Comparison of carbapenem MIC for NDM-producing Enterobacterales by different AST methods.

Introduction: This study compared the performance of MIC test strip (ETEST), automated AST card (Vitek 2) and broth microdilution (BMD) in determining carbapenem susceptibility and MIC values of NDM-producing Enterobacterales. Methods: NDM-producing Enterobacterales recovered from clinical specimens were included. The presence of blaNDM was confirmed by PCR. Identification of bacterial isolates was done by MALDI-TOF. Phenotypic susceptibility to three carbapenems (ertapenem, imipenem and meropenem) was tested by BMD, ETEST and Vitek 2. MIC values were interpreted in accordance with CLSI M100 (2022 edition). Using BMD as the reference standard, the essential agreement (EA), categorical agreement (CA), very major error (VME) and major error (ME) rates were evaluated. Results: Forty-seven NDM-producing Enterobacterales isolates were included, 44 of which were Escherichia coli. The EA of Vitek 2 was 97.9% for ertapenem, 25.5% for meropenem and 42.6% for imipenem. Using Vitek 2, there were 0% VMEs across all three carbapenems tested. The EA of ETEST was 53.2% for ertapenem, 55.3% for imipenem and 36.2% for meropenem. The rates of VMEs for ETEST were high too (ertapenem 8.5%, meropenem 36.2%, imipenem 26.1%). The MIC values obtained from Vitek 2 were consistently higher than those from BMD, while MICs from ETEST were consistently lower than those from BMD. Conclusions: The VME rate for ETEST was unacceptably high when BMD was used as the standard for comparison. Vitek 2 had acceptable EA and CA for ertapenem when BMD was used as the standard for comparison. For meropenem and imipenem, neither of the methods (ETEST, Vitek 2) showed acceptable EA and CA when compared with BMD.

The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids.

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culture-negative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml.

Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

Application of digital PCR to determine the reliability of Xpert Xpress SARS-CoV-2 assay with envelope (E) gene negative and nucleocapsid (N2) gene positive results.

This study used digital polymerase chain reaction (dPCR) to determine whether envelope (E) gene-negative and nucleocapsid (N2) gene-positive (E-N+) results obtained with the Cepheid Xpert Xpress SARS-CoV-2 assay are reliable. Using droplet digital PCR results as a reference, 18 of 22 E-N+ samples with a low viral load (81.8%) were identified as true positives.

Evaluation of the Xpert Xpress SARS-CoV-2/Flu/RSV Assay for Simultaneous Detection of SARS-CoV-2, Influenza A and B Viruses, and Respiratory Syncytial Virus in Nasopharyngeal Specimens.

Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (flu A), influenza B (flu B), and respiratory syncytial virus (RSV) have overlapping clinical presentations, but the approaches to treatment and management of infections caused by these viruses are different. Therefore, rapid diagnosis in conjunction with infection prevention measures is important to prevent transmission of the diseases. Recently, a new Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4-in-1) assay enables the detection and differentiation of SARS-CoV-2, flu A, flu B, and RSV in upper respiratory tract specimens. In this study, we evaluated the performance of the Xpert 4-in-1 assay by comparing it with that of the Xpert Xpress SARS-CoV-2 and Xpert Xpress Flu/RSV assays for the detection of the four viruses in nasopharyngeal (NP) specimens. A total of 279 NP specimens, including 66, 56, 64, and 53 specimens positive for SARS-CoV-2, flu A, flu B, and RSV, respectively, were included. The Xpert 4-in-1 assay demonstrated high concordance with the comparator assays, with overall agreement for SARS-CoV-2, flu A, flu B, and RSV at 99.64%, 100%, 99.64%, and 100%, respectively, and a high Cohen's kappa (κ) value ranging from 0.99 to 1.00, indicating an almost perfect correlation between assays. The cycle threshold value association between positive samples also showed a good correlation between assays. In conclusion, the overall performance of the Xpert 4-in-1 assay was highly comparable to that of the Xpert SARS-CoV-2 and Xpert Flu/RSV assays for the detection and differentiation of SARS CoV-2, flu A, flu B, and RSV in NP specimens.

Deep throat saliva as an alternative diagnostic specimen type for the detection of SARS-CoV-2.

