Early diagnosis by mNGS could improve the prognosis for severe leptospirosis patients

doi:10.21203/rs.3.rs-2108428/v1

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Early diagnosis by mNGS could improve the prognosis for severe leptospirosis patients

https://doi.org/10.21203/rs.3.rs-2108428/v1

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PurposesTo evaluate the diagnostic efficiency of metagenomics next generation sequencing (mNGS) assay in severe leptospirosis and its role on prognosis.

Methods We retrospectively reviewed the medical records of patients with confirmed leptospirosis from Jan. 1, 2019 to Dec. 31, 2021 from three hospitals. Detailed data including socio-demographic data, clinical manifestations, laboratory examination results, CT images etc. were collected and analyzed.

Results Six severe cases were included in this study. All were diagnosed by positive mNGS results. Three of the patients were also polymerase chain reaction examined and presented negative results. Five patients were diagnosed by mNGS assay within two or three days and antibiotics were down-graded to ceftriaxone in them. All of them were cured finally. One case, who was diagnosed by mNGS late on day 5, died.

Conclusions mNGS is highly efficient in diagnosing leptospirosis and could possibly reduce the mortality rate if used at an early stage.

General Microbiology

Metagenomics next generation sequencing

Leptospirosis

Diagnosis

Prognosis

Treatment

Leptospirosis is a tropical disease world-widely and the leading zoonotic cause of morbidity and mortality. It is estimated that 1.03 million people suffer from and 59,800 die from the disease annually. Reported mortality of leptospirosis varies from about 4%-50% in severe cases [1, 2]. Even intensive care unit supported, the case fatality rate of severe leptospirosis can be agonizing high. In a report from Sri Lanka, forty-two severe patients with confirmed diagnosis of leptospirosis presented a case fatality rate as high as 44.4%[3]. The mortality remained 4% at the lowest even patients were intensive care unit admitted, mechanical ventilation supported, and supplementary treatment administrated in Australia[4]. Apparently, to further reduce the mortality is clinically urgent and meaningful considering the high morbidity of the disease.

Many factors are involved in the prognosis of the disease yet which one plays the most important or crucial remains unknown. Researches demonstrated that the prognosis of leptospirosis depended on whether a diagnosis was made promptly, antibiotics timely applied, important organ function protected, etc[5]. Theoretically, the management and treatments are usually controllable after admission. Therefore, early diagnosis and followed adequate treatment should deserve more attentions as a postponed diagnosis could yield delayed treatment and treatment failure[6].

Unfortunately, the delay of diagnosis of leptospirosis is rather common. Patients usually start with fever and other non-specific symptoms, which are often overlapped with that of other febrile illnesses. Therefore, the diagnosis could not be established by clinical manifestations merely and a confirmative laboratory examination is usually needed. However, conventional laboratory tests for leptospirosis are not efficient in whole. Leptospira culture is technically difficult due to the demand of special growth media[7]and the long incubation time needed. Serological test like microscopic agglutination test (MAT) can detect against live leptospirosis reference serovars in the blood sample but poorly efficient. Besides, the detection of serum antibody of leptospirosis could not tell a acute infection from previous silent ones[5]. Polymerase chain reaction (PCR) can detect leptospira in blood and urine at a relatively early stage, allowed an increasing of diagnosis by 16.1% compared with the traditional serologic tests[8], especially for patients in the early stage without micro-agglutination detectable antibodies[9]. But due to the limitation of variable primes, PCR remained an unsatisfactory method either. Even combined with MAT, the diagnostic efficiency was fairly poor[5].

Metagenomics next generation sequencing (mNGS) is a new technology for detecting unknown micro pathogens. With an extreme high efficiency, it has been used for identifying pathogens and performed prominent in determining complicated and rare infections. For example, mNGS performed well in diagnosing pneumocystis jiroveci pneumonia in patients after organ transplantation[10]. However, there are rare studies regarding the use of mNGS in leptospirosis diagnosis, except sporadic single case reports recently presented their successful endeavor in this field[5].

