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Abstract Background Kocuria, previously classified into the genus of Micrococcus, is commonly found on human skin. Two species, K. rosea and K. kristinae, are etiologically associated with catheter-related bacteremia. Case presentation We describe the first case of K. kristinae infection associated with acute cholecystitis. The microorganism was isolated from the bile of a 56-year old Chinese man who underwent laparoscopic cholecystectomy. He developed post-operative fever that resolved readily after levofloxacin treatment. Conclusion Our report of K. kristinae infection associated with acute cholecystitis expands the clinical spectrum of infections caused by this group of bacteria. With increasing number of recent reports describing the association between Kocuria spp. and infectious diseases, the significance of their isolation from clinical specimens cannot be underestimated. A complete picture of infections related to Kocuria spp. will have to await the documentation of more clinical cases.

A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak. [ABSTRACT FROM AUTHOR]

This study investigated the transmission dynamics of meticillin-resistant Staphylococcus aureus (MRSA) in a tertiary referral surgical unit with 300 beds. All adult patients were actively screened for MRSA by culture at hospital admission and twice weekly thereafter during hospitalisation from 1 October to 31 December 2008. The colonisation pressure per 1000 patient-days and the incidence density of nosocomial MRSA transmission per 1000 colonisation-days were calculated for the different spa types of MRSA. In total, 6619 nasal swabs were obtained from 2289 patients. One-hundred and forty-eight (7%) patients had MRSA in nasal swabs at admission screening, of which 68/148 (46%) were residents of elderly care homes. Fifty-two of 2141 (2%) patients had conversion of nasal MRSA carriage status from negative to positive during hospitalisation. Among the 200 patients with MRSA, spa types t1081 and t037 were found in 99 (50%) and 30 (15%) patients, respectively. The colonisation pressure per 1000 patient-days was 40.9 for t0181, 22.2 for t037 and 26.3 for the less common spa types. The incidence densities of nosocomial MRSA transmission per 1000 colonisation-days were significantly higher for t1081 (28.5 vs 4.0, P<0.01) and t037 (21.5 vs 4.0, P=0.03) compared with the less common spa types. Proactive screening of MRSA in patients from elderly care homes and targeted isolation of these patients, especially those carrying spa types with high transmissibility, are important for the control of MRSA in hospitals. [ABSTRACT FROM AUTHOR]

Multidrug-resistant tuberculosis (MDR-TB) has posed a serious threat to global public health, and antimicrobial peptides (AMPs) have emerged to be promising candidates to tackle this deadly infectious disease. Previous study has suggested that two AMPs, namely D-LAK120-A and D-LAK120-HP13, can potentiate the effect of isoniazid (INH) against mycobacteria. In this study, the strategy of combining INH and D-LAK peptide as a dry powder formulation for inhalation was explored. The antibacterial effect of INH and D-LAK combination was first evaluated on three MDR clinical isolates of Mycobacteria tuberculosis (Mtb). The minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indexes (FICIs) were determined. The combination was synergistic against Mtb with FICIs ranged from 0.25 to 0.38. The INH and D-LAK peptide at 2:1 mole ratio (equivalent to 1: 10 mass ratio) was identified to be optimal. This ratio was adopted for the preparation of dry powder formulation for pulmonary delivery, with mannitol used as bulking excipient. Spherical particles with mass median aerodynamic diameter (MMAD) of around 5 µm were produced by spray drying. The aerosol performance of the spray dried powder was moderate, as evaluated by the Next Generation Impactor (NGI), with emitted fraction and fine particle fraction of above 70 % and 45 %, respectively. The circular dichroism spectra revealed that both D-LAK peptides retained their secondary structure after spray drying, and the antibacterial effect of the combination against the MDR Mtb clinical isolates was successfully preserved. The combination was found to be effective against MDR Mtb isolates with KatG or InhA mutations. Overall, the synergistic combination of INH with D-LAK peptide formulated as inhaled dry powder offers a new therapeutic approach against MDR-TB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering., Methods: A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance., Results: In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/μL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases., Conclusions: Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care., (© American Association for Clinical Chemistry 2023.)

Sensitive detection of Mycobacterium tuberculosis (TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford nanopore technologies (ONT) MinION sequencing allows rapid and simple sample preparation for sequencing. Its recently developed adaptive sequencing selects reads from targets while allowing real-time base-calling to achieve sequence enrichment or depletion during sequencing. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods using ONT MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human (95%) and other microbial DNA. Adaptive sequencing with readfish and UNCALLDE achieved a 3.9-fold and 2.2-fold enrichment compared to the control run. We provide a simple automatic analysis framework to support the detection of TB for clinical use, openly available at https://github.com/HKU-BAL/ONT-TB-NF . Depending on the patient's medical condition and sample type, we recommend users evaluate and optimize their workflow for different clinical specimens to improve the detection limit., (© 2023. The Author(s).)