Nasopharyngeal swabs (NPS) are widely accepted as specimens for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the current pandemic of coronavirus disease 2019. However, the collection procedures for NPS specimens causes sneezing and coughing in most patients, which generate droplets or aerosol particles that are hazardous to the healthcare workers collecting these specimens. In this study, 95 patient-matched paired deep throat saliva (DTS) and NPS specimens from 62 patients were analyzed. Samples were tested for SARS-CoV-2 by reverse-transcription polymerase chain reaction (RT-PCR). The rates of detection for DTS (53.7%) and NPS (47.4%) samples were comparable (P = .13). It is important to note that the patients should be clearly instructed or supervised during DTS collection. In conclusion, SARS-CoV-2 detection by RT-PCR was equivalent in DTS and NPS specimens.

Evaluation on testing of deep throat saliva and lower respiratory tract specimens with Xpert Xpress SARS-CoV-2 assay.

BACKGROUND: Xpert® Xpress SARS-CoV-2 assay is only validated on nasopharyngeal specimens for detection of SARS-CoV-2. Other specimen types such as deep throat saliva (DTS), also known as posterior oropharyngeal saliva and lower-respiratorytract specimens (LRT) including sputum, tracheal aspirate and bronchoalveolar lavage are not validated. These non-validated specimen types, however, do have significant diagnostic value. OBJECTIVE: Evaluate the performance of Xpert Xpress SARS-CoV-2 assay for detection of SARS-CoV-2 from DTS and LRT specimens. METHODS: 162 specimens from 158 patients with suspected COVID-19 disease were tested with Xpert Xpress SARS-CoV-2 assay. These included 120 DTS and 42 LRT specimens i.e. 35 sputum, 6 tracheal aspirate and one bronchoalveolar lavage. Results were compared to those by the TIB-Molbiol LightMix® SarbecoV E-gene assay. RESULTS: Xpert Xpress SARS-CoV-2 assay has satisfactory performance when compared with reference method. The positive percent agreement (PPA) of DTS and LRT specimens were 98.86 % & 100 % respectively while the negative percent agreement (NPA) was 100 % for both DTS and LRT specimens. CONCLUSIONS: This study demonstrated with appropriate sample pre-treatment, Xpert Xpress SARS-CoV-2 assay can be used to test on non-validated specimen types including DTS & LRT specimens.

Emergence of Carbapenem-Resistant Acinetobacter baumannii ST195 Harboring blaOXA-23 Isolated from Bacteremia in Hong Kong.

Aims: The aim of the study was to analyze the epidemiology of Acinetobacter baumannii and investigate the genetic characteristics of carbapenem-resistant A. baumannii (CRAB) isolates isolated from blood cultures in a regional hospital in Hong Kong. Results: Twenty blood culture isolates were collected from a regional hospital in Hong Kong from 2014 to 2017. Twenty isolates were grouped into five existing sequence types (STs) and five new STs within the following prevalence: ST195 was predominant with a prevalence of 45% (n = 9), followed by ST373 and ST447 (10%; n = 2 each), and ST176 and ST345 (5%; n = 1 each). Resistance to carbapenem antibiotics was 55% (n = 11). Six carbapenem-resistant isolates harbored blaOXA-23 genes and ISAba1 mobile elements. Polymerase chain reaction confirmed that ISAba1 is located upstream to the blaOXA-23 genes, suggesting an association between ISAba1 and blaOXA-23 genes with carbapenem resistance. Conclusion: This study is the first to report the emergence of CRAB ST195 harboring blaOXA-23 in Hong Kong.

Admission Screening of Methicillin-Resistant Staphylococcus aureus with Rapid Molecular Detection in Intensive Care Unit: A Three-Year Single-Centre Experience in Hong Kong.

Background. The admission screening of methicillin-resistant Staphylococcus aureus (MRSA) by rapid molecular assay is considered to be an effective method in reducing the transmission of MRSA in intensive care unit (ICU). Method. The admission screening on patients from ICU once on their admissions by BD GeneOhm MRSA assay has been introduced to Prince of Wales Hospital, Hong Kong, since 2008. The assay was performed on weekdays and reported on the day of testing. Patients pending for results were under standard precautions until the negative screening results were notified, while contact precautions were implemented for MRSA-positive patients. In this study, we compared the MRSA transmission rate in molecular screening periods (2008 to 2010) with the historical culture periods (2006 to 2007) as control. Results. A total of 4679 samples were tested; the average carriage rate of MRSA on admission was 4.45%. By comparing with the historical culture periods, the mean incidence ICU-acquired MRSA infection was reduced from 3.67 to 1.73 per 1000 patient bed days. Conclusion. The implementation of admission screening of MRSA with molecular method in intensive care unit could reduce the MRSA transmission, especially in the area with high MRSA prevalence situation in Hong Kong.