Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, and is one of widespread acute febrile illness throughout the world, with fatality rate ranging as high as 20-25% in some regions (Leptospirosis worldwide, 1999). Leptospirosis is mostly transmitted to humans by ambient water contaminated with the urine of wild and domestic mammals that have been chronically colonized by Leptospira (Bharti et al., 2003). Specific occupational activities (e.g., farming, veterinary medicine, military training), recreational immersion in water, poor living conditions, and seasonal rainfall in the tropics are commonly connected with the disease (Bharti et al., 2003). In general, human leptospirosis is prevalent in east/southeast Asia, as well as South America (Ricaldi et al., 2013). However, in temperate zones, such as Europe, North America, and Africa (Hartskeerl et al., 2011; Biggs et al., 2013; Dupouey et al., 2014), it has been reported that the incidence of leptospirosis is increasing, due to global warming, hurricanes, or heavy rains with floods and proliferation of urban rodents (Levett, 2001; Pijnacker et al., 2016).

Even though leptospirosis is a serious but underappreciated infectious tropical illness, it continues to be a major public health concern throughout the world. In recent decades, there is a trend towards increasing incidence in many countries (Lee et al., 2016; Holla et al., 2018; Sasmal et al., 2019) including India, South Korea and America. In some endemic areas (Zhang et al., 2012) of China, there are periodically small-scale local outbreaks of human leptospirosis. Leptospirosis presents a diverse array of clinical manifestations, including jaundice, renal failure, thrombocytopenia, and possibly lethal pulmonary hemorrhage (Levett, 2001). Meanwhile, leptospirosis shares common clinical signs with many acute febrile diseases, such as influenza, dengue fever or malaria (Tubiana et al., 2013), causing various degrees of infections (Doudier et al., 2006). Additionally, some clinical symptoms of leptospirosis have been confirmed to be closely related to death, including oliguria, shock, altered mental status, dyspnea, pulmonary infiltrates, and more (Ko et al., 1999; Doudier et al., 2006; Wang et al., 2020). However, there is currently no consensual definition or diagnostic criteria for severe and different forms of leptospirosis. Leptospirosis has been classified in a variety of methods, ranging from clinical manifestations to genetic (Marquez et al., 2017) determination. Therefore, more insight on clinical manifestations, risk factors, and outcomes of leptospirosis is warranted.

This retrospective multicenter analysis was conducted to determine the association between clinical phenotypes of leptospirosis, laboratory findings, treatment strategies and prognosis.

Leptospirosis is a zoonosis of protean manifestations (Peter, 1982) and can be easily misdiagnosed. Our study found a significantly higher incidence of leptospirosis in male than in female, which was consistent with the results of most previous studies (Kawaguchi et al., 2008). This might be related not only to occupation and exposure (Alvarado-Esquivel et al., 2015; Lau et al., 2016), but also to gender-based genetic and immune susceptibility (Puca et al., 2018). The age of onset was concentrated around the age of 50, which might be associated with more frequent exposure to risk factors for leptospirosis such as farming and fishing in this age group (Puca et al., 2018). These demographic features were less helpful for diagnosis.

Furthermore, the symptoms spectrum of leptospirosis is extremely broad, and there is no good clinical method to differentiate patients with leptospirosis by demographics, clinical symptoms, laboratory testing, and prognosis. In the current study, we conducted a MCA analysis based on clinical features, laboratory findings and outcomes, and identified three subtypes of populations with differences in ICU admission and mortality rate, which could be effective indicators for prognosis of patients with leptospirosis. Traditionally, distinct clinical syndromes of leptospirosis have been considered to be linked to specific serogroups. However, this view was questioned by some authorities (Edwards and Domm, 1960; Alexander et al., 1963; Feigin and Anderson, 1975), and more intense study over the past 50 years has refuted this hypothesis. In humans, severe leptospirosis is frequently but not invariably caused by serovars of the icterohaemorrhagiae serogroup. The infection of the specific serovars largely depend on the geographic location and the ecology of local maintenance hosts.

