[Evaluation of a Newly Developed Campylobacter Antigen Detection Kit for Patients with Enteritis].

The newly developed rapid diagnostic test (RDT, DK14-CA1, Denka Seiken Co., Ltd.) to detect Campylobacter antigen was evaluated using fecal specimens of patients with enteritis. The RDT is an immunochromatographic assay using colored latex and can detect Campylobacter antigen (C. jejuni and C. coli) from patients' stool samples within 15 minutes. A total of 227 stool samples obtained from patients with enteritis were examined and the results were compared with conventional culture methods. Overall sensitivity, specificity, accuracy and positive predictive value (PPV) were 75.6%, 98.6%, 89.9% and 97.0% respectively. Among 53 severe cases defined with their clinical findings, sensitivity, specificity, accuracy and PPV were 82.1%, 100%, 90.6% and 100% respectively. Mean time to obtain the result with the RDT was 7 minutes whereas the culture method took 2.2 days. This study revealed the usefulness of the newly developed RDT as a rapid detection tool for Campylobacter antigen. Although the RDT has a little lower sensitivity compared with culture method, the simple and rapid test can contribute to treatment decisions for patients with enteritis and can be used at the patient's bedside and in outpatient clinics.

Emergence in Japan of an HIV-1 variant associated with transmission among men who have sex with men (MSM) in China: first indication of the International Dissemination of the Chinese MSM lineage.

A survey of HIV-1 strains circulating in the Tokyo-Kanagawa metropolitan area of Japan during 2004 to 2011 (n = 477) identified six Japanese males (patients 1 to 6), who harbored viruses with genome segments derived from a distinct CRF01_AE variant uniquely found among men who have sex with men (MSM) in China (designated CN.MSM.01-1). These six HIV infections were diagnosed in 2010 and 2011 among MSM (3 of 75) and men with unknown risk factors (3 of 63) and differed from the vast majority of HIV infections among MSM in Japan, which are overwhelmingly characterized by subtype B (239 of 246 [97.2%]). Approximately one-third (91 of 239 [38.1%]) of subtype B strains from MSM in Japan belong to a large monophyletic cluster (designated JP.MSM.B-1). In addition, we identified a smaller subtype B cluster (n = 8) (designated JP.MSM.B-2) that also contains strains from two Chinese MSM living in Japan. Interestingly, patients 5 and 6 were found to be coinfected with CRF01_AE (CN.MSM.01-1) and subtype B (JP.MSM.B-2 or JP.MSM.B-1) variants that are unique to the HIV-1 epidemics among MSM in China and Japan, respectively. Our study demonstrates for the first time the effect of the expanding HIV epidemic among MSM in China on transmission in neighboring countries and shows the ongoing mixing of CRF01_AE and subtype B lineages unique to HIV-1 that cocirculate in MSM populations in East Asia. This finding highlights the importance of strengthening epidemiological surveillance in the region and the need for effective measures to limit transmission among MSM in East Asia.

[Evaluation of a new vesion of the human immunodeficiency virus antigen and antibody combination assay with improved sensitivity in HIV-1 p24 antigen detection].

