Thyroid abscess associated with thyrotoxicosis caused by Yersinia enterocolitica subsp. palearctica in a patient with follicular adenoma of the thyroid gland.

BACKGROUND: Yersinia enterocolitica is a gram-negative zoonotic bacterial pathogen that is typically transmitted via the fecal-oral route. The most common clinical manifestation of a Y. enterocolitica infection is self-limited gastroenteritis. Although various extraintestinal manifestations of Y. enterocolitica infection have been reported, there are no reports of thyroid abscesses. CASE PRESENTATION: An 89-year-old Japanese man with follicular adenoma of the left thyroid gland was admitted to our hospital with a 2-day history of fever and left neck pain. Laboratory tests revealed low levels of thyroid stimulating hormone and elevated levels of free thyroxine 4. Contrast-enhanced computed tomography showed low-attenuation areas with peripheral enhancement in the left thyroid gland. He was diagnosed with thyroid abscess and thyrotoxicosis, and treatment with intravenous piperacillin-tazobactam was initiated after collecting blood, drainage fluid, and stool samples. The isolated Gram-negative rod bacteria from blood and drainage fluid cultures was confirmed to be Y. enterocolitica. He was diagnosed with thyroid abscess and thyrotoxicosis due to be Y. enterocolitica subsp. palearctica. The piperacillin-tazobactam was replaced with levofloxacin. CONCLUSION: We report a novel case of a thyroid abscess associated with thyrotoxicosis caused by Y. enterocolitica subsp. palearctica in a patient with a follicular thyroid adenoma.

Seroprevalence kinetics of SARS-CoV-2 antibodies in pediatric patients visiting a hospital during COVID-19 pandemic in Japan.

Children infected with SARS-CoV-2 are often asymptomatic or have mild symptoms. The studies on the seroprevalence kinetics of SARS-CoV-2 antibodies in children are limited. We conducted a cross-sectional survey of the positive rate of the SARS-CoV-2 IgG in pediatric patients without suspected COVID-19 infection between January 2007 and March 2022. We defined the serum samples from the pre-pandemic and pandemic groups (1st-6th waves). Totally, 2557 samples were collected and no samples from the pre-pandemic group or the 1st-4th waves were positive for IgG. There were 4/661 and 16/373 positives at the 5th and 6th waves, respectively. At the 5th wave, the prevalence of IgG was 1.3% in children aged 1-4 years. At the 6th wave, in children <1 year of age, the prevalence was 4.0%, and 2.4%, 5.3%, 5.2% and 10% in age groups 1-4, 5-9, 10-14 and 15-18 years, respectively. In conclusions, the pre-pandemic samples were negative, and the IgG positivity increased during the later period of the pandemic.

Evaluation of lateral flow devices for postmortem rabies diagnosis in animals in the Philippines: a multicenter study.

Expansion of the use of lateral flow devices (LFD) for animal rabies diagnosis can help mitigate the widespread underreporting of rabies. However, this has been hindered by the limited number and small sample size of previous studies. To overcome this limitation, we conducted a multicenter study with a larger sample size to assess the diagnostic accuracy of the ADTEC LFD for postmortem rabies diagnosis in animals. Thirteen governmental animal diagnostic laboratories in the Philippines were involved in this study, and 791 animals suspected of having rabies were tested using both the direct fluorescence antibody test (DFAT) and ADTEC LFD between August 2021 and October 2022. The LFD demonstrated a sensitivity of 96.3% [95% confidence interval (CI): 94.1%-97.9%] and a specificity of 99.7% (95% CI: 98.4%-100%). Notably, false-negative results were more likely to occur in laboratories with lower annual processing volumes of rabies samples in the previous years (adjusted odds ratio 4.97, 95% CI: 1.49-16.53). In this multicenter study, the high sensitivity and specificity of the LFD for the diagnosis of animal rabies, compared to that of the DFAT, was demonstrated, yet concerns regarding false-negative results remain. In areas with limited experience in processing rabies samples, it is essential to provide comprehensive training and careful attention during implementation.

Bacillus subtilis Bacteremia from Gastrointestinal Perforation after Natto Ingestion, Japan.

