5-year horizon. Model inputs were from published sources and included CRE mortality with COL (41%), CAZ-AVI's absolute risk reduction in CRE mortality (23%), daily cost of CAZ-AVI ($926), risk of nephrotoxicity with COL (42%) and probability of discharge to long-term care (LTC) following CRE infection (56%). Outcomes included quality adjusted life-years (QALYs), costs, and incremental cost-effectiveness ratios (ICER; $/QALY). 1-way and probabilistic sensitivity analyses were performed and ICERs were compared to willingness to pay standards of $100,000/QALY and $150,000/QALY.Results: In the base case, CAZ-AVI had an ICER of $95,000/QALY. At a $100,000/QALY threshold, results were sensitive to a number of variables including: the probability and cost of LTC, quality of life following CRE infection, CAZ-AVI's absolute risk reduction in mortality, all-cause mortality, daily cost of CAZ-AVI, and healthcare costs after CRE infection. The ICER did not exceed $150,000/QALY after varying all model inputs across a wide range of plausible values. In probabilistic sensitivity analysis, CAZ-AVI was the optimal strategy in 59% and 99% of simulations at $100,000/QALY and $150,000/QALY threshold, respectively.Conclusion: CAZ-AVI is a cost-effective treatment for CRE bacteremia and pneumonia based on accepted willingness to pay standards in the US.
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Dolor Abdominal/etiología , Criptococosis/diagnóstico , Cryptococcus neoformans/aislamiento & purificación , Huésped Inmunocomprometido , Linfoma de Células B Grandes Difuso/complicaciones , Antineoplásicos Inmunológicos/efectos adversos , Criptococosis/complicaciones , Diagnóstico Diferencial , Resultado Fatal , Glucocorticoides/efectos adversos , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos XAsunto(s)
Cefalea/etiología , Coriomeningitis Linfocítica/diagnóstico , Virus de la Coriomeningitis Linfocítica/aislamiento & purificación , Viaje , Adulto , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/microbiología , Angiografía por Tomografía Computarizada , Diagnóstico Diferencial , Femenino , Humanos , Inmunoglobulina G/sangre , Coriomeningitis Linfocítica/complicaciones , Meningitis Bacterianas/diagnósticoAsunto(s)
Hepatitis B/diagnóstico , Hepatitis D/diagnóstico , Ictericia/etiología , Diagnóstico Diferencial , Hepatitis B/complicaciones , Hepatitis C Crónica/complicaciones , Hepatitis D/complicaciones , Dependencia de Heroína/complicaciones , Dependencia de Heroína/tratamiento farmacológico , Humanos , Hepatopatías/diagnóstico , Masculino , Persona de Mediana Edad , Abuso de Sustancias por Vía Intravenosa/complicaciones , UltrasonografíaAsunto(s)
Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B Crónica/diagnóstico , Adulto , Biomarcadores/sangre , Femenino , Hepatitis B , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/sangre , Humanos , Pruebas Serológicas , Transaminasas/sangreAsunto(s)
Antibacterianos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Tigeciclina , Receptores de Trasplantes , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Tigeciclina/uso terapéuticoRESUMEN
CRISPR technology has recently emerged as a powerful biosensing tool for sensitive and specific nucleic acid detection when coupled with isothermal amplification (e.g., recombinase polymerase amplification (RPA)). However, it remains a challenge to incorporate isothermal amplification into CRISPR detection in a one-pot system due to their poor compatibility. Here, we developed a simple CRISPR gel biosensing platform for human immunodeficiency virus (HIV) RNA detection by combining reverse transcription-recombinase polymerase amplification (RT-RPA) reaction solution with a CRISPR gel. In our CRISPR gel biosensing platform, CRISPR-Cas12a enzymes are embedded into the agarose gel, providing a spatially separated but connected reaction interface with the RT-RPA reaction solution. During isothermal incubation, the RT-RPA amplification occurs initially on the CRISPR gel. When RPA products are sufficiently amplified and reach the CRISPR gel, the CRISPR reaction occurs in the whole tube. With the CRISPR gel biosensing platform, we successfully detected down to 30 copies of HIV RNA per test within 30 min. Furthermore, we validated its clinical utility by detecting HIV clinical plasma samples, achieving superior performance compared with the real-time RT-PCR method. Thus, our one-pot CRISPR gel biosensing platform demonstrates great potential for rapid and sensitive molecular detection of HIV and other pathogens at the point of care.