Clinical manifestations of tuberculosis range from asymptomatic infection to a life-threatening disease such as tuberculous meningitis (TBM). Recent studies showed that the spectrum of disease severity could be related to genetic diversity among clinical strains of Mycobacterium tuberculosis (Mtb). Certain strains are reported to preferentially invade the central nervous system, thus earning the label "hypervirulent strains".However, specific genetic mutations that accounted for enhanced mycobacterial virulence are still unknown. We previously identified a set of 17 mutations in a hypervirulent Mtb strain that was from TBM patient and exhibited significantly better intracellular survivability. These mutations were also commonly shared by a cluster of globally circulating hyper-virulent strains. Here, we aimed to validate the impact of these hypervirulent-specific mutations on the dysregulation of gene networks associated with virulence in Mtb via multi-omic analysis. We surveyed transcriptomic and proteomic differences between the hyper-virulent and low-virulent strains using RNA-sequencing and label-free quantitative LC-MS/MS approach, respectively. We identified 25 genes consistently differentially expressed between the strains at both transcript and protein level, regardless the strains were growing in a nutrient-rich or a physiologically relevant multi-stress condition (acidic pH, limited nutrients, nitrosative stress, and hypoxia). Based on integrated genomic-transcriptomic and proteomic comparisons, the hypervirulent-specific mutations in FadE5 (g. 295,746 C >T), Rv0178 (p. asp150glu), higB (p. asp30glu), and pip (IS 6110 -insertion) were linked to deregulated expression of the respective genes and their functionally downstream regulons. The result validated the connections between mutations, gene expression, and mycobacterial pathogenicity, and identified new possible virulence-associated pathways in Mtb .

An in-house-developed target amplicon sequencing by next-generation sequencing technology (TB-NGS) enables simultaneous detection of resistance-related mutations in Mycobacterium tuberculosis (MTB) against 8 anti-tuberculosis drug classes. In this multi-center study, we investigated the clinical utility of incorporating TB-NGS for rapid drug-resistant MTB detection in high endemic regions in southeast China. From January 2018 to November 2019, 4,047 respiratory specimens were available from patients suffering lower respiratory tract infections in Hong Kong and Guangzhou, among which 501 were TB-positive as detected by in-house IS6110-qPCR assay with diagnostic sensitivity and specificity of 97.9 and 99.2%, respectively. Preliminary resistance screening by GenoType MTBDR plus and MTBDR sl identified 25 drug-resistant specimens including 10 multidrug-resistant TB. TB-NGS was performed using MiSeq on all drug-resistant specimens alongside 67 pan-susceptible specimens, and demonstrated 100% concordance to phenotypic drug susceptibility test. All phenotypically resistant specimens with dominating resistance-related mutations exhibited a mutation frequency of over 60%. Three quasispecies were identified with mutation frequency of less than 35% among phenotypically susceptible specimens. They were well distinguished from phenotypically resistant cases and thus would not complicate TB-NGS results interpretations. This is the first large-scale study that explored the use of laboratory-developed NGS platforms for rapid TB diagnosis. By incorporating TB-NGS with our proposed diagnostic algorithm, the workflow would provide a user-friendly, cost-effective routine diagnostic solution for complicated TB cases with an average turnaround time of 6 working days. This is critical for timely management of drug resistant TB patients and expediting public health control on the emergence of drug-resistant TB., Competing Interests: OM, S-HY, J-XC and QH were employed by KingMed Diagnostics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leung, Tam, Ng, Lao, Shek, Ma, Yu, Chen, Han, Siu and Yam.)

The emergence of multidrug-resistant strains and hyper-virulent strains of Mycobacterium tuberculosis are big therapeutic challenges for tuberculosis (TB) control. Repurposing bioactive small-molecule compounds has recently become a new therapeutic approach against TB. This study aimed to identify novel anti-TB agents from a library of small-molecule compounds via a rapid screening system. A total of 320 small-molecule compounds were used to screen for their ability to suppress the expression of a key virulence gene, phop , of the M. tuberculosis complex using luminescence ( lux )-based promoter-reporter platforms. The minimum inhibitory and bactericidal concentrations on drug-resistant M. tuberculosis and cytotoxicity to human macrophages were determined. RNA sequencing (RNA-seq) was conducted to determine the drug mechanisms of the selected compounds as novel antibiotics or anti-virulent agents against the M. tuberculosis complex. The results showed that six compounds displayed bactericidal activity against M. bovis BCG, of which Ebselen demonstrated the lowest cytotoxicity to macrophages and was considered as a potential antibiotic for TB. Another ten compounds did not inhibit the in vitro growth of the M. tuberculosis complex and six of them downregulated the expression of phoP/R significantly. Of these, ST-193 and ST-193 (hydrochloride) showed low cytotoxicity and were suggested to be potential anti-virulence agents for M. tuberculosis .

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.

