Human Adenovirus B7d-Associated Urethritis after Suspected Sexual Transmission, Japan.

Outbreaks of acute respiratory disease associated with human adenovirus (HAdV) B7d have been reported, including fatal cases in the United States. In 2018, we detected HAdV-B7d in a patient with urethritis, probably transmitted through sexual contact. Infectious HAdV-B7d was excreted in urine and gargle for >10 days after the disappearance of symptoms.

Characteristic of slow growth in cell culture of adenovirus type 54 causing nationwide outbreak epidemic keratoconjunctivitis in Japan.

PURPOSE: To characterize the virological features of adenovirus type 54 (Ad54) causing nationwide outbreak of severe epidemic keratoconjunctivitis (EKC) in Japan, we comparatively analysed the viral propagation phenotype of Ad54 and other Ads: type 37 (Ad37), 64 (Ad64), and 5 (Ad5), in A549 cells quantitatively. STUDY DESIGN: Laboratory investigation. METHODS: We compared the growth rate of Ads using copy numbers and cytopathic effect observation during propagation in A549 cell lines. Expressions of mRNA of E1 gene were also calculated and compared. Phylogenetic analysis of the region, including putative promoter of E1 gene and E1 open reading frame (ORF), were performed. RESULTS: Increases in viral loads, growth rate, and viral propagation were slower for Ad54 than for other Ads. The expression level of the E1 gene per infected cell was lower for Ad54 than for other Ad types on post-infection day 1. Phylogenetic analysis of the E1 gene putative promoter and ORF revealed Ad54 was the closest to Ad type 8. CONCLUSION: The propagation of Ad54 in A549 is slow compared with Ad37, Ad64 and Ad5. This slow propagation could have been caused by slow genomic replication resulting from delayed viral entry or E1 transcription initiation. The EKC caused by Ad54 needs more attention because the slow propagation of Ad54 may contribute to prolonged disease duration.

Pediatric Infections by Human mastadenovirus C Types 2, 89, and a Recombinant Type Detected in Japan between 2011 and 2018.

Between 2011 and 2018, 518 respiratory adenovirus infections were diagnosed in a pediatric clinic in Shizuoka, Japan. Detection and typing were performed by partial sequencing of both hexon- and fiber-coding regions which identified: adenovirus type 1 (Ad-1, n = 85), Ad-2 (n = 160), Ad-3 (n = 193), Ad-4 (n = 18), Ad-5 (n = 27), Ad-11 (n = 2), Ad-54 (n = 3), and Ad-56 (n = 1). Considering previous reports of the circulation of an endemic recombinant Ad-2, e.g., Ad-89, 100 samples typed as Ad-2 were randomly selected for further molecular typing by sequencing the penton base-coding region. Despite the high nucleotide sequence conservation in the penton base- coding region, 27 samples showed 98% identity to Ad-2. Furthermore, 14 samples showed 97.7% identity to Ad-2 and 99.8% identity to Ad-89, while the remaining 13 samples showed an average 98% pairwise identity to other Ad-C types and clustered with Ad-5. The samples typed as Ad-89 (n = 14) and as a recombinant Ad type (P5H2F2) (n = 13) represented 27% of cases originally diagnosed as Ad-2, and were detected sporadically. Therefore, two previously uncharacterized types in Japan, Ad-89 and a recombinant Ad-C, were shown to circulate in children. This study creates a precedent to evaluate the epidemiology and divergence among Ad-C types by comprehensively considering the type classification of adenoviruses.

Infectious human adenoviruses are shed in urine even after disappearance of urethral symptoms.