The performance of a new version of the HIV p24 antigen and antibody combination assays (Genscreen Ultra HIV Ag-Ab) was evaluated by comparing it with three other fourth-generation enzyme immunoassays (Architect HIV Ag/Ab Combo assay, VIDAS HIV DUO Quick and Genscreen Plus HIV Ag-Ab). The assays were examined with 200 HIV positive samples, 1,000 HIV negative samples, 30 samples (28 positives including 24 samples of subtype A, B, B', C, D, F, G, B/D, CRF01_AE in HIV-1 group M, one sample of HIV-1 group O, three samples of HIV-2 and two negatives) of one worldwide HIV performance panel, 59 samples of ten HIV-1 seroconversion panels and the WHO international standard HIV-1 p24 antigen. Both the sensitivity and specificity of Genscreen Ultra HIV Ag-Ab were 100%. All of the 28 positive samples in the worldwide HIV performance panel were positive. The days of the earliest detection in the ten seroconversion panels were the same in three assays (Genscreen Ultra HIV Ag-Ab, Architect HIV Ag/Ab combo assay and VIDAS HIV DUO Quick). Genscreen Plus HIV Ag-Ab which is a former version of the Genscreen Ultra HIV Ag-Ab detected the earliest positive sample one bleed slower than the other three assays in 5 of 10 seroconversion panels. The p24 antigen limit of detection was determined in two ways, using the WHO international standard and three samples from HIV-1 antigen panels; the values obtained were 1IU/mL and 3.5-9.9 pg/mL for Genscreen Ultra HIV Ag-Ab, 1U/mL and 7.1-9.9 pg/mL for Architect HIV Ag/Ab combo assay, 0.5IU/mL and 4.0-7.1 pg/mL for VIDAS HIV DUO Quick, and 32.0-56.5 pg/mL for Genscreen Plus HIV Ag-Ab. In this study, we have shown that Genscreen Ultra HIV Ag-Ab has the sensitivity, specificity and p24 antigen limit of detection that is equal to those of two typical fourth-generation assays. This assay can be considered useful and reliable for HIV screening.

Plasmid-mediated resistance to cephalosporins in Salmonella enterica serovar Typhi.

This study characterized a cephalosporin-resistant Salmonella enterica serovar Typhi isolate. The organism possessed a plasmid encoding the CTX-M-15 extended-spectrum-beta-lactamase. This plasmid is the determinant for the phenotype of cephalosporin resistance and is transferrable among Enterobacteriaceae.

[Evaluation of new fourth-generation human immunodeficiency virus antigen and antibody detection assay with enzyme-linked fluorescent immunoassay].

We evaluated the fourth-generation HIV screening assay VIDAS HIV DUOII (DUOII) based on ELFA for simultaneous detection of anti-HIV-1 and anti-HIV-2 antibodies and HIV-1 p24 antigen through comparison with other HIV antigen-antibody detection assays. Materials were 1228 HIV-negative specimens, 95 HIV-antibody-positive specimens, and HIV commercial panels. The specificity of DUOII was 99.8% and sensitivity 100%, detecting all of HIV-1 group M subtype A, B, B', C, D, A/E, F, G, B/D, HIV-1 group O, and HIV-2. The sensitivity test to HIV-1 p24 antigen was 5pg/ mL, higher than other assays. DUOII was equivalent to or superior in detecting results earlier than other assays in an evaluation using 10 commercial HIV-1 seroconversion panels of primary infection. DUOII detects anti-HIV IgM antibody, so no negative sample was found in the second window between p24 antigen disappearance and raised anti-HIV IgG antibody. DUOII has sufficient specificity and sensitivity for HIV screening, and detects primary infection sooner than other assays. These results indicate that DUOII is useful and reliable in HIV screening.

Analysis of a long-term discrepancy in drug-targeted genes in plasma HIV-1 RNA and PBMC HIV-1 DNA in the same patient.

Drug-resistance genotypes were investigated in a patient under treatment with anti-HIV drugs. Since the drug resistance-associated mutations in plasma HIV-1 RNA and proviral DNA in peripheral blood mononuclear cells (PBMCs) were inconsistent, changes were followed over time, and the discrepancy was shown to persist for a long period. In plasma HIV-1 RNA, D67N, K70R, T215Y, and Y188L were present in the reverse transcriptase (RT) region, and two primary mutations, I84V and L90M, were noted in the protease (Pro) region. In contrast, in proviral DNA, no drug resistance-associated mutations were found in the RT region, and mutations such as L90L/M were only infrequently present in the Pro region. This situation persisted for more than 3 years. In addition, sequencing analysis of the V3 loop in the envelope gene showed that non-syncytium-inducing/macrophage-tropic viruses contribute to acquisition of drug resistance. In this study, drug-resistant viruses were produced primarily at macrophages, and drug-sensitive viruses were maintained in PBMCs as a reservoir.

Diagnosis of febrile illnesses in returned travelers using the PC software GIDEON.