We report a case of Bacillus subtilis variant natto bacteremia from a gastrointestinal perforation in a patient who ingested natto. Genotypic methods showed the bacteria in a blood sample and the ingested natto were the same strains. Older or immunocompromised patients could be at risk for bacteremia from ingesting natto.

Evaluation of a real-time mobile PCR device (PCR 1100) for the detection of the rabies gene in field samples.

BACKGROUND: The Philippines is ranked among the top countries with 200-300 annual deaths due to rabies. Most human rabies cases have been reported in remote areas, where dog surveillance is inadequate. Therefore, a strategy to effectively improve surveillance in remote areas will increase the number of detections. Detecting pathogens using portable real-time reverse transcription-polymerase chain reaction (RT-PCR) has the potential to be accepted in these areas. Thus, we aimed to develop an assay to detect the rabies virus (RABV) genome by combining the robust primer system LN34 with the PicoGene PCR1100 portable rapid instrument targeting RABV RNA (PCR1100 assay). METHODS: Procedures were optimised using an LN34 primer/probe set, KAPA3G Plant PCR Kit (KAPA Biosystems), FastGene Scriptase II (NIPPON Genetics), and an artificial positive control RNA. RESULTS: Positive control RNA showed an analytical limit of detection of 10 copies/µL without false positivity, generating results in approximately 32 min. Compared to dFAT or RT-qPCR using field samples, the sensitivity and specificity of the PCR1100 assay were 100%, and even lower copy numbers (approximately 10 copies/µL) were detected. CONCLUSIONS: This study demonstrated that the developed assay can detect rabies RNA in field samples. Because dog-mediated rabies is endemic in remote areas, the rapidity, mobility, and practicality of the PCR1100 assay as well as the high sensitivity of the LN34 system make it an ideal tool for the confirmation of rabies in these areas.

Molecular Analysis of Rabies Virus Using RNA Extracted from Used Lateral Flow Devices.

Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.

Implications of the antiviral drug favipiravir on rabies immunoglobulin for post-exposure prophylaxis of rabies in mice model with category III-like exposures.

Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), with almost 100% mortality if proper post-exposure prophylaxis (PEP), consisting of rabies immunoglobulin (RIG) and rabies vaccine, is not applied in a timely manner. However, this is challenged by the limited availability of RIG, especially in resource-constrained countries. In this study, we assessed the scope of the antiviral drug favipiravir to treat rabies-infected mice as an alternative to RIG. Category III-like wounds were induced in RABV-challenged mice treated with favipiravir instead of RIG in the PEP regimen. The use of favipiravir followed by rabies vaccine provided complete protection against rabies-related death in 100% of mice, even after RABV propagated to the central nervous system during infection. Additionally, the virus-neutralizing antibody titer in the favipiravir and vaccine group was significantly higher than that of the RIG and vaccine recipients. The use of favipiravir with rabies vaccine seemingly prevents fatal outcomes and even rescues the cases that already express clinical symptoms. A clinical trial of this approach is warranted, especially in countries with low RIG availability.

Dengue Infections during COVID-19 Period: Reflection of Reality or Elusive Data Due to Effect of Pandemic.

The outbreak of coronavirus disease 2019 (COVID-19) devastated the overall health management strategy of most countries. In this scenario, the present study provided insights into the possible impact of the COVID-19 pandemic on dengue infection. This ecological study retrieved data from WHO/Government reporting system from 22 major dengue epidemic countries. Incidence of dengue infections during the pre-COVID-19 time (2015~2019) and COVID-19 period (2020~2021) was compared. A correlation between the dengue and COVID-19 cases and predicted dengue incidence in 2022 was calculated using the linear regression equation. Data indicated that dengue incidences across the studied area decreased by 16% during the pandemic period (2.73 million vs. 2.29 million; p < 0.05) than the same reported in pre-COVID-19 time. Although countries in Latin America reported more cases than Asia, a positive correlation (r = 0.83) between dengue and COVID-19 cases was observed in Asia. Prediction analysis warned that specific preparation for dengue management is needed in some countries of both regions in 2022 to contain the upsurge in incidences. Due to the similar nature of symptoms of dengue and COVID-19, a state of confusion will be prevailing during the ongoing pandemic. Therefore, comprehensive and evidence-based scientific approaches were warranted at all levels.

