DOI: https://doi.org/10.21203/rs.3.rs-2236214/v1
Hemorrhagic fever with renal syndrome (HFRS) caused by Orthohantavirus (OHV) and scrub typhus (ST) caused by Orientia tsutsugamushi (OT) are two infectious diseases prevalent in southwest China. Rodents are the natural host and the main source of the two diseases. OT infection to humans is usually resulted from bite of an infective chigger mite on rodents, and OHV is transmitted through contact or inhalation of aerosols and secretions from infected rodent. For infectious diseases, the use of antibiotics and hormones is essential, so the diagnosis of complex situations is not easy under the action of drugs. Clinically, fever is the first symptom of these two diseases, and most of them are accompanied by common symptoms such as chills and headaches. The clinical symptoms of these two diseases are very similar, so it is easy to diagnose and miss diagnosis.
In this case, a 44-year-old male famer with a history of working in coal transportation was admitted to the hospital because of respiratory symptoms accompanied by fever, headache, and skin rashes on his body. Biochemical and urinalysis revealed the hepatic and renal injury. The subsequent molecular testing confirmed he suffered from HFRS and scrub typhus simultaneously that the serological and clinical diagnosis could not identify the cause of infection before. Such case has not been reported in Yunnan Province before.
As the clinical findings of HFRS and scrub typhus are similar and usually unspecific, the clinical diagnosis should be made with a combination of serological and etiological approaches. In the endemic areas of the two diseases, potential HFRS and ST patients should be precisely distinguished with more tests, which is critical for developing proper treatment plans.
China has the second highest incidence rate of pulmonary tuberculosis (PT) in the world. In most cases of PT, there are few, if any, clinical symptoms or signs in the first contact with Mycobacterium tuberculosis [1]. PT develops gradually with mild symptoms until the disease progresses to moderate or severe illness. The most common systemic symptom is fever which is low at the beginning, with "night sweats" in some cases. Patients often develop a cough and sputum due to mild hemoptysis with progressive disease. Shortness of breath usually occurs in the advanced stages of the disease due to lesions of the lungs and the lung parenchyma, or some form of tracheobronchial obstruction. As primary pulmonary lesions are usually located below the pleural area, a ruptured pleural cavity can lead to tuberculous pleuritis with pleural effusion and chest pain with typical nonspecific pleuritis symptoms [2].
Scrub typhus (ST), also known as bush typhus or tsutsugamushi disease, is a zoonotic vector-borne disease caused by Orientia tsutsugamushi (OT), which is usually transmitted by the bite of a chigger mite and leads to illness from non-specific symptoms, such as fever, headache, swollen lymph nodes, abdominal pain, a rash or scab at the site of the bite, to severe diseases such as bronchitis, pneumonia, myocarditis, and meningitis [3, 4]. The diagnosis of scrub typhus is mainly based on laboratory tests including serological analysis and some molecular assays targeting specific genes specific of OT. ST is prevalent predominantly in South and Southeast China, followed by the southwestern regions including the provinces of Yunnan and Sichuan [5, 6]. Karp and Gilliam genotypes of OT are prevalent to the south of the Yangtze River, while Kawasaki and Gilliam genotypes are mainly distributed to the north of the Yangtze River [7, 8]. Especially, Karp, Kato and Gilliam genotypes are the most prevalent ones in Yunnan Province [9].
Hemorrhagic fever with renal syndrome (HFRS), a rodent-borne disease caused by Orthohantavirus(OHV), is characterized by fever, renal failure and hemorrhagic symptoms[10]. It is predominantly prevalent in Europe and Asia, and China, accounting for the 90% of all global cases. Hantaan Orthohantavirus (HTNV) and Seoul Orthantavirus (SEOV) are the two major strains of OHV associated with HFRS in China which are transmitted by the black threaded mouse (Apodemus agrarius) and brown house mouse (Rattus norvegicus), respectively [11]. The incidence of HFRS shows a seasonal trend with the peaks from May to July and from December to January in China [12].
Here, we report a rare patient with BT co-infected by SEOV and Gilliam genotype of OT in Yunnan Province.
