Clinical characteristics of acute Q fever patients in South Korea and time from symptom onset to serologic diagnosis.

BACKGROUND: Acute Q fever usually presents as a nonspecific febrile illness, and its occurrence is rapidly increasing in South Korea. This study investigated the clinical characteristics of acute Q fever patients in South Korea and the time from symptom onset to serologic diagnosis. The clinical courses were examined according to antibiotic treatment. METHODS: Data of patients diagnosed with acute Q fever at Chungbuk National University Hospital between January 2015 and February 2018 were retrospectively collected. Demographic and epidemiologic data were reviewed. The time from symptom onset to serologic diagnosis by an immunofluorescence assay (IFA) was analyzed. Clinical courses and the percentage of patients with a high phase I immunoglobulin G titer (≥ 1:1024) were compared between patients administered antibiotics with anti-Coxiella burnetii activity and patients not administered such antibiotics. RESULTS: Forty-eight patients (median age: 51.5 years) were included. Most were male (95.8%) and had no history of animal contact (91.7%). The median time from illness onset to serologic diagnosis was 21 days. Thirty-nine patients received antibiotics with anti-C. burnetii activity. The length of hospital stay and fever duration did not significantly differ between patients who received antibiotics with anti-C. burnetii activity (7 and 15 days) and those who did not (5 and 8 days) (P = 0.110 and P = 0.137, respectively). The percentage of patients with a high phase I immunoglobulin G titer (≥ 1:1024) did not significantly differ between patients who received antibiotics with anti-C. burnetii activity and those who did not (P = 0.340). CONCLUSIONS: Most acute Q fever patients had a nonspecific febrile illness with mild elevation of transaminases and no history of animal contact or occupational risk. The time from symptom onset to a positive IFA test was longer than the fever duration in most acute Q fever patients. Consequently, it may be difficult for clinicians to serologically diagnose acute Q fever. However, inappropriate antibiotic treatment was not associated with prolongation of symptoms or progression to chronic Q fever.

Recent Increase of Human Granulocytic Anaplasmosis and Co-Infection with Scrub Typhus or Korean Hemorrhagic Fever with Renal Syndrome in Korea.

We report 17 patients with human granulocytic anaplasmosis between January 2015 and September 2018 at two tertiary university hospitals in Korea. Monthly incidence peaked in May and June. Among these patients, we identified three who were co-infected with scrub typhus, and one patient with hemorrhagic fever with renal syndrome.

Clinical and Genetic Features of Coxiella burnetii in a Patient with an Acute Febrile Illness in Korea.

Although Q fever is an important zoonotic infection with a worldwide distribution, no human isolates of Coxiella burnetii have been identified in Korea. For the first time, we identified the nucleotide sequence of C. burnetii from a 32-year-old man with an acute febrile illness in Korea. Diagnosis of acute Q fever was confirmed by seroconversion using indirect immunofluorescence antibody assays. Phylogenetic analysis demonstrated high sequence similarity (99.6%-100%) with C. burnetii 16S rRNA sequences identified from the reservoir. These results are the first genetic analysis of C. burnetii in a human case of Q fever in Korea.

Seroreactivity and Risk Factors Associated with Human Brucellosis among Cattle Slaughterhouse Workers in South Korea.

The prevalence rate of human brucellosis in high-risk populations, as well as their risk factors, have not been well understood in South Korea. In this cross-sectional study, we investigated the seroreactivity and risk factors associated with human brucellosis among South Korean cattle slaughterhouse workers. We enrolled 922 subjects working in 71 slaughterhouses across the country in 2012. A structured questionnaire was used to obtain data from the subjects, following which blood samples were collected and tested using the microagglutination test; serum titers ≥ 1:20 were considered reactive. Independent risk factors were identified using multivariate logistic regression analysis with backward elimination. Overall, 62 of 922 participants (6.7%) exhibited seroreactivity for brucellosis, and 0.4% had a seroprevalence at a dilution of 1:160. Multivariate analysis revealed that the risk factors for human brucellosis seroreactivity included large-scale slaughtering (≥100 cattle per day; odds ratio (OR), 5.41; 95% confidence interval (CI), 2.95⁻9.91) and medium-scale slaughtering (50⁻99 cattle per day; OR, 2.53; 95% CI, 1.16⁻5.51). Moreover, the risk of brucellosis infection was significantly lower among slaughterhouse workers who always wear protective glasses (OR, 0.27; 95% CI, 0.11⁻0.69) than in those who sometimes or rarely wore such glasses. Regular and consistent use of personal protective equipment, especially protective glasses, should be encouraged among cattle slaughterhouse workers to reduce brucellosis infection.

