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Cytokine response in human leptospirosis with different clinical outcomes: a systematic review

Background Leptospirosis is a neglected zoonotic disease which is a major challenge for clinicians and public health professionals in tropical countries. The cytokine storm during the second (immune) phase is thought to be a major contributory factor for the leptospirosis disease severity. We aim to summarize evidence for cytokine response in leptospirosis at different clinical outcomes. Methods A systematic review was carried out to examine the cytokine response in leptospirosis patients using relevant scientific databases. Reference lists of the selected articles were also screened. Quality of the selected studies was assessed by using the National Institutes of Health Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. Results Of the 239 articles retrieved in the initial search, 18 studies fulfilled the selection criteria. India and Thailand have produced the highest number of studies (17% each, n = 3). The majority were comparative cross-sectional studies (72%, n = 13). Overall the quality of the selected studies was fair regardless of few drawbacks such as reporting of sample size and the lack of adjustment for confounders. Microscopic agglutination test (67% - 12/18) and enzyme-linked immunosorbent assay (50% - 9/18) were commonly used for the confirmation of leptospirosis and the measurement of cytokines respectively. IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α levels were found to be significantly higher in severe than in mild leptospirosis. There were equivocal findings on the association between IL-1β, TNF-α and IL-10/TNF-α ratio and disease severity. Conclusions Leptospirosis had a wide-range of elevated cytokines. However, prospective studies in-relation to the onset of the symptom are required to better understand the pathophysiology of cytokine response in leptospirosis.

still not fully described; however; the innate immunemediated complications are described. Several molecules of the infectious agents like lipopolysaccharides, hemolysins, outer membrane protein and the glycolipoprotein have been implicated with the disease [3,9]. It is proposed that the pathogenesis of leptospirosis is driven through the cytokine response [10].
Many viral infections show an abnormal immune response and cytokine production [11]. Dengue hemorrhagic fever (DHF) is closely linked to the host innate and adaptive immune response. IL-6, IL-8, IL-10, IP-10, MCP-1, TNF-α and vascular endothelial growth factors (VEGF-A) promote clinical manifestations of DHF [12]. Pathogenesis of Ebola virus is influenced by multiple factors out of which excessive inflammatory response is a significant feature [13]. The potential source of inflammatory cytokines during multiple viral hemorrhagic fever infections could vary. Dendritic cells secrete minimal amounts of cytokines since they are targeted by multiple viruses (arenaviruses, bunyaviruses, filoviruses, and flaviviruses). However, monocytes and macrophages appear to be playing a dominant role in secreting inflammatory cytokines during these infections [11]. Severe systemic clinical manifestations of gram-positive sepsis are also attributed to the non-specific pathogen-associated molecular pattern effect of peptidoglycan and lipoteichoic acid which trigger toll-like receptor 2 and induce inflammation. Similarly, lipopolysaccharides has its inflammatory effect through trigger toll-like receptor 4 [14]. The function of the immune system is to eliminate the microbial pathogen by producing pro-inflammatory agents such as cytokines. However, tissue injury could be possible with excessive production of cytokines [15]. We aim to systematically review the previous evidence on cytokine response in leptospirosis at different clinical outcomes.

Eligibility criteria
Full-text articles which reported cytokine response in human leptospirosis were included in this review. Reports in the non-English language were excluded. Reports were not excluded based on the year of publication or patient population.

Information sources and search strategy
The studies published before 31st of August 2018 were identified through systemic searches of electronic databases and cross-checking reference lists of the selected articles. Cochrane-Library, Google-Scholar, Google, PubMed, Science-Direct, Trip and Web of Science were the databases searched using a string of keywords ( Fig. 1). Appropriate Medical Subjects Heading (MeSH) and other relevant terms were used to identify published studies.

Study selection
IS performed a comprehensive literature search. All selected titles and abstracts were independently screened by IS and DR. Full-text articles were assessed when the abstract was unclear. The selected studies were independently reviewed by SA to confirm eligibility.

Data collection process, data items and data analysis
Year of publication, demographic details, study design, methods of leptospirosis confirmation, types and methods of cytokine analysis and outcomes were extracted from the selected studies. The Population, Intervention, Comparator and Outcome (PICO) were patients with leptospirosis / severe disease / fatal outcome, cytokine analysis, healthy individuals/patients with mild disease/ survivors and the cytokine levels respectively. SI units were used for presenting data. The data were analysed using Microsoft Excel (Additional file 1). Descriptive statistics were used to describe the data. The Cochrane guideline for systematic review was followed [16]. The review is presented according to the PRISMA -Preferred Reporting Items for Systematic Reviews guidelines [17]. Quality assessment of the selected studies was done using the National Institutes of Health Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies (Additional file 2) [18]. Graphs were drawn using the GraphPad Prism 8 software [19].