A 44-year-old male patient, residing in a village of Yunnan Province, was admitted to Xiangyun County People's Hospital in March 2021. He had a history of working in coal transportation. He visited the local Center for Disease Control and a hospital for recurrent cough and sputum for six years. He was diagnosed with PT, tuberculous bronchial stenosis, and silicosis. He was discharged with a recommendation for anti-tuberculosis treatment and given an anti-tuberculosis HRZE regimen (H: isoniazid, R: rifampin, Z: pyrazinamide, E: ethambutol), but the patient did not take the medication according to the doctor's advice every day. After taking anti-tuberculosis drugs on his own for five days, he was admitted to the hospital because of headache and fever for two days with rash, and a high body temperature up to 39.4℃. Rashes were observed on the face, head, neck, chest and abdomen, and limbs, distributing in patches with size ranging from a pinpoint to a grain of rice, which led to pruritus and a red halo at the base without rupture, separated by normal skin. The lips and mouth were slightly cyanotic, and the breath sounds of both lungs were coarse, with scattered moist rales. The other examination results were unremarkable.
By laboratory examination, normal level of blood cells were observed as follows: white blood cells (7.3×109/L), hemoglobin (155g/L), platelets (202×109/L), neutrophil percentage (90.6%) and lymphocyte count (7.8×109/L). Biochemical tests showed impaired liver function: elevated aspartate aminotransferase(AST)(50U/L), elevated gamma-glutamyl transferase༈GGT༉(64U/L), slightly lower prealbumin (145.5 mg/L), but normal alanine aminotransferase༈ALT༉(21U/L). Inflammatory biomarker tests showed an elevated calcitonin inogen (0.63 ng/mL) and elevated C-reactive protein (59 mg/L). Coagulation analysis showed a prolonged prothrombin time of 13.2s. Plasma fibrinogen was elevated at 4.80g/L. Routine urinalysis showed that the patient was positive (+) for urine protein; positive (+) for blood cell; and positive (++) for urinary ketones, suggesting the presence of impairment of renal function.
No acid-fast bacilli were detected by pooling the patient's nocturnal sputum, immediate sputum and morning sputum for testing. According to the patient's chest computed tomography (CT) scan (bilateral lungs and mediastinum) findings, the scattered nodular and lamellar shadows in both lungs were considered as tuberculosis most likely, and the lesions in the lingual segment of the upper lobe of the left lung and the lower lobe of the left lung were probably infectious lesions (Fig. 1A;Figure 1C). The patient was tested for the antibodies (IgM and IgG) against OHV due to the patient from the national foci of HFRS, but the results were negative.
On the first day after admission, the patient suspended his anti- tuberculosis treatment and was given an acetaminophen oral suspension. Subsequently, his temperature decreased and anti-infective treatment s were provided with methylprednisolone sodium succinate. The blood gas analysis of the patient showed that the pH value was 7.48, the oxygen partial pressure was 53.2 mmHg, the oxygen saturation was 90.1%, and the potassium ion (K+) was 2.8 mmol/L. The presence of electrolyte disorders (hypokalemia, hyponatremia) was treated with sodium chloride and potassium chloride injections. On the second day after admission, the patient's temperature fluctuated between 38.1–38.7 ° C, and he still had headache, cough, expectoration and other uncomfortable symptoms. For the symptoms of headache and rash, he took rotundine and ebastine tablets for symptomatic treatment. Later examination showed that the rash on the patient's face, head, chest and neck, and limbs had subsided, and the patient felt relief from the headache. On the third day after admission, the patient continued with symptomatic treatment for anti-infection and cough suppression. The patient sometimes had fever, discomfort, cough and expectoration with little sputum, which was yellow-white sputum. On the Fourth day after admission, the patient was discharged after the rash and fever subsided at the request of the patient and his family.
In cases where serological and clinical examinations could not identify the specific cause of infection, rash and fever, we performed retrospectively etiological testing of the whole blood and serum from the collected patent previously. Nucleic acid (DNA/RNA) extraction kit (TIANGEN, China) was used to extract viral nucleic acid from the patient whole blood and serum, and the gene sequence was amplified by one-step polymerase chain reaction(PCR)method using universal primers of OHV according to reference[13].PCR amplification was performed using the 56-kDa type specific antigen (TSA) gene of OT according to references [14, 15]. The agarose gel electrophoresis experiment was carried out under the imager (Fig. 1E).The PCR products were purified by gel cutting and sent to a sequencing company (Shanghai Sangon Biotech) for sequencing. The 362bp sequence of OHV༈accession no.OP392989༉and the 172bp DNA sequence of OT were obtained from serum samples. Then primers (ICRA-F2:5 '- CCTCAGTATAATGCCC-3' and ICR8A-R: 5 '- TCCTGCATGACGCTGCAA-3') were designed to obtain 449bp DNA sequence of tsutsugamushi (accession no.OP392990).
Nucleotide sequence similarity searches in the public databases were assessed by the Basic Local Alignment Search Tool, implemented in the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/blast/), using BLASTn, and BLASTn optimized for highly similar sequences (MEGABLAST) and BLASTp, algorithms. In BLAST, it was 92.54% and 97.69% that the highest identity of nucleotide(nt) and amino acid(aa) compared the OHV sequence(OP39298)in this study to SEOV L0199 strain(HQ992814) and SEOV Rn-SHY17 ( ADR32120.1 ). The highest nt and aa identity compared the obtained OT sequence༈OP392990༉to Gilliam genotype of HZ01034 strain (MT258795.1) and Orientia tsutsugamushi str. Gilliam (KJV51889.1) was 96.88%, 93.96% respectively.
Phylogenetic trees were analyzed for the obtained OHV sequences (362bp) and OT sequences (449bp), and the related sequences retrieved in the Genbank database. The each sequence set was aligned by Clustal-X, and phylogenetic relationships were reconstructed using MEGA X for the initial trees obtained by the maximum likelihood neighbor joining method. In the nucleotide substitution models, the K2 + I and T92 + G models were selected for Bootstrap analysis using 1000 replicates to improve the confidence level of the phylogenetic tree, respectively (Fig. 1F-G).The results showed that the patient was infected with SEOV of Orthohantavirus and Gilliam genotype of O. tsutsugamushi.
Compared to the CT images of the patient's admission (Fig. 1A-D), the discharge review showed that the patient was effectively treated and recovered in the hospital. Mediastinal lymph nodes in the CT images decreased from 12.6 mm to 9.67 mm after treatment and discharge, which also indicated an inflammatory response in the mediastinal lymph nodes due to the presence of pulmonary tuberculosis and silicosis. The lung lesions seemed to be typical tuberculosis infection. From a large area with blurred boundaries, there was a degree of reduction in the lesion and a clearer peripheral lesion after treatment. If evidence of other infections in the patient was obtained without etiological testing, the patient would have an initial diagnosis of tuberculosis or pulmonary infection.
Since fever is the most common symptom of systemic toxicity in tuberculosis, most of which are low fever for long periods, but the patient in the case sustained high temperature. Combined with the laboratory test data, it cannot be excluded that the patient had fever and rash due to other causes. Patients may experience exacerbations or poor treatment outcomes because they do not follow the principles of early, combined, moderate, regular, and full course of tuberculosis treatment regimens for taking medications. Among many drugs, the most commonly used first-line anti tuberculosis drugs (streptomycin, isoniazid, rifampicin, piracetam, ethambutol) are clinically effective. The incidence of adverse reactions in conventional doses is low, and liver injury is mainly caused in the form of elevated ALT. The patient did not raise ALT, while AST and GCT were elevated, and there were no other adverse reactions such as gastrointestinal reactions and neurological damage. The patient's past medical history and living habits should not be overlooked in clinical practice, because the ignored factors can have an impact on the development of the disease[16].
However, rifampicin is currently used to treat scrub typhus in addition to tuberculosis [17]. Several studies on scrub typhus have also shown that rifampicin which can shorten the time of fever and better control the concurrent inflammation in the patient's lungs is more effective than chloramphenicol [18, 19]. In that case, rifampin, together with anti-tuberculosis drugs, controlled the progression of scrub typhus. Although serology remains the most extensive diagnostic method of Rickettsiosis, molecular methods are complementary to serology. The combination of PCR and serology for acute disease can improve the diagnosis rate and make the diagnosis as clear as possible. The clinical manifestations of rickettsial disease are sometimes atypical and may be similar to other febrile diseases. It is not uncommon for such non-specific symptoms to be misdiagnosed and missed. In many cases, physicians may only consider infection of O. tsutsugamushi unless the patient has characteristic clinical signs such as eschar [20]. The eschar had not found in this case. The reported case had been infected by Gilliam type of O. tsutsugamushi by PCR.
