Case
On April 22, 2018, a 60-year-old man was hospitalized with fever. His vital signs were as follows: blood pressure as 140/90 mm Hg, heart rate of 88 beats per minute, respiratory rate of 18 times per minute, and a temperature of 39.2°C. He had no underlying disease. History-taking revealed that he had been mowing for three days, 13 days prior to hospitalization, and had manifested symptoms including headache, fatigue, nausea, and myalgia for six days before hospitalization. In an effort to alleviate the symptoms, he took medications acquired from a pharmacy. However, he had fever for three days before hospitalization and the symptoms had worsened, which prompted his hospital visit.
On admission, hematological and biochemical results revealed white blood cell count of 7780 cells/µL (normal range: 4800–10,800; neutrophils 83.3%), platelet count of 68,000 cells/µL (normal range: 130,000–450,000), hemoglobin level of 14.9 g/dL (normal range: 12–18), serum blood urea nitrogen level of 14.8 mg/dL (reference range: 8.0–20), and creatinine level of 0.83 mg/dL (reference range: 0.5–1.3).
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were higher than the normal range at 144/183 IU/L (reference range: 5–40/5–40), but prothrombin time, total bilirubin, and C-reactive protein levels were within the normal range. PCR and antibody tests were performed to detect influenza A/B, Leptospira, Hantavirus, and Orientia tsutsugamushi, but none were detected. Blood was cultured and no bacterial growth was detected.
After hospitalization, the patient related that he had found ticks on his body and clothes while showering. However, he could not recall the moment of being bitten by ticks. Hence, we collected tick samples from the location of mowing. PCR targeting vector-borne infectious diseases was carried out on the four ticks collected. The results were compared with the PCR result of the blood from the patient.
DNA extraction, PCR detection, and molecular identification of vector-borne infectious diseases
The four ticks collected were first washed with 70% ethanol, then with sterile distilled water, and air dried. The life cycle stages and species of the ticks were morphologically classified using microscopy and standard taxonomic keys (10). The ticks were ground using a FastPrep®-24 Classic instrument (MP Biomedicals, Solon, OH, USA) and stored at −80 °C until used for DNA extraction.
Genomic DNAs were extracted from 300 µL of the patient’s blood sample and from the ground tick lysate (150 µL) using the QIAamp Tissue & Blood Mini Kit (Qiagen, Hilden, Germany) by following the manufacturer’s instructions. DNA was eluted into 50 μL TE (10 mM Tris·Cl, 0.5 mM EDTA, pH 9.0) buffer and stored at −20 °C until PCR amplification was carried out.
Nested-PCR (N-PCR) and real-time PCR were performed to diagnose Q fever, rickettsial disease, Lyme disease, and anaplasmosis in the blood specimen and the four ticks. The htpAB-associated repetitive element (IS1111) and 16S ribosomal RNA (16S rRNA) gene were targeted to detect Coxiella sp. (11, 12). For 16S rRNA N-PCR, we designed three additional primers (16s1s-tF0, 16s1st-R0, and Cox16SF3) based on the C. burnetii 16S rRNA sequence (GenBank Accession No. CP001019) to make one contig after DNA sequencing. To detect the presence of Rickettsia DNA, the outer membrane protein A gene (ompA) of the spotted fever group Rickettsia species and the 23S rRNA gene of Rickettsia (panrickettsia) were targeted (13-15). The CTP synthase (pyrG) gene was targeted to detect Borrelia species (16). The heat shock protein gene (groEL) and the ankyrin-related protein gene (ankA) were targeted to detect A. phagocytophilum (17, 18). All PCR primers and probes used for detecting bacterial pathogens, PCR conditions, and product sizes are given in Table 1.
Conventional PCR (C-PCR) was performed in 20 μL reaction volumes using the AccuPowerR PCR PreMix (Bioneer, Daejeon, Korea). Each PCR mixture consisted of 16 μL distilled water, 1 μL each primer (10 pmol/μL), and 2 μL genomic DNA as the template DNA. For N-PCR, the reaction mixture was identical to that used in C-PCR, except that the first PCR product was used as template DNA and the N-PCR primers were used. With each PCR run, a positive and a negative control (molecular grade water) were included.
All amplifications were performed in an AB thermal cycler (Applied Biosystem, Foster City, CA, USA). The amplified products were separated by 1.2% agarose gel electrophoresis and visualized by staining with ethidium bromide (Bioneer).
Real-time PCR was performed in 20 μL reaction volumes, which consisted of 5 µL genomic DNA, 4 μL LightCycler TaqMan Master mix (Roche Diagnostics, Indianapolis, IN, USA), 1 μL of each primer (10 pmol/μL), 1 μL probe (5 pmol/ μL), and 8 µL distilled water. The real-time PCR was carried out in an ExicyclerTM 96 Real-Time Quantitative Thermal Block (Bioneer).
Sequencing and phylogenetic analysis
The amplified PCR products were purified using QIAquick PCR purification kits (Qiagen) and directly sequenced with the PCR primers at Cosmogenetech Co, Ltd. (Daejeon, Korea). The resulting sequences obtained in this study were examined using the BlastN program from NCBI (Bethesda, MD, USA) to identify the bacteria present.
The concatenating 16S rRNA sequences were aligned and analyzed for homology comparisons using DNASTAR-Lasergene v6 software (DNASTAR, Madison, WI, USA) and the NCBI BlastN network service.
The phylogenetic tree was constructed using ClustalW of the MegAlign Program and the Tree Explorer program (DNASTAR) based on the alignments of positive gene sequences using the neighbor-joining method. Bootstrap analysis (1,000 replicates) was performed to improve the confidence level of the phylogenetic tree according to the Kimura 2-parameter method. Pairwise alignments were performed with an open-gap penalty of 10 and a gap extension penalty of 0.5.
IFA
To detect IgG and IgM antibodies specific to Q fever, spotted fever, Lyme disease, and anaplasmosis, IFAs were performed at the Korea Centers for Disease Control and Prevention (KCDC) as described previously (19).
Ethics approval and consent to participate
The study was approved by the Ethics in Human Research Committee of Chosun University Hospital (IRB No. 2013-10-001-018). The patient provided written informed consent to participate in the study.