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Infecciones por VIH , Transcripción Reversa , Humanos , Sensibilidad y Especificidad , ARN Viral/genética , Recombinasas/genética , Infecciones por VIH/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
INTRODUCTION: Tebipenem is a potential option for the treatment of a range of infections because of its oral dosing coupled with the safety profile of the ß-lactam antimicrobial class. OBJECTIVES: To evaluate tebipenem in vitro activity against a challenge set of clinical Enterobacterales collected from outpatient and community settings. METHODS: 618 Enterobacterales isolates were submitted by 11 geographically dispersed U.S medical centers that processed cultures from affiliated outpatient centers in 2022. Susceptibility tests for tebipenem and comparator agents were performed by broth microdilution. Extended-spectrum-ß-lactamase (ESBL)-like isolates were identified phenotypically. Multidrug-resistant isolates were non-susceptible to ≥1 agent in ≥3 antimicrobial classes. Genotypic testing (CarbaR) was conducted on select isolates. RESULTS: Isolates (59% Escherichia coli) were recovered from patients seen predominantly in urology/nephrology (24%), nursing home/long-term care (21%), and ambulatory/primary care (21%) clinics. Comparator agent susceptibility rates against all isolates were as follows: levofloxacin (67.5%), amoxicillin/clavulanate (73.6%), cefixime (70.4%), cefpodoxime (70%), cephalexin (61.7%), ceftriaxone (74.4%), cefazolin (63.8%), ertapenem (97.6%), meropenem (99.7%), nitrofurantoin (64.9%), and sulfamethoxazole/trimethoprim (70.9%). Overall, 90.3% (558/619) of isolates were inhibited at a tebipenem MIC of ≤0.125 mg/L (MIC50/90, 0.016/0.125 mg/L), including 85.7% inhibition of ESBL-phenotype isolates (n=161; MIC50/90, 0.03/0.25 mg/L), 86.3% of levofloxacin and sulfamethoxazole/trimethoprim co-resistant isolates (n=95; MIC50/90, 0.016/0.25 mg/L) and 84.3% of multidrug-resistant isolates (n = 172; MIC50/90, 0.03/0.25 mg/L). Carbapenemase genes were observed in 2 ESBL-phenotype isolates with a tebipenem MIC of ≥0.5 mg/L. CONCLUSION: Relative to common oral comparators, these data demonstrate excellent tebipenem in vitro activity against Enterobacterales isolated from patients receiving care in outpatient settings, including urology clinics and nursing homes.
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Antibacterianos , Levofloxacino , Humanos , Estados Unidos , Antibacterianos/farmacología , Pacientes Ambulatorios , Escherichia coli , beta-Lactamasas/genética , Casas de Salud , Sulfametoxazol , Trimetoprim , Pruebas de Sensibilidad MicrobianaRESUMEN
Hospital-acquired pneumonia and ventilator-associated pneumonia that are caused by multidrug resistant (MDR) pathogens represent a common and severe problem with increased mortality. Accurate diagnosis is essential to initiate appropriate antimicrobial therapy promptly while simultaneously avoiding antibiotic overuse and subsequent antibiotic resistance. Here, we discuss the main conventional phenotypic diagnostic tests and the advanced molecular tests that are currently available to diagnose the primary MDR pathogens and the resistance genes causing pneumonia.
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Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for early diagnosis and timely medical treatment of coronavirus disease 2019. Here, we describe a digital warm-start CRISPR (dWS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The dWS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate and reliable digital quantification of SARS-CoV-2. By targeting SARS-CoV-2's nucleoprotein gene, the dWS-CRISPR assay is able to detect down to 5 copies/µl SARS-CoV-2 RNA in the chip. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples. Moreover, it has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction. Thus, the dWS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.