Here, we present the outcomes of a group of severe cases who were diagnosed at different stage of the disease using mNGS and treated against leptospira. We believe the conclusions of this study would be a meaningful reference for diagnosing and treating leptospirosis in practice.

Methodology

We reviewed retrospectively the illness records about confirmed leptospirosis cases in the past three years from three hospitals. Data were collected from medical record bank and anonymity was ensured at all stages. The study was carried out in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of The Affiliated First Hospital of USTC (Approval No: 2022-RE-067). The demand for patient consent was waived due to the nature of a retrospective study.

Data collection

The medical records of patients with confirmed leptospirosis in the Affiliated First Hospital of USTC, Wanbei Coal and Power Group General Hospital, and The First Affiliated Hospital of Anhui University of Science and Technology from Jan. 1, 2019 to Dec. 31, 2021 were traced. Detailed data including socio-demographic data, clinical manifestations, laboratory examination results including full blood count, extent of organ involvement like hepatic and renal function parameters, coagulation profile, C-reactive protein (CRP), procalcitonin (PCT), arterial blood gas analysis, diagnostic method used, treatment including antibiotic therapy, mechanical ventilation support, outcomes, etc were collected. Laboratory results on the first day of presentation or the first time after admission were extracted. The CT images were retrieved by searching the CT data bank. CT images were defined to the images on the first day or at the first time and images after treatment or before discharge.

Sample Processing and DNA Extraction

Volume of 5 ml of blood was drawn from patients and stored at room temperature for 3-5 min and then centrifuged at 4,000 rpm for 10 min at 4℃. Plasma samples were obtained and transferred to new sterile tubes. Volumes of 2.5 ml of bronchoalveolar lavage fluid(BALF) were collected from patients.Approximately, 0.6-ml sample and 250ml 0.5mm glass bead were attached to a horizontal platform on a vortex mixer and agitated vigorously at 2,800–3,200 rpm for 30 min. Then, 7.2 ml lysozyme was added for wall-breaking reaction. DNA was extracted from 0.3 ml of plasma/BALF using the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH, Beijing, China), according to the manufacturer’s operational manual. The extracted DNA specimens were prepared for the DNA libraries construction [11].

DNA Library Construction and Sequencing
DNA library was constructed through DNA fragmentation, end-repair, adapter-ligation, and PCR amplification using MGIEasy Cell-free DNA Library PrepSet (MGI Tech). Agilent 2100 was used for quality control of the DNA libraries. Qualified libraries were pooled; DNA Nanoball (DNB) was made using single-stranded DNA circles and sequenced by MGISEQ-2000/NextSeq 550 platform [12].

Bioinformatics Analysis

High-quality sequencing data were generated by removing low-quality, short reads (length < 35 bp) [13],followed by computational subtraction of human host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment[14] .The remaining data by removal of low-complexity reads were classified by simultaneously aligning with Pathogen Metagenomics Database (PMDB), including bacteria, fungi, viruses, and parasites. The classification reference databases were downloaded from NCBI..The coverage ratio and the depth of each microorganism were calculated using BEDTools[15]. The criteria for an infectious pathogen by mNGS were defined routinely.

In total, six patients with confirmed diagnosis of severe leptospirosis were included in this study: four from the Affiliated First Hospital of USTC, one from Wanbei Coal and Power Group General Hospital, and one from The First Affiliated Hospital of Anhui University of Science and Technology. The demographic features of the patients are shown in Table 1. It is noteworthy that within two or three days after admission, five of the six patients were diagnosed as leptospirosis by mNGS assay, promptly treated with aimed antibiotics, admitted to intensive care unit,, mechanical ventilation supported, and gradually fully recovered, while case 5, who was diagnosed on day five after admission, died although ICU admitted.

Clinical manifestations

Six patients were all adults aged about sixty. All patients sought medical treatment within a week. Of them, two patients had a history of contacting river water in the past two weeks as washing injured hand in the river and fishing, separately. Fever, chest distress, and dyspnea presented in all the patients. Shock was seen in five of the patients. Hemoptysis appeared in four cases. Jaundice happened in two patients. Specific symptoms of leptospirosis like conjunctival suffusion were seen in case 4 and myalgia in case 1, 5, and 6, respectively. All patients were ranked the severe cases clinically. The symptoms and signs of patients are summarized in Table 1.