BACKGROUND: The prompt and accurate diagnosis of febrile illnesses should have the highest priority when dealing with returned travelers. However, traditional diagnostic procedures aided by collecting information from printed materials may have drawbacks. Here, we conducted a retrospective study to evaluate the diagnostic capability of the software, Global Infectious Disease and Epidemiology Network (GIDEON). METHOD: We recruited a total of 98 febrile travelers in whom an infectious disease diagnosis had been confirmed by microbiology and/or serology. The presence or absence of symptoms/signs and laboratory abnormalities, travel destination, entry and departure dates, and the date of onset were input into updated versions of GIDEON. RESULTS: Overall, the correct diagnoses appeared on the differential diagnosis lists for 91% of the cases and ranked first for 52%. A correct diagnosis could be excluded from the differential diagnostic list by the presence of symptoms and signs irrelevant to the disease, which was demonstrated most clearly in a case of Lassa fever. We also found that a correct diagnosis can be listed lower than expected, probably due to the irrelevant database. CONCLUSIONS: Improvements are required at the level of the developer and users are required to have adequate knowledge of infectious diseases for best use of the program. Despite these limitations, we believe that GIDEON is a novel and potentially powerful tool in infectious disease diagnosis.

[The current status of infectious enteritis in Japan--reports of the "Research Group for Infectious Enteric Diseases, Japan" in the last 5 years (1996-2000)].

The patients or carriers with infectious enteritis admitted to the Hospitals for infectious diseases in the last 5 years (1996-2000) were studied. The total number of cases admitted in each year were 969, 1,113, 981, 637 and 573 respectively. A total of 1,527 Shigella spp. strains including 1,078 strains from overseas travelers' cases were isolated. The isolates of Salmonella spp. excluding S. Typhi and S. Paratyphi A were 562 in number. A total of 61 Vibrio cholerae O1 strains including 44 strains from overseas travelers was isolated. These V. cholerae O1 strains were all of El Tor type. Entamoeba histolytica, Giardia lamblia, Cryptosporidium parvum and Isospora belli were detected in 225, 46, 3 and 3 cases respectively. Abdominal pain, nausea and vomiting were frequently observed in the cases caused by Vibrio parahaemolyticus. The highest body temperature and the highest frequency of bowel movements were revealed in the cases caused by Salmonella spp. Bloody stool was observed in 55.3% of the cases due to Escherichia coli, in 40.5% of the cases due to Campylobacter spp. and in 24.1% of cases due to Shigella spp. As for shigellosis and salmonellosis, the clinical symptoms were more serious in the domestic cases than those in travelers. OFLX-resistant strains accounted for 1.7% of Shigella spp. isolates. No strains of Salmonella spp. were resistant to OFLX. The incidence of drug-resistant isolates of Campylobacter jejuni were 26.0% for OFLX and 2.5% for EM.

Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR.

The level of human immunodeficiency virus type 1 (HIV-1) proviral DNA is likely to be an important marker of the long-term effectiveness of highly active antiretroviral therapy. A new method was developed for quantifying HIV-1 group M proviral DNA using TaqMan real-time PCR, in which degenerate primers and an MGB probe were used to resolve the difference in amplification efficiencies among different subtypes. The present assay provided good linearity and accuracy in the range of 4-5000 copies of proviral DNA in 0.5microg of cellular DNA. The intra-assay and inter-assay coefficients were <31.6% and <30.1%, respectively. In 19 HIV-1 clinical isolates of six subtypes (A, B, C, CRF01_AE, F, and G), quantitation values by the real-time PCR assay matched closely those by Poisson distribution analysis of PCR results at endpoint dilution (R(2)=0.988). This assay is characterized by the use of degenerate primers and having been validated by comparing with a Poisson distribution-based assay. The present real-time PCR assay is highly sensitive, linear, reproducible, accurate, and independent of group M subtypes. The assay will be useful for studying the relationship between HIV-1 proviral loads and the long-term efficacy of antiretroviral therapy for subtype B as well as non-B subtype strains.

DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolone resistance-determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A.

The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.