A 75-year-old male patient from Yongjia county (Zhejiang province, China) visited our hospital [i.e., Wenzhou Central Hospital (WCH)/The Second Affiliated Hospital of Shanghai University] on 16 November 2021 (defined as day 0) with the manifestation of fever accompanied by expectoration for 6 days and diarrhea for 5 days.
The patient had visited a local clinic and received oral medication (the detail was unknown) 6 days ago due to fever and paroxysmal cough with yellow sticky phlegm. The patient had a maximum body temperature of 39.0 C and felt no chills then (i.e., 6 days ago). One day later, the patient started to have an episode of watery diarrhea with a stool frequency of four to five times per day, which is accompanied by pains in bilateral thighs and shanks. He then visited the Second Affiliated Hospital of Wenzhou Medical University on the same day (i.e., 5 days ago), where he was administered with piperacillin sodium and tazobactam sodium [4.5 g, intravenous (I.V.) infusion every 8 h (quaque octa hora, q8h)], levofloxacin [0.5 g, I.V. infusion one a day (quaque die, qd)], and fluid infusion.
A chest CT on day 0 showed binary pulmonary infection, which was in suspicion of pulmonary tuberculosis (PTB) ( Figure 1A ). Given the pulmonary infection and acute renal failure, the patient was admitted to our hospital the next day and subsequently transferred to the infectious intensive care unit (ICU) of our hospital for further diagnosis and treatment.
On admission, the patient had an ear temperature of 37.2 C, a pulse rate of 83 beats per minute, a blood pressure of 100/54 mmHg, and a respiratory rate of 18 breaths per minute. At that time, he was conscious and mentally normal. Physical examination showed that both of his pupils were round and equal in size (3 mm in diameter). There was no jaundice, scleral icterus, spider angioma, or palmer erythema observed. The patient's neck was supple, trachea was at the midline of the neck, and the patient had no jugular vein engorgement. Abnormal breathing sounds was auscultated in bilateral lungs of the patient, and occasional moist rale in the right lower lobe. In addition, the patient had normal heart rhythm and no pathological murmurs of valves on auscultation. His belly was soft, and there was no (rebound) tenderness as revealed by palpation. The liver and spleen were not palpable. There was no leg edema. The bilateral pathological reflexes were negative.
The result of initial laboratory examination (day 1) was as follows: white blood cell count (WBC), 11.43 x 109 cells/L; neutrophils % (neut. %), 0.924%; hemoglobin (HB), 88 g/L; platelets (PLT), 86 x 109 cells/L; C-reactive protein (CRP), 165.53 mg/L; total protein, 52.8 g/L; albumin, 26.6 g/L; bilirubin, 22.1 mumol/L; and creatinine, 464 mumol/L ( Table 1 ).
Anti-infectious treatment was empirically administered with sulperazone (2.0 g, I.V. infusion, q8h) and moxifloxacin (0.4 g, I.V. infusion, qd) on day 1, combined with supportive care for relieving cough, reducing sputum, and regulating intestinal flora and fluid infusion. The patient still manifested with persistent fever, chest distress, and tachypnea. Oxygen was then supplied at a rate of 6 L/min using an oxygen mask to maintain an oxygen saturation of 95%. The patient had less urine and progressively elevated creatinine. He was then given methylprednisolone [40 mg, I.V. infusion bis in die (bid)] on day 2 to enhance the anti-inflammatory effect and reduce pulmonary exudation. Continuous renal replacement therapy (CRRT) was simultaneously applied. On day 3, the patient produced blood-tinged sputum. Xpert MTB RIF using sputum identified the presence of rifampicin-sensitive Mycobacterium tuberculosis complex (MTBC) (data not shown). In association with the chest CT result ( Figure 1A ), the diagnosis of PTB was confirmed. The therapeutic strategy was then altered to rifampicin [0.45 g, by mouth (per os, P.O.) every morning (quaque mane, qm)], isoniazid tablets (0.3 g, P.O., qm), ethambutol tablet [0.75 g, P.O., ter in week (tiw)], and pyrazinamide tablets (1.5 g, P.O., tiw). After CRRT, the level of serum creatinine decreased during day 2 and day 3. However, the patient became restless and delirious at the night of day 3. Since he was unable to communicate with, tolerate being in bed, or cooperate to the treatment, CRRT was forced to be terminated and sedation was applied.
