Clinical perspectives of Treponema pallidum subsp. Endemicum infection in adults, particularly men who have sex with men in the Kansai area, Japan: A case series.

Bejel, caused by Treponema pallidum subsp. Endemicum (TEN), is a locally transmitted disease among children and juveniles in hot and dry regions. The number of adult cases of TEN infection outside of endemic areas has recently increased. We clinically examined five cases of TEN infection among adult cases previously reported in Japan. TEN infection mainly developed among young to middle-aged men who have sex with men (MSM). The clinical features of cases of TEN infection were similar to those of primary- and secondary-stage T. pallidum subsp. pallidum (TPA) infection. Genital lesions were common as the primary lesion. The clinical features and laboratory parameters of cases of TEN infection were similar to those of TPA infection. Most of the isolated strains had the A2058G mutation in 23S rDNA, which is responsible for resistance to macrolides. We also performed the systemic literature review of the TEN cases outside the endemic countries. The recent reported cases diagnosed with molecular methods shared the clinical features, occurred in young-to middle-aged sexually active persons in urban areas of developed countries and often accompanied with genital lesions, which were distinct from the classic description of bejel. This case series and the literature review provides important clinical insights and will contribute to the clinical detection of this rarely identified disease in developed countries. The surveillance of treponematoses, including TEN infection, using molecular diagnostic techniques is also warranted in developed countries, for the purpose of grasping the epidemic situation and control the local transmission.

Bejel, a Nonvenereal Treponematosis, among Men Who Have Sex with Men, Japan.

Bejel, an endemic treponematosis caused by infection with Treponema pallidum subspecies endemicum, has not been reported in eastern Asia and the Pacific region. We report local spread of bejel among men who have sex with men in Japan. Spread was complicated by venereal syphilis.

Circulation of Distinct Treponema pallidum Strains in Individuals with Heterosexual Orientation and Men Who Have Sex with Men.

Human treponematosis is caused by various pathogenic Treponema pallidum subspecies, including T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and Treponema carateum The global prevalence of syphilis has been increasing since the 2000s. Men account for more than 90% of the cases, with the majority being men who have sex with men (MSM). In Japan, the increase in the number of syphilis patients began in 2011, a 10-year delay from the global trend. In 2017, a total of 5,829 syphilis cases (3,934 men and 1,895 women) were reported, with an outstanding increase in cases among young adult women; the number reported for women age 15 to 20 years was 1,100. Hence, a molecular epidemiological study was conducted on circulating T. pallidum strains using two strain typing methods, the enhanced CDC method and sequencing-based molecular typing. Clinical specimens from 95 adults suspected of syphilis were collected from September 2013 to August 2017 in Osaka, Japan. T. pallidum DNA was detected in specimens from 25 males and 11 females, including seven MSM. The majority of the heterosexual patients (66.7% and 90.9% of males and females, respectively) were positive for 14d/f-SSR8. In contrast, the genotypes identified in the MSM group were significantly divergent. T. pallidum subsp. endemicum was notably identified in two MSM patients. Macrolide-sensitive or Nichols-like strains were significantly associated with the MSM group. These data suggest that distinct T. pallidum strains were circulating in the heterosexual and MSM groups. Our findings imply that independent factors may contribute to the increased syphilis prevalence in heterosexual and MSM populations.

Clonal expansion and spread of the ceftriaxone-resistant Neisseria gonorrhoeae strain FC428, identified in Japan in 2015, and closely related isolates.