We identified three clusters: mild leptospirosis (cluster 1), respiratory leptospirosis (cluster 2), and hepato-renal leptospirosis (cluster 3).The current study did not identify a subtype of neurological leptospirosis, while, notably, we discovered that respiratory and hepato-renal leptospirosis had a higher prevalence of altered awareness and CNS symptoms than mild leptospirosis. Therefore, we speculated that neuro-leptospirosis was implicated in the kind of respiratory and hepato-renal leptospirosis due to the low incidence of neuro-leptospirosis, and the neurological symptoms were concealed by pulmonary or digestive symptoms, and the limitation of statistical approach. Studies suggested that neurological symptoms occurred in 10-15% of cases and were often neglected, so we should consider leptospirosis-associated neurological symptoms in areas where leptospirosis is endemic (Puca et al., 2017; Nabity et al., 2020). Mild leptospirosis of Cluster 1, which accounted for 45.3% of our patients, had the best prognosis among three clusters in this study, which was highly accordance with anicteric leptospirosis. The anicteric leptospirosis commonly begins abruptly and frequently presents with vague symptoms such as headache, myalgia (particularly in the calf muscles), ocular abnormalities, fever, nausea, vomiting, rash and epistaxis. Puca E et al. also showed that ocular and cutaneous involvement of leptospirosis usually did not cause serious complications and death (Puca et al., 2016). Cluster 2, mainly characterized by respiratory failure, had the high rate of endotracheal intubation that required ICU admission, was comparable with a pattern previously described in severe leptospirosis (Gouveia et al., 2008; Herath et al., 2019). The incidence of respiratory leptospirosis was about 10% in Brazil and French (Gouveia et al., 2008; Miailhe et al., 2019), compared to 29.5% in our population. Cluster 3 was distinguished by acute liver and kidney failure in contrast with slightly lower incidence of respiratory and circulatory failure. Second, as a temperate zone, there were still some outbreaks and a high incidence of leptospirosis in Hunan. Leptospirosis is a neglected zoonosis that causes considerable public health concerns throughout the world, particularly in Southeast Asian countries such as China, Indonesia, Vietnam, Malaysia, Cambodia and South Korea (Zhang et al., 2012; Loan et al., 2015; Zhang et al., 2019). It was discovered that leptospirosis morbidity rates varied across China’s provinces, with the warm and moist areas of the southern and central China being the predominant regions of leptospirosis infection (Zhang et al., 2020). Frequent rainfall can flush leptospirosis-contaminated soil into bodies of water, increasing the risk of human exposure to leptospirosis.

Notably, this study revealed that 40% of hospitalized leptospirosis cases in a temperate zone was adequately severe to transfer to ICU ward, 23.2% of patients required endotracheal intubation and longer ICU stays. The death rate for icteric leptospirosis has been reported to be as high as 5–15% (Levett, 2001) and 30–70% for leptospirosis-associated pulmonary hemorrhage syndrome (Dolhnikoff et al., 2007; Gouveia et al., 2008), compared to 20.8% and 60.7%, respectively, in our population. Leptospira interrogans serogroup Icterohaemorrh agiae serovar Lai (Hu et al., 2014) was the most common pathogen in Chinese leptospirosis patients, which was also the causative pathogen in 75% of the pulmonary diffuse hemorrhagic leptospirosis. The 15.8% mortality rate was slightly higher than the mean case fatality ratio of 6.85% (Costa et al., 2015), and the majority of leptospirosis deaths was due to renal failure and/or gastrointestinal, pulmonary, or cerebral hemorrhage (Vijayachari et al., 2008; Dong and Chen, 2021) caused by extensive capillary damage including extensive renal necrosis and glomerular atrophy. Moreover, leptospira increased the permeability and engorgement of blood vascular cells (Narayanavari et al., 2015; Matsunaga and Haake, 2018; Zavala-Alvarado et al., 2020). Given the prevalence and predominance of L. interrogans in China, the mortality rate was significantly higher than in other countries.