Background: The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics., Methods: A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports., Results: Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities., Conclusion: Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: This study is to elucidate the disinfection effect of ozone producing low-pressure Hg vapor lamps against human pathogens. Ozone producing low-pressure Hg vapor lamps emit mainly 254 nm ultraviolet light C (UVC) with about 10% power of Vacuum-ultraviolet (VUV) light at 185 nm. The combination of UVC and VUV can inactivate airborne pathogens by disrupting the genetic materials or generation of reactive oxygen species, respectively. In this study, inactivation of common bacteria including Escherichia coli ATCC25922 (E. coli), Extended Spectrum Beta-Lactamase-producing E. coli (ESBL), Methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis (MTB), and that of influenza A viruses H1N1 and H3N2 under the radiation from ozone producing low-pressure Hg vapor lamps was examined. Log reduction values at different treatment durations were determined., Methods: In vitro tests were carried out. Various bacterium and virus suspensions were added onto nitrocellulose filter papers and subjected to the illumination from ozone producing low-pressure Hg vapor lamps. The extents of pathogen inactivation at different illumination times were investigated by conducting a series of experiments with increasing duration of illumination. log10 reduction in CFU/ml and reduction at log10(TCID 50 ) were respectively measured for bacteria and viruses. The disinfection effectiveness of this type of lamps against the pathogens under the environment with a moderate barrier to light was therefore evaluated., Results: Ozone producing low-pressure Hg vapor lamp successfully inactivated these human pathogens. Nevertheless, among these pathogens, disinfection of MTB required more intense treatment. In the best tested situation, 3-log10 inactivation of pathogens can be achieved with ≤10 min of VUV treatment except MTB which needed about 20 min. This demonstrated the high resistance against UV disinfection of MTB., Conclusions: Following the criteria that valid germicidal results can be reflected with 3-log10 inactivation for bacteria, 4-log10 inactivation for viruses and 5-log10 inactivation for MTB, most of the bacteria required ≤10 min of VUV treatment, 20 min for the influenza viruses while MTB needed about 30 min VUV treatment. This indicated that VUV light is an effective approach against different environmental microorganisms.

An in-house-developed pncA sequencing assay for analysis of pyrazinamide (PZA) resistance was evaluated using 162 archived Mycobacterium tuberculosis complex (MTBC) isolates with phenotypic PZA susceptibility profiles that were well defined by analysis of Bactec MGIT 960 PZA kit and PZase activity data. Preliminary results showed 100% concordance between pncA sequencing and phenotypic PZA drug susceptibility test (DST) results among archived isolates. Also, 637 respiratory specimens were prospectively collected, and 158 were reported as MTBC positive by the Abbott Realti m e MTB assay (96.3% sensitivity [95% confidence interval {CI}: 92.2% to 98.7%]; 100% specificity [95% CI: 99.2% to 100.0%]). Genotypic and phenotypic PZA resistance profiles of these 158 MTBC-positive specimens were analyzed by pncA sequencing and Bactec MGIT 960 PZA kit, respectively. For analysis of PZA resistance, pncA sequencing detected pncA mutations in 5/5 (100%) phenotypic PZA-resistant respiratory specimens within 4 working days. No pncA mutations were detected among PZA-susceptible specimens. Combining archived isolates with prospective specimens, 27 were identified as phenotypic PZA resistant with pncA mutation. Among these 27 samples, 6/27 (22.2%) phenotypic PZA-resistant strains carried novel pncA mutations without rpsA and panD mutations. These included 5 with mutations (a deletion [Del] at 383T [Del383T], Del 380 to 390, insertion of A [A Ins] at position 127, A Ins at position 407, and G Ins at position 508) in pncA structural genes and 1 with a mutation (T-12C) at the pncA promoter region. All six of these strains had no or reduced PZase activities, indicating that the novel mutations might confer PZA resistance. Additionally, 25/27 phenotypic PZA-resistant strains were confirmed multidrug-resistant tuberculosis (MDR-TB) strains. As PZA is commonly used in MDR-TB treatment regimens, direct pncA sequencing will rapidly detect PZA resistance and facilitate judicious use of PZA in treating PZA-susceptible MDR-TB., (Copyright © 2019 American Society for Microbiology.)

In 2017, we identified a Clostridium difficile strain HKCD4 that caused community-acquired fulminant colitis in a previously healthy child. Phylogenetically, it belonged to clade 2, sequence type 67 and was resistant to fluoroquinolone and tetracycline. The strain was pathogenicity locus and binary toxin positive. It has a mutation in the trehalose repressor treR leading to the L172I substitution that was previously reported in the epidemic ribotype 027 lineage. HKCD4 has a tcdB sequence that shared very high identities with 3 highly virulent reference strains. It has a CpG depleted genome that is characteristic of hypervirulent C. difficile. The emergence of ST67 lineage with molecular feature of hypervirulence in the community is concerning and emphasizes the need for full characterization of strains causing severe disease in patients without classical risk factors., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: Over the past years, an increasing trend was noticed for non-B and non- CRF01&#95;AE HIV-1 strains prevalence in Hong Kong., Objective: In this study, we aimed at using the available HIV-1 pol sequences collected from 1994 to 2013 through our local antiretroviral resistance surveillance program to investigate the molecular epidemiology and evolution of HIV-1 minority subtypes in Hong Kong. We also aimed at investigating their potential association and impact of those transmission risk groups., Methods: A total of 2,315 HIV-1 partial pol sequences were included. HIV-1 genotypes were determined by REGA Genotyping Tool and phylogenetic analysis with reference sequences. The viral evolutionary rates and time of the most common ancestor (tMRCA) were estimated by Bayesian Markov Chain Monte Carlo (MCMC) interference., Results: Apart from the two prevalent HIV-1 genotypes in Hong Kong (subtype B,41.6%, CRF01&#95;AE,40.5%), phylogenetic analysis revealed a broad viral diversity including CRF07&#95;BC(5.1%), subtype C(4.5%), CRF02&#95;AG(1.1%), CRF08&#95;BC(0.8%), subtype A1(0.8%), subtype G(0.4%), subtype D(0.4%), CRF06&#95;cpx(0.4%), subtype F(0.1%), CRF12&#95;BF(0·04%) and other recombinants(4.5%). The top five minority subtypes were further analyzed which demonstrated distinct epidemiological and phylogenetic patterns. Over 70% of subtypes A1, C and CRF02&#95;AG infections were circulated among non-Chinese Asians or African community in Hong Kong and were mainly transmitted between heterosexual regular partners. Instead, over 90% of CRF07&#95;BC and CRF08&#95;BC patients were Chinese. An epidemic cluster was identified in CRF07&#95;BC and estimated to expand from 2002 onwards based on skyline plot and molecular clock analysis., Conclusion: Our results highlighted the emergence of CRF07&#95;BC epidemic in local MSM community, public health interventions targeting the community should be further enhanced to tackle the epidemic., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Molecular epidemiology of Clostridium difficile infection (CDI) has been extensively studied in North America and Europe; however, limited data on CDI are available in the Asia-Pacific region. A multicentre retrospective study was conducted in this region. C. difficile isolates were subjected to multilocus sequence typing (ST) and antimicrobial susceptibility testing. Totally, 394 isolates were collected from Hangzhou, Hong Kong, China; Busan, South Korea; Fukuoka, Japan; Singapore; Perth, Sydney, Australia; New York, the United States. C. difficile isolates included 337 toxin A-positive/B-positive/binary toxin-negative (A + B + CDT - ), 48 A - B + CDT - , and nine A + B + CDT + . Distribution of dominant STs varied geographically with ST17 in Fukuoka (18.6%), Busan (56.0%), ST2 in Sydney (20.4%), Perth (25.8%). The antimicrobial resistance patterns were significantly different among the eight sites ( χ 2 = 325.64, p < 0.001). Five major clonal complexes correlated with unique antimicrobial resistances. Healthcare-associated (HA) CDI was mainly from older patients with more frequent antimicrobial use and higher A - B + positive rates. Higher resistance to gatifloxacin, tetracycline, and erythromycin were observed in HA-CDI patients ( χ 2 = 4.76-7.89, p = 0.005-0.029). In conclusion, multiple C. difficile genotypes with varied antimicrobial resistance patterns have been circulating in the Asia-Pacific region. A - B + isolates from older patients with prior antimicrobial use were correlated with HA-CDI.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is the deadliest infectious disease and the associated global threat has worsened with the emergence of drug resistance, in particular multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). Although the World Health Organization (WHO) End-TB Strategy advocates for universal access to antimicrobial susceptibility testing, this is not widely available and/or it is still underused. The majority of drug resistance in clinical MTB strains is attributed to chromosomal mutations. Resistance-related mutations could also exert certain fitness cost to the drug-resistant MTB strains and growth fitness could be restored by the presence of compensatory mutations. Understanding these underlying mechanisms could provide an important insight into TB pathogenesis and predict the future trend of MDR-TB global pandemic. This review covers the mechanisms of resistance in MTB and provides a comprehensive overview of current phenotypic and molecular approaches for drug susceptibility testing, with particular attention to the methods endorsed and recommended by the WHO., (© 2018 Asian Pacific Society of Respirology.)

DNA vaccines have been extensively studied as preventative and therapeutic interventions for various infectious diseases such as tuberculosis, HIV/AIDS and influenza. Despite promising progresses made, improving the immunogenicity of DNA vaccine remains a technical challenge for clinical development. In this study, we investigated a tuberculosis DNA vaccine BER opt , which contained a codon-optimized fusion immunogen Ag85B-ESAT-6-Rv2660c for enhanced mammalian cell expression and immunogenicity. BER opt immunization through in vivo electroporation in BALB/c mice induced surprisingly high frequencies of Ag85B tetramer + CD8 + T cells in peripheral blood and IFN-γ-secreting CD8 + T cells in splenocytes. Meanwhile, the BER opt vaccine-induced long-lasting T cell immunity protected BALB/c mice from high dose viral challenge using a modified vaccinia virus Tiantan strain expressing mature Ag85B protein (MVTT-m85B) and the virulent M. tb H37Rv aerosol challenge. Since the BER opt DNA vaccine does not induce anti-vector immunity, the strong immunogenicity and protective efficacy of this novel DNA vaccine warrant its future development for M. tb prevention and immunotherapy to alleviate the global TB burden., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To perform a prospective evaluation on the diagnostic performance of an in-house developed, duplex nested IS6110 real-time Polymerase-Chain-Reaction (PCR) assay (IS6110-qPCR assay) for rapid pulmonary TB diagnosis., Methods: A total of 503 sputum specimens were prospectively collected from July 2016 to November 2016. Diagnostic accuracy and optimal cut-off Cycle-threshold (Ct) value for IS6110-qPCR assay was determined by Receiver Operating Characteristic (ROC) curve. Using the optimal cut-off Ct, diagnostic performance of IS6110-qPCR assay was assessed with reference to both bacteriological and clinical information. Meanwhile, limit of detection (LOD) was calculated using Mycobacterium tuberculosis H37Rv as reference strain., Result: ROC curve analysis of IS6110-qPCR assay showed a high Area Under the Curve (AUC) value (0.948) with optimal Ct value at 24.140. Prospective analysis of IS6110-qPCR assay with cut-off Ct = 24.140 showed a high overall sensitivity and specificity of 97.2% and 99.7%, respectively. No cross reactivity was observed among all non-tuberculous mycobacteria specimens in this study. LOD analysis on MTB-spiked sputum showed an average detection limit of 5.0 CFU/mL at Ct = 23.18 (±SD, 0.57)., Conclusion: IS6110-qPCR assay is a highly accurate and cost-effective assay developed for primary screening of suspected TB cases, which is particularly suitable for regions with limited resources but high TB burden., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Pre-exposure prophylaxis (PrEP) targeting high-risk men who have sex with men (MSM) has been shown to be a cost-effective HIV control measure. However, the approach could be a challenge in low HIV incidence places with a low proportion of high-risk MSM. To examine the impact of PrEP in such setting in Asia, we developed an epidemic model and conducted cost-effectiveness analysis using empirical multicentre clinical and HIV sequence data from HIV-infected MSM in Hong Kong, in conjunction with behavioural data of local MSM. Without PrEP, the HIV incidence (per 100 person-years) would increase from 1.1 to 1.6 between 2011 and 2021. PrEP could avert 3-63% of total new infections in a five-year period (2017-2021), the variability of which depends on the implementation strategies and combination with test-and-treat. However, under current market drug price in 2016, the incremental cost per quality-adjusted life-year gained (QALYG) of PrEP (USD1583136/QALYG) is almost 3 times higher than test-and-treat intervention alone (USD396874/QALYG). Assuming 93% fall of PrEP drug price and in combination with test-and-treat, putting 30% of MSM on non-targeting PrEP would be more feasible, cost-effective (USD268915/QALYG), and could avert more new infections (40%). PrEP could contribute to HIV epidemic control in a low incidence place.

Background: Drug resistance in Mycobacterium tuberculosis (MTB) is one of the major challenges in tuberculosis (TB) treatment. However, known mutations cannot explain all of the cases of resistance and little research has focused on the relationship between insertions / deletions (indels) and drug resistance., Results: Here, we retrieved whole genome sequencing data of 743 drug-resistant MTB strains and 367 pan-susceptible strains from TB patients from the public domain to identify novel genomic markers of drug resistance. A total of 20 region markers containing genes and intergenic regions (IGRs) with significant statistical correlation with antibiotic resistance were revealed, four of which have been previously reported to be associated with drug resistance. In addition, 83 point markers containing frameshift (FS) mutations and IGR indels were also identified independently based on differences in their incidence rates between drug-sensitive and -resistant strains. Among the 83 point markers, eight indels were detected in known drug-associated genes or IGRs. Furthermore, the overlap between 20 region markers and 83 point markers further indicated their associations with drug resistance. The markers identified were involved in essential bacterial metabolic functions, including cell wall and transmembrane transporter functions. A strong correlation between FS mutations and mutations in DNA repair genes including I21V in alkA, R48G in mutT4 and P2R in nth was also found., Conclusions: This study identified a set of novel genetic markers with FS mutations and IGR indels associated with MTB drug resistance, which greatly broadens the pool of mutations related to MTB drug resistance. This insight may be important in identifying novel mechanisms of drug resistance in MTB.

Background: Development of improved therapeutics against tuberculosis (TB) is hindered by an inadequate understanding of the relationship between disease severity and genetic diversity of its causative agent, Mycobacterium tuberculosis . We previously isolated a hypervirulent M. tuberculosis strain H112 from an HIV-negative patient with an aggressive disease progression from pulmonary TB to tuberculous meningitis-the most severe manifestation of tuberculosis. Human macrophage challenge experiment demonstrated that the strain H112 exhibited significantly better intracellular survivability and induced lower level of TNF-α than the reference virulent strain H37Rv and other 123 clinical isolates. Aim: The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent M. tuberculosis strains of the same phylogenetic clade isolated in other global regions. Methods: A low-virulent M. tuberculosis strain H54 which belonged to the same phylogenetic lineage (L2) as strain H112 was selected from a collection of 115 clinical isolates. Both H112 and H54 were whole-genome-sequenced using PacBio sequencing technology. A comparative genomics approach was adopted to identify mutations present in strain H112 but absent in strain H54. Subsequently, an extensive phylogenetic analysis was conducted by including all publically available M. tuberculosis genomes. Single-nucleotide-polymorphisms (SNPs) and structural variations (SVs) common to hypervirulent strains in the global collection of genomes were considered as potential genetic determinants of hypervirulence. Results: Sequencing data revealed that both H112 and H54 were identified as members of the same sub-lineage L2.2.1. After excluding the lineage-related mutations shared between H112 and H54, we analyzed the phylogenetic relatedness of H112 with global collection of M. tuberculosis genomes ( n = 4,338), and identified a novel phylogenetic clade in which four hypervirulent strains isolated from geographically diverse regions were clustered together. All hypervirulent strains in the clade shared 12 SNPs and 5 SVs with H112, including those affecting key virulence-associated loci, notably, a deleterious SNP ( rv0178 p. D150E) within mce1 operon and an intergenic deletion (854259&#95; 854261delCC) in close-proximity to phoP . Conclusion: The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent M. tuberculosis . The causative role of these mutations in mycobacterial virulence should be validated in future study.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c ( lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c ( cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA , and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The clinical workflow of using chromogenic agar and matrix-assisted laser desorption ionization time-of-fight mass spectrometry (MALDI-TOF MS) for Clostridium difficile identification was evaluated. The addition of MALDI-TOF MS identification after the chromID C. difficile chromogenic agar culture could significantly improve the diagnostic accuracy of C. difficile., (Copyright © 2016. Published by Elsevier B.V.)

Haemophilus influenzae is associated with severe invasive disease, while Haemophilus haemolyticus is considered part of the commensal flora in the human respiratory tract. Although the addition of a custom mass spectrum library into the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system could improve identification of these two species, the establishment of such a custom database is technically complicated and requires a large amount of resources, which most clinical laboratories cannot afford. In this study, we developed a mass spectrum analysis model with 7 mass peak biomarkers for the identification of H. influenzae and H. haemolyticus using the ClinProTools software. We evaluated the diagnostic performance of this model using 408 H. influenzae and H. haemolyticus isolates from clinical respiratory specimens from 363 hospitalized patients and compared the identification results with those obtained with the Bruker IVD MALDI Biotyper. The IVD MALDI Biotyper identified only 86.9% of H. influenzae (311/358) and 98.0% of H. haemolyticus (49/50) clinical isolates to the species level. In comparison, the ClinProTools mass spectrum model could identify 100% of H. influenzae (358/358) and H. haemolyticus (50/50) clinical strains to the species level and significantly improved the species identification rate (McNemar's test, P < 0.0001). In conclusion, the use of ClinProTools demonstrated an alternative way for users lacking special expertise in mass spectrometry to handle closely related bacterial species when the proprietary spectrum library failed. This approach should be useful for the differentiation of other closely related bacterial species., (Copyright © 2017 American Society for Microbiology.)

Aims: Rapid and accurate diagnostic assays with simultaneous microbial identification and drug resistance detection are essential for optimising treatment and control of tuberculosis., Methods: We developed a novel multiplex (TRIOL, Tuberculosis-Rifampicin-Isoniazid-Ofloxacin-Luminex) assay using the Luminex xMAP system that simultaneously identifies Mycobacterium tuberculosis and detects resistance to first-line and second-line anti-tuberculous drugs, and compared its performance with that by PCR sequencing, using phenotypic drug susceptibility testing as the gold standard., Results: Identification of M. tuberculosis by the TRIOL assay was highly sensitive (100%) and specific (100%). The overall drug-specific specificities were excellent (100%). The overall sensitivity of the TRIOL assay was lower than that of the PCR-sequencing assays (72.4% vs 82.8%) because of a lower sensitivity of detecting rifampicin resistance (71.4% vs 92.9%). The sensitivity of detecting isoniazid and ofloxacin resistance was as good as the PCR-sequencing assays. Importantly, the TRIOL assay did not miss any mutations that were included in the assay. All of the resistant isolates that were missed had uncommon mutations or unknown resistance mechanisms that were not included in the assay., Conclusions: The TRIOL assay has higher throughput, lower cost and is less labour intensive than the PCR-sequencing assays. The TRIOL assay is advantageous in having the capability to detect resistance to multiple drugs and an open-architecture system that allows addition of more specific primers to detect uncommon mutations. Inclusion of additional primers for the identification of non-tuberculous mycobacteria, spoligotyping and improvement of rifampicin resistance detection would enhance the use of the TRIOL assay in future clinical and epidemiological studies., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background and Objective: The tuberculin skin test (TST), T-Spot.TB (T-Spot) and QuantiFERON-TB Gold-In Tube (QFT) were compared in diagnosing latent tuberculosis infection (LTBI) among human immunodeficiency virus (HIV)-infected persons., Methods: Human immunodeficiency virus-infected persons without previous history of tuberculosis or LTBI were simultaneously tested by TST, T-Spot and QFT annually and followed up for tuberculosis., Results: Among 110 HIV-infected subjects with 85% previous TST screening coverage, 75% on anti-retroviral therapy, well-preserved median CD4 count (414/μL) and low median viral load (<75/μL), baseline TST, T-Spot and QFT were positive in 5.5%, 5.6% and 4.9%, respectively, with almost complete discordance of positive results. Among 91 (83%), 66 (60%) and 26 (24%) subjects successfully undergoing the first, second and third annual retesting, TST, T-Spot and QFT were, respectively, positive in 11/123 (8.9%), 13/173 (7.5%) and 21/182 (11.5%) on retesting, with similar discordance of positive results. There was no significant association with the concurrent CD4 count or viral load. Conversion occurred in 11/123 (8.9%), 8/160 (5.0%) and 18/168 (10.7%) of TST, T-Spot and QFT, respectively, and none was associated with changes in CD4 count or viral load. More than half of the positive T-SPOT and QFT results reverted to negative on follow-up. None of these tests picked up the single case of culture-confirmed tuberculosis observed after 798 person-years of follow-up., Conclusion: Major discordance in positive results, high reversion rates and low tuberculosis incidence among test-positive subjects cast serious doubt on the utility of the currently available LTBI tests in the annual screening of HIV-infected persons in an intermediate tuberculosis burden area., (© 2016 Asian Pacific Society of Respirology.)

Following HIV/AIDS, tuberculosis (TB) continues to be the second most deadly infectious disease in humans. The global TB prevalence has become worse in recent years due to the emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains, as well as co-infection with HIV. Although Bacillus Calmette-Guérin (BCG) vaccine has nearly been used for a century in many countries, it does not protect adult pulmonary tuberculosis and even causes disseminated BCG disease in HIV-positive population. It is impossible to use BCG to eliminate the Mycobacterium tuberculosis (M. tb) infection or to prevent TB onset and reactivation. Consequently, novel vaccines are urgently needed for TB prevention and immunotherapy. In this review, we discuss the TB prevalence, interaction between M. tb and host immune system, as well as recent progress of TB vaccine research and development., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01&#95;AE, CRF07&#95;BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01&#95;AE patients, 100% CRF07&#95;BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is rapidly emerging in Asia, but information on the temporal relationship between the increase in macrolide resistance and changes in strain types is scarce. Between 2011 and 2014, M. pneumoniae infection was diagnosed by PCR as part of routine care in a health care region in Hong Kong. Testing was initiated by clinicians, mainly in patients with suspected M. pneumoniae pneumonia. Specimens positive for M. pneumoniae were retrospectively investigated by macrolide resistance genotyping and a four-locus (Mpn13 to -16) multilocus variable-number tandem-repeat analysis (MLVA) scheme. The overall percentage of M. pneumoniae-positive specimens was 17.9%, with annual rates ranging from 9.8% to 27.2%. The prevalence of MRMP had rapidly increased from 13.6% in 2011 to 30.7% in 2012, 36.6% in 2013, and 47.1% in 2014 (P = 0.038). Two major MLVA types, 4-5-7-2 and 3-5-6-2, accounted for 75% to 85% of the infections each year. MLVA types 4-5-7-2 and 3-5-6-2 predominated among macrolide-resistant and macrolide-sensitive groups, respectively. The increase in MRMP was mainly caused by increasing macrolide resistance in the prevalent MLVA type 4-5-7-2, changing from 25.0% in 2011 to 59.1% in 2012, to 89.7% in 2013, and to 100% in 2014 (P < 0.001). In conclusion, increasing MRMP in Hong Kong was linked to a single MLVA type, which was both prevalent and increasingly resistant to macrolides., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: A multicenter study was conducted to evaluate the diagnostic performance and the time to identifcation of the Verigene Blood Culture Test, the BC-GP and BC-GN assays, to identify both Gram-positive and Gram-negative bacteria and their drug resistance determinants directly from positive blood cultures collected in Hong Kong., Methods and Results: A total of 364 blood cultures were prospectively collected from four public hospitals, in which 114 and 250 cultures yielded Gram-positive and Gram-negative bacteria, and were tested with the BC-GP and BC-GN assay respectively. The overall identification agreement for Gram-positive and Gram-negative bacteria were 89.6% and 90.5% in monomicrobial cultures and 62.5% and 53.6% in polymicrobial cultures, respectively. The sensitivities for most genus/species achieved at least 80% except Enterococcus spp. (60%), K.oxytoca (0%), K.pneumoniae (69.2%), whereas the specificities for all targets ranged from 98.9% to 100%. Of note, 50% (7/14) cultures containing K.pneumoniae that were missed by the BC-GN assay were subsequently identified as K.variicola. Approximately 5.5% (20/364) cultures contained non-target organisms, of which Aeromonas spp. accounted for 25% and are of particular concern. For drug resistance determination, the Verigene test showed 100% sensitivity for identification of MRSA, VRE and carbapenem resistant Acinetobacter, and 84.4% for ESBL-producing Enterobacteriaceae based on the positive detection of mecA, vanA, blaOXA and blaCTXM respectively., Conclusion: Overall, the Verigene test provided acceptable accuracy for identification of bacteria and resistance markers with a range of turnaround time 40.5 to 99.2 h faster than conventional methods in our region.