OBJECTIVES: Ceftriaxone resistance in Neisseria gonorrhoeae is a major public health concern globally because a high-dose (1 g) injection of ceftriaxone is the only remaining option for empirical monotherapy of gonorrhoea. The ceftriaxone-resistant gonococcal strain FC428, cultured in Osaka in 2015, is suspected to have spread nationally and internationally. We describe the complete finished genomes of FC428 and two closely related isolates from Osaka in 2015, and examine the genomic epidemiology of these isolates plus three ceftriaxone-resistant gonococcal isolates from Osaka and Hyogo in 2016-17 and four ceftriaxone-resistant gonococcal isolates cultured in 2017 in Australia, Canada and Denmark. METHODS: During 2015-17, we identified six ceftriaxone-resistant gonococcal isolates through our surveillance systems in Kyoto, Osaka and Hyogo. Antimicrobial susceptibility testing (six antimicrobials) was performed using Etest. Complete whole-genome sequences of the first three isolates (FC428, FC460 and FC498) from 2015 were obtained using PacBio RS II and Illumina MiSeq sequencing. The three complete genome sequences and draft genome sequences of the three additional Japanese (sequenced with Illumina MiSeq) and four international ceftriaxone-resistant isolates were compared. RESULTS: Detailed genomic analysis suggested that the Japanese isolates (FC428, FC460, FC498, KU16054, KM383 and KU17039) and the four international MLST ST1903 isolates from Australia, Canada and Denmark formed four linked subclades. CONCLUSIONS: Using detailed genomic analysis, we describe the clonal expansion of the ceftriaxone-resistant N. gonorrhoeae strain FC428, initially identified in 2015 in Japan, and closely related isolates. FC428 and its close relatives show some genomic diversity, suggesting multiple genetic subclades are already spreading internationally.

New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan.

We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3'-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread.

Treatment failure with 2 g of azithromycin (extended-release formulation) in gonorrhoea in Japan caused by the international multidrug-resistant ST1407 strain of Neisseria gonorrhoeae.

OBJECTIVES: Antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally. We report the first verified treatment failure of gonorrhoea with 2 g of azithromycin (extended-release formulation) in Japan and characteristics of the corresponding N. gonorrhoeae isolates. METHODS: Pre- and post-treatment isolates (n = 4) were investigated by Etest for antimicrobial susceptibility. The isolates were examined for molecular epidemiology by multilocus sequence typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multiple-locus variable-number tandem repeat analysis (MLVA), and for the presence of azithromycin resistance determinants (23S rRNA gene mutations, erm genes and mtrR mutations). RESULTS: All isolates were resistant to azithromycin (MIC 4 mg/L) and ciprofloxacin, but remained susceptible to cefixime, ceftriaxone and spectinomycin. All isolates were assigned to MLST ST1901 and NG-MAST ST1407 and three of four isolates possessed MLVA profile 8-3-21-16-1. All isolates contained the previously described C2599T mutation (N. gonorrhoeae numbering) in all four 23S rRNA alleles and the previously described single-nucleotide (A) deletion in the mtrR promoter region. CONCLUSIONS: This verified treatment failure occurred in a patient infected with an MLST ST1901/NG-MAST ST1407 strain of N. gonorrhoeae. While this international strain commonly shows resistance or decreased susceptibility to multiple antimicrobials, including extended-spectrum cephalosporins, the strain reported here remained fully susceptible to the latter antimicrobials. Hence, two subtypes of azithromycin-resistant gonococcal MLST ST1901/NG-MAST ST1407 appear to have evolved and to be circulating in Japan. Azithromycin should not be recommended as a single antimicrobial for first-line empirical treatment of gonorrhoea.

Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance.

In 2009, the first high-level ceftriaxone-resistant Neisseria gonorrhoeae strain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) of Neisseria gonorrhoeae isolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193 N. gonorrhoeae isolates were collected and the MICs (µg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 µg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore, N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 µg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic type penA alleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of the penA mosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistant N. gonorrhoeae strain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.

[Evaluation of an immunochromatographic fourth generation test for the rapid diagnosis of acute HIV infection].

