The use of multi-pronged screening strategy to understand the epidemiology of carbapenemase-producing Enterobacteriaceae in Hong Kong: transition from epidemic to endemic setting.

A multi-pronged carbapenemase-producing Enterobacteriaceae (CPE) screening strategy was implemented in Hong Kong West healthcare network. Of 199,192 fecal specimens from 77,194 patients screening from 1 July 2011 to 30 June 2019, the incidence of CPE per 1000 patient admission significantly increased from 0.01 (2012) to 1.9 (2018) (p<0.01). With appropriate infection control measures, the incidence of nosocomial CPE per 1000 CPE colonization day decreased from 22.34 (2014) to 10.65 (2018) (p=0.0094). Exposure to wet market for purchasing raw pork (p=0.007), beef (p=0.017), chicken (p=0.026), and vegetable (p=0.034) for >3 times per week significantly associated with community acquisition of CPE. Strategic CPE control measures should be implemented in both the hospital and the community.

Assessment of SARS-CoV-2 Immunity in Convalescent Children and Adolescents.

Persistence of protective immunity for SARS-CoV-2 is important against reinfection. Knowledge on SARS-CoV-2 immunity in pediatric patients is currently lacking. We opted to assess the SARS-CoV-2 adaptive immunity in recovered children and adolescents, addressing the pediatrics specific immunity towards COVID-19. Two independent assays were performed to investigate humoral and cellular immunological memory in pediatric convalescent COVID-19 patients. Specifically, RBD IgG, CD4+, and CD8+ T cell responses were identified and quantified in recovered children and adolescents. SARS-CoV-2-specific RBD IgG detected in recovered patients had a half-life of 121.6 days and estimated duration of 7.9 months compared with baseline levels in controls. The specific T cell response was shown to be independent of days after diagnosis. Both CD4+ and CD8+ T cells showed robust responses not only to spike (S) peptides (a main target of vaccine platforms) but were also similarly activated when stimulated by membrane (M) and nuclear (N) peptides. Importantly, we found the differences in the adaptive responses were correlated with the age of the recovered patients. The CD4+ T cell response to SARS-CoV-2 S peptide in children aged <12 years correlated with higher SARS-CoV-2 RBD IgG levels, suggesting the importance of a T cell-dependent humoral response in younger children under 12 years. Both cellular and humoral immunity against SARS-CoV-2 infections can be induced in pediatric patients. Our important findings provide fundamental knowledge on the immune memory responses to SARS-CoV-2 in recovered pediatric patients.

Assessment of population susceptibility to upcoming seasonal influenza epidemic strain using interepidemic emerging influenza virus strains.

Seasonal influenza virus epidemics have a major impact on healthcare systems. Data on population susceptibility to emerging influenza virus strains during the interepidemic period can guide planning for resource allocation of an upcoming influenza season. This study sought to assess the population susceptibility to representative emerging influenza virus strains collected during the interepidemic period. The microneutralisation antibody titers (MN titers) of a human serum panel against representative emerging influenza strains collected during the interepidemic period before the 2018/2019 winter influenza season (H1N1-inter and H3N2-inter) were compared with those against influenza strains representative of previous epidemics (H1N1-pre and H3N2-pre). A multifaceted approach, incorporating both genetic and antigenic data, was used in selecting these representative influenza virus strains for the MN assay. A significantly higher proportion of individuals had a ⩾four-fold reduction in MN titers between H1N1-inter and H1N1-pre than that between H3N2-inter and H3N2-pre (28.5% (127/445) vs. 4.9% (22/445), P < 0.001). The geometric mean titer (GMT) of H1N1-inter was significantly lower than that of H1N1-pre (381 (95% CI 339-428) vs. 713 (95% CI 641-792), P < 0.001), while there was no significant difference in the GMT between H3N2-inter and H3N2-pre. Since A(H1N1) predominated the 2018-2019 winter influenza epidemic, our results corroborated the epidemic subtype.

Measuring non-polyaminated lipocalin-2 for cardiometabolic risk assessment.