Diagnosis and molecular characteristics of human infections caused by Anaplasma phagocytophilum in South Korea.

Human granulocytic anaplasmosis (HGA) is a tick borne infection caused by Anaplasma phagocytophilum. HGA cases in South Korea have been identified since the first report in 2014. In this study, we investigated the serological response in 594 clinical samples of patients with acute febrile illness and molecular characteristics of A. phagocytophilum clinical isolates obtained from HGA patients. In serological test for A. phagocytophilum, 7.91% (47/594 cases) were positive for IgG and Ig M and 13 of 47 cases showed seroconversion. In the detection rate of the 16S rRNA, msp2(p44), and ankA, genes were showed 3.68% (14/380 cases) for A. phagocytophilum-specific 16S rRNA gene. Phylogenetic analysis of three clinical isolates demonstrated high sequence similarity (98.58-100%) with A. phagocytophilum 16S rRNA sequences identified from public databases. Analysis of the msp2(p44) gene showed highly variable similarity rates (7.24-98.85%) even within isolated countries and host ranges. These results provide clues into the bacterial characterization of A. phagocytophilum originating from Korean patients, providing useful guidance for treatment and improving clinical outcomes.

Seroreactivity and Risk Factors Associated with Coxiella burnetii Infection among Cattle Slaughterhouse Workers in South Korea.

Q fever, caused by Coxiella burnetii, is a zoonotic disease that is an occupational hazard to people who work in close contact with animals or their carcasses. A nationwide serologic study among cattle slaughterhouse workers who were presumed to be at risk of having C. burnetii infection in South Korea was performed to investigate the seroreactivity of C. burnetii infection and identify related risk factors. Out of 1017 cattle slaughterhouse workers in South Korea, 923 (90.8%) participated in this cross-sectional study. Samples were tested for immunoglobulin G (IgG) and M (IgM) antibodies against phase II C. burnetii via indirect immunofluorescence assay. The overall seroreactivity, defined as IgG or IgM antibody titer cutoffs ≥1:16, was 9.1% (84/923). Additionally, a significant association was found between the seroreactivity of C. burnetii infection and performing carcass evisceration work (odds ratio, 2.36; 95% confidence interval, 1.39⁻4.03) in multivariate analysis. To diminish C. burnetii infection, cattle slaughterhouse workers need to take precautions during the evisceration process.

Clinical Isolation of Anaplasma phagocytophilum in South Korea.

We report the first isolation of Anaplasma phagocytophilum in South Korea. A 61-year-old woman presented with a 6-day history of fever, headache, and myalgia. Initial investigation showed neutropenia and thrombocytopenia. We diagnosed human granulocytic anaplasmosis by microscopic examination and serologic testing. The patient recovered fully without antibiotic therapy. The isolate was obtained from the patient's blood by cell culture and mouse inoculation. Its identity was confirmed by an immunofluorescence assay, sequencing of the 16S rRNA gene, msp2 (p44), and ankA genes, and staining and electron microscopy of morulae of A. phagocytophilum in cultured human promyelocytic leukemia HL-60 cells.

Case Report: Polymerase Chain Reaction Testing of Tick Bite Site Samples for the Diagnosis of Human Granulocytic Anaplasmosis.

Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium. Until now, the utility of tick-bite site samples for HGA diagnosis has not been reported. Using a patient's buffy coat and tick-bite site crust samples, we performed polymerase chain reaction (PCR) testing using Ehrlichia- or Anaplasma-specific primers. PCR with buffy coat and crust samples obtained before doxycycline administration was positive. Six days after doxycycline administration, PCR with the buffy coat sample was negative but PCR with a crust tissue sample from the tick-bite site remained positive. This is the first case to suggest that crust tissue at the tick-bite site may be useful for early HGA diagnosis in patients who have already been treated with antibiotics such as doxycycline.

Evaluation and Selection of Multilocus Variable-Number Tandem-Repeat Analysis Primers for Genotyping Brucella abortus Biovar 1 Isolated from Human Patients.