Studies have shown that early treatment of HFRS with antiviral and hormone treatment attenuates direct viral tissue damage and immunity damage, reduces capillary permeability, suppresses cytokine production and release, and suppresses inflammation [21]. In the case of the patient, the nonspecific manifestation of HFRS was in basic remission after hormonal and antiviral therapy. It was negative that the antibody against Orthohantavirus in this case, because the antibody has not been produced or the antibody level has not reached the detectable amount in the early stage of the illness. Therefore, patients with negative serological test results should also be diagnosed in conjunction with other clinical indicators (such as urine volume, platelets, white blood cells, procalcitonin, etc.). And pathogenic tests avoid misdiagnosis and delay[22].
As a seasonal rodent-borne disease, there are certain objective conditions for co-infection of HFRS and scrub typhus, and external environmental factors, including climatic factors, which may play an important role in disease transmission[23].In this case, the presence of HFRS and scrub typhus were not initially considered based on the patient's clinical symptoms and serologic test. The case may be considered as a fever of unknown origin or a pulmonary infection. For natural epidemic areas where these two diseases exist at the same time, serological and etiological examinations should be carried out for patients with HFRS or scrub typhus as far as possible to maximize the diagnostic rate, reduce missed cases of co-infection, and provide early treatment to avoid complications caused by delayed treatment or poor treatment results.
Ethics approval and consent to participate
This research was approved by the Medical Ethics Committee of Dali University under number DLDXLL2018008 and was obtained with the informed consent of all participants. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Consent for publication
Written informed consent was obtained from the patient for publication of this case report.
Availability of data and materials
All the sequences in this manuscript can be obtained from NCBI database (https://www.ncbi.nlm.nih.gov) via the following accession numbers: XYPatient2S-OT (OP392990); XYPatient2S-HV (OP392989); reference OT sequences (MT258795.1, GU120166.1, HQ718430.1, HQ660213.1, JQ898371.1, JQ898391.1, EF213083.1, AF516948.1, MW460713.1, MW464203.1, DQ852664.1, DQ852664.1, MW495582.1, AY222635.1, JQ898380.1); reference HV sequences (HE801635.1, JN831952.1, MT024592.1, M63194.1, MN183133.1, AJ005637.1, MK386161.1, KT885047.1, AF336826.1, HQ992814.1, KP900346.1, EF190551.1, NC 043074.1).
Competing interests
The authors declare that they have no competing interests.
Funding
This study was funded by the National Natural Science Foundation of China (81874274); Yunnan high-level health and family planning technical personnel training project (L-2017027); Doctoral research start-up fee project of Dali University (KYBS2018004); and Cross-border prevention, control, and quarantine innovation group of zoonosis in Dali University (ZKPY2019302).
Authors' contributions
Hao Huang and Yunzhi Zhang wrote the main manuscript text. Hao Huang and Yichen Kong detected patient samples. Hongmin Yin, Zi Yang, Tilian Ren collected data. All authors read and approved the final manuscript.All authors contributed to the follow-up of the patient, as well as preparation and submission of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Thanks to Lian Qin, Chunyu Wang from Xiangyun People's Hospital for helping with the sample collection and data collation.
Authors' information
Authors and Affiliations
Hao Huang, Yichen Kong, Hongmin Yin, Zi Yang
1. Institute of Preventive Medicine，School of Public Health，Dali University，Yunnan Key Laboratory of Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan（Cultivation），Key Laboratory for Cross-border Control and Quarantine of Zoonoses in Universities of Yunnan Province，Dali 671000，China
Tilian Ren
2. Department of infection, Xiangyun people's Hospital, Dali Prefecture, Yunnan Province, China
Corresponding author
Correspondence to Yunzhi Zhang and Tilian Ren.