BACKGROUND: Urethritis is a common sexually transmitted disease, and human adenoviruses (HAdVs) have been found to be associated with nonchlamydial nongonococcal urethritis. However, the level and viability of HAdV in the urine of patients with urethritis remain unclear. METHODS: Male patients with urethritis and an asymptomatic group were screened using their First-void urine (FVU) for urethritis-related pathogens to identify those with HAdV DNA. FVU and gargle fluid were collected from all patients including from those in the asymptomatic group. A swab of eye discharge was also collected from patients with eye symptoms. The pharyngeal and/ or ocular fluid was also screened only in cases in which FVU was positive for HAdV DNA. HAdVs were isolated using A549 cell lines and typed by sequencing, and viral shedding during 2 years was quantified using real-time PCR. The prevalence of HAdV was assessed in the urethritis and asymptomatic groups, and viral load, isolated HAdV types, and urethral symptoms were compared between the groups. RESULTS: The positive detection rate of HAdV DNA was significantly higher in the urethritis group than in the asymptomatic group. Of 398 patients with urethritis, HAdV was isolated in all 32 cases (23 cases in which only HAdV DNA was detected with a mean of 2 × 109 copies/mL in urine samples). Of 124 control cases, one had HAdV monoinfection. The most frequently detected HAdV type was 56, followed by types 37 and 64. Regarding the relationship between symptoms and isolated HAdVs, the virus was isolated for up to 12 days after urethritis symptoms disappeared. CONCLUSIONS: HAdVs were significantly detected and isolated from the FVU of patients with urethritis. Furthermore, high levels of infectious HAdVs are excreted in urine for a long period even after urethritis symptoms disappear.

Evaluation of a silver-amplified immunochromatography kit for adenoviral conjunctivitis.

OBJECTIVE: To compare and evaluate the sensitivity of a newly developed silver-amplified immunochromatography (SAI) kit with various immunochromatography (IC) kits for adenoviruses based on the detection limit (copies/test). METHODS: An SAI kit and four ophthalmic IC kits were evaluated. The detection limits of the five kits were determined using the limiting dilution method for 15 conjunctivitis-associated adenoviruses (adenoviruses 1, 2, 3, 4, 5, 7, 8, 11, 37, 53, 54, 56, 64, 81, and 85). The detection limits were presented as numerical values as determined by real-time polymerase chain reaction (PCR). RESULTS: The detection limit of the SAI kit for the adenovirus types ranged from 1.0 × 103 -5.0 × 10 4 copies/test (geometric mean, 4.7 × 10 3 ). SAI had a 10-250-fold lower detection limit than the four IC kits for all adenoviruses studied. There were also differences in detection limits among the adenovirus types for each kit. DISCUSSION: The detection limit of the SAI kit was drastically reduced because the silver-amplification reaction increased the color development sensitivity. The results revealed the high sensitivity of SAI for detecting adenoviruses and suggested its usefulness for conjunctivitis examination.

Species differences in circulation and inflammatory responses in children with common respiratory adenovirus infections.

Human adenoviruses (HAdVs) cause severe inflammatory respiratory infections, but previous epidemiological studies lacked analysis of the characteristics of the inflammation. Consecutive patients <13 years old with acute febrile illness during a 2-year period were tested. HAdV strains were isolated from nasopharyngeal swabs, and molecular identification was performed by hexon, fiber, and species-specific PCR methods. Blood inflammatory markers, including the white blood cell (WBC) count, CRP, and 29 cytokines, were measured. A total of 187 patients were enrolled, and HAdV types were identified from 175 patients (93.5%). Species C (types 2, 1, 5, and 6, in order of frequency) was most common at 37.1%, followed by B (type 3) at 30.9% and E (type 4) at 26.9%. Species C was detected predominantly in 1-year-old, whereas B and E were in older ages. Species C and B had seasonal circulation patterns, but E was found in only one season during the 2-year study period. The WBC count was highest in patients with species C. Eleven of the 29 tested serum cytokines were detected. Seven kinds, including G-CSF, IL-6, and TNF-α, were elevated in species C infections, whereas IL-10 was lowest in species C. Species differences in inflammatory responses, especially regarding serum cytokines were described in common pediatric HAdV infections. Species C causes the strongest inflammatory responses in young children.

[Laboratory Diagnosis of Human Adenovirus for Surveillance in Japan in 2014].

Objective: This study sought to clarify how laboratory testing of human mastadenovirus (adenovirus) is conducted in local public health institutes (LPHI) as part of Japanese adenovirus surveillance. Methods: Questionnaires on adenovirus surveillance were distributed to LPHIs (n=77) between February and May, 2014. Results: The participation response rate was 100%. During this period, adenoviral cultures were conducted at 65 institutes (84%) while 49 (64%) used neutralization testing. PCR diagnoses were conducted at 58 institutes (75%). The national adenovirus diagnosis manual (http://www.nih.go.jp/niid/ja/labo-manual.html), which includes methods of viral culture and PCR for penton, hexon, and fiber-coding regions, was used in 68% of the LPHIs. Ocular samples were diagnosed at 36 (47%) LPHIs. Conclusion: Most LPHIs conduct adenovirus surveillance in accordance with the national diagnosis manual. Both PCR and viral cultures were used for adenovirus surveillance in the majority of LPHIs during the surveyed period.