AIMS: Lipocalin-2 is a pro-inflammatory molecule characterized by a highly diversified pattern of expression and structure-functional relationships. In vivo, this molecule exists as multiple variants due to post-translational modifications and/or protein-protein interactions. Lipocalin-2 is modified by polyamination, which enhances the clearance of this protein from the circulation and prevents its excessive accumulation in tissues. On the other hand, animal studies suggest that non-polyaminated lipocalin-2 (npLcn2) plays a causal role in the pathogenesis of obesity-associated medical complications. The present study examined the presence of npLcn2 in samples from healthy volunteers or patients with cardiac abnormalities and evaluated npLcn2 as a biomarker for cardiometabolic risk assessment. METHODS AND RESULTS: Immunoassays were developed to quantify npLcn2 in blood and urine samples collected from 100 volunteers (59 men and 41 women), or venous plasma and pericardial fluid samples obtained from 37 cardiothoracic surgery patients. In healthy volunteers, npLcn2 levels in serum are significantly higher in obese and overweight than in lean subjects. After adjustment for age, gender, smoking, and body mass index (BMI), serum npLcn2 levels are positively correlated with heart rate, circulating triglycerides, high-sensitivity C-reactive protein (hsCRP), and creatinine in plasma. The npLcn2 levels in urine are significantly increased in subjects with metabolic syndrome and positively correlated with BMI, heart rate, circulating triglycerides, and urinary aldosterone. In cardiothoracic surgery patients, the circulating concentrations of npLcn2 are higher (more than two-fold) than those of healthy volunteers and positively correlated with the accumulation of this protein in the pericardial fluid. Heart failure patients exhibit excessive expression and distribution of npLcn2 in mesothelial cells and adipocytes of the parietal pericardium, which are significantly correlated with the elevated plasma levels of npLcn2, total cholesterol, and creatinine. CONCLUSIONS: Quantitative and qualitative evaluation of npLcn2 in human biofluid samples and tissue samples can be applied for risk assessment of healthy individuals and disease management of patients with obesity-related cardiometabolic and renal complications.

Application of a dual target PCR-high resolution melting (HRM) method for rapid nontuberculous mycobacteria identification.

Species differentiation of nontuberculous mycobacteria (NTM) has long been a difficult task in clinical laboratories. This study demonstrated and evaluated a simple and cost-effective method using the real-time PCR with high-resolution melting (PCR-HRM) analysis technique, which could differentiate at least 14 different medically related NTM.

Strategic measures for the control of surging antimicrobial resistance in Hong Kong and mainland of China.

Antimicrobial-resistant bacteria are either highly prevalent or increasing rapidly in Hong Kong and China. Treatment options for these bacteria are generally limited, less effective and more expensive. The emergence and dynamics of antimicrobial resistance genes in bacteria circulating between animals, the environment and humans are not entirely known. Nonetheless, selective pressure by antibiotics on the microbiomes of animal and human, and their associated environments (especially farms and healthcare institutions), sewage systems and soil are likely to confer survival advantages upon bacteria with antimicrobial-resistance genes, which may be further disseminated through plasmids or transposons with integrons. Therefore, antibiotic use must be tightly regulated to eliminate such selective pressure, including the illegalization of antibiotics as growth promoters in animal feed and regulation of antibiotic use in veterinary practice and human medicine. Heightened awareness of infection control measures to reduce the risk of acquiring resistant bacteria is essential, especially during antimicrobial use or institutionalization in healthcare facilities. The transmission cycle must be interrupted by proper hand hygiene, environmental cleaning, avoidance of undercooked or raw food and compliance with infection control measures by healthcare workers, visitors and patients, especially during treatment with antibiotics. In addition to these routine measures, proactive microbiological screening of hospitalized patients with risk factors for carrying resistant bacteria, including history of travel to endemic countries, transfer from other hospitals, and prolonged hospitalization; directly observed hand hygiene before oral intake of drugs, food and drinks; and targeted disinfection of high-touch or mutual-touch items, such as bed rails and bed curtains, are important. Transparency of surveillance data from each institute for public scrutiny provides an incentive for controlling antimicrobial resistance in healthcare settings at an administrative level.

Viral lung infections: epidemiology, virology, clinical features, and management of avian influenza A(H7N9).

PURPOSE OF REVIEW: The avian influenza A(H7N9) virus has jumped species barrier and caused severe human infections. Here, we present the virological features relevant to clinical practice, and summarize the epidemiology, clinical findings, diagnosis, treatment, and preventive strategies of A(H7N9) infection. RECENT FINDINGS: As of 18 February 2014, A(H7N9) virus has caused 354 infections in mainland China, Taiwan, and Hong Kong with a case-fatality rate of 32%. Elderly men were most affected. Most patients acquired the infection from direct contact with poultry or from a contaminated environment, although person-to-person transmission has likely occurred. A(H7N9) infection has usually presented with severe pneumonia, often complicated by acute respiratory distress syndrome and multiorgan failure. Mild infections have been reported in children and young adults. Nasopharyngeal aspirate and sputum samples should be collected for diagnosis, preferably using reverse transcriptase-PCR. Early treatment with neuraminidase inhibitors improved survival, but the efficacy of antivirals was hampered by resistant mutants. The closure of live poultry markets in affected areas has significantly contributed to the decline in the incidence of human cases. SUMMARY: The emergence of A(H7N9) virus represents a significant health threat. High vigilance is necessary so that appropriate treatment can be instituted for the patient and preventive measures can be implemented.

Molecular diagnosis in clinical parasitology: when and why?

Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also be used for monitoring these recipients or highly immunosuppressed patients, so that early preemptive treatment can be given for reactivated parasitic infections while the parasitic burden is still low.

Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and fungi was recently introduced in microbiology laboratories. This technology could greatly improve the clinical management of patients and guidance for chemotherapy. In this study, we used a commercial MALDI Sepsityper extraction method to evaluate the performance of two commercial MALDI-TOF MS systems, the Vitek MS IVD (bioMérieux) and the Microflex LT Biotyper (Bruker Daltonics) for direct bacterial identification in positive blood cultures. In 181 monomicrobial cultures, both systems generated genus to species level identifications for >90% of the specimens (Biotyper, 177/181 [97.8%]; Vitek MS IVD, 167/181 [92.3%]). Overall, the Biotyper system generated significantly more accurate identifications than the Vitek MS IVD system (P = 0.016; 177 versus 167 out of 181 specimens). The Biotyper system identified the minority species among polymicrobial blood cultures. We also compared the performance of an in-house extraction method with that of the Sepsityper on both MALDI-TOF MS systems. The in-house method generated more correct identifications at the genus level than the Sepsityper (96.7% versus 93.5%) on the Biotyper system, whereas the two methods exhibited the same performance level (88.0% versus 88.0%) on the Vitek MS IVD system. Our study confirmed the practical advantages of MALDI-TOF MS, and our in-house extraction method reduced the reagent cost to $1 per specimen, with a shorter turnaround time of 3 h, which is highly cost-effective for a diagnostic microbiology service.

The use of high-resolution melting analysis for rapid spa typing on methicillin-resistant Staphylococcus aureus clinical isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hong Kong for three decades. This study evaluated the practical use of high-resolution melting (HRM) real-time PCR analysis on MRSA staphylococcal Protein A (spa) typing on local MRSA isolates. Among 55 clinical MRSA isolates collected in 2011, 12 different spa types were observed by the conventional PCR-sequencing method including the locally predominant spa type t1081 and two locally predominant community acquired MRSA spa types t019 and t437. By using the HRM method, it could differentiate all 12 spa genotypes by distinct melting curves and HRM difference plot analysis. These two methods demonstrated 100% concordance whereas the HRM method required only 3h of turnaround time and one-fifth of reagent cost compared to the conventional method. Our study confirmed that the cost effective and rapid HRM typing approach is practically useful for MRSA community transmission monitoring and nosocomial outbreak control in Hong Kong.

Analysis of multilocus sequence typing schemes for 35 different bacteria revealed that gene loci of 10 bacteria could be replaced to improve cost-effectiveness.

Although multilocus sequence typing (MLST) has been widely used for bacterial typing, the contribution of the gene loci to the discriminatory power of each MLST scheme is unknown. We analyzed the discriminatory powers of 36 MLST schemes using all combinations of the 7 loci and contributions of each locus to the schemes. In 10 schemes, sequencing 6 loci can achieve the discriminatory powers of 7 loci. For the other 26 schemes, the median marginal increase in discriminatory power when 7 instead of 6 loci were used is 0.0004. Sequencing the 7 loci of 50 strains each of Pseudomonas aeruginosa and Acinetobacter baumannii revealed that the discriminatory power for P. aeruginosa was 0.9861 when either 6 (without trp) or 7 loci were used and that for A. baumannii was 0.9363 when 5, 6, or 7 loci were used. Genes that have no additional or minimal contribution to the overall discriminatory powers should be replaced.

Natural selection retains overrepresented out-of-frame stop codons against frameshift peptides in prokaryotes.

BACKGROUND: Out-of-frame stop codons (OSCs) occur naturally in coding sequences of all organisms, providing a mechanism of early termination of translation in incorrect reading frame so that the metabolic cost associated with frameshift events can be reduced. Given such a functional significance, we expect statistically overrepresented OSCs in coding sequences as a result of a widespread selection. Accordingly, we examined available prokaryotic genomes to look for evidence of this selection. RESULTS: The complete genome sequences of 990 prokaryotes were obtained from NCBI GenBank. We found that low G+C content coding sequences contain significantly more OSCs and G+C content at specific codon positions were the principal determinants of OSC usage bias in the different reading frames. To investigate if there is overrepresentation of OSCs, we modeled the trinucleotide and hexanucleotide biases of the coding sequences using Markov models, and calculated the expected OSC frequencies for each organism using a Monte Carlo approach. More than 93% of 342 phylogenetically representative prokaryotic genomes contain excess OSCs. Interestingly the degree of OSC overrepresentation correlates positively with G+C content, which may represent a compensatory mechanism for the negative correlation of OSC frequency with G+C content. We extended the analysis using additional compositional bias models and showed that lower-order bias like codon usage and dipeptide bias could not explain the OSC overrepresentation. The degree of OSC overrepresentation was found to correlate negatively with the optimal growth temperature of the organism after correcting for the G+C% and AT skew of the coding sequence. CONCLUSIONS: The present study uses approaches with statistical rigor to show that OSC overrepresentation is a widespread phenomenon among prokaryotes. Our results support the hypothesis that OSCs carry functional significance and have been selected in the course of genome evolution to act against unintended frameshift occurrences. Some results also hint that OSC overrepresentation being a compensatory mechanism to make up for the decrease in OSCs in high G+C organisms, thus revealing the interplay between two different determinants of OSC frequency.