Mamie Hui

Rapid Multiplex Nested PCR for Detection of Respiratory Viruses

ABSTRACT Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.

Biases in human papillomavirus genotype prevalence assessment associatedwith commonly used consensus primers

Consensus primers targeting human papillomaviruses (HPVs) have biases in sensitivity toward certain HPV types. We applied 3 primer sets (GP5+/6+, MY09/11, PGMY09/11) in parallel on 120 Chinese cervical cancer specimens. GP5+/6+ exhibited a poor sensitivity for HPV52, for which the prevalence among squamous cell cervical cancer was underestimated from 14.6% to 0%. The fact that HPV52 should rank second in prevalence among squamous cell cervical carcinoma in Hong Kong could be missed if GP5+/6+, a worldwide commonly used primer set, was selected for HPV detection. Biases in HPV type‐specific sensitivity may result in misprioritization of vaccine candidates.

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong

Abstract Objectives To characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA). Methods The MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products. Results Resistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci. Conclusions These findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

Effects of cathelicidin and its fragments on three key enzymes of HIV-1.

Cathelicidins exhibit anti-HIV activity but it is not known if they reduce the activity of enzymes crucial to the life cycle of the retrovirus. It is shown in this investigation that human cathelicidin LL37 and its fragments LL13-37 and LL17-32 inhibited HIV-1 reverse transcriptase dose-dependently with an IC50 value of 15μM, 7μM, and 70μM, respectively. The three peptides inhibited HIV-1 protease with a weak potency, achieving 20-30% inhibition at 100μM. The mechanism of inhibition was protein-protein interaction as revealed by surface plasmon resonance. The peptides were devoid of the ability to inhibit translocation of HIV-1 integrase, which has been labeled with green fluorescent protein, into the nucleus. The peptides did not exert toxicity on human peripheral blood mononuclear cells.

Epidemiology of respiratory syncytial virus infection among paediatric patients in Hong Kong: seasonality and disease impact

In a 5-year retrospective survey of respiratory syncytial virus (RSV) infections among hospitalized children, 1340 cases were identified of which, 98.4% were children < 5 years old with a male:female ratio of 1.5: 1. Most cases occurred from April to September showing a significant positive correlation with temperature and relative humidity. Community-acquired infections accounted for 92.5% of the cases with a mean hospital stay of 5 days. The estimated annual incidence of RSV infection requiring hospitalization was 2.5/1000 children < 5 years old with a mortality of 0.15% among hospitalized cases. On average, 248 children were admitted each year to the 1400-bed acute regional hospital accounting for an expenditure of HK S1.94 ((approximately US +/-0.25) million for hospitalization costs which equates to an annual cost in excess of HK

Presence of human herpesviruses 6, 7, and 8 DNA sequences in normal brain tissue

6.67 (approximately US

Immunofluorescence Assay for Serologic Diagnosis of SARS

0.86) million for the whole of Hong Kong. An RSV vaccine should be a priority.

Antifungal action of human cathelicidin fragment (LL13–37) on Candida albicans

The three novel human herpesviruses (HHV) 6, 7, and 8 are predominantly, but not exclusively, lymphotropic. In an attempt to elucidate their neurotropism in vivo, viral DNA sequences present in fresh autopsy cortical brain tissues obtained from 84 consecutive Chinese subjects (mean age, 66.9 years; range, 21–98 years) were detected by a nested polymerase chain reaction. These patients were apparently immunocompetent and free of clinical signs of viral diseases. HHV‐6 DNA was detected in 36 of 84 (42.9%) patients, and the DNA‐positive and ‐negative groups did not show a significant difference in age or sex distribution. Of the 36 HHV‐6 DNA‐positive cases, 9 (25%) were variant A and 27 (75%) were variant B. In view of the lower prevalence of variant A than variant B in the adult population, the two variants may share a comparable neuroinvasive potential. HHV‐7 and HHV‐8 DNA were detected respectively in three and two patients. The low positive rates of HHV‐7 and HHV‐8 may represent a relatively lower neuroinvasive potential of the viruses. Alternatively, the localization of HHV‐7 and HHV‐8 may be more restricted and the sampled cortical tissues may not represent the most abundant site of persistence in the nervous system. The results provide molecular evidence of the presence of the three newly identified herpesviruses in brain tissue. The pathogenic role for HHV‐7 and HHV‐8, as with HHV‐6, in neurological diseases should not be overlooked. J. Med. Virol. 59:491–495, 1999.

Photodynamic inactivation of bacteria and viruses using two monosubstituted zinc(II) phthalocyanines

We evaluated a virus-infected cell-based indirect immunofluorescence assay for detecting anti–severe acute respiratory syndrome-associated coronavirus (SARS-CoV) immunoglobulin (Ig) G antibody. All confirmed SARS cases demonstrated seroconversion or fourfold rise in IgG antibody titer; no control was positive. Sensitivity and specificity of this assay were both 100%. Immunofluorescence assay can ascertain the status of SARS-CoV infection.

Meta-Analysis on Prevalence and Attribution of Human Papillomavirus Types 52 and 58 in Cervical Neoplasia Worldwide

Human cathelicidin LL37 and its fragments LL13-37 and LL17-32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13-37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13-37. LL13-37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13-37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13-37. Bimane-labeled LL13-37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37 impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13-37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13-37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.