Cytokines response in leptospirosis treatment
More than five doses of antimicrobial agents (penicillin, ceftriaxone, doxycycline) significantly lowered IL-6 and IL-8 levels, when compared to lower doses. IL-8 levels were negatively associated with the total number of antimicrobial doses administered (penicillin, ceftriaxone, doxycycline) and also with the number of ceftriaxone doses administered [21]. Extended sustained low-efficiency dialysis via hemodiafiltration significantly decreased the post-admission levels of IL-17, IL-7, and MCP-1 compared to the dialysis via hemodialysis among patients with acute kidney injury or acute respiratory distress syndrome following leptospirosis [30].

Discussion
The review had data from studies which included leptospirosis patients with varying degree of severity. Although, the review produced valuable data on the possible role of cytokines in leptospirosis disease severity, there were equivocal findings with regards to IL-1β, TNF-α and IL-10/TNF-α ratio among the selected studies. Failure to measure soluble TNF receptor-1 (sTNFR1) might have led to low levels of TNF-α in some studies, as the bound TNF-α will not be included in the analysis [37]. IL-1β, IL-6 and TNF-α were proposed to be pro-inflammatory cytokines which enhance the inflammatory response in leptospirosis [10]. However, the present review found IL-1β to be low among leptospirosis patients, IL-6 to be elevated and TNF-α to be equivocal. The above findings could be due to the variation in the time of sample collection in relation to the onset of the symptoms. Nevertheless, results among the complicated leptospirosis showed elevated levels of IL-6 and TNF-α which shows a higher degree of pro-inflammatory action.
In contrast, IL-10 is thought to be the most important antiinflammatory cytokine which restores the immunological homeostasis in leptospirosis [10]. In addition, IL-4 and IL-13 restrict the inflammatory response [10]. The present review revealed elevated levels of the above-mentioned anti-inflammatory cytokines among leptospirosis patients and patients with complicated disease. The findings suggest that anti-inflammatory response is also expected to increase in complicated leptospirosis. Therefore, a low IL-10 / TNF-α is most probable among patients with complicated disease. Differential diagnosis for leptospirosis includes DHF, chikungunya, hanta virus and malaria infections. Similar to the present review, IL-6 [38], IL-8 [38], IL-10 [38,39], IP-10 [38], MCP-1 [38], TNF-α [40,41] and VEGF [39]  were found to be elevated in DHF, a condition common to almost all endemic settings for leptospirosis. IL-6, IL-12, IL-1RA, IP-10, and MCP-1 were also found to be elevated in chikungunya [42][43][44]. Hanta virus infection, a disease which can mimic pulmonary leptospirosis, had shown elevation of GM-CSF, IL-2, IL-6, IL-10, IL-12P40, IL-17 and VEGF [45]. Fatal outcomes from Plasmodium falciparum malaria showed elevated levels of IL-6, IL-10 and TNF-α than in survival which was comparable to the results on leptospirosis [46]. Cerebral malaria was found to have high levels of TNF-α in fatal outcomes [46]. Moreover, Ebola viral infection showed elevation of pro-inflammatory cytokines IL-6 and IL-8 in non-survivors similar to the present review [47,48]. It is interesting to note that IL-6 was found to be elevated in leptospirosis, DHF, chikungunya, hanta virus, malaria and Ebola infections. However, cytokine response alone cannot be used to differentiate the above conditions.
Both Papa et al. [33] and Kyriakidis et al. [24] showed that the day of illness is a determinant of cytokine levels. The above fact emphasizes the need of cytokine selection in relation to the day of illness, if we are to use the cytokine levels for disease monitoring. The selected studies were heterogeneous, and the methods used for the diagnosis of leptospirosis and measurement of cytokines varied between studies, making it impossible for proper evidence synthesis. Although, the quality assessment revealed few drawbacks such as reporting of sample size and the lack of adjustment for confounders, overall the quality was fair. Nevertheless, the review points out the importance of clinical assessment as the cytokine response in leptospirosis is still poorly understood, and published literature are highly inadequate to produce a clear picture.

Conclusions
A wide-range of cytokines were found in the circulation during a leptospirosis infection. Especially, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α levels were significantly higher in severe disease. However, prospective studies with minimal sampling bias, standard diagnosis and optimum cytokine measurement in-relation to the onset of the symptom are required to better understand the pathophysiology of cytokine response in leptospirosis.
Additional file 1. Datasheet of the review on cytokine response in human leptospirosis, 2019. This provides the data extracted from the review.
Additional file 2. Summary of NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. This provides the summary of quality assessment for the selected studies.
Abbreviations DHF: Dengue hemorrhagic fever; MAT: Microscopic agglutination test; MeSH: Medical subjects heading; PICO: Population, intervention, comparison and outcome; PRISMA: Preferred reporting items for systematic reviews and meta-analyses; VEGF: Vascular endothelial growth factors