Distribution of Severe Fever with Thrombocytopenia Syndrome Virus and Antiviral Antibodies in Wild and Domestic Animals in Oita Prefecture, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging life-threatening infectious disease caused by the tickborne SFTS virus (SFTSV), first identified in China in 2009 and then in Japan in 2013. Human SFTS cases were reported to be concentrated in western Japan, but the epidemiological conditions of SFTSV infection in a specific region are still obscure. We performed an epidemiological study of SFTSV in Oita Prefecture on the island of Kyushu, located in western Japan. For our research, we collected sera from wild and domestic animals (deer, wild boars, raccoons, cats, and dogs) and ticks from January 2010 to November 2020 in Oita. The anti-SFTSV antibody positivity rate of deer in 2014 was significantly higher than that in 2011 (65% versus 27%, P < 0.001). The anti-SFTSV antibody positivity rates of deer, wild boars, raccoons, wild dogs, domestic dogs, and domestic cats were 55%, 12%, 27%, 1.8%, 0.53%, and 1.4%, respectively. Moreover, RT-PCR could not detect SFTSV in any tick sample. Of the six areas of Oita Prefecture, only the Eastern area showed no incidence or possibility of SFTSV infection among wild and domestic animals, ticks, and human beings. Further investigation is required to assess whether local seroepidemiology in animals will help assess the risk of SFTSV infections in inhabitants.

Household survey on owned dog population and rabies knowledge in selected municipalities in Bulacan, Philippines: A cross-sectional study.

BACKGROUND: Despite the effort to eradicate rabies in the Philippines, human rabies cases have not decreased in the past decade. Rabid dogs pose the most significant hazard in the countries with the highest burden of rabies, and 70% rabies vaccine coverage is recommended for dogs in high-risk areas. Ascertaining the owned dog population and community knowledge on rabies can help improve vaccine coverage and information campaigns. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional survey in six randomly selected communities (five urban, one rural) in Central Luzon, Philippines. We first conducted the complete mapping of 9,173 households and then randomly selected 727 households. More than half (54.1%) of the households owned dogs (1.21 dogs/household). In the 727 households, we identified 878 owned dogs and 3256 humans. According to these results, the dog-to-human ratio was approximately 1:3.7. Only 8.8% of households reported a history of dog bite in 2019. Among dog-owning households, 31% reported that they allow their dogs to roam freely. Of the recorded dogs, 35.9% have never been vaccinated, and only 3.5% were spayed or castrated. Factors associated with lower rabies knowledge include (1) no education aOR: 0.30 (0.16-0.59), and (2) only primary school education aOR: 0.33 (0.22-0.49). In contrast, factors associated with higher knowledge include (1) owning a dog and not allowing them to roam freely aOR: 2.01 (1.41-2.87) and (2) owning a dog and allowing them to roam freely aOR: 1.84 (1.17-2.92), when compared to those with no dogs. CONCLUSIONS/SIGNIFICANCE: We identified a larger dog population in the community than the usual estimates (1:10), suggesting that annual vaccine needs in the Philippines must be reassessed. Our survey shows a relatively good understanding of rabies; however, awareness of the concept of rabies as a disease, and how animals and humans can acquire it, is lacking.

Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis.

The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.

Immunohistochemical detection of virus antigen in the nasal planum of rabid dogs.

The rabies virus is one of the most neurotropic of all viruses infecting mammals. During the terminal phases of infection, the virus spreads to peripheral tissues, including the skin. The external skin of the nose, called the nasal planum, is a sensory organ where numerous nerve bundles and terminal nerves are distributed. Therefore, the nasal planum is expected to serve as a postmortem diagnostic material. However, the distribution of rabies virus antigens in the nasal planum in rabid animals has not yet been studied. In this study, the nasal planum was obtained from 45 rabid dogs. In all rabid dogs, the viral antigen was detected in the peripheral nerve tissues, Merkel cells, and squamous cells. The viral antigen in the epidermis exhibited three patterns: first, a diffuse positive pattern from the basal layer to the squamous layer; second, a reticular positive pattern along the cell membrane in the squamous layer; and third, a basal layer pattern of the epidermis. In the dermis, viral antigens were detected more often in lamellated corpuscles just beneath the rete pegs. These results suggest that the nasal planum could serve as a useful alternative source for postmortem diagnosis in rabies endemic countries.

