Rapid adaptation and continuous performance evaluation of SARS-CoV-2 envelope gene (E-gene) real-time RT-PCR assays to support the hospital surge in test demand.

We describe the timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with an on-going evaluation to ensure the provision of quality service within the time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, the provision of round-the-clock service is feasible despite the test is of high complexity. The thermal cycling duration of the adapted LightMix E-gene and WHO E-gene is shortened by half and one hour respectively and allows the number of runs to double when 24-h round-the-clock service is provided. An increase in testing capacity could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system, and to optimize utilization of the isolation beds.

Complete sequencing of the FII plasmid pHK01, encoding CTX-M-14, and molecular analysis of its variants among Escherichia coli from Hong Kong.

OBJECTIVES: We characterized plasmids encoding CTX-M-14 ß-lactamase originating from Escherichia coli isolates recovered from patients with uncomplicated cystitis or individuals with faecal colonization in Hong Kong from 2002 to 2004. METHODS: Plasmids carrying CTX-M-14 were studied by conjugation, replicon typing, S1 nuclease-PFGE and plasmid PCR-restriction fragment length polymorphism (RFLP). The complete sequence of pHK01, a 70 kb plasmid encoding CTX-M-14 from an E. coli strain, was determined and the results compared with reference plasmids and aligned with GenBank data. RESULTS: The blaCTX-M-14 plasmids could be transferred in 23 of 44 E. coli strains tested. Among the 23 transconjugants, the replicon types of the CTX-M-14-encoding plasmid were FII (n=13), I1-Iγ (n=4), F1B (n=2), FII and I1-Iγ (n=1), K (80 kb, n=1) and undetermined (n=2). Plasmid pHK01 (FII replicon) shares a high degree of homology with R100 except mainly for a 11 kb variable region containing blaCTX-M-14 (with an upstream ISEcp1 and a downstream truncated IS903), an iron transport system, an outer membrane protein (malB, maltoporin) and a putative toxin-antitoxin plasmid stability system (yacABC). It was highly related to blaCTX-M-14 (pKF3-70) and blaCTX-M-24 (pEG356) plasmids reported from mainland China in 2006 and Vietnam in 2007, respectively. Subtyping by a plasmid PCR-RFLP scheme showed that 10 of the 13 FII plasmids originating from isolates collected by multiple laboratories exhibited either identical or highly similar profiles. CONCLUSIONS: This study showed that narrow host-range FII plasmids play important roles in the dissemination of CTX-M-14. FII plasmids closely related to pHK01 have disseminated widely in the Hong Kong community.

Antimicrobial resistance in Escherichia coli outpatient urinary isolates from women: emerging multidrug resistance phenotypes.

This study evaluated the antimicrobial resistance profile of outpatient urinary Escherichia coli isolated from women obtained throughout Hong Kong during 2004-2005. Of 1067 single patient isolates analyzed, 60.1% were resistant to ampicillin, 34% were resistant to co-trimoxazole, and 22.1% were resistant to ciprofloxacin. Thirty-four (6.6%) of 519 isolates in 2004 and 55 (10%) of 548 isolates in 2005 were extended-spectrum beta-lactamase (ESBL) producers with a CTX-M phenotype. Rates of non-beta-lactam resistance and ESBL production were strongly influenced by patient age. The age-stratified rates for dual co-trimoxazole and ciprofloxacin resistance and for ESBL production were 10.9% and 7.6% in women aged 18-35 years, 13% and 6.9% in women aged 36-50 years, 20.4% and 8.8% in women aged 51-64 years, and 23.7% and 11.8% in women aged > or =65 years, respectively. Nitrofurantoin and fosfomycin remain active against >90% of the isolates, irrespective of the resistance phenotypes for other drugs. Our results documented the emergence of problematic resistance phenotypes among community urinary E. coli and highlight the need to explore strategies for their containment.

Community emergence of CTX-M type extended-spectrum beta-lactamases among urinary Escherichia coli from women.

