Territorywide Study of Early Coronavirus Disease Outbreak, Hong Kong, China.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10-3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.

Transmission Patterns of Co-Circulation of Omicron Sub-Lineages in Hong Kong SAR, China, a City with Rigorous Social Distancing Measures, in 2022.

OBJECTIVE: This study aimed to characterize the changing landscape of circulating SARS-CoV-2 lineages in the local community of Hong Kong throughout 2022. We examined how adjustments to quarantine arrangements influenced the transmission pattern of Omicron variants in a city with relatively rigorous social distancing measures at that time. METHODS: In 2022, a total of 4684 local SARS-CoV-2 genomes were sequenced using the Oxford Nanopore GridION sequencer. SARS-CoV-2 consensus genomes were generated by MAFFT, and the maximum likelihood phylogeny of these genomes was determined using IQ-TREE. The dynamic changes in lineages were depicted in a time tree created by Nextstrain. Statistical analysis was conducted to assess the correlation between changes in the number of lineages and adjustments to quarantine arrangements. RESULTS: By the end of 2022, a total of 83 SARS-CoV-2 lineages were identified in the community. The increase in the number of new lineages was significantly associated with the relaxation of quarantine arrangements (One-way ANOVA, F(5, 47) = 18.233, p < 0.001)). Over time, Omicron BA.5 sub-lineages replaced BA.2.2 and became the predominant Omicron variants in Hong Kong. The influx of new lineages reshaped the dynamics of Omicron variants in the community without fluctuating the death rate and hospitalization rate (One-way ANOVA, F(5, 47) = 2.037, p = 0.091). CONCLUSION: This study revealed that even with an extended mandatory quarantine period for incoming travelers, it may not be feasible to completely prevent the introduction and subsequent community spread of highly contagious Omicron variants. Ongoing molecular surveillance of COVID-19 remains essential to monitor the emergence of new recombinant variants.

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.

A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2.

The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/µL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.

A cross-sectional survey of snake oral bacterial flora from Hong Kong, SAR, China.

OBJECTIVE: To determine the pattern of oral bacterial flora and their sensitivity to antibiotics in freshly captured native snakes in Hong Kong SAR, People's Republic of China. METHODS: Healthy native snakes were captured and kept in a designated centre. Snake species were identified by experienced herpetologists. Mouth swabs were taken by the veterinarian using strict aseptic techniques. The snakes were released back to the wild immediately after the above procedure. Swabs were sent for microbiological studies of bacterial culture and antibiotic sensitivity. RESULTS: 47 venomous snakes of the families Colubridae, Elapidae and Viperidae and 53 non-medically important snakes were captured. 406 bacterial isolates of 72 different species were cultured: these included gram negative and positive bacterial species and also anaerobic bacterial species. With the exception of the white-lipped pit viper (Cryptelytrops albolabris), venomous snakes harboured more pathogenic bacteria and total bacteria species compared to the non-medically important species. Of the venomous snakes, the Chinese cobra (Naja atra) harboured the largest number of bacterial species. In the present study, all gram negative bacteria associated with wound infection were sensitive to levofloxacin, netilmicin and piperacillin/tazobactam. Many gram negative bacteria in the study were not sensitive to cefuroxime axetil. Amoxicillin/clavulanic acid was an appropriate choice to cover Enterococcus faecalis and anaerobes. CONCLUSION: In the presence of wound infection from snakebite injury in Hong Kong, first line empirical antibiotics include amoxicillin/clavulanic acid plus levofloxacin. Prophylactic antibiotics may be considered in selected cases of Chinese cobra (N. atra) bite, otherwise prophylactic antibiotics are not recommended in snakebite unless tissue necrosis is present.

The use of polymerase chain reaction assay versus conventional methods in detecting neonatal chlamydial conjunctivitis.

PURPOSE: To compare the performance of polymerase chain reaction versus conventional methods (cell culture and direct immunofluorescent assay) in diagnosing neonatal chlamydial conjunctivitis and their correlations to the severity of conjunctivitis. METHODS: Consecutive cases of neonatal conjunctivitis were recruited over a year. Both eyes were clinically graded according to the severity of conjunctivitis and investigated using the three aforementioned chlamydial tests. Neonatal chlamydial conjunctivitis was assumed if one of these three tests was positive and there was clinical improvement after treatment. Sensitivity and specificity of each of the tests were analyzed. RESULTS: Three hundred sixty-eight sets of chlamydial tests were done for 184 neonates. The percentage of positive results was 93.8% and 71.9% for polymerase chain reaction and conventional methods, respectively. Using positive results in either cell culture or direct immunofluorescent assay as a standard to diagnose neonatal chlamydial conjunctivitis, the sensitivity and specificity of polymerase chain reaction were 92.0% and 97.7%, respectively. If we used polymerase chain reaction as a standard, the sensitivity and specificity of cell culture were 73.3% and 99.7%, respectively. A discrepancy was noted in the number of positive results between polymerase chain reaction and conventional methods in milder disease. CONCLUSIONS: Polymerase chain reaction might have a higher sensitivity and similar specificity in diagnosing neonatal chlamydial conjunctivitis compared to conventional methods, and it has an additional advantage as a diagnostic tool in mild disease.