Validation of serum apolipoprotein A1 in rabies virus-infected mice as a biomarker for the preclinical diagnosis of rabies.

Rabies is a type of acute fetal encephalitis caused by rabies virus (RABV). While it becomes incurable after symptom onset, it can be prevented by post-exposure prophylaxis (PEP) during the long incubation period. While preclinical diagnosis aids the appropriate PEP administration, it is mostly nonfeasible owing to the absence of viremia or a specific antibody response during the incubation period. Here, an attempt was made to identify a serum biomarker for the preclinical diagnosis of rabies. Using the serum from a mouse inoculated intramuscularly (i.m.) with 5 × 105 focus-forming units (FFU) of recombinant RABV expressing red firefly luciferase (1088/RFLuc) immediately before symptom onset, two-dimensional differential gel electrophoresis was conducted, followed by mass spectrometry, and it was confirmed that apolipoprotein A1 (ApoA1) was up-regulated. ELISA showed that the serum ApoA1 and specific antibody levels increased during the incubation period and on the day of symptom onset. Since a lower infectious dose can be used to induce the unstable and long incubation period generally observed in natural infection, the ApoA1 level in mice inoculated i.m. with 103 FFU of 1088/RFLuc was examined by monitoring viral dynamics using in vivo imaging. The serum ApoA1 and specific antibody levels were up-regulated in 50% and 58.3% of mice exhibiting robust RABV replication, respectively, but not in mice exhibiting weak RABV replication. In addition, it was reported that ApoA1 was found to be a biomarker for neuronal damage. Additional biomarker candidates will be needed for the effective preclinical diagnosis of rabies.

Background and descriptive features of rabies-suspected animals in Central Luzon, Philippines.

BACKGROUND: The Philippines is one of the major endemic countries for canine rabies in Southeast Asia. However, detailed description and analysis of laboratory-confirmed animal rabies are limited. Highly accurate surveillance requires a thorough understanding of the target area-specific problems and obstacles. Therefore, we aim to describe and analyze the rabies suspect animals in Central Luzon, Philippines, to clarify the characteristics of management and clinical signs by conducting interviews with the owners. METHODS: We prospectively collected information on the rabies suspect animals submitted to the Regional animal laboratory in Central Luzon through passive laboratory-based rabies surveillance between 1st April 2019 and 30th September 2020. We performed active interviews directly or telephonically with the owner. The direct fluorescent antibody test was performed on the hippocampus, brain stem, and cerebellum for laboratory confirmation. Descriptive statistics were used to characterize the number of rabies cases according to management methods and characteristics of suspected animals during the observation period. Clinical symptoms of suspected rabid animals were analyzed by univariate logistic regression analysis. RESULTS: There were 292 sample submissions during the study period. Of these, 160 were positive for dFAT. Samples of pet animals (85.3%) provided by owners or their acquaintances (59.2%) accounted for the majority of laboratory confirmed cases. Case mapping showed that more rabies-suspected cases were sent from areas near the regional laboratory than from those far from the laboratory, despite the incidence of rabies being high in these areas. The management and clinical symptoms of 227 animal cases showed that most owners were managing their animals at home and were allowing them to roam outside (69.6%) and be unvaccinated (78.9%). Rabid animals were more likely to manifest aimless running, restlessness, and agitation. CONCLUSIONS: Our study provided some features of animals with laboratory-confirmed rabies in Central Luzon. However, most of the samples were submitted from areas near the rabies diagnosis laboratory, and the number of samples submitted from remote areas was low. To improve the surveillance capacity, it is necessary to increase sample submissions from remote areas.

Evaluation of the diagnostic accuracy of lateral flow devices as a tool to diagnose rabies in post-mortem animals.

Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings; however, the use of LFDs for diagnosis remains controversial because some commercially available kits show low sensitivity. Therefore, we compared the diagnostic efficacy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) using fresh samples and investigated the diagnostic accuracies. To do so, we evaluated rabies-suspected samples submitted to the Regional Animal Disease Diagnostic Laboratory III, Philippines. Furthermore, we conducted real-time RT-PCR and sequencing to measure the accuracy of field laboratory diagnosis. The total number of animals submitted during this study period was 184 cases, including negative control samples. Of these, 53.9% (84 cases) were positive in the dFAT. Dogs were the most common rabies-suspected animal (n = 135). The sensitivities of the ADTEC and Bionote kits were 0.88 (74 cases) and 0.95 (80 cases), respectively. The specificity of both kits was 1.00 (100 cases). Furthermore, the sensitivity and specificity of the ADTEC kit after directly homogenizing the samples in assay buffer without dilution in phosphate-buffered saline (ADTEC kit DM) were 0.94 (79 cases) and 1.00 (100 cases), respectively. By contrast, there were no positive results using the Elabscience kit among all dFAT-positive samples. The sensitivity and specificity of LFDs make these tests highly feasible if properly used. Therefore, LFD tests can be used to strengthen the surveillance of rabies-infected animals in endemic and resource-limited settings.

Reevaluation of the efficacy of favipiravir against rabies virus using in vivo imaging analysis.

Rabies virus (RABV) is a highly neurotropic virus and the causative agent of rabies, an encephalitis with an almost 100% case-fatality rate that remains incurable after the onset of symptoms. Favipiravir (T-705), a broad-spectrum antiviral drug against RNA viruses, has been shown to be effective against RABV in vitro but ineffective in vivo. We hypothesized that favipiravir is effective in infected mice when RABV replicates in the peripheral tissues/nerves but not after virus neuroinvasion. We attempted to clarify this point in this study using in vivo bioluminescence imaging. We generated a recombinant RABV from the field isolate 1088, which expressed red firefly luciferase (1088/RFLuc). This allowed semiquantitative detection and monitoring of primary replication at the inoculation site and viral spread in the central nervous system (CNS) in the same mice. Bioluminescence imaging revealed that favipiravir (300 mg/kg/day) treatment commencing 1 h after intramuscular inoculation of RABV efficiently suppressed viral replication at the inoculation site and the subsequent replication in the CNS. However, virus replication in the CNS was not inhibited when the treatment began 2 days after inoculation. We also found that higher doses (600 or 900 mg/kg/day) of favipiravir could suppress viral replication in the CNS even when administration started 2 days after inoculation. These results support our hypothesis and suggest that a highly effective drug-delivery system into the CNS and/or the enhancement of favipiravir conversion to its active form are required to improve favipiravir treatment of rabies. Furthermore, the bioluminescence imaging system established in this study will facilitate the development of treatment for symptomatic rabies.

Statistical analysis of the usefulness of follicle-sinus complexes as a novel diagnostic material for canine rabies.

In the present study, follicle-sinus complexes (FSCs) were harvested from the muzzle skin of 123 dogs with suspected canine rabies, and the sensitivity and specificity of FSC analysis were compared with those of brain tissue immunohistochemistry analysis. In the FSCs, viral antigen was detected from Merkel cells. Sensitivity was 97.3%, specificity was 100%, and the coefficient κ was 0.88. These results reconfirm that FSCs are very useful for the postmortem diagnosis of canine rabies, and suggest that 5 FSCs can yield results that are almost equivalent to those derived from brain tissue analysis in rabid dogs.

Follicle sinus complexes (FSCs) in muzzle skin as postmortem diagnostic material of rabid dogs.

Recently, we reported that follicle-sinus complexes (FSCs) in the muzzle skin are useful for postmortem diagnosis of rabid dogs. Here, we compared the sensitivity and specificity of detecting the viral antigen in the brain and FSCs of 226 suspected rabid dogs, and assessed whether the FSC harbored the virus genome and particles. The viral antigen was detected in 211 of 226 samples with 100% sensitivity and specificity. Viral RNA and particles were observed in the cytoplasm of Merkel cells (MCs). These results suggest that MCs are targets of virus infection and FSCs are useful material for diagnosing rabies.