CRISPR-on-beads: A simple and sensitive assay for detection of methicillin-resistant staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-beta-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct.

Staphylococcus aureus is one of the main pathogens causing hospital and community-acquired infections, in particular, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) cause a higher mortality rate than those caused by methicillin-sensitive strains, which poses a serious global public health problem. Therefore, rapid and ultrasensitive detection of patients with clinical MRSA infection and timely control of infection are essential. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) based on nucleic acid detection methods are well-known for its high specificity and sensitivity and programmability. Here, we successfully proposed a method based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) through fluorescent readout to achieve accurate identification and highly sensitive detection of MRSA in clinical samples. Results showed that the limit of detection (LoD) of the RAA-Cas12a method could reach 10 copies/μl at 60 min of reaction. Specificity tests showed that the method could distinguish MRSA from clinically common bacteria. The results of RAA-Cas12a were consistent with that of antimicrobial susceptibility tests (AST) and polymerase chain reaction (PCR) in 83 clinical samples. These results indicated that the detection method based on RAA-Cas12a has high sensitivity and specificity, and provides important value for rapid detection of MRSA.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

Five decades of MRSA: controversy and uncertainty continues

ABSTRACTIn order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies μL−1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy μL−1). The LOD of both platforms reached 4 × 103 fg/μL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA.IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/μL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

3D-printed electrochemical loop-mediated isothermal amplification (E-LAMP): A miniaturized and portable platform to detect methicillin-resistant Staphylococcus aureus (MRSA).

BackgroundStaphylococcus aureus (S. aureus), including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), is an eminent human pathogen that can colonize the human host and cause severe life-threatening infections. The development of a reliable, simple and rapid assay for detecting S. aureus and identifying MRSA is important for diagnosis and follow-up treatment.MethodsA novel molecular diagnosis technique, named multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor (m-LAMP-LFB), was applied to detect all S. aureus species and identify MRSA. Two sets of primers were designed based on the femA gene (S. aureus-specific gene) and the mecA gene (encoding penicillin-binding protein 2a), and the multiple-LAMP products were analyzed using LFB. The m-LAMP-LFB amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of the m-LAMP-LFB method were tested in the current study, and the multiple-LAMP-LFB technology was applied to detect the MSSA and MRSA strains from clinical samples.ResultsThe S. aureus- and MRSA-specific primers based on the femA and mecA genes allowed the multiple-LAMP technology to detect S. aureus and MRSA, respectively. The multiple-LAMP conditions were optimized at 63 °C for 40 min. The full process, including genomic DNA template preparation, LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the multiple-LAMP assay for femA and mecA detection was 100 fg of genomic DNA template per reaction. The specificity of m-LAMP-LFB detection was 100 %, and no cross-reactions to non-S. aureus strains were observed.ConclusionThe multiple-LAMP-LFB technique developed in the current study is a reliable, simple, rapid, specific and sensitive method to identify MSSA and MRSA infections for appropriate antibiotic therapy.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

Establishment and evaluation of a rapid nucleic acid detection method for methicillin-resistant Staphylococcus aureus

MRSA screening: can one swab be used for both culture and rapid testing? An evaluation of chromogenic culture and subsequent Hain GenoQuick PCR amplification/detection

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid

To describe methicillin-resistant Staphylococcus aureus (MRSA) control in a hospital, including a surgical intensive care unit (SICU) outbreak. Prospective surveillance of newly identified patients with MRSA. Barrier isolation (disposable gloves for direct contact with patient or immediate environment) was used for the routine care of hospitalized MRSA patients as of October 1991. Beginning in 1992, MRSA isolates were typed by restriction endonuclease enzyme analysis of plasmid DNA (REAP) and/or pulsed-field gel electrophoresis of genomic DNA (PFGE). Surveillance information and MRSA typing were used concurrently to identify nosocomial case clustering, confirm cross-infection, and support a need for additional outbreak control interventions. University-affiliated public hospital. Patients with newly identified MRSA colonization or infection from 1991 through 1993 and epidemiologically associated staff providing care to eight SICU patients in an outbreak. Barrier isolation for affected and unaffected patients in and admitted to the SICU institution when the outbreak was identified and cross-infection confirmed. Anterior nares cultures of staff in contact with outbreak cases for detection of MRSA colonization. Fifty-six hospitalized patients with community-acquired MRSA and 80 patients with nosocomial MRSA colonization or infection were identified during the 3 years. After the introduction of barrier isolation, the annual frequency of new nosocomial MRSA cases decreased and only one outbreak (eight cases in the SICU) caused by type-related isolates occurred. The other 35 nosocomial cases of MRSA during 1992 and 1993 were not epidemiologically related or were caused by isolates with different types. The SICU outbreak ended after instituting barrier isolation for all patients (with and without MRSA) in and admitted to the unit. Six colonized SICU staff were identified. All outbreak cases had identical or related MRSA types by PFGE and REAP. Staff isolates were different from case isolates by typing, and staff were not restricted and not given treatment for colonization. After more than 6 months of follow up, no further outbreaks of MRSA in the SICU or elsewhere in the hospital occurred despite returning to barrier isolation for affected patients only. MRSA in hospitals and outbreaks of MRSA in ICUs can be controlled by surveillance and minimal barrier interventions. REAP or PFGE typing of MRSA can be used to support or refute the presence of cross-transmission. Typing also may be helpful when planning and assessing the effectiveness of interventions directed at endemic, as well as outbreak, MRSA control.

Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles