Sabrina Wai-Chi To

Phylodynamics of HIV-1 subtype B among the men-having-sex-with-men (MSM) population in Hong Kong.

The men-having-sex-with-men (MSM) population has become one of the major risk groups for HIV-1 infection in the Asia Pacific countries. Hong Kong is located in the centre of Asia and the transmission history of HIV-1 subtype B transmission among MSM remained unclear. The aim of this study was to investigate the transmission dynamics of HIV-1 subtype B virus in the Hong Kong MSM population. Samples of 125 HIV-1 subtype B infected MSM patients were recruited in this study. Through this study, the subtype B epidemic in the Hong Kong MSM population was identified spreading mainly among local Chinese who caught infection locally. On the other hand, HIV-1 subtype B infected Caucasian MSM caught infection mainly outside Hong Kong. The Bayesian phylogenetic analysis also indicated that 3 separate subtype B epidemics with divergence dates in the 1990s had occurred. The first and latest epidemics were comparatively small-scaled; spreading among the local Chinese MSM while sauna-visiting was found to be the major sex partner sourcing reservoir for the first subtype B epidemic. However, the second epidemic was spread in a large-scale among local Chinese MSM with a number of them having sourced their sex partners through the internet. The epidemic virus was estimated to have a divergence date in 1987 and the infected population in Hong Kong had a logistic growth throughout the past 20 years. Our study elucidated the evolutionary and demographic history of HIV-1 subtype B virus in Hong Kong MSM population. The understanding of transmission and growth model of the subtype B epidemic provides more information on the HIV-1 transmission among MSM population in other Asia Pacific high-income countries.

Increased genetic diversity of HIV-1 circulating in Hong Kong

HIV-1 group M strains are characterized into 9 pure subtypes and 48 circulating recombinant forms (CRFs). Recent studies have identified the presence of new HIV-1 recombinants in Hong Kong and their complexity continues to increase. This study aims to characterize the HIV-1 genetic diversity in Hong Kong. Phylogenetic analyses were performed by using HIV-1 pol sequences including protease and partial reverse transcriptase isolated from 1045 local patients in Hong Kong from 2003 to 2008. For the pol sequences with unassigned genotype, the evidence of recombination was determined by using sliding-window based bootscan plots and their env C2V3 region were also sequenced. Epidemiological background of these patients was further collected. The pol phylogenetic analyses highlighted the extent of HIV-1 genetic diversity in Hong Kong. Subtype B (450/1045; 43.1%) and CRF01_AE (469/1045; 44.9%) variants were clearly predominant. Other genotypes (126/1045; 12.1%) including 3 defined subtypes, 10 CRFs, 1 unassigned subtype and 33 recombinants with 11 different mosaic patterns were observed. Recombinants of subtype B and CRF01_AE were mainly found among local Chinese MSM throughout 2004 to 2008, while the CRF02_AG and subtype G recombinants were circulating among non-Chinese Asian population in Hong Kong through heterosexual transmission starting from 2008. Our study demonstrated the complex recombination of HIV-1 in Hong Kong and the need in developing surveillance system for tracking the distribution of new HIV-1 genetic variants.

Performance Evaluation of the Verigene Gram-Positive and Gram-Negative Blood Culture Test for Direct Identification of Bacteria and Their Resistance Determinants from Positive Blood Cultures in Hong Kong

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, bla OXA and bla CTXM 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.

HLA-B*5701 genetic screening among HIV-1 infected patients in Hong Kong: is this a practical approach in Han-Chinese?

Abacavir hypersensitivity is associated with the presence of human leukocyte antigen (HLA)-allele B*5701. However, the cost and workload of routine HLA-B*5701 pretreatment screening is relatively heavy. This study aimed to determine the prevalence of the HLA-B*5701 allele in the HIV-positive population under care in Hong Kong. Blood samples from 1264 HIV-1 infected patients in Hong Kong were collected between 2007 and 2011 for this study. HLA-B*5701 screening of the study group was determined by in-house polymerase chain reaction (PCR) followed by confirmation using the AlleleSEQR(®) HLA-B PCR/Sequencing Kit (Celera Corporation for Abbott, San Francisco, USA). HLA-B*5701 carriers were identified among 3% of Caucasians, 1% of non-Chinese Asians and 0.5% of Han-Chinese in Hong Kong. Our findings revealed that HLA-B*5701 pretreatment screening might not be necessary for the local Han-Chinese population due to its low prevalence.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing

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.

Performance comparison of an in-house integrase genotyping assay versus the ViroSeq™ Integra48, and study of HIV-1 integrase polymorphisms in Hong Kong

BACKGROUND Integrase inhibitors are recently prescribed to multi-class drug resistant HIV-1 patients in Hong Kong. Unlike pol gene, there are no FDA-approved genotypic resistance tests available for int. Limited studies compared the performance between an in-house and commercial integrase genotyping system. Information on baseline polymorphisms was also insufficient in our region. OBJECTIVES To compare integrase genotyping data obtained from an in-house and ViroSeq™ Integra48 assay, and to illustrate integrase polymorphisms on HIV-1 B and non-B subtypes in Hong Kong. STUDY DESIGN A total of 283 HIV-1 patients were recruited during 2006-2012 in Hong Kong. All samples were genotyped by an in-house assay for pol and int separately, and 46 of them were further genotyped by ViroSeq™ Integra48. Polymorphisms and resistance mutations were analyzed by Stanford HIV Drug Resistance Database. RESULTS The included patients were mainly infected by HIV-1 subtype B (38.9%) and CRF01_AE (43.1%), followed by CRF07_BC (5.3%), C (3.9%), CRF02_AG (2.8%), D (1.4%), CRF08_BC (1.1%) or others (3.5%). Of 46 samples genotyped by ViroSeq™ and the in-house assays, all major and minor resistance mutations were concordant. Integrase major resistance mutations were identified in two CRF01_AE raltegravir-treated patients. Integrase minor resistance mutations were observed in subtypes B and CRF01_AE. CONCLUSIONS With 25% of the commercial cost, the in-house integrase genotyping assay managed to regenerate over 96% concordant results as good as the RUO ViroSeq™ assay. Further investigations are required to understand the effect on integrase minor mutations, which are present in many subtype B and CRF01_AE samples.

Pharmacogenetic screening: HLA‐B*5701 vs. CYP2B6 G516T

AWC Lin, W-C Yam, H-Y Lam, S To, D Chan, KCW Chan and S-S Lee Special Preventive Programme, Centre for Health Protection, Department of Health, Hong Kong, Department of Microbiology, Queen Mary Hospital, Faculty of Medicine, The University of Hong Kong, Hong Kong, Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong and Department of Microbiology, The Chinese University of Hong Kong, Hong Kong

Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

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_AE, CRF07_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_AE patients, 100% CRF07_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.

Utilization of a Duplex HybProbe Real-Time PCR to Detect and Estimate IL-28B Polymorphisms Prevalence among HIV/HCV Co-infected Patients in Hong Kong

Conventional treatment for chronic HCV infection relies on the combination of peg-interferon and ribavirin therapy. Both interleukin-28B (IL-28B) polymorphisms and HCV genotypes serve as the strongest predictive values for therapeutic prognosis. The treatment regimens for HIV/HCV co-infected patients are more complex and dependent on various host immune and viral factors. A rapid and cost-effective IL-28B genotyping tool is therefore crucial to assist clinicians on better patient management. This study aimed to evaluate the performance of a newly developed HybProbe duplex real-time PCR assay in detecting IL-28B polymorphisms on rs12979860 and rs8099917, and to estimate the prevalence of IL-28B polymorphisms among HIV/HCV co-infected patients in Hong Kong. A total of 88 HIV/HCV co-infected patients were recruited at the Integrated Treatment Centre during 2009 to 2014. IL- 28B polymorphisms on rs12979860 and rs8099917 were determined by an in-house HybProbe assay with melting curve analysis. For assay evaluation, IL-28B polymorphisms of 46 samples with diverse HIV/HCV genotypes were confirmed by Sanger sequencing. Both in-house HybProbe assay and sequencing results for IL28B polymorphisms determination were completely concordant. Among the 88 HIV/HCV co-infected, the frequency of rs12979860 wildtype (C/C) was 88.6%, heterozygous mutant (C/T) was 9.1% and remaining 2.3% homozygous mutant (T/T). The prevalence of IL-28B polymorphisms in rs8099917 was slightly differed, which had 90.9% wild-type (T/T), 6.8% heterozygous mutant (G/T) and 2.3% homozygous mutant (G/G). This novel duplex assay could allow clinicians to make early decision on treatment option for HIV/HCV co-infected patients by detecting rs12979860 and rs8099917 polymorphisms simultaneously.