| BackgroundScrub typhus, resulting from Orientia tsutusgamushi infection, is a disease of natural focus characterized by fever, eschar or skin, ulcer, rash, and lymphopathy. It is transmitted to humans by the bite of an infected larval mite. Scrub typhus is an ancient disease in China. It was confined to the south of Yangtze River of China before 1986. Summer was the epidemic season. Karp, Kato, and JG strains of O.tsutsugamushi could be the causative strains of summer type scrub typhus. The severity of the complications caused by the above three strains is usually of high level. After the year of 1986, new epidemic zones were continuously confirmed in northern China. As epidemic seasons in northern China are autumn and winter, scrub typhus in Beijing, Tianjin, Jiangsu province, Shanxi province, Anhui province, Henan province, and Shandong province, is also called autumn-winter type scrub typhus. According to previous reports, Gilliam was the main serotype and Kawasaki was the main genotype in northern China.The infection severity of scrub typhus patients vary from self-limiting to fatal, which may relate to the genotypes of O.tsutsugamushi patients infected with. Severe complications including meningoencephalitis, acute respiratory distress syndrome (ARDS), hepatitis, renal failure, hearing impairment, pancreatic, and even death were reported in previous studies. It would be of great value to clarify the factors related to severe scrub typhus. We provided a detailed panel of clinical and laboratory profiles caused by these genotypes, helping promote clinical awareness facilitating its diagnosis and providing reference for comparative study.Objective1. To clarify the predominant strains of O.tsutsugamushi in Shandong province in recent years, to confirm the gene sequences of the 56kDa type-specific antigen (TSA), and to disclose the homology between the predominant strains in Shandong province and national references strains of O.tsutsugamushi.2. To clarify the clinical characteristic of scrub typhus patients caused by the predominant strains of O.tsutsugamushi in Shandong province.3. To identify the risk factors associated with severe scrub typhus, in order to provide a reference for clinical decision-making.MethodsAccording to surveillance data from Shandong Diseases Reporting Information System (SDRIS), we used a structured questionnaire to record their demographic information, hematologic laboratory tests, clinical manifestations and complications, and outcome of scrub typhus patients who were from the monitoring hospitals in Yiyuan County, Zibo city, and Mengyin County, Linyi city, etal. Besides, EDTA blood samples and eschar biopsies were collected before antibiotics administration. DNA was extracted from whole blood samples and eschar biopsies to be tested by nested PCR targeting the 56kDa TSA gene of O.tsutsugamushi. Complete or partial sequences encoding 56kDa TSA of the references and prototype strains were obtained from GenBank. Multiple sequence alignment and analysis of the genetic relationships between O. tsutsugamushi strains in this study and reference strains were performed with Mega5.0 software using ClustalW algorithm, and the resulting dendrogram was constructed with Mega5.0 software using the neighbor-joining method. Sequence homologies were calculated using MegAlign of Lasergene7.1 software. SPSS 17.0 was used for statistical processing. We used logistic regression analysis to explore risk factors associated with severe scrub typhus. The odds ratio (OR) with 95% confidence intervals (95% CIs) was presented to examine statistical significance.Results1. Distribution of the genotypes of O. tsutsugamushi in patientsDNA was prepared from acute-phase whole blood or eschar acquired from 43 scrub typhus patients. After PCR amplification and sequencing, the sequences of O. tsutsugamushi together with other international sequences were used for phylogenetic analysis. All of the 43 sequences were distributed within the Kawasaki clade with the nucleotide sequence identities ranging from 95.1% to 96.4% with Kawasaki strain, and 98.5% to 100% to each other. The rest four sequences:SH1002, SH1307, SH1306, and SH1309 were not identical to other strains in the tree, standing for four new sequences.2. The molecular characteristics of the 56kDa TSA of Kawasaki-related strainThe open reading frame (ORF) ragion of this study corresponds to 80.11% of the whole gene. There are 1273 bp in length, composed of four VD (variable domain, VD) regions. The amount of aa of each region are 25,22,28, and 39. The aa consistencies of VDⅠ, VDⅡ, VDⅣ region between JIANG3 and Kawasaki are 96%,81.82%, 94.87%, respectively, which is higher than that of JIANG3 and Karp, JIANG3 and Kato, and JIANG3 and Gilliam. The aa consistency of VDⅢ region between JIANG3 and Gilliam is higher than that of JIANG3 and Kawasaki, JIANG3 and Karp, and JIANG3 and Kato. Phylogenetic analysis based on the DNA sequences of VDⅠ, VDⅡ, VDⅣ regions indicates JIANG3 is distributed in the same clade with Kawasaki, while phylogenetic analysis based on the DNA sequences of VDⅣ indicates JIANG3 is distributed in the same clade with Gilliam. Phylogenetic trees based on the sequences of each VD indicate the same results with aa analysis based on each VD.3. The clinical characteristics of O. tsutsugamushi infectionScrub typhus caused by Kawasaki-related O.tsutsugamushi is characterized by fever, eschar or skin ulcer, headache, rashes, chill, and fatigue. The incidence of eschar was high (97.7%) in our patients and only one patient had no eschar presentation. Groin, perineum, axilla, and waist were the most preferential sites for eschar formation. Leukocytosis (white blood cell [WBC]>10×109/L) and leucopenia (WBC<4×109/L) were found in 18.6% and 4.7% of 43 patients, respectively. Decreased neutrophil (the percentage of neutrophil<77%) and thrombocytopenia (platelet count<100×109/L) occurred in 35.9% and 23.1% of 39 patients. Erythrocyte sedimentation rate was 18.43 ±11.62 mm/h.4. Risk factors associated with severe scrub typhusA univariate logistic regression indicated that the significant factors for severe scrub typhus were as follows:(1) duration of illness before effective antibiotic therapy, (2) lymphadenopathy, (3) the presence of rash, (4) Platelet counts<100×109/L, (5) neutrophil counts<50%, (6) lymphocyte counts>40%, (7) total bilirubin>24μmol/L, (8) direct bilirubin>8.6μmol/L. Multivariable unconditional logistic regression demonstrated that the following four factors were significantly associated with the severe scrub typhus:(1) duration of illness before effective antibiotic therapy (OR=2.287, P=0.027,95%CI=1.096-4.770), (2) lymphadenopathy (OR=2.438, P=0.030,95%CI=1.090-5.458), (3) the presence of rash (OR=3.694, P=0.014,95% CI=1.300-10.495), (4) Platelet counts<100×109/L (OR=2.226, P=0.050,95%CI =1.002-4.946).Conclusions1. All of the 43 sequences were distributed within the Kawasaki clade with the nucleotide sequence identities ranging from 95.1% to 96.4% with Kawasaki strain, which indicates that the predominant strains of O.tsutsugamushi in Shandong province is Kawasaki-related genotypes. However, four new sequences:SH1002, SH1307, SH1306, and SH1309 were identified, which meant there was gene mutation of 56kDa TSA genes of O. tsutsugamushi in Shandong province.2. The main variation between Kawasaki-related strain and Kawasaki strain is located at the VDⅢ.3. Kawasaki-related strains of O.tsutsugamushi could lead to a high incidence of eschar.4. Multivariable unconditional logistic regression demonstrated that the duration of illness before effective antibiotic therapy, lymphadenopathy, the presence of rash, and platelet counts<100×109/L were significantly associated with the severe complications of scrub typhus. |
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