Laboratory examinations

Blood routine examinations revealed significant abnormalities in the peripheral blood. White blood cell count and neutrophils increased remarkably in all patients. In all cases, anemia to some degree was noted. Severe thrombocytopenia appeared in most cases except case 4. Biochemistry examinations showed mild to severe multi-organ dysfunction in each patient. Blood gas analysis revealed lowered PaO2/FiO2 (mmHg) and respiratory failure. All patients presented a rather high CRP level. Urine erythrocytes were seen in almost all cases. See Table 2.

mNGS was performed in five of the patients within two days after admission. mNGS revealed leptospiral infection and the diagnose of leptospirosis was established within two or three days after admission. For case 5, mNGS was performed late on day four and got the positive result on day five after admission. It is worth noting that, in case 1, 3 and 6, both mNGS and PCR assays were performed, and mNGS presented positive result of leptospira infection while PCR reported negative. Besides, mNGS reported aspergillus oryzae and roseomonas as the co-morbidity pathogens in case 1 and 3, respectively. Routine blood culture and sputum culture did not reveal any significant pathogenic microorganisms. See Table 3.

Image abnormalities

All patients had abnormal chest images at admission. Typical pulmonary inflammation and hemorrhage as bilateral diffuse infiltration and patch shadows were noted. After treatment, chest images ameliorated in all patients. The changes were in accordance with the alleviation of clinical presentations synchronously. Figure 1 showed the representative changes of CT image in a patient before and after treatment.

Treatment procedures and outcomes

Five of the six patients were diagnosed within two or three days after admission and immediately treated accordingly. When mNGS results revealed leptospira as the pathogen of infection, modulation and downgrade of antibiotics were immediately administered. The patients were given ceftriaxone immediately. Broad spectrum antibiotics were ceased. In case 1, cefoperazone sulbactam was replaced by ceftriaxone and an extra antibiotics voriconazole was added due to a suspected concomitant infection. In case 2, 3, 4, and 6, antibiotics was down-graded from carbapenems to ceftriaxone. All patients were admitted into intensive care unit and received invasive mechanical ventilation for 3 to 6 days. In addition, other supportive treatments were also given separately depending on the individual conditions of each patient. The duration of hospitalization ranged from 6 to 18 days. Five patients were finally cured and completely recovered. Unfortunately, case 5, who was diagnosed on day 5, was an exception. The patient had shock, dyspnea, hemoptysis, and very high BNP levels. He was initially diagnosed as severe community acquired pneumonia with MODs, septic shock and treated with meropenem and moxifloxacin. The diagnosis of leptospirosis was determined by mNGS late on day five and the patient died on that day before the diagnosis reports. See Table 4.

Leptospirosis is a widely distributed zoonosis with considerable fatality[16]. A certain part of patients will develop severely and even die at a high fatality rate[3, 17], especially in the un-treated patients[18]. Many factors are involved in the prognosis of the disease[19]. Among them, early diagnosis is an important one. Unfortunately, conventional laboratory diagnostic methods are limited in efficiency and promptness. In this study, mNGS was proved highly efficient in diagnosing leptospirosis. The diagnosis was quickly established within one or two days using mNGS in most of the patients. Treatment was modulated consequently right after the diagnosis establishment and those patients were all cured.