In this study, 11 patients were diagnosed using metagenomic next-generation sequencing (mNGS) of clinical samples including blood or alveolar lavage fluid. Several diagnostic methods have been used to confirm leptospirosis infection including pathogen culture, pathogen-specific antibodies detection, and genetic way such as mNGS. However, several limitations exist for above testing, for example: (1) Leptospirosis culture is less helpful and not commonly used for clinical diagnosis since it is technically difficult to culture due to the demand of fastidious culture media and long incubation time (Rodriguez et al., 2014); (2) The traditional antibody test of MAT is the most commonly used method to confirm leptospiraosis infection (Sykes et al., 2011), while identifying infecting serovar is difficult based on the cross-reactive MAT tests due to laboratory variation and differences in host-specific humoral immune responses (Markovich et al., 2012); (3) Several ELISAs for detecting IgM, IgG, or both antibodies have been developed to detect specific antibodies in leptospirosis, however, it has not been priorly recommended due to lower sensitivity compared to MAT test; (4) Genetic identification, such as mNGS, have become widely available for the diagnosis of leptospirosis although it is relatively expensive than traditional methods (Harkin et al., 2003). However, false-negative results can be avoided when facing low bacterial loads due to disease phase, complicated immune response, or administration of antimicrobial drugs (Levett et al., 2005; Waggoner and Pinsky, 2016). Thus, to some extends, mNGS, as a method allowing for high-throughputand massively parallel sequencing, benefits leptospirosis patients a lot, particular for those patients with suspicious symptoms, negative traditional testing, or emergent conditions, thus avoiding inadequate diagnosis, delayed treatment, and increased morbidity and mortality. Over the past five years, mNGS testing has been widely used for clinical diagnosis of microbiology examinations (Simner et al., 2018) due to the advantages of quicker diagnosis, higher sensitivity, and even detecting pathogen subtypes as well. Therefore, to some extends, mNGS can avoid some disadvantages of traditional testing, and represent as a promising supplementary tool for some uncommon pathogen diagnosis.

In the current study, we identified several possible predictors, such as dyspnea and neutrophil count, for ICU admission in hospitalized leptospirosis. However, no significant predictors were determined to be associated with mortality of leptospirosis in the current investigation, which might be due to the small sample size. Age has already been shown as a significant predictor of leptospirosis mortality (Chang et al., 2005). Oliguria, hypotension and abnormal chest auscultation were all found to be independent predictors of severe leptospirosis mortality in a Polynesian cohort (Doudier et al., 2006). According to a French study, the following variables including dyspnea, oliguria, increased white blood cell count, repolarization abnormalities on electrocardiograms and alveolar infiltrates on chest radiographs were reported to be associated with leptospirosis mortality (Dupont et al., 1997). Additionally, a Thai cohort demonstrated that hypotension, oliguria, hyperkalaemia, and presence of pulmonary rales were linked to the mortality of leptospirosis. While oliguria has been frequently shown to be related to mortality in numerous earlier studies, our findings are different, and we believe they are justified. Patients in the current study were admitted to tertiary care referral centers with fully functional haemodialysis units, and the patients with AKI were dialyzed whenever indicated. This, we believe, is the cornerstone of leptospirosis care and is likely to have resulted in a decrease in AKI mortality.

In the current study, we identified three phenotypes of leptospirosis with distinct clinical features and prognosis, which would assist clinicians in recognizing leptospirosis-like illness while also providing prognostic predictors. Additionally, dyspnea and neutrophil count were found to be independent risk factors for severe leptospirosis progression. When patients are infected with leptospira, it is prudent to consider more rigorous surveillance and therapy.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by The institutional review boards (IRBs) in Xiangya Hospital of Central South University (No. 202104005). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

All authors contributed to the study conception and design. Material preparation was performed by DL, HL. Data collection and analysis were performed by DL, HL and other authors. The first draft of the manuscript was written by DL and HL. Supervision was performed by PP and YAZ. All authors contributed to the article and approved the submitted version.

This work was supported by Natural Science Foundation of China (No.82100037, No.81770080), National Key R&D Program of China (No. 2016YFC1304204), National Science Foundation for Post-doctoral Scientists of China (No. 2021TQ0375), Hunan Outstanding Postdoctoral Innovative Talents Program (No.2021RC2018), Key R&D Prpgram of Hunan Province (No.2022SK2038) and Youth Foundation of Xiangya Hospital (No.2020Q06).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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