Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.
Aims: Use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis (TB PCR) as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. Methods: A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. TB PCR was performed on pulmonary and extrapulmonary specimens from these patients. The sensitivity of TB PCR was analysed. Results: Of the 155 patients, 144 fitted the clinical diagnosis of tuberculosis, and 112 of them were culture positive for M tuberculosis . Sixty (58.3%) patients with clinical features suggestive of tuberculosis received antituberculosis treatment based on positive TB PCR alone. Of 224 clinical specimens (138 pulmonary and 86 extrapulmonary) sent for TB PCR, 148 (99 pulmonary and 49 extrapulmonary) were positive in 117 patients. Of the 690 clinical specimens sent for culture, 279 were positive for M tuberculosis in 112 patients. The diagnostic sensitivity of TB PCR was 75.9% (85 of 112) and 81.3% (117 of 144) in patients with culture confirmed and clinically diagnosed tuberculosis, respectively. Using culture as the gold standard, the overall sensitivity of TB PCR was 78.3%, and for pulmonary and extrapulmonary specimens it was 82.3% and 72.0%, respectively. Conclusions: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.
To determine the frequency of highly active antiretroviral therapy resistance mutations in the viral pol gene of human immunodeficiency virus-1 (HIV-1) genotypes that circulate in Hong Kong, by means of an in-house HIV-1 genotyping system.Retrospective study.Two HIV clinics in Hong Kong.A modified in-house genotyping resistance test was used to sequence the partial pol gene in 1165 plasma samples from 965 patients. The performance of our test was cross-compared with the US Food and Drug Administration-approved ViroSeq HIV-1 genotyping system. The results of genotyping were submitted to the Stanford HIV-1 drug resistance database for analysis.The cost-effective in-house genotypic resistance test (US$40) demonstrated comparable performance to the US Food and Drug Administration-approved ViroSeq system. The detection limit of this in-house genotypic resistance test could reach 400 copies/mL for both HIV-1 subtype B and CRF01_AE, which were the predominant genotypes in Hong Kong. Drug resistance mutations were detected only in post-treatment samples from treatment-failure patients. However, there was no significant difference in the frequency of drug resistance mutations between subtype B and CRF01_AE.Our cost-effective in-house genotypic resistance test detected no significant difference in drug resistance-related mutations frequencies between HIV-1 subtype B and CRF01_AE in Hong Kong. A drug resistance-related mutations database for different HIV-1 genotypes should be established in Hong Kong to augment guidance for HIV treatment.
AIMS--To evaluate the usefulness of two IS6110 based typing methods, an amplityping assay and restriction fragment length polymorphism (RFLP) analysis, for fingerprinting respiratory isolates of Mycobacterium tuberculosis. METHODS--For amplityping, a pair of primers which amplify the intervening sequence between the repetitive insertion sequence IS6110 was used to generate a banding pattern which was confirmed by hybridisation. This assay was compared with conventional chromosomal DNA RFLP typing in the evaluation of 110 epidemiologically diverse isolates. RESULTS--Polymerase chain reaction (PCR) amplityping generated a single pattern in Hong Kong Chinese strains, but two and four diverse patterns in Filipino and Vietnamese strains, respectively, and could be completed within four days. When compared with chromosomal DNA RFLP typing, which took three weeks to complete, four different RFLP patterns could be seen among the Chinese strains, while seven patterns were found in the Filipino and Vietnamese strains. No change in amplityping or RFLP patterns was found in 36 sequential isolates from the same patients after anti-tuberculosis treatment for up to 12 months, despite the emergence of resistance in three of these strains. No specific amplityping or RFLP pattern could be related to different patterns of drug susceptibility. CONCLUSION--PCR amplityping could be used initially as a rapid typing method to distinguish strains originating from different localities. This could be important for investigation of outbreaks of tuberculosis--for example, in refugee camps.