OBJECTIVES: Brucellosis is the most common bacterial zoonosis in the world. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is a molecular method for genotyping bacterial species. Brucella abortus biovar I was isolated from most of the brucellosis-suspected patients in Korea. This study was conducted to investigate the ability of various MLVA primers that are used for molecular typing B. abortus isolates and for analyzing their epidemiological data. METHODS: A total of 80 human isolates of B. abortus biovar I isolated from human patients and the reference strain were used for MLVA. Genetic diversity was determined by calculating the Simpson's diversity index (DI) of each VNTR locus. The Brucella strains were subcultured 30 times to determine the stability of each locus. The DNA of the strains cultivated in each passage was extracted and subjected to MLVA for further investigation. RESULTS: The 15 VNTR loci were selected based on high DI values. The DIs of the 15 VNTR loci showed considerable discrimination power ranging from 59% for Bruce 43 to 87% for Bruce 22. Bruce 09, Bruce 11, Bruce 16, Bruce 42, and Bruce 43 were confirmed to remain stable in vitro among the 15 VNTR loci selected. CONCLUSION: The results of this study suggest that the five loci subsets may be a useful epidemiological tool for investigating B. abortus biovar 1 outbreak.

Application of the microagglutination test for serologic diagnosis of human brucellosis.

OBJECTIVES: Brucellosis is one of the most common zoonoses in the world, and occurs mainly in farmers, slaughterhouse workers, and veterinarians via direct or indirect contact with infected animals or their products. The clinical symptoms of human brucellosis are nonspecific, such as fever, headache, chills, and sweating. Diagnosis and treatment of brucellosis requires laboratory tests. Although the serum tube agglutination test (SAT) is the standardized gold method, it is laborious, time consuming, and requires a number of reagents. A microagglutination test (MAT) variant of the SAT or enzyme-linked immunosorbent assay (ELISA) is recommended for serological diagnoses. For the simple and rapid diagnosis of brucellosis, the MAT was standardized using samples for the SAT to define positive and negative categories, and we then compared the sensitivity and specificity of the MAT and ELISA. METHODS: Thirty SAT-positive sera and 60 SAT-negative sera were used in this study. Antibody titers of ≥1:160 were considered positive readings in both the SAT and MAT. Brucella abortus antigens and Brucella-positive control antiserum were used in the SAT and MAT. ELISAs of IgM and IgG were performed according to the manufacturers' instructions. RESULTS: The titers of the MAT differed according to antigen concentration. The optimal concentration of B abortus antigen was determined to compare the sensitivity and specificity between the MAT and SAT. The sensitivity and specificity of the MAT were 93.3% and 96.7%, respectively, for IgG with reference to ELISA, and 96.7% and 98.3%, respectively, for IgM. CONCLUSIONS: The optimal concentration of antigen for the MAT was 1:10. The MAT is less time consuming and requires less antigen and serum than the SAT. The results of the MAT showed good agreement with those of ELISA. The results of this study suggest that the MAT could be useful for diagnosis of brucellosis.

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus.

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 µg of HA antigens and complete heterologous protection required over 5.0 µg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 µg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.

Protection of pregnant mice, fetuses and neonates from lethality of H5N1 influenza viruses by maternal vaccination.

The highly pathogenic H5N1 influenza viruses are one of candidates for the next pandemic. Information on protective immunity for pregnant animals by vaccination against the H5N1 influenza virus is limited. Here, we show that the immunization of pregnant mice with inactivated H5N1 influenza vaccine protects them, their fetuses, and their infant mice from H5N1 influenza viruses. Pregnant mice immunized with two doses of H5N1 influenza vaccine were protected from homologous infections of H5N1 influenza viruses with no viruses detected in fetuses, and that they were protected upto 30% from heterologous infections of H5N1 influenza viruses with viruses detected in fetuses. The infant mice born to mothers immunized with H5N1 influenza vaccine were fully protected from infections of H5N1 influenza viruses for upto 4 weeks of age. The protection of infant mice was closely related to the presence of IgG2a antibody in lung, heart, and rectum tissues. Our results suggest that maternal vaccination may be critical for protecting pregnant animals, their fetuses, and their infant mice from lethal infections of H5N1 influenza viruses.

Phylogenic analysis of reassorted avian influenza viruses isolated from Korean domestic ducks from 2005 to 2007.

Ducks have been regarded as animals that can perpetuate most avian influenza viruses since they generally do not show the clear clinical signs such as death and reduced body weight when they are infected. Here, we characterized two H3N2 and one H3N6 avian influenza viruses isolated from ducks on the local farms in Korea from 2005 to 2007. Genetic analysis of these viruses showed that most segments of isolates except NP genes belonged to Eurasian lineage. NP genes of two H3N2 isolates, A/Duck/Korea/S71/07, and A/Duck/Korea/S72/07 belonged to North American lineage. Our results suggest that the genetic reassortment among avian influenza viruses can occur in domestic ducks.