On day 4, lumbar puncture was conducted with a CSF opening pressure of 50 mmH2O. CSF examination was as follows: WBC, 2 x 106 cells/L; protein, 0.30 g/L; chloride, 127 mmol/L; and glucose, 5.2 mmol/L. There was neither Cryptococcus neoformans nor MTBC (via Xpert MTB RIF) detected. The samples of CSF, urine, and plasma prepared on day 4 were subjected to mNGS, by which the DNA sequences of L. interrogans (without MTBC) were detected with read numbers of 4, 9, and 2, respectively (reported on day 6;  Table 2 ,  Supplementary Figures 1A-C ). This was in accordance with the positive screening for Leptospira in blood samples of the patient that were collected on day 3 and detected using the Serion ELISA classic Leptospira immunoglobulin G (IgG) kit by the Wenzhou Center for Disease Control and Prevention (reported on day 4 after samples being sent for mNGS). Therefore, the diagnosis of leptospirosis was verified. In combination with the heightened inflammation in bilateral lungs shown by the chest CT screen on day 4 ( Figure 1B ) as well as mental changes of the patient developed during the administration of sulperazone and moxifloxacin, the Jarisch-Herxheimer reaction (JHR) and leptospirosis-induced encephalopathy were of high suspicion. Accordingly, the dosage of methylprednisolone was increased to 80 mg from 40 mg, I.V., bid; sulperazone and moxifloxacin were replaced with penicillin (800,000 IU, I.V., q8h; the initial dose was 400,000 IU) from day 4. On day 6, the patient's consciousness improved, so the sedation was gradually terminated (see  Table 1  for detailed laboratory tests). The chest CT of day 6 showed decreased infection foci and less amount of pleural effusion in the lung, in comparison to that of day 4 ( Figures 1B, C ). To further confirm the result of Xpert MTB RIF and CT, the sputum of the patient was also prepared for mNGS on day 6, from which the DNA sequences corresponding to MTBC (without L. interrogans) were detected with a read number of 4 (reported on day 8;  Table 2 ,  Supplementary Figure 1D ). The mNGS result of sputum was consistent with the Xpert MTB RIF result of day 3. On day 13, patchy shadows with high density were roughly absorbed as revealed by chest CT ( Figure 1D ); thus, the patient was transferred to the general ward of the infectious disease department for subsequent treatment. The detailed clinical course of the patient during hospitalization is shown in  Figure 2 .
After sequential operations, including the sample preparation of microbial cell wall disruption via bead beating (for CSF and urine, not for plasma), DNA extraction and library construction (PCR-free) using an NGS Automatic Library Preparation System (Hangzhou Matridx Biotechnology Co., Ltd., Zhejiang, China), NGS on an Illumina NextSeq 550Dx (single end, 50 bp), and the data processing of removing short reads (<35 bp), low-quality and low-complexity reads, and high-quality sequencing data were generated. The generated data were then separately aligned to a human-specific database constructed from Homo sapiens sequences in NCBI nucleotide (nt) databases (for eliminating human sequences) using bowtie2 v2.3.5.1, and a manual-curated microbial genome database for taxonomy classification using kraken2 (confidence = 0.5) and bowtie2 v2.3.5.1. The microbial genome database contained 11,704 viral genome sequences, 11,162 bacterial genome sequences, 229 parasite genomes, and 1,324 fungal genomes relevant to human infectivity. Moreover, the reads classified to L. interrogans or MTBC were subsequently aligned to a relevant microbial genome (i.e., L. interrogans or MTBC) using BLASTN v2.10.1+ for further validation ( Supplementary Figure 1D ).