The clinical manifestations of leptospirosis could vary from mild illness to severe life-threatening ones. In most patients, the presentations are mild and self-limiting. But in some others it could be severe or even mortal[16], yielding a rather high mortality. Patients developed MODs could die at 44.4% even admitted to critical care settings[5]. Patients complicated by pulmonary hemorrhages and acute respiratory distress syndrome (ARDS) could have a very high mortality even respiratory support like extracorporeal membrane oxygenation was applied[20]. In a report from Australia, 37/55 (67%) patients developed moderate to severe acute respiratory distress syndrome, 32/55 (58%) had pulmonary hemorrhage. Even intensive care unit admitted, mechanical ventilation supported, 4% patients still died[5]. In our study, all patients presented MODs symptoms like shock, dyspnea, lowered PaO2/FiO2 (mmHg) and respiratory failure, jaundice etc. Laboratory examinations revealed MODs symbols including thrombocytopenia, acute liver and kidney injuries, coagulation parameters and blood gas abnormalities. Most patients presented CT abnormalities like diffuse infiltration and patch shadows, implying an existing pulmonary inflammation and hemorrhage, as shown in Fig. 1A and B. These indicated that these patients were in severe status in disease degree.

Research pointed out that early diagnosis was crucial for the prognosis of the patients and a delayed antibiotic therapy might increase the fatality[5]. However, the diagnosis of leptospirosis remains a challenge because the diagnosis mainly depends on laboratory examinations instead of clinical manifestations. Unfortunately, conventional examination methods including ELISA, dual MAT, and PCR are rather limited in their diagnostic efficiency[5]. A large scale study enrolled 1,734 cases of confirmed 591 (34.1%) and 297 (17.1%) “probable” leptospirosis revealed that whole blood quantitative PCR (qPCR) identified the most cases (322/540 (60%) ) but missed 40, acute sample MAT missed 80% (409/510); paired serum sample MAT missed 58% (98/170)[5]. In our study, PCR was conducted in three patients and revealed negative results. Apparently, although those techniques could facilitate the diagnosis in some cases, they are not efficient in whole. Better methods are urgent needed[21].

mNGS, a molecular diagnostic tool developed in recent years has been used in clinical practice[22, 23]. Research showed that mNGS was highly efficient in revealing rare pathogens[5]. It has been used especially in complicated infection diagnostics to identify the genomic characterization of various specific pathogen directly from various different specimens[24]. Recently, a few researchers tried mNGS in diagnosing leptospirosis. In a report of a case of patient, the researchers diagnosed leptospirosis successfully using mNGS although the patient died eventually[25]. In another case report, the researchers established the diagnosis of leptospirosis using mNGS assay and cured the patient successfully[5]. In our study, six cases of patients were all diagnosed by mNGS assay after admission. The results were often obtained quickly a day after blood or BALF sample collection. It is noteworthy that three of the patients were also examined by PCR but revealed negative results.

Many factors affect the prognosis of patients. Due to the limited sensitivity of all three conventional diagnostic tests, clinicians had been suggested with an empirical treatment for suspected cases of leptospirosis[26]. In fact, appropriate antibiotic administration is one of the crucial factors that deciding the prognosis of the patients. Once the diagnosis was established, adequate antibiotics treatment are needed promptly besides other routine measures like intensive care support if necessary. Doxycycline and penicillin was standard therapy in early leptospirosis treatment while ceftriaxone was considered the antibiotics of choice in late and severe disease in the past decades[7]. In our study, ceftriaxone was given immediately when the diagnosis was established in the five early diagnosed patients. Two of the patients down-graded the antibiotics from imipenem-cilastatin sodium, one from meropenem and vancomycin, acyclovir, and the other case from meropenem to ceftriaxone, see table 4. After treatment, clinical manifestations of the patients were significantly attenuated. The CT image abnormalities alleviated in consistent with the attenuation of severity of dyspnea, chest tightness, hemoptysis, and abnormal blood gas changes, as shown by Fig. 1.

Apparently, an in time diagnosis is beneficial to adequate antibiotics choice. The down-grade of antibiotics from broad-spectrum antibiotics could possibly lower the risks to multi-resistance to antibiotics and opportunistic secondary infections, and reduce financial expenses in these patients. On the early diagnose and adequate antibiotics administration, five of the patients were cured and discharged eventually. Contrary to those patients, case 5 was diagnosed as leptospirosis late on day 5 after admission. He had been treated with meropenem and moxifloxacin after admission and died on day five. In summary of these cases, it is reasonable to deduce that early diagnosis and appropriate antibiotics administration are crucial to the prognosis of the disease. Substantially, although the case number was somewhat limited, our study demonstrated the potency of mNGS for diagnosing leptospirosis and significance of early diagnosis.