"Streptococcus milleri" endocarditis caused by Streptococcus anginosus.

Unlike other viridans streptococci, members of the "Streptococcus milleri group" are often associated with abscess formation, but are only rare causes of infective endocarditis. Although it has been shown that almost all S. intermedius isolates and most S. constellatus isolates, but only 19% of S. anginosus isolates, were associated with abscess formation, no report has addressed the relative importance of the 3 species of the "S. milleri group" in infective endocarditis. During a 5-year period (April 1997 through March 2002), 6 cases of "S. milleri" endocarditis (out of 377 cases of infective endocarditis), that fulfil the Duke's criteria for the diagnosis of infective endocarditis, were encountered. All 6 "S. milleri" isolates were identified as S. anginosus by 16S ribosomal RNA (rRNA) gene sequencing. Three patients had underlying chronic rheumatic heart disease and 1 was an IV drug abuser. Five had monomicrobial bacteremia, and 1 had polymicrobial (S. anginosus, S. mitis, Granulicatella adiacens, and Slackia exigua) bacteremia. Two patients died. None of the 6 isolates were identified by the Vitek system (GPI) or the API system (20 STREP) at >95% confidence. All 6 isolates were sensitive to penicillin G (MIC 0.008-0.064 microg/mL), cefalothin, erythromycin, clindamycin, and vancomycin. Accurate identification to the species level, by 16S rRNA gene sequencing, in cases of bacteremia caused by members of the "S. milleri group", would have direct implication on the underlying disease process, hence guiding diagnosis and treatment. Infective endocarditis should be actively looked for in cases of monomicrobial S. anginosus bacteremia, especially if the organism is recovered in multiple blood cultures.

Direct detection of isoniazid-resistant Mycobacterium tuberculosis in respiratory specimens by multiplex allele-specific polymerase chain reaction.

This study evaluated the feasibility of using 2 multiplex allele-specific polymerase chain reaction (MAS-PCR) assays targeting 2 mutations (codon 315 of the katG gene and the 15th nucleotide preceding the mabA-inhA operon) to directly detect isoniazid (INH)-resistant Mycobacterium tuberculosis in cultured isolates and respiratory specimens. A total of 203 M. tuberculosis isolates and 487 respiratory specimens were investigated. The MAS-PCR assays successfully amplified all M. tuberculosis isolates and acid-fast bacilli smear-positive specimens while only 49.2% of the smear-negative specimens exhibited positive MAS-PCR results. The MAS-PCR assays identified 83.4% and 79.2% of the resistant strains in the culture isolates and respiratory specimens, respectively. All the inferred genotypes were in complete accordance with subsequent DNA sequence analyses. This study suggested the application of our improved MAS-PCR protocols to provide the rapid identification of INH-resistant M. tuberculosis directly in respiratory specimens. The technical simplicity, short turnaround time, and low cost of this molecular strategy should facilitate routine diagnostic services in developing areas with a high prevalence of drug-resistant tuberculosis.

A comparative study on bacterial cultures of urine samples obtained by clean-void technique versus urethral catheterization.

UNLABELLED: Contamination during urine collection causes difficulty in diagnosing infantile urinary tract infection (UTI). Though considered a gold-standard, suprapubic aspiration is traumatic and not always successful. Catheterization and clean void technique were often preferred but their relative usefulness has not been compared. OBJECTIVES: To compare the culture results of clean void urine (CVU) and catheter urine (CathU) from children below 2-years old known to have no UTI. We tested whether the false-positive rates of CVU were significantly different from that of CathU. METHODS: Paired CVU and CathU samples were collected from asymptomatic children admitted for micturiting cystourethrogram, and tested for leucocytes and nitrite, and bacterial culture using standard laboratory methods. RESULTS: Culture results for 98 children (82 boys, 16 girls; aged 6 +/- 4.3 months) were analysed. Analysis by presence/absence of growths showed good agreement between CVU and CathU for boys (Kappa 0.42) but poor for girls (Kappa 0.18). When analysed by colony counts, agreement was poor with CVU yielding more counts than CathU (Kappa 0.1 for boys and 0.04 for girls). If all mixed growth results were considered as contaminants, the false positive rates for CathU and CVU were similar whether the cut-off was defined as 10(3), 10(4) or 10(5)/mL. If mixed growth was believed to cause UTI, CathU produced less false-positive rates than CVU, though both rates were unacceptably high. CONCLUSION: In uncircumcized boys, both CVU and CathU were prone to contamination. Though the contaminating bacterial counts were lower in CathU culture, the false-positive rates were high with the lower cut-off CFUs. Contrary to previous recommendations, CathU should be interpreted similar to CVU to avoid false positive diagnosis of UTI.

Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles.

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the "gold standard," we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification.