Ho-Yin Lam

Molecular Epidemiological Study of Hiv-1 Crf01_ae Transmission in Hong Kong

Objectives:The objective of this study was to investigate the transmission history of the HIV-1 CRF01_AE epidemics in Hong Kong between 1994 and 2007. Methods:A total of 465 HIV-1 CRF01_AE pol sequences were derived from an in-house or a commercial HIV-1 genotyping system. Phylogenies of CRF01_AE sequences were analyzed by the Bayesian coalescent method. Results:CRF01_AE patient population included 363 males (78.1%) and 102 females (21.9%), whereas 65% (314 of 465) were local Chinese. Major transmission routes were heterosexual contact (63%), followed by intravenous drug use (IDU) (19%) and men having sex with men (MSM) (17%). From phylogenetic analysis, local CRF01_AE strains were from multiple origins with 3 separate transmission clusters identified. Cluster 1 consisted mainly of Chinese male IDUs and heterosexuals. Clusters 2 and 3 included mainly local Chinese MSM and non-Chinese Asian IDUs, respectively. Chinese reference isolates available from China (Fujian, Guangxi, or Liaoning) were clonally related to our transmission clusters, demonstrating the epidemiological linkage of CRF01_AE infections between Hong Kong and China. The 3 individual local transmission clusters were estimated to have initiated since late 1980s and late 1990s, causing subsequent epidemics in the early 2000s. Conclusions:This is the first comprehensive molecular epidemiological study of HIV-1 CRF01_AE in Hong Kong. It revealed that MSM contact is becoming a major route of local CRF01_AE transmission in Hong Kong. Epidemiological linkage of CRF01_AE between Hong Kong and China observed in this study indicates the importance of regular molecular epidemiological surveillance for the HIV-1 epidemic in our region.

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.

Clinical evaluation of the new high-throughput Luminex NxTAG Respiratory Pathogen Panel assay for multiplex respiratory pathogen detection

ABSTRACT A broad range of viral and bacterial pathogens can cause acute respiratory tract infection. For rapid detection of a broad respiratory pathogen spectrum, multiplex real-time PCR is ideal. This study evaluated the performance of the new Luminex NxTAG Respiratory Pathogen Panel (NxTAG-RPP) in comparison with the BioFire FilmArray Respiratory Panel (FA-RP) or singleplex real-time PCR as reference. A total of 284 clinical respiratory specimens and 3 influenza A/H7N9 viral culture samples were tested. All clinical specimens were processed and analyzed in parallel using NxTAG-RPP and the reference standard method. The H7N9 viral culture samples were tested using NxTAG-RPP only. Overall, the NxTAG-RPP demonstrated ≥93% sensitivity and specificity for all respiratory targets except human coronavirus OC43 (HCoV-OC43) and HCoV-HKU1. The H7N9 virus was detected by the influenza A virus matrix gene target, while other influenza A virus subtyping gene targets in the panel remained negative. Complete concordance between NxTAG-RPP and FA-RP was observed in 98.8% (318/322) of positive results (kappa = 0.92). Substantial agreement was found for most respiratory targets, but significant differences were observed in human metapneumovirus (P = 0.001) and parainfluenza virus type 3 (P = 0.031). NxTAG-RPP has a higher sample throughput than FA-RP (96 samples versus 1 sample per run) while the turnaround times for NxTAG-RPP and FA-RP were 5 h (up to 96 samples) and 1 h (for one sample), respectively. Overall, NxTAG-RPP demonstrated good diagnostic performance for most respiratory pathogens. The high sample throughput with reasonable turnaround time of this new assay makes it a suitable multiplex platform for routine screening of respiratory specimens in hospital-based laboratories.

Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

ABSTRACT Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

Molecular Epidemiology and Divergence of HIV Type 1 Protease Codon 35 Inserted Strains among Treatment-Naive Patients in Hong Kong

This study reported the prevalence and pattern of viral replication-associated HIV-1 protease codon 35 amino acid insertions among treatment-naive patients in Hong Kong. The transmission and divergence date of these inserted strains was also investigated. The pol gene of 264 local HIV-1 isolates was sequenced and phylogenetic analysis was performed. The transmission history of protease codon 35-inserted HIV-1 strains in Hong Kong was estimated by the Bayesian coalescent method. This insertion was detected in 12 (4.55%) among 264 treatment-naive subtype B HIV-1 patients in Hong Kong, which was 20-times higher than the prevalence in the western countries. Among these strains, eight carried a glutamic acid (GAA) insertion (E35E_E), two carried an aspartic acid (GAC) insertion (E35E_D), and two carried a glycine (GGA) insertion (E35E_G). E35E_D and E35E_E insertions were the first to be reported. All the 12 inserted sequences clustered in the same lineage of the phylogenetic tree, indicating the possibility of transmission of this insertion. Epidemiological investigation revealed the major route of infection for this inserted strain in Hong Kong was associated mainly among homosexual Chinese males. The evolutionary rate of these inserted strains was similar to other subtype B HIV-1 strains. Through coalescent-based analysis, the divergence date of the protease codon 35-inserted strains in Hong Kong was 1995. Our findings demonstrate the epidemic pathways of viral fitness-related HIV-1 protease codon 35-inserted isolates in Hong Kong. The effect of these novel insertions on viral fitness and drug susceptibility requires further investigation.

High prevalence of primary Enfuvirtide (ENF) resistance-associated mutations in HIV-1-infected patients in Hong Kong

BACKGROUND Enfuvirtide (ENF) is a viral fusion inhibitor used in patients failing highly active antiretroviral therapy (HAART). Mutations associated with ENF resistance have been identified within amino acid positions 36-45 of gp41. As ENF will be introduced to Hong Kong, an understanding of the prevalence of naturally occurred ENF resistance mutations is important before implementation of ENF treatment. OBJECTIVES To investigate the prevalence of ENF resistance-associated mutations in the HR1 and HR2 of HIV-1 strains obtained from ENF-naïve patients. STUDY DESIGN HIV-1 strains isolated from 185 patients (156 antiretroviral treatment [ART]-naïve and 29 HAART-experienced) were screened for ENF resistance-associated mutations using RT-PCR and DNA sequencing. RESULTS Primary mutations were detected in 19.4% of HARRT-experienced patients and 20.5% of ART-naïve patients. G36D was encountered most frequently and more prevalent in non-B subtypes. N42S, L54M and V69I were the major polymorphisms detected. N42S and L54M were predominant in CRF01_AE and subtype B, respectively. V69I was found in all samples harboring G36D. In three longitudinal samples from an ENF-treated patient, G36D was detected after ENF treatment for 6 months and the mutation persisted after termination of ENF for 6 months. CONCLUSIONS The high prevalence of ENF resistance-associated mutations in HARRT-experienced and ART-naïve patients identified in this study highlights the importance of mutation screening before ENF therapy in Hong Kong. Our findings from the ENF-treated patient showed that G36D mutation persisted as long as 6 months after ENF withdrawal. Phenotypic assays will be necessary to confirm the influence of this mutation to ENF susceptibility.

Combined factors V and VIII deficiency (F5F8D) in a Chinese family due to compound heterozygosity for nonsense mutations of the LMAN1 gene

through the germinal centre programming process. This is particularly important in understanding the biology of the disease, but also may have clinical implications. The potential development of a new, more aggressive clone should be considered in patients who demonstrate significant change in their clinical course. This also suggests that longitudinal IGHV study may be necessary in some patients especially when there is a change in the clinical course.

Clinical evaluation of real-time PCR assays for rapid diagnosis of SARS coronavirus during outbreak and post-epidemic periods

Abstract Background: The protocols of WHO network laboratories facilitated development of rapid diagnosis for SARS coronavirus (CoV) using reverse transcription (RT)-PCR assays. However, several reports have shown that conventional and real-time PCR assays were very specific for SARS CoV but lack sensitivity depending on the assay, specimen, and time course of disease. Objective: To evaluate an automatic nucleic acid extraction system and two standardized real-time PCR assays for rapid diagnosis of SARS CoV during outbreak and post-epidemic periods in Hong Kong. Study design: Specimens from clinically suspected SARS patients collected during outbreak and post-epidemic periods were tested by an automatic nucleic acid extraction system followed by our first generation conventional RT-PCR and two standardized real-time PCR assays (Artus GmbH, Germany and Roche Diagnostics, Germany). Paired serum samples were assayed for increasing titer against SARS CoV. Results: In the SARS epidemic, Artus and Roche PCR assays exhibited sensitivities of 87% and 85% for respiratory specimens (n =64), 91% and 88% for stool (n =44), and 82% for urine (n =29). A specificity of 100% was exhibited by both PCR assays except Artus attained only a 92% specificity for stool. For post-epidemic period, no SARS CoV was identified among 56 respiratory specimens by all PCR assays. Inhibitors to PCR assays were detected at an average rate of 7–8% among 202 clinical specimens. Conclusion: This study highlights the high throughput and performance of automatic RNA extraction in coordination with standardized real-time PCR assays suitable for large-scale routine diagnosis in case of future SARS epidemic. As no SARS CoV was detected among specimens collected during post-epidemic period, the positive predictive value of real-time PCR assays for detection of SARS CoV during low epidemic requires further evaluation.

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

Evaluation of the molecular Xpert Xpress Flu/RSV assay vs. Alere i Influenza A & B assay for rapid detection of influenza viruses

A new FDA-approved Xpert Xpress Flu/RSV assay has been released for rapid influenza virus detection. We collected 134 nasopharyngeal specimens to compare the diagnostic performance of the Xpert assay and the Alere i Influenza A & B assay for influenza A and B virus detection. The Xpert assay demonstrated 100% and 96.3% sensitivity to influenza A and influenza B virus respectively. Its specificity was 100% for both viruses. The Alere i assay demonstrated slightly lower sensitivity but similar specificity to the Xpert Xpress assay. Although the Xpert assay (30 min) required longer processing time than the Alere assay (15 min), the handling procedure of the Alere assay was more complicated than the Xpert assay. As the GenXpert system has higher throughput than the Alere system, it is more suitable for hospital clinical laboratories. Overall, the new Xpert Xpress Flu/RSV assay is a reliable and useful tool for rapid influenza detection.