The early diagnosis of human immunodeficiency virus (HIV) infection is important to provide effective antiviral treatment and to prevent transmission of HIV. One of the key issues to achieve this goal is to shorten the so-called "diagnostic window period" when the humoral immune response toward the virus is not fully developed during the acute phase of HIV-1 infection. In 2008, the Espline HIV Ag/Ab test kit (E4G, Fujirebio Inc. Japan) was marketed in Japan belonging to the fourth generation of HIV test kits characterized by its ability to detect both viral antigens (Ag) and anti-HIV-1/2 antibodies (Ab). E4G is the first and only fourth generation immunochromatographic HIV test kit approved in Japan at present. To evaluate its performance to diagnose acute HIV infection (AHI), E4G was compared with fourth generation Ag/Ab ELISA test kits, a third generation PA test kit, WB and real-time PCR for the testing of 25 AHI clinical specimens. E4G detected HIV infection in 18/25 specimens (sensitivity : 72.0%), of which the viral Ag was detected in only 2 specimens (8.0%) bearing a viral load > 10 million copies/mL. No spesimens were simultaneously reactive to both Ag and Ab against HIV. The third generation PA achieved a positive score of 17/ 25 specimens (68.0%), which was almost the same as the E4G figure. In contrast the fourth generation Ag/ Ab ELISA scored all the 25 AHI specimens positive (sensitivity : 100%). Overall, although having the merit of offering a rapid diagnostic test for HIV infection, E4G does not provide a sensitivity in AHI diagnosis superior to test kits currently available.

Increasing seroprevalence but waning herd immunity against measles after elimination: Longitudinal seroepidemiology of measles in Osaka Prefecture, Japan, 2003-2020.

Japan is one of the countries conducting longitudinal serosurveillance of vaccine-preventable diseases. We conducted surveillance of the local measles-specific antibody titer, calculated the effective reproduction number (Re), and compared data of four terms: term 1, 2003-2006 (before the introduction of the second shot of measles-containing vaccine); term 2, 2007-2010 (early term toward measles elimination); term 3, 2011-2014 (later term toward measles elimination); and term 4, 2015-2020 (after elimination of measles in Japan). Approximately 250 sera from volunteers aged 0 to ≥ 40 years were collected and examined for measles-specific IgG using the gelatin particle agglutination (PA) method annually from 2003 to 2020. Seroprevalence and the geometric mean of the PA antibody titer were examined by term. Re was calculated using the age-dependent proportion immune and contact matrix for each term. Of the 4,716 sera, 886 in term 1, 1,217 in term 2, 1,069 in term 3, and 1,544 in term 4 were collected. The seroprevalence gradually increased from term 1 (88.3% CI 86.0-90.3) to term 4 (95.7% CI 94.6-96.7), and the seroprevalence of term 1 was significantly lower than those of other terms (Fisher's exact test, p < 0.001), with PA titer ≥ 16 as positive. By contrast, PA antibody titers significantly decreased from term 1 (median 1,024) to term 4 (median 256) (Mann-Whitney U test, p < 0.001). With the protection level (PA titer ≥ 128 and ≥ 256) as positive, Re gradually increased from term 1 (1.8 and 2.3) to term 4 (2.5 and 4.8, respectively). Waning levels of measles antibodies potentially increase the measles susceptibility in Osaka, Japan. This trend might imply a limitation of vaccine-induced immunity in the absence of a natural booster for wild strains after measles elimination. This study provides a cue for maintaining continuous measles elimination status in the future.

Synthesis, anti-HIV and anti-oxidant activities of caffeoyl 5,6-anhydroquinic acid derivatives.

In our continued research on chlorogenic acid analogues and derivatives with improved bioactivity, we have synthesized some caffeoyl 5,6-anhydroquinic acid derivatives. The 1,7 acetonides of chlorogenic acid (15), and of the mono-caffeoyl 5,6-anhydroquinic acids (7-8) showed appreciable anti-HIV activity. The 3,4-dicaffeoyl 5,6-anhydroquinic acid (12) exhibited an anti-HIV activity twice as that of 3,5-dicaffeoylquinic acid (22). The caffeoyl 5,6-anhydroquinic acid derivatives displayed potent anti-oxidant activities. The mono-caffeoyl 5,6-anhydroquinic acids (10-11) were more than twice stronger than chlorogenic acid (21) on SOD-like activity.

Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus.

We evaluated the antiviral activity of a chlorine dioxide gas solution (CD) and sodium hypochlorite (SH) against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. CD at concentrations ranging from 1 to 100 ppm produced potent antiviral activity, inactivating >or= 99.9% of the viruses with a 15 sec treatment for sensitization. The antiviral activity of CD was approximately 10 times higher than that of SH.

First isolation of human adenovirus type 85 by molecular analysis of adenoviruses in cases of urethritis.

Introduction. Human adenovirus (HAdV) has been reported as a potential cause of sexually transmitted urethritis.Aim. We aimed to investigate HAdVs associated with urethritis in Osaka, Japan through molecular characterization.Methodology. Urine samples were obtained from male patients with urethritis from 2015 to 2018. Molecular analysis of the isolated strains and follow-up real-time polymerase chain reaction analyses of the clinical samples were performed.Results. The isolates were classified into five types belonging to species D (18 cases) or E (one case). HAdV-85 (species D) was detected for the first time in a urethritis case. Follow-up examination demonstrated that HAdV was isolated from urine samples half a month after the first sampling in four cases, and that viral DNA could be detected after 1 month in two cases.Conclusion. The HAdV types detected from urethritis cases were related to respiratory and ocular HAdV infections, while a novel HAdV type identified as a cause of conjunctivitis also causes urethritis. Sexual contact should be avoided for 1 month after HAdV genital infection.

Estimating HIV-1 incidence in Japan from the proportion of recent infections.

The first step of the UNAIDS/WHO 90-90-90 targets to encourage early diagnosis with treatment for the control of HIV-1 epidemic is to achieve 90% HIV-1 diagnosis in infected individuals. In Japan, approximately 30% of newly reported cases have been annually identified by AIDS onset, implying that substantial numbers of HIV-1-infected individuals remain undiagnosed. However, the proportion of undiagnosed cases has not yet been determined. In the present study, the proportion of recent HIV-1 infections to newly-diagnosed cases was determined from 2006 to 2015 using a recent infection assay for three metropolitan areas in Japan: Tokyo, Osaka, and Fukuoka. Estimated median periods between infection and diagnosis were 1.0, 1.8, and 2.9 years for Tokyo, Osaka, and Fukuoka, respectively. Estimation of annual HIV-1 incidence by a back-calculation method using these data as well as HIV/AIDS national surveillance data indicated proportions of undiagnosed to new HIV-1 infections from 2006 to 2015 to be 18% in Tokyo, 22% in Osaka, 38% in Fukuoka, and 28% in Japan. This is the first report estimating HIV-1 incidence in Japan using a serological biomarker in combination with national HIV/AIDS surveillance data.

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

Comparative evaluation of the Geenius HIV 1/2 Confirmatory Assay and the HIV-1 and HIV-2 Western blots in the Japanese population.

Accurate diagnosis of earlier HIV infection is essential for treatment and prevention. Currently, confirmation tests of HIV infection in Japan are performed using Western blot (WB), but WB has several limitations including low sensitivity and cross-reactivity between HIV-1 and HIV-2 antibodies. To address these problems, a new HIV testing algorithm and a more reliable confirmation and HIV-1/2 differentiation assay are required. The Bio-Rad Geenius HIV-1/2 Confirmatory Assay (Geenius) has recently been approved and recommended for use in the revised guidelines for diagnosis of HIV infection by the Center for Disease Control and Prevention (USA). We made comprehensive comparison of the performance of Geenius and the Bio-Rad NEW LAV BLOT 1 and 2 (NLB 1 and 2) which are WB kits for HIV-1 and HIV-2, respectively, to examine if Geenius is a suitable alternative to these WB assays which are now being used in HIV testing in Japan. A total of 166 HIV-1 positive samples (146 from patients with established HIV-1 infection and 20 from patients with acute infection), five HIV-1 seroconversion panels containing 21 samples and 30 HIV-2 positive samples were used. In addition, a total of 140 HIV negative samples containing 10 false-positives on screening tests were examined. The sensitivity of Geenius and NLB 1 for HIV-1 positive samples was 99.3% and 98.6%, respectively. Geenius provided more positive results in the samples from acute infections and detected positivity 0 to 32 days earlier in seroconversion panels than NLB 1. NLB 2 gave positive results in 12.3% of HIV-1 positive samples. The sensitivity of both Geenius and NLB 2 for HIV-2 positive samples was 100%. The specificity of Geenius, NLB 1 and NLB 2 was 98.5%, 81.5% and 90.0%, respectively. Geenius is an attractive alternative to WB for confirmation and differentiation of HIV-1 and HIV-2 infections. The adaptation of Geenius to the HIV testing algorithm may be advantageous for rapid diagnosis and the reduction of testing costs.