The automated high-throughput Abbott RealTime MTB real-time PCR assay has been recently launched for Mycobacterium tuberculosis complex (MTBC) clinical diagnosis. This study would like to evaluate its performance. We first compared its diagnostic performance with the Roche Cobas TaqMan MTB assay on 214 clinical respiratory specimens. Prospective analysis of a total 520 specimens was then performed to further evaluate the Abbott assay. The Abbott assay showed a lower limit of detection at 22.5 AFB/ml, which was more sensitive than the Cobas assay (167.5 AFB/ml). The two assays demonstrated a significant difference in diagnostic performance (McNemar's test; P = 0.0034), in which the Abbott assay presented significantly higher area under curve (AUC) than the Cobas assay (1.000 vs 0.880; P = 0.0002). The Abbott assay demonstrated extremely low PCR inhibition on clinical respiratory specimens. The automated Abbott assay required only very short manual handling time (0.5 h), which could help to improve the laboratory management. In the prospective analysis, the overall estimates for sensitivity and specificity of the Abbott assay were both 100 % among smear-positive specimens, whereas the smear-negative specimens were 96.7 and 96.1 %, respectively. No cross-reactivity with non-tuberculosis mycobacterial species was observed. The superiority in sensitivity of the Abbott assay for detecting MTBC in smear-negative specimens could further minimize the risk in MTBC false-negative detection. The new Abbott RealTime MTB assay has good diagnostic performance which can be a useful diagnostic tool for rapid MTBC detection in clinical laboratories.

Human immunodeficiency virus (HIV)-1 epidemics in Asian countries are driven by varying exposures. The epidemiology of the regional pandemic has been changing with the spread of HIV-1 to lower-risk populations through sexual transmission. Common HIV-1 genotypes include subtype B and circulating recombinant form (CRF) 01&#95;AE. Our objective was to use HIV-1 genotypic data to better quantify local epidemics. TASER-M is a multicenter prospective cohort of HIV-infected patients. Associations between HIV exposure, patient sex, country of sample origin and HIV-1 genotype were evaluated by multivariate logistic regression. Phylogenetic methods were used on genotypic data to investigate transmission relationships. A total of 1086 patients from Thailand, Hong Kong, Malaysia and the Philippines were included in analyses. Proportions of male patients within countries varied (Thailand: 55.6%, Hong Kong: 86.1%, Malaysia: 81.4%, Philippines: 93.8%; p < 0.001) as did HIV exposures (heterosexual contact: Thailand: 85.7%, Hong Kong, 46.2%, Malaysia: 47.8%, Philippines: 25.0%; p < 0.001). After adjustment, we found increased subtype B infection among men who have sex with men, relative to heterosexual-reported exposures (odds ratio = 2.4, p < 0.001). We further describe four transmission clusters of eight to 15 treatment naïve, predominantly symptomatic patients (two each for subtype B and CRF01&#95;AE). Risk-group subpopulations differed with respect to the infecting HIV-1 genotype. Homosexual exposure patients had higher odds of being infected with subtype B. Where HIV-1 genotypes circulate within countries or patient risk-groups, local monitoring of genotype-specific transmissions may play a role in focusing public health prevention strategies. Phylogenetic evaluations provide complementary information for surveillance and monitoring of viruses with high mutation rates such as HIV-1 and Ebola., (Copyright © 2015. Published by Elsevier Taiwan.)

Background: Differentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system., Methods: This study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone., Results: The percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification., Conclusions: The additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)