A sixty-year-old male farmer, residing on the northern coast of Hainan Island, was admitted to the Second Affiliated Hospital of Hainan Medical University on December 8, 2018. He presented with a high fever (38.4–40.0 °C), chills, headache, myalgia, and cough for six days. A typical 5 × 5 mm eschar was observed on the lateral side of the left waist (Fig. 1A). Laboratory examination showed normal white blood cell level (9.6 × 109 /L) and elevated levels of lactate dehydrogenase (266 U/L), procalcitonin (0.46 ng/ml), C-reactive protein (100.23 mg/L), alanine aminotransferase (60 U/L), gamma-glutamyl transferase (238 U/L), and alkaline phosphatase (168 U/L). A CT scan showed multilobar interstitial infiltrates and a small right pleural effusion. Rapid immunochromatographic diagnostic tests for O. tsutsugamushi antibodies (Colloidal Gold, WANTAI Inc., China) showed positive IgG and weakly positive IgM. Thus, the patient was diagnosed with scrub typhus complicated by pulmonary involvement and hepatic dysfunction. He was treated with oral doxycycline 100 mg twice daily for 14 days. His symptoms subsided during his five-day hospitalization.
Patient’s DNA samples were isolated from both venous blood and eschar swabs collected on the day of his admission. Nested polymerase chain reaction (PCR) was then performed to amplify the hypervariable regions of the tsa56 gene using primers described previously [8]. Primers 34 and 55 were used in the first round of PCR with 2 μl DNA template and primers 10 and 11 were used in the second PCR amplification to generate a 408–453 base-pair fragment with 2 μl of the product from the first round as the template. A high-fidelity DNA polymerase (KOD One PCR Master Mix, TOYOBO) was used in all PCR steps in this report. The thermal cycling conditions for both PCRs were 98 °C for 5 min, followed by 30 cycles of 98 °C for 10 s, 58 °C for 5 s, 68 °C for 5 s, and a final elongation step at 68 °C for 1 min. The nested PCR products were sequenced via Sanger sequencing. Its results revealed that the target sequences of blood and eschar samples were identical (GenBank accession no. MZ856313). Sequencing results were also found to be related to the reference O. tsutsugamushi Karp genotype (GenBank accession no. LS398548.1) with 99.33% identity at the nucleotide level, and were completely identical to a Karp genotype strain reported in Taiwan (GenBank accession no. MW464199.1). To avoid the sequence variability of the tsa56 gene affecting primer annealing in some strains, such as some strains of Kato, Shimokoshi and TA686, another pair of primers (5’-AATGTCTGC GTTGTCGTTGCC-3') and (5’-ATAGCGCACACCTACACTTGC-3') were designed to detect nearly full-length tsa56 coding DNA sequence (CDS) region, based on total 325 sequences of tsa56 gene collected from the National Center for Biotechnology Information [9, 10] The PCR products were cloned into pLB vectors and the colonies of each sample were randomly selected for Sanger sequencing. The sequencing results have confirmed that there is indeed only one tsa56 genotype (GenBank accession No. ON568725) via the results of 35 colonies from the blood and 36 colonies from the eschar samples.
Similar to the tsa56 gene, seven canonical highly conserved genes, as previously selected and reported in the PubMLST database (https://pubmlst.org/), were also subjected to nested PCR amplification, followed by Sanger sequencing [3, 10]. Different from the single tsa56 genotype, the sequencing results from both the blood and eschar samples exhibited several double peaks at multiple sites in five of the seven conserved genes (gpsA, mdh, nrdB, ppdk, and sucD) (Fig. 1B–D), which suggests the patient could be co-infected by at least two different O. tsutsugamushi strains. To confirm this discovery, the nested PCR products of gpsA, mdh, nrdB and sucD were also cloned into the pLB vectors. Thirty to forty colonies of each gene were randomly selected for Sanger sequencing. The sequencing results have showed that it is indeed a co-infection. Both the gpsA and mdh gene fragments had two different genotypes in the original nested PCR samples (Fig. 1C, D). And both nrdB and sucD gene fragments had three genotypes, including a major genotype, a minor genotype and another minor genotype mutated from the major genotype (Additional file 1: Fig. S1). There is no significant difference observed in the ratio of the mixed genotypes between the blood and eschar samples, suggesting the evaluations are consistent and the discoveries are very reliable.
Furthermore, the patient’s PCR sequencing results of the conserved genes were compared with all current publicly available typing sequences of 225 O. tsutsugamushi isolates from South Korea, Laos, Thailand, Japan, Myanmar, and New Guinea in the PubMLST database [10]. Our results have showed that there were three new single nucleotide polymorphism (SNP) sites discovered that were not reported in any previous sequence types, including two in the mdh gene and one in nrdB gene. Moreover, we discovered eight new sequence types of alleles that were new combinations of previously reported SNP sites, including one for the gpsA gene, one for the nuoF gene, two for the mdh gene, two for the nrdB gene and two for the sucD gene.