Prescription surveillance and polymerase chain reaction testing to identify pathogens during outbreaks of infection.

Syndromic surveillance, including prescription surveillance, offers a rapid method for the early detection of agents of bioterrorism and emerging infectious diseases. However, it has the disadvantage of not considering definitive diagnoses. Here, we attempted to definitively diagnose pathogens using polymerase chain reaction (PCR) immediately after the prescription surveillance system detected an outbreak. Specimens were collected from 50 patients with respiratory infections. PCR was used to identify the pathogens, which included 14 types of common respiratory viruses and Mycoplasma pneumoniae. Infectious agents including M. pneumoniae, respiratory syncytial virus (RSV), rhinovirus, enterovirus, and parainfluenza virus were detected in 54% of patients. For the rapid RSV diagnosis kit, sensitivity was 80% and specificity was 85%. For the rapid adenovirus diagnosis kit, no positive results were obtained; therefore, sensitivity could not be calculated and specificity was 100%. Many patients were found to be treated for upper respiratory tract infections without the diagnosis of a specific pathogen. In Japan, an outbreak of M. pneumoniae infection began in 2011, and our results suggested that this outbreak may have included false-positive cases. By combining syndromic surveillance and PCR, we were able to rapidly and accurately identify causative pathogens during a recent respiratory infection outbreak.

Cultivation for 21 days should be considered to isolate respiratory adenoviruses from samples containing small numbers of adenoviral genomes.

Adenovirus types 1, 2, and 3 can usually be isolated in only a short time, although occasionally it may take longer. This phenomenon has been explained empirically as being due to the viral load in the sample, although to date there has been no experimental confirmation of this. In this study we therefore tried to establish a correlation between the quantity of respiratory adenovirus genome in the clinical sample and the time required for its isolation. The correct choice of sensitive cell line is important for this purpose, thus we compared the sensitivity of three different cell lines (HeLa, A549, and RD), and found A549 to be the most sensitive to adenoviruses 1-3. Stored clinical samples (n=21) containing adenoviruses 1-3 were diluted to make solutions containing between 10 and 10(8) copies/microL of adenovirus genome (n=242). These diluted clinical samples were then inoculated into A549 cells, which were cultivated for 21 days and the results compared to the number of viral genomes in each cultivated sample. Adenoviruses could be isolated from all samples (41/41) containing >/=10(6) copies/microL within 6 days, whereas samples containing 10 and 10(2) copies/microL required cultivation for 12.6+/-3.8 and 11.2+/-3.8 days (mean+/-S.D.), respectively, before adenoviruses could be isolated. A cultivation time of 21 days should therefore be considered for the isolation of respiratory adenoviruses from samples containing <10(3) adenovirus genome copies/microL.

Novel high-speed real-time PCR method (Hyper-PCR): results from its application to adenovirus diagnosis.

PCR, including real-time PCR, usually requires at least 1 h to obtain results. To shorten this time, a novel real-time PCR method (Hyper-PCR) was developed. This method utilizes high-speed DNA polymerase and a thin disc-type reaction vessel that can quickly alter the temperature of the reaction mixture in a newly developed PCR machine. Reactions capable of amplifying adenovirus (Ad) DNA can be completed within 11 min (3 temperature steps, 55 cycles). Hyper-PCR can detect 3.1-18.0 DNA copies/reaction of Ad types 1-4, 7, 8, 11, 15, 19, and 37. Hyper-PCR and conventional real-time PCR were applied to diagnose 147 clinical samples, and the results were compared. Hyper-PCR had a sensitivity of 100% (73/73) and a specificity of 100% (74/74), using conventional real-time PCR as the gold standard. Our newly developed PCR method, Hyper-PCR, was able to diagnose Ad infection within 17 min (not including the time for genome extraction). The thermocycling time of the novel PCR is faster than that of previously available PCR applications, and this method is thought to be potentially applicable to clinical and environmental diagnostics, where rapid diagnosis is important.