We determined the occurrence of the single nucleotide polymorphisms (SNPs) -403A/G and -28C/G in the promoter region of RANTES in 1082 Chinese blood donors from northern and southern China and 249 HIV patients from southern China. Compared to healthy adults, Chinese AIDS patients had a significantly higher frequency of the -403G allele and haplotype I, -403G/-28C (P < 0.05), and a lower frequency of the -403A/A genotype (P < 0.01). Symptomatic patients had a higher frequency of the -28G allele and a lower frequency of the -28C/C genotype (P < or = 0.01). The plasma RANTES level was significantly lower in blood donors homozygous for haplotype I than in those who were homozygous for haplotypes II and III (P < 0.05). The frequency of the -403G allele was found to be higher in Chinese than in indigenous Africans, but lower than in Caucasians, Hispanics, and African Americans. The frequency of the -28G allele was comparable in Chinese and Japanese; this allele is rare in other ethnic groups. Results suggest that -403G may be associated with increased susceptibility to HIV infection, while -28G may be associated with advanced disease progression. The impact of SNPs on HIV infection appears to be unique in Chinese.
Transmitted HIV resistance is of both clinical and public health importance. Baseline genotypic resistance testing was performed for HIV-1-infected treatment-naive patients who were newly diagnosed between 2003 and 2007 and attended the government HIV clinic in Hong Kong. International AIDS Society-USA mutation figures and the Stanford resistance interpretation algorithm were used to identify resistance mutations and drug susceptibility, respectively. The pattern and factors associated with resistance were examined. The presence of one or more IAS-USA resistance mutations was found in 26 (3.6%) of 731 patients over the 5-year study period. Overall, protease inhibitor (PI) resistance mutations were most common (16), followed by nucleoside reverse transcriptase inhibitors (NRTIs) (8) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) (3). Resistance to drugs in one, two, and three classes was present in 25 (3.4%), 1 (0.1%), and 0, respectively. Seventy-eight (10.7%) had strains of reduced susceptibility, as predicted by the Stanford algorithm to display at least low-level resistance to one or more drugs of the three classes. Intermediate or high-level resistance was found in 1.6% overall, and in descending order for NRTIs, PIs, and NNRTIs. There was no temporal trend of increase in resistance. Sex between men, Chinese ethnicity, and lower baseline CD4 were associated with harboring resistant strains as elucidated by either method. We conclude that transmitted HIV-1 drug resistance is uncommon in up to two decades of antiretroviral therapy in Hong Kong. The situation has to be continually monitored for any change in significance.
In the present study, T-Spot.TB and the tuberculin skin test (TST) were compared in the screening of latent tuberculosis infection among silicotic patients. A conditional probability model was used to compare the potential clinical utilities of T-Spot.TB and TST performed on 134 silicotic subjects from December 1, 2004 to January 31, 2007. Data from a historical cohort were also reanalysed for further comparison. Agreement with T-Spot.TB was best using a TST cut-off of 10 mm. Age ≥65 yrs independently predicted a tuberculin reaction <10 mm (odds ratio = 3), but not a negative T-Spot.TB response. Lower measures of agreement were observed among current smokers and those aged ≥65 yrs. Tuberculin reaction size was well correlated with both early secretary antigenic target 6 and culture filtrate protein 10 spot counts, except among current smokers. Within the current estimates of sensitivity (88–95%) and specificity (86–99%) for T-Spot.TB, the positive likelihood ratio for T-Spot.TB test would be substantially higher (6.29–95.0 versus 1.65–1.94) and negative likelihood ratio substantially lower (0.05–0.14 versus 0.32–0.41) than the corresponding ratios for the tuberculin test. A low tuberculosis risk differential was similarly observed between tuberculin-negative and untreated tuberculin-positive subjects in the historical cohort. T-Spot.TB is likely to perform better than tuberculin test in the screening of latent tuberculosis infection among silicotic subjects.
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