OBJECTIVES: To conduct a territory-wide study of extended-spectrum beta-lactamases (ESBLs) among community isolates of urinary Escherichia coli from women in Hong Kong. METHODS: Up to 50 consecutive single-patient E. coli isolates, collected from 13 laboratories in 2004, were studied. The ESBLs were characterized by PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by PFGE and phylogenetic group PCRs. RESULTS: Forty-two ESBL producers were found among 600 consecutive isolates tested. The ESBL prevalence was 7.3% (15/205) for women aged 18-35 years, 5% (11/219) for women aged 36-50 years, 6.3% (4/63) for women aged 51-64 years and 10.6% (12/113) for women aged >or=65 years (P=0.3). The ESBL-producing isolates were often multidrug-resistant and CTX-M-14 was found in 37 isolates, CTX-M-15 in 3 isolates and CTX-M-3 in 2 isolates. PFGE revealed no significant clusters among the ESBL producers. Overall, CTX-M-14 producers were significantly more likely to belong to group D than non-ESBL producers [18/37 (48.6%) versus 13/57 (22.8%), P=0.009]. However, 7 of 13 (53.8%) CTX-M-14 producers from women aged 18-35 years represented phylogenetic group B2, compared with 7 of 24 (29.2%) for women of all other ages (P=0.1). CONCLUSIONS: The study documented the community emergence of CTX-M as the predominant ESBL type among urinary isolates from women. The spread of CTX-M enzymes among isolates from young women is concerning and deserves close monitoring.

Escherichia coli producing CTX-M beta-lactamases in food animals in Hong Kong.

A study was conducted to evaluate the occurrence of extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli from fecal samples of healthy food animals in Hong Kong. Rectal or cloacal swabs were obtained from cattle, pigs, chicken, ducks, geese, and pigeons in slaughterhouses or wholesale markets over a 5- month period in 2002. Antibiotic-containing medium was used for selective isolation of potentially ESBL-producing E. coli. Of 734 samples analyzed, six (2%) from pigs, three (3.1%) from cattle, and one (3%) from pigeons had E. coli strains with the ESBL phenotype. The ESBL content for the 10 isolates include CTXM- 3 (n = 4), CTX-M-13 (n = 3), CTX-M-14 (n = 2), and CTX-M-24 (n = 1). In five isolates, the bla (CTX-M) gene was encoded on transferable plasmids (60 or 90 kb), and the gene was found to transfer to E. coli (J53 or JP995) with frequencies of 10(7) to 10(3) per donor cells. The ten isolates had five distinct pulsotypes with some clonal spread. However, the isolates from the different kinds of animals were not clonally related. These findings imply that bacteria of animal origins may serve as reservoirs of some ESBL genes.

Occurrence and molecular analysis of extended-spectrum {beta}-lactamase-producing Proteus mirabilis in Hong Kong, 1999-2002.

OBJECTIVES: A study was conducted to evaluate the occurrence and characterization of extended-spectrum beta-lactamases (ESBLs) among blood isolates of Proteus mirabilis collected over a 4 year period in Hong Kong. METHODS: Production of ESBLs among 99 consecutive and non-duplicate isolates was evaluated by the double-disc synergy test. The ESBLs were characterized by isoelectric focusing and PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by the Dienes test and PFGE. RESULTS: ESBLs were identified in 13 isolates, from none in 1999-2000 and up to 18.5% (5/27) in 2001 and 25.8% (8/31) in 2002. The ESBL-producing isolates were more resistant to ceftriaxone than to ceftazidime, and were more likely than non-ESBL-producers to have resistance to ciprofloxacin (76.9% versus 14%) and gentamicin (38.5% versus 9.3%). The ESBL content included CTX-M-13 (n=8), CTX-M-14 (n=3), SHV-5 (n=2), TEM-11 (n=1), and an unidentified ESBL with a pI of 7.5. The Dienes test revealed that the genetic background in the 99 isolates was highly heterogeneous, with 54 distinct types among 92 isolates and seven were non-typeable. Among the 13 ESBL-producing isolates, five different backgrounds, including one cluster (Dienes-pulsotype A) with nine isolates, were identified by both Dienes test and PFGE, thus suggesting both clonal and multi-clonal spread of the CTX-M enzymes. CONCLUSIONS: Our findings indicate the emergence of CTX-M enzymes among P. mirabilis in Hong Kong. More ESBL screening of this species is required to improve their recognition.