Performance and quality assurance of genotypic drug-resistance testing for human immunodeficiency virus type 1 in Japan.

Highly active antiretroviral therapy (HAART) can suppress human immunodeficiency virus type 1 (HIV-1) replication and plasma HIV-1 to below detectable levels. However, HAART becomes ineffective when drug-resistant viruses emerge during HAART. Monitoring drug-resistance mutations in viruses is necessary for selecting new drugs or therapies effective at inhibiting such HIV-1 variants. Most laboratories in Japan perform the tests using in-house protocols. However, the quality of these tests has never been assessed. Our study assessing the accuracy and reliability of HIV-1 genotypic drug-resistance testing in 15 laboratories in Japan revealed that the quality was very high (97.3% accurate). The errors, though rare, were caused by human errors, poor electropherograms, and the use of inadequate primers. Here, we propose troubleshooting procedures to improve testing accuracy and reliability in Japan.

Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan.

The increasing prevalence of drug-resistant HIV transmission has become a critical epidemic in the world today. Studies in developed countries reported 8-27% of newly diagnosed HIV/AIDS patients are infected by drug-resistant strains. To determine the prevalence of drug-resistant HIV-1 among newly diagnosed cases in Japan, eight HIV/AIDS clinical centers, three public health laboratories and the National Institute of Infectious Diseases conducted a nationwide survey. Between January 2003 and December 2004, 575 newly diagnosed HIV/AIDS patients with both acute and chronic infections were enrolled in the study. Twenty-three cases, including three recently infected patients, were infected with HIV-1 having major drug-resistance mutations, including M41L, D67N, L100I, K103N, V106A, M184I, M184V, L210W, and revertant mutations at the 215 codon in reverse transcriptase and M46I in protease encoding regions. In this newly diagnosed population, we also clarified the prevalence of hepatitis virus coinfection, which was 8.8% for HBV and 4.3% for HCV. In conclusion, the drug-resistant transmission rate was 4.0% in Japan. Although this rate is significantly lower than that of other developed countries, this rate almost reaches the threshold at which baseline genotypic resistance testing would be cost-effective for all infected persons before initiating therapy.

Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan.

We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6'-N-aminoglycoside acetyltransferase gene [aac(6')-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC >or= 16 microg/ml), amikacin (MIC >or= 64 microg/ml), and ciprofloxacin (MIC >or= 4 microg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6')-Iae gene and the AAC(6')-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with >or=70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6')-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia.

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.

Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase.

We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.