[Two cases of HIV-1 acute infection with antibody negative by immunochromatography method].

We found two cases of HIV-1 acute infection, confirmed by nucleic amplification test (NAT) and/or RT-PCR, with HIV-1 antibody negative by immunochromatography (IC) method but weakly positive by particle agglutination (PA) test. These cases suggested that IC method was less sensitive than PA test in the detection of acute infections. It is necessary to execute the post counseling that considers the possibility of the acute infection in public health centers and testing places where IC method is used for the screening test. It is also important to recommend taking the following re-examination after a certain period to a person who seems to have had a chance of infection in a short time before testing.

A cluster of rapid disease progressors upon primary HIV-1 infection shared a novel variant with mutations in the p6gag/pol and pol/vif genes.

Few studies have described the etiologic factors associated with rapid AIDS onset during primary HIV-1 infection. Our molecular epidemiological study identified a cluster of individuals infected with HIV-1 variants characterized by novel mutations in the p6 and pol/vif genes during 2011 and 2013 in Osaka, Japan. Individuals positive for the novel HIV-1 variant showed rapid disease progression, suggesting a role of viral mutations in the fostering of the clinical course of HIV-1 infection.

Identification of Novel Recombinant Forms of Hepatitis B Virus Generated from Genotypes Ae and G in HIV-1-Positive Japanese Men Who Have Sex with Men.

The rare hepatitis B virus (HBV) genotype G (HBV/G) coinfects HIV-1-positive individuals along with HBV/A and generates recombinants. However, the circulation of HBV A/G recombinants remains poorly understood. This molecular epidemiologic study examined HBV A/G recombinants in Japanese HIV-1-positive men who have sex with men (MSM). Initially, blood specimens submitted for confirmatory tests of HIV infection in Osaka and Tokyo, Japan, from 2006 to 2013 were examined for HIV-1, and HIV-1-positive specimens were screened for HBV. Among 817 specimens from HIV-1-positive individuals, HBsAg was detected in 59 specimens; of these, HBV/Ae (alternatively A2), a subgenotype of HBV/A prevalent in Europe and North America, was identified in 70.2%, HBV/C in 17.5%, and HBV/G in 10.5%, and HBV/E in 1.8% according to the core gene sequence. The full-length genome analysis of HBV was performed on HBV/G-positive specimens because some HBV A/G recombinants were historically overlooked by genotyping based on a partial genome analysis. It revealed that five of the specimens contained novel Ae/G recombinants, the core gene of which had a high sequence similarity to HBV/G. Detailed analyses showed that novel recombinants were coinfected with HBV/Ae in a recombinant-dominant fashion. No major drug-resistant mutations were found in the newly identified HBV Ae/G recombinants. Some of the individuals asymptomatically coinfected with HIV/HBV suffered mild liver injury. This study demonstrated that novel Ae/G HBV recombinants were identified in Japanese HIV-1-positive MSM. The pathogenicity of novel HBV Ae/G recombinants should be examined in a future longitudinal study. Surveillance of such viruses in HIV-1-positive individuals should be emphasized.