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Técnicas Biosensibles , COVID-19 , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Técnicas de Amplificación de Ácido Nucleico , ARN Viral , SARS-CoV-2RESUMEN
The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.
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COVID-19 , Pandemias , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/genética , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.
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Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Betacoronavirus/genética , COVID-19 , Prueba de COVID-19 , Sistemas CRISPR-Cas , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Genes Virales , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pandemias , Neumonía Viral/diagnóstico , Sistemas de Atención de Punto , ARN Viral/análisis , ARN Viral/genética , SARS-CoV-2 , Sensibilidad y Especificidad , Proteínas Virales/análisis , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Candida tropicalis is a virulent fungal pathogen for which echinocandins are the primary therapy. Emergence of resistance to echinocandins of C. tropicalis carries potentially ominous therapeutic implications. METHODS: We describe herein two patients with breakthrough C. tropicalis fungemia during echinocandin therapy, characterize their molecular mechanism of resistance, and systematically review 13 previously reported cases of echinocandin-resistant C. tropicalis bloodstream infections (BSIs) and other diseases. RESULTS: Among these 15 patients with echinocandin-resistant C. tropicalis infections, the median age was 61 years (ages 28-84 years) and 13 (86%) were immunocompromised. Thirteen (86%) of all patients had a history of pervious or concurrent exposure to echinocandins. Isolates of C. tropicalis from 11 cases, including the two index cases, underwent DNA sequencing of the FKS1 gene for mutations known to confer echinocandin resistance. The amino acid substitution Ser654Pro was shown in four cases, while other FKS1 mutations encoded Ser80S/Pro, Phe641Leu, Phe641Ser, Ser80S/Pro substitutions. These mutational events were not associated with collateral increases in minimum inhibitory concentrations to antifungal triazoles and amphotericin B. Overall mortality in patients with echinocandin-resistant C. tropicalis infections was 40%. Among those six patients who died, two received monotherapy with voriconazole, one was treated with fluconazole, one remained on caspofungin, and two were switched to liposomal amphotericin B. Nine patients (60%) survived after being treated with an antifungal agent other than an echinocandin. CONCLUSIONS: Emergence of resistance to echinocandins by C. tropicalis, occurs during antifungal therapy, is associated with high mortality, is mediated by a diverse range of FKS1 mutations, retains in vitro susceptibility to triazoles and amphotericin B, and constitutes an emerging threat to patients with hematological malignancies.
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Enfermedades Cerebelosas/etiología , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/complicaciones , Adulto , Antivirales/uso terapéutico , Encéfalo/patología , Enfermedades Cerebelosas/diagnóstico , Enfermedades Cerebelosas/tratamiento farmacológico , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Imagen por Resonancia Magnética , Resultado del TratamientoRESUMEN
Burkholderia cepacia complex (Bcc) is an emerging pathogen causes life-threatening infections in cystic fibrosis patients. This narrative review aims to highlight an update on the epidemiology, the microbiological diagnostic methods, the antimicrobial resistance mechanisms, and the most current as well the promising therapeutic options for Bcc. Although Bcc plays a role in crop growth due to its fungicidal activity and pesticide metabolizer, it has been frequently implicated in outbreaks in the United States and Worldwide due to contaminated medicines or medical devices. Phenotypic methods of microbial identification often lack sensitivity. Consequently, the diagnosis is mainly based on molecular testing which is critical for species identification. Disc diffusion is a poorly reproducible method for antibiotic susceptibility testing of Bcc when compared to broth microdilution, agar dilution or Etest. Bcc is naturally resistant to many antimicrobialagents including carboxypenicillins, polymyxin, and often, aminoglycosides due to the efflux pump activity. Treatment is often based on antimicrobial susceptibility data. However, trimethoprim-sulfamethoxazole, meropenem, doripenem, doxycycline, minocycline, and ceftazidime have been shown to be among the most active antimicrobial agents to date. Further studies are needed to compare the current antimicrobial agents and to study new inhibitors of the different efflux pumps implicated in the resistance of Bcc to antibiotics. Furthermore, harmonization of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines is highly important to fight against the rapidly emerging resistant bacteria e.g. Bcc.