Limitations are obvious in this report. This is a retrospective study. Factors involved in deciding the treatment outcomes could not been assessed concisely and accurately. Secondly, the number of patients was fairly limited and the severity of each patient could be different. We could not come to a strong and solid conclusion about the role of in time treatments on the prognosis in those cured and the one died. Thirdly, mNGS method needs specific equipments and somewhat expensive, which will limit its application in the resource limited settings. Apparently, although most patients were diagnoses by mNGS and subsequently cured successfully in this study, prospective studies enrolling a significant number of patients would be more convincing.

In summary, compared with the conventional diagnostic methods, mNGS is a promising method which is sensitive, highly efficient, and prompt in diagnosing leptospirosis. The technique could help physicians to establish the diagnosis accurately and timely, take proper treatment measures, and possibly reduce the mortality rate. A timely diagnosis and prompt specific treatment might reduce the death rate of leptospirosis substantially. Prospective studies enrolling considerable number of patients are expected.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (82000018), the Fundamental Research Fund for the Central Universities (WK9110000089).

Author contribution Xuqin Jiang and Xiaodong Mei designed the study. Hui Jiang, Yingying Xiong , Ming Fang and Bei Geng took responsibility for the integrity of data and accuracy of the data analysis. Xuqin Jiang wrote the draft of the paper. Xiaodong Mei and Huimei Wu revised and edited the paper. All authors contributed to writing or reviewing the paper.

Data availability Not applicable.

Code availability Not applicable.

Ethics approval The study was carried out in accordance with the Declaration of Helsinki and was approved by the Medical Ethics Committee of The Affiliated First Hospital of USTC (Approval No: 2022-RE-067) .The demand for patient consent was waived due to the nature of a retrospective study.

Consent to participate Written informed consent was obtained from all individuals involved in the study.

Consent for publication All authors consent to publish.

Conflict of interest The authors declare no competing interests.

Table 1 Demographic features and clinical presentations of the patients

	Case1	Case2	Case3	Case4	Case5	Case6
Gender	Male	Male	Male	Male	Male	Female
Days from onset to admission	6	2	3	6	3	6
Age (yr)	63	77	58	60	62	55
Underlying diseases	Malaria	No	Hypertension, Cerebral infarction	No	No	No
Consciousness	Alert	Light coma	Light coma	Alert	Alert	Coma
Shock	Yes	Yes	Yes	No	Yes	Yes
Fever	Yes	Yes	Yes	Yes	Yes	Yes
Chest distress	Yes	Yes	Yes	Yes	Yes	Yes
Dyspnea	Yes	Yes	Yes	Yes	Yes	Yes
Hemoptysis	Yes	No	No	Yes	Yes	Yes
Diarrhea	No	No	Yes	Yes	No	No
Conjunctival suffusion	No	No	No	Yes	No	No
Myalgia	Yes	No	No	No	Yes	Yes
Arthralgia	Yes	No	No	No	Yes	No
Jaundice	No	Yes	No	No	Yes	No

Table 2 Laboratory examinations of the patients

	Case1	Case2	Case3	Case4	Case5	Case6	Ref. values
Blood routine
White blood cell (×10⁹/L )	19.87	12.04	10.04	15.40	14.44	14.88	3.50 to 9.50
Neutrophil count(×10⁹/L )	17.50	10.70	9.10	12.80	11.95	13.9	1.80 to 6.30
Hemoglobin (g/L)	108.0	66.0	120.0	75.0	117	107	130.0 to 175.0
Platelets (×10⁹/L )	34	7	33	120	24	67	125-350
Biochemistry examinations
ALT (U/L)	23.3	47.0	64.0	57.0	52.0	286.5	5.0-50.0
AST (U/L)	46.5	186.0	100.8	85.0	114.0	495.9	15.0-45.0
GGT (U/L)	41.0	30.0	35.9	45.0	44	46.2	10.0-60.0
Albumin (g/L)	35.7	24.2	28.3	25.6	21.0	40.7	40.0-55.0
Creatinine (umol/L)	164.0	230.0	134.1	89.0	302.0	405.7	57.0-111.0
Urea nitrogen (mmol/L)	28.80	15.30	7.90	9.10	19.60	18.60	3.10-8.00
Total bilirubin (umol/L)	21.6	67.5	27.3	17.0	44.0	14.0	3.4-21.0
Direct bilirubin (umol/L)	10.30	58.60	17.30	10.20	32.00	9.60	0.00-5.00
Indirect bilirubin (umol/L)	11.30	8.90	10.00	6.80	/	2.00	0.00-18.00
LDH (U/L)	1269	381	363	459	303	2766.2	120-250
CK U/L	136	3642	1180	98	497	940	39-308
Coagulation parameters
Prothrombin time(seconds)	11.60	16.30	15.00	11.20	13.60	12.60	8.00-14.00
APTT (seconds)	20.30	54.50	39.20	43.90	33.80	35.70	20.00-40.00
INR	0.99	1.42	1.30	0.86	1.17	1.08	> 1.0
Troponin I (ug/L)	0.096	0.130	2.24	0.020	<0.05	1.710	0.000-0.034
Arterial blood gases analysis
PH	7.495	7.2	7.26	7.439	7.05	7.395	7.35-7.45
PaO2 (mmHg)	72.2	45.9	91.30	58.1	180	90.3	70-100
PaCO2 (mmHg)	36.7	41.6	35.20	16.3	27	33.2	32-45
PaO2/FiO2 (mmHg)	180.5	218.6	130.4	96.8	180.0	180.6	>300
Others
CRP (mg/L)	210.46	216.26	256.78	135.86	242.5	165.7	0.00 to 10.00
PCT (ng/ml)	26.020	11.176	>100	0.558	18.23	>100	0.000-0.500
Urine erythrocyte (/ul)	48.0	2915.0	799.9	/	296	182	0-17
NT-proBNP (pg/ml)	4305	28727	5579	1440	12179.9	8490	0-125,0-900.0
CD3+CD4+%	22.9	/	35.4	23.8	/	37.5	28.5-60.5
P-ANCA	Negative	Negative	/	Negative	/	Negative	Negative
C-ANCA	Negative	Negative	/	Negative	/	Negative	Negative

Table 3 Microbiological examination results of the patients

	Case1		Case2	Case3	Case4	Case5	Case6
mNGS
Samples used		BALF	Blood	Blood	BALF	Blood	Blood
Reads for leptospira		8	188233	22	1	2	7
Results judgment		Positive	Positive	Positive	Positive	Positive	Positive
Others reported		Aspergillus	Negative	Roseomonas	Negative	Negative	Negative
Days before sampling		1	0	1	2	4	1
Days before diagnosis		2	1	2	3	5	2
Blood PCR assay results		Negative	/	Negative	/	/	Negative
Sputum culture		Negative	Negative	Negative	Negative	Negative	Negative
Blood culture		Negative	Negative	Negative	Negative	Negative	Negative

Table 4 Treatment modulation base on mNGS results and outcome of the patients

	Case1	Case2	Case3	Case4	Case5	Case6
Initial antibiotics regime	Cefoperazone sulbactam	Imipenem-Cilastatin Sodium	Imipenem-Cilastatin Sodium	Meropenem, Vancomycin, Acyclovir	Meropenem, Moxifloxacin	Meropenem
Modulation on mNGS	Ceftriaxone, Voriconazole	Ceftriaxone	Ceftriaxone	Ceftriaxone	/	Ceftriaxone
Admitted to ICU	Yes	Yes	Yes	Yes	Yes	Yes
Invasive ventilation	Yes	Yes	Yes	Yes	Yes	Yes
Ventilation time (days)	3	6	3	6	5	6
Days of hospitalization	12	13	6	18	5	16
Outcomes	Cured	Cured	Cured	Cured	Died	Cured

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Early diagnosis by mNGS could improve the prognosis for severe leptospirosis patients

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