Clinical presentation and laboratory findings: key indicators for early diagnosis and severity prediction of leptospirosis in Sri Lanka

Abstract

Background

The diagnosis and treatment of leptospirosis in high-burden, low-resource settings pose a challenge due to the limited availability of confirmatory diagnostics. Therefore, the initiation of treatment relies primarily on the clinical assessment of patients suspected of having leptospirosis in resource-poor settings. This study aimed to both early diagnostic indicators and predictors of disease severity.

Methods

A total of 170 patients suspected of having leptospirosis admitted to selected hospitals in Western Province, Sri Lanka were enrolled. MAT, qPCR, and culture were used for confirmatory diagnosis. Patients with confirmed leptospirosis were grouped as severe and non severe based on presenting with at least one organ failure and/or dysfunction.

Results

Out of 170 leptospirosis suspected patients, 79 (46.47%) were confirmed by PCR (n = 49), MAT (n = 44) and/or culture (n = 2). Myalgia, oliguria, elevated neutrophils, serum creatinine, serum urea, SGOT, SGPT and CRP, decreased lymphocytes, haemoglobin and platelets had a significant association with confirmed leptospirosis. Of patients with confirmed leptospirosis, 51 were categorized as non severe and 28 were categorized as severe. Leptospirosis severity was correlated with decreased haemoglobin, lymphocyte and platelet counts, and elevated WBC, neutrophil, K⁺, SGOT, SGPT, serum urea, serum creatinine, total and direct bilirubin and CRP. A bacterial load between 1.21 × 10² to 1.26 × 10⁶ (median 3,326) Leptospira/ml was reported with no significant association between severity of the disease and leptospiraemia.

Conclusion

Clinical and basic laboratory findings play a crucial role in supporting the diagnosis of leptospirosis and predicting disease severity, particularly in tropical, resource-poor settings.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-13042-x.

Background

Leptospirosis is a zoonotic infection, while treatable with antibiotics, poses a significant challenge due to the difficulty in early detection, as its symptoms often mimic those of other tropical diseases. Sri Lanka’s tropical climate, combined with widespread agricultural practices and rapid urbanization, creates an ideal environment for the survival and transmission of leptospirosis. As a result, Sri Lanka experiences a persistent seasonal occurrence of leptospirosis, with reported case fatality rates ranging from 1.9% to 33.3%, underscoring the urgent need for effective diagnostic and preventive measures [1].

Leptospirosis typically follows a biphasic course, beginning with an acute bacteraemic phase and progressing to an immune phase. Most patients present with an abrupt febrile illness accompanied by headache, chills, intense myalgia, conjunctival suffusion, anorexia, arthralgia, and malaise [2]. Although many infections remain subclinical or mild, approximately 10% progress to severe disease with multiorgan failure and significant mortality [3]. Early antibiotic therapy is critical, highlighting the need for timely diagnosis and the identification of clinical and laboratory predictors of severe illness. While the mechanisms driving severe leptospirosis remain incompletely understood, bacterial virulence factors and pathogen load have been proposed as major contributors [4–7]. Some studies suggest that a threshold bacterial load of 10⁴ Leptospira/ml correlates with worse outcomes [8, 9], whereas others report no clear association between bacterial burden and disease severity [10].

Sri Lanka, a resource-poor country, faces significant challenges in the early diagnosis of leptospirosis due to limited diagnostic facilities. The Centres for Disease Control and Prevention (CDC) recommends the use of the Microscopic Agglutination Test (MAT) and PCR as confirmatory tests, while serological tests to detect IgM is considered supportive tests for early detection. However, in Sri Lanka, diagnostic facilities for confirmation of leptospirosis are restricted to a central laboratory at the Medical Research Institute, Colombo. This limitation poses a major challenge in providing timely laboratory diagnoses, particularly in rural and remote areas where the disease is most prevalent.

To address this challenge, it is crucial to explore whether haematological and biochemical parameters commonly used in routine investigations can aid in supporting early clinical diagnosis of leptospirosis. Leptospirosis may result in a range of clinical manifestations, including hepatic and renal dysfunction, inflammation, and tissue damage, many of which are reflected in changes to laboratory parameters [11]. These investigations could offer clinicians a potential diagnostic clue, helping to distinguish leptospirosis from other prevalent febrile illnesses, especially in resource-poor settings [12].

The aim of this study was to evaluate both early diagnostic indicators and predictors of disease severity, evaluate key clinical characteristics and fundamental laboratory parameters, essential for distinguishing leptospirosis from other acute febrile illnesses and to identify haematological and biochemical predictors of severe disease.

Methods

Study design

A descriptive, cross-sectional hospital-based study was conducted in a cohort of patients with clinically suspected leptospirosis in the Western Province of Sri Lanka from January to December 2017. Patients presenting with clinically suspected leptospirosis admitted to the medical wards were recruited for the study after obtaining informed written consent. The inclusion criteria of this study were based on the “suspected case definition” given in Communicable Disease Epidemiology Profile, Sri Lanka, World Health Organization [13]. Patients less than 18 years of age were excluded from the study.

Study sample

Blood samples were collected on the day of admission to the medical ward or the first day of clinical suspicion of leptospirosis. Blood was aliquoted to a plain tube for serum separation and to an ethylene diamine tetraacetic acid (EDTA) tube for DNA extraction. All specimens were transported at 4 °C. Serum was separated and stored at -80 °C until further processing.

Laboratory confirmation of leptospirosis

Microscopic agglutination test (MAT), culture isolation and quantitative polymerase chain reaction (qPCR) were used to confirm leptospirosis in patients. MAT was carried out using a panel of 15 reference Leptospira strains at the Medical Research Institute, Sri Lanka, which is the National Reference Laboratory. A MAT titre of ≥ 1:320, the cut-off value recommended by the National Reference Laboratory for diagnosing leptospirosis in Sri Lanka, was considered positive. DNA was extracted from EDTA whole blood specimens using a QIAamp DNA blood mini kit (QIAGEN GmbH, Germany) following the manufacturer’s instructions. Quantitative PCR (Bio-Rad CFX96™ (Bio-Rad, USA)) based on EvaGreen technology was performed to measure the level of leptospiraemia, using secYIVF and secYIV [14, 15]. Leptospira was isolated from patients’ blood by inoculating into semisolid EMJH medium and confirmed by Leptospira flaB PCR as previously described by the same authors (Nisansala et al., 2018) [16].

A patient was considered as having “confirmed leptospirosis” if positive by one of the above three tests, while patients who were enrolled into the study due to clinical suspicion of leptospirosis but were negative for any of these tests were considered as “unconfirmed leptospirosis.”

Data collection

A pretested questionnaire was used to record patient information (Questionnaire 1). Data on socio-demographic profile (e.g.: age, gender), exposure risk (e.g.: occupational risk, history of exposure to water bodies, animals), clinical features, basic laboratory investigations including haematological and biochemical results, management and patient outcome were obtained. Data were extracted from the medical records of each patient after obtaining informed consent. Reference values of laboratory investigations were consistent with the medical laboratory of the relevant hospital.

Severity of leptospirosis was determined according to the National guidelines on management of leptospirosis, Sri Lanka [17]. Patients were defined as having severe leptospirosis if presented with at least one organ failure and/or dysfunction (acute kidney injury (AKI), myocarditis, pulmonary haemorrhage and liver dysfunction). AKI was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria, myocarditis according to the presence of one or more of the following criteria: suggestive symptoms and signs, such as shortness of breath, chest pain, palpitations, crackles and/or hypotension and/or electrocardiogram (ECG) abnormalities such as arrhythmias, ST segment/T wave changes, bundle branch block and/or wall motion abnormalities on echocardiography, pulmonary hemorrhage based on the presence of one or more of the following criteria: oxygen saturation < 94%, suggestive symptoms, such as cough, breathlessness, haemoptysis, respiratory rate > 30/min, crackles and wheezes on auscultation of the lungs and lung parenchymal involvement on chest radiograph and liver dysfunction based on the presence of one or more of the following: jaundice, tender hepatomegaly, AST or ALT increased more than thrice the upper limit of normal and raised serum bilirubin, serum alkaline phosphatase or serum gamma-GT.

Statistical analysis

Data were analysed with Statistical Package for the Social Sciences (SPSS) version 21.0 and GraphPad Prism version 7.0 and p values less than (<) 0.05 were considered statistically significant. Categorical variables were expressed as proportions and compared using a two-sample proportion test. The distribution of continuous variables was determined by the Shapiro‒Wilk test. Continuous variables were expressed as the mean ± standard deviation (SD) (data with normal distribution) and compared using the unpaired t test or were expressed as median ± inter quartile range (IQR) (data with skewed distribution) and compared using Mann‒Whitney U test, as appropriate.

Results

Of the 170 patients initially suspected of having leptospirosis, those who tested negative were excluded. This resulted in 79 confirmed cases, comprising 51 patients in the non-severe group and 28 patients in the severe group who were included in the study. (Fig. 1).

Fig. 1.

Flow diagram of patient enrollment and classification. Patients clinically suspected of leptospirosis were enrolled, laboratory confirmation was performed using MAT, qPCR, and/or culture, and confirmed cases were classified into two groups based on severity. Severe leptospirosis was defined according to the National Guidelines on Management of Leptospirosis, Sri Lanka [17]

Disease confirmation

Of the confirmed cases, 44 (56%) were positive by MAT, 49 (64%) were positive by qPCR, and 2 (2.5%) were positive by culture isolation as previously reported by the same authors [16]. Two culture positives were sequenced and submitted to NCBI GenBank (Accession number SLUSJ_182 - MH243436 and SLUSJ__183 - MH243437).

Among the confirmed leptospirosis patients, 14 tested positive by both qPCR and MAT, while one patient was positive by culture and MAT, and another patient was positive by culture and qPCR. According to qPCR, bacterial load on admission ranged between 1.21 × 10² to 1.26 × 10⁶ with a median of 3,326 Leptospira/ml (IQR 7,450.83). When considering bacterial burden, 23 patients (47%) had a bacterial load < 3,000 Leptospira/ml while 10 patients (20%) had a bacterial load > 10,000 Leptospira/ml.

MAT was done using a panel of 15 serovars of which, 10 were reactive for one or more serum samples. The highest MAT titre was reported against L. interrogans serovar Bakeri, strain LT79 in Tarassovi, which accounted for 36 (82%) of positive serum samples.

Two positive culture isolates were obtained and motile live spirochetes could be observed after two weeks of incubation [16]. The DNA of those two cultures were positive for flaB nested PCR and both were identified as L. interrogans by sequencing.

Participant profile

Among the 79 leptospirosis confirmed patients (69 male, 10 female), the mean (SD) age was 45 ± 16 years. Approximately 31 (40%) patients were between 40 and 59 years of age. The prevalence of leptospirosis was significantly higher in males than females (p < 0.00) in this study cohort. More than half the population 46 (58.22%) reported exposure to paddy or agricultural fields while 17 (21.51%) were exposed to domestic sewage and 6 (7.59%) were exposed to flooding or rain water.

When clinical features were considered among leptospirosis confirmed patients, 77 (97.46%) patients had fever. Other common symptoms found in the study sample were headache (n = 51, 64.55%), myalgia (n = 72, 91.13%), arthralgia (n = 41 51.89%), vomiting (n = 37, 46.83%) and anuria/oliguria (n = 34, 43.03%). Myalgia (p = 0.01) and anuria/oliguria (p = 0.03) had a significant association with patients having confirmed leptospirosis compared to patients without a confirmed diagnosis of leptospirosis. (Table 01).

Table 1.

Comparison of clinical parameters between patients with and without a confirmed diagnosis of leptospirosis (%)

Clinical features	Confirmed diagnosis of leptospirosis a (n = 79) (%)	Without a confirmed diagnosis of leptospirosis b (n = 91) (%)	p value*
Fever	97.46	96.70	0.77
Headache	64.55	67.03	0.73
Myalgia	91.13	76.92	0.01*
Arthralgia	51.89	52.74	0.91
Chills and rigors	39.24	52.74	0.07
Lethargy	21.51	19.78	0.78
Conjunctival suffusions	21.51	17.58	0.51
Cough	35.44	38.46	0.68
Dyspnea	10.12	7.69	0.57
Nausea	24.05	18.68	0.39
Vomiting	46.83	43.95	0.70
Diarrhea	34.17	42.85	0.24
Abdominal pain	30.37	28.57	0.79
Icterus	16.45	16.48	0.99
Anuria/oliguria	43.03	27.47	0.03*

^a Confirmed diagnosis of leptospirosis included patients having symptoms consistent with leptospirosis and positive for any confirmatory assay

^b Patients negative for any confirmatory assay (which includes presumptive and unconfirmed cases) *p < 0.05: Two-sample proportion test

Among the patients included in the study, those with confirmed leptospirosis demonstrated distinct laboratory abnormalities when compared with unconfirmed cases. Significantly elevated levels of neutrophils (p = 0.01), serum creatinine (p < 0.001), serum urea (p = 0.00), SGOT (p = 0.00), SGPT (p = 0.02), and C-reactive protein (p = 0.00) were observed in the confirmed group. Conversely, haemoglobin (p = 0.03) and platelet counts (p = 0.02) were markedly lower in patients with confirmed leptospirosis. These findings highlight the value of routine laboratory parameters in differentiating leptospirosis from other febrile illnesses on admission (Tables 1 and 2).

Table 2.

Comparison of biochemical and haematological parameters between patients with and without confirmed leptospirosis

Haematological/biochemical parameter [reference value]	Confirmed diagnosis of leptospirosis a (n = 79)	Without a confirmed diagnosis of leptospirosis b (n = 91)	p value
Median (IQR)
WBC (109/L) [4–11]	10.55 (6.92)	9.44 (6.82)	0.17
Neutrophils (%) [40–75]	85.35 (14.63)	79.80 (18.89)	0.01*
Lymphocytes (%) [10–45]	9.30 (12.00)	11.90 (12.95)	0.02*
Serum creatinine (µmol/L) [60–120]	122.05 (93.23)	96.40 (42.50)	< 0.00*
Serum urea (mmol/L) [2.5–6.5]	10.30 (14.75)	7.50 (4.69)	0.00*
Na+ [136–145]	135.65 (8.70)	136.45 (4.77)	0.31
AST/SGOT (U/L) [10–40]	60.30 (58.20)	42.45 (36.78)	0.00*
ALT/SGPT (U/L) [13–31]	70.50 (73.27)	47.50 (35.30)	0.02*
Total bilirubin (µmol/L) [66–83]	19.88 (47.57)	17.88 (25.02)	0.31
Direct bilirubin (µmol/L) [5–21]	9.20 (32.53)	8.19 (10.84)	0.38
CRP (mg/L) [< 0.5]	171.00 (131.20)	122.60 (142.10)	0.00*
	Mean ± SD
Hb (mmol/L) [Male 14.0-17.5 Female 12.3–15.3]	11.70 (1.99)	12.32 (1.61)	0.03*
Platelet (106/L) [150–450]	98.05 (58.20)	118.50 (62.38)	0.02*
K+ [3.5–5.2]	4.24 (0.67)	4.10 (1.00)	0.59

^a Confirmed diagnosis of leptospirosis included patients having symptoms consistent with leptospirosis and positive for any confirmatory assay

^b Patients negative for any confirmatory assay (which includes presumptive and unconfirmed cases) *p < 0.05: Mann‒Whitney U test for median comparison, unpaired t test for mean comparison

Complications associated with severe leptospirosis

A total of 28 (35.4%) confirmed leptospirosis patients were identified as severe leptospirosis according to the National guideline on management of leptospirosis [17], as described in the methodology section. Among these, 19 (67.8%) experienced acute kidney injury (AKI), 10 (35.7%) had pulmonary hemorrhage, 10 (35.7%) showed liver dysfunction, and 2 (7.1%) developed myocarditis. Of the patients with severe disease, 8 (28.6%) required ICU care, and 6 deaths occurred, resulting in a case fatality rate of 21.4%.

Compared to patients with severe disease, those without sever disease had significantly lower hemoglobin (p = 0.03), lymphocyte counts (p = 0.00), and platelet counts (p = 0.04), along with higher levels of WBC (p = 0.00), neutrophils (p = 0.00), potassium (K⁺) (p = 0.02), liver enzymes (SGOT, p = 0.01; SGPT, p = 0.00), serum urea (p < 0.001), serum creatinine (p < 0.001), total and direct bilirubin (p = 0.00), and CRP (p = 0.01) (Table 3).

Table 3.

Comparison of the basic laboratory results of confirmed leptospirosis patients with and without severe disease

Haematological/bioche mical parameter	Organ involvement /dysfunction with leptospirosis (myocarditis, liver insufficiency, AKI, pulmonary haemorrhage)		p value
	Yes (n = 28)	No (n = 51)
Median (IQR)
WBC (106/L)	12.49 (6.30)	9.21 (5.97)	0.00*
Neutrophils (%)	88.40 (6.99)	79.30 (23.43)	0.00*
Lymphocytes (%)	6.60 (7.5)	15.75 (17.36)	0.00*
Serum creatinine (µmol/L)	313.80 (383.42)	99.15 (43.58)	< 0.00*
Serum urea (mmol/L)	20.90 (15.87)	7.20 (6.94)	< 0.00*
Na+	133.20 (11.25)	135.70 (7.25)	0.90
AST/SGOT (U/L)	81.75 (72.77)	51.50 (56.80)	0.01*
ALT/SGPT (U/L)	101.30 (100.20)	53.15 (52.30)	0.00*
Total bilirubin (µmol/L)	55.20 (105.10)	12.26 (9.83)	0.00*
Direct bilirubin (µmol/L)	32.12 (69.74)	5.46 (6.14)	0.00*
CRP (mg/L)	190.20 (71.40)	152.80 (163.85)	0.01*
Mean ± SD
Hb (mmol/L)	11.06 ± 0.45	12.07 ± 0.24	0.03*
Platelet (106/L)	80.04 ± 1.78	108.20 ± 7.86	0.04*
K+	4.50 ± 0.13	4.06 ± 0.12	0.02*

*p < 0.05: Mann‒Whitney U test for median comparison, independent student t test for days comparison

Based on the findings of this study, several laboratory indicators appearing within 3–5 days of illness were observed in patients who progressed to severe leptospirosis. Among them haematological abnormalities including thrombocytopenia, decrease haemoglobin and leucocytosis were noted along with elevated neutrophils and lymphocytopenia. Further elevation of total and direct bilirubin, SGPT and SGOT levels was observed. Reflecting systemic inflammation, a marked elevation of CRP was seen. Further elevated serum creatinine, serum urea together with hyperkalemia were evidence of AKI. Collectively, these early clinical signs and laboratory parameters highlight their importance in identifying patients at risk of developing severe disease.

Association of bacterial load with leptospirosis

Real-time qPCR was positive in 49 patients. Of these, 28 had uncomplicated infections, 12 developed multiorgan failure, 6 presented with AKI, and one patient each had liver dysfunction and pulmonary haemorrhage. The median bacterial load was lower in patients with complications, but the difference between those with and without complications was not statistically significant (p = 0.93). Among the 7 qPCR-positive patients who required ICU care, the median bacterial load was 4,492.12 Leptospira/ml (IQR: 9,059.63; range: 158.13–140,063.30). In the 4 fatal qPCR-positive cases, the median bacterial load was 8,512.53 Leptospira/ml (IQR: 104,121.96; range: 2,805.60–140,063.30).

Identification of Leptospira serovars by microscopic agglutination test

Among the 44 MAT-positive patients, the most frequent antibody response was against L. interrogans serovar bakeri, strain LT79 (Tarassovi serogroup), which was present in 36 patients. Few patients were identified with two serovars including serovars rathnapura, bangkinang, pyrogenes, and australis, L. interrogans serovar Poi (Javanica serogroup), and serovars Cynopteri and bangkinang.

Discussion

Early and accurate diagnosis of leptospirosis is critical for guiding patient management. In resource poor settings specially in developing countries where the infection is recurrent pathogen-specific tests such as PCR, culture, or MAT are largely confined to reference laboratories. Therefore, disease laboratory confirmation of leptospirosis is a challenge causing inevitable delays in disease confirmation. Consequently, clinical presentation has been useful in differentiating leptospirosis from non-leptospirosis patients. Further, routine laboratory markers were useful indicators for early identification of patients at risk for severe disease.

Key laboratory indicators included hematological changes such as thrombocytopenia, reduced hemoglobin, leukocytosis with neutrophilia, and lymphocytopenia. Liver involvement was reflected by elevated SGPT, SGOT, and bilirubin levels, while systemic inflammation was evident from markedly raised CRP. AKI was indicated by increased serum creatinine, serum urea and hyperkalemia. Collectively, these early laboratory changes provide a clinically useful profile for identifying patients at risk of severe disease, enabling timely supportive care, closer monitoring, or early escalation to higher-level facilities. Similar findings were reported in a case series study from Sri Lanka [18] underscoring the potential value of routine laboratory tests in assisting clinicians to differentiate leptospirosis from other tropical diseases, particularly in resource-limited settings. Integrating these markers into routine early assessment could improve patient outcomes, particularly where confirmatory diagnostics are delayed or unavailable.

In the leptospirosis-confirmed study cohort, nearly all patients (97.46%) presented with fever. Other common symptoms included myalgia, headache, arthralgia, vomiting, and anuria/oliguria. Most studies conducted in Sri Lanka have identified fever as the most prevalent clinical feature of leptospirosis [19–23], with the exception of one study that found headache to be the most common clinical feature in acute leptospirosis patients [24]. Additionally, jaundice and conjunctival injection were identified as common clinical signs in a study by Gunawardhana and Sellahewa at the National Hospital of Sri Lanka [22]. Consistent with our findings, headache and myalgia were also frequently observed during a flood-associated outbreak in Anuradhapura, Sri Lanka [25]. Among all the clinical features, only myalgia and anuria/oliguria showed a significant association with leptospirosis in this study. These two symptoms were also notably prevalent in leptospirosis-confirmed patients in a study by Rajapakse et al. [23]. Another study highlighted icterus and albuminuria as significant features in leptospirosis patients compared to those without a confirmed diagnosis [19]. While myalgia is generally considered a nonspecific symptom that can resemble other endemic infections, this study emphasizes that the presence of either myalgia or anuria/oliguria when accompanied by other supportive evidence, should prompt clinicians to consider leptospirosis as a likely diagnosis. Severe leptospirosis develops in approximately 10% of infected patients, with a case fatality rate ranging from 5 to 15%. The present study found that acute kidney injury (AKI) was the most common complication of leptospirosis, followed by pulmonary haemorrhage and liver impairment. In a study by Gunawardena and Sellahewa involving 45 patients, 11 had AKI, and 14 had acute lung injury [22]. Additionally, AKI was the most frequent complication observed in studies by Agampodi et al. during leptospirosis outbreaks in 2008 and 2011. Apart from AKI, the 2008 outbreak also saw myocarditis as the only other complication, whereas both myocarditis and liver involvement were noted in the 2011 outbreak. Neither study reported pulmonary haemorrhage among the patients during these outbreaks [25, 26]. These observations suggest that the virulence of the infecting serovar may vary, leading to differing complications in patients. A recent study conducted at the Teaching Hospital in Karapitiya, Sri Lanka, found a significantly higher percentage of AKI and severe pulmonary haemorrhage syndrome in leptospirosis patients. Complications such as liver failure and myocarditis were also more prominent in this study [27]. This discrepancy could be attributed to the fact that the study was conducted at a major tertiary care hospital in the Southern Province of Sri Lanka, where complicated cases are often concentrated due to the transfer of severe patients from peripheral hospitals.

In the present study, nearly all haematological and biochemical parameters showed significant differences between the two groups (those with severe disease and those without), with the exception of sodium (Na⁺) levels. A study conducted in India also found similar results, reporting lower haemoglobin and platelet levels, as well as higher leukocyte counts, in severe patients compared to those with mild disease [28]. Likewise, a Sri Lankan study noted lymphopaenia and thrombocytopaenia in leptospirosis-confirmed patients, along with leukocytosis, elevated neutrophil counts, and low haemoglobin levels in patients with severe disease [12]. Furthermore, Daher et al. reported elevated serum urea, creatinine, and bilirubin levels among thrombocytopenic leptospirosis patients, consistent with the findings of the present study [29]. A case series from Sri Lanka also emphasized the prognostic value of serial haematological and biochemical monitoring in leptospirosis [18]. In resource-limited settings, routine haematological and biochemical tests, including full blood counts, renal function tests, C-reactive protein, and erythrocyte sedimentation rate, can provide valuable supporting evidence for risk stratification and the early management of the disease [18].

The current study found no evidence to suggest a direct correlation between bacterial load and disease severity in leptospirosis, similar with the findings reported by Agampodi et al. [10]. On the other hand, previous studies by Truccolo et al. and Segura et al. identified a bacterial load of 10^4 Leptospira/ml in blood as a critical factor linked to severe pulmonary complications and case fatality [8, 9]. The timing of sample collection is crucial when performing qPCR to determine bacterial load, as specimens should ideally be collected during the peak of bacteraemia, when bacterial levels are highest [30]. In this cohort, samples were collected between 2 and 14 days post-onset. Prior antibiotic use could also be a significant confounding factor affecting bacteraemia levels. Moreover, several factors, including geographical location, the source of transmission, and differences in virulence of the infecting Leptospira species and the circulating serovars or strains may also contribute to these observations. It is important to note that this study was conducted among patients admitted to the medical wards of two hospitals in the Western Province of Sri Lanka. As a result, the study primarily included patients with more severe symptoms, while those with mild to moderate symptoms are more likely to seek care through outpatient visits. Detection of bacterial load is further influenced when patients with prior antibiotic use, admissions after days 5–7 of onset of illness and / or the patient being in the immune phase of infection. Among diagnostic limitations, low culture positivity (2/79) was a challenge which reinforced the significant role of prior antibiotic usage and multiple factors associated with culture based techniques. Additionally, a limitation of the study is the use of a single sample for the microscopic agglutination test (MAT) to confirm leptospirosis. In Sri Lanka the cut off titre for MAT has been validated at 1:320 by the national reference laboratory [17]. Therefore, although paired sera are ideal for accurate serological confirmation of leptospirosis, a single titre of > 1:320 was considered in confirming leptospirosis as obtaining paired sera was not feasible due to early patient discharge and the lack of follow-up facilities.

Although the association between the leptospirosis serovar and disease severity has not been investigated in this study, previous studies have suggested an association between serovars and severe disease. According to this study Leptospira interrogans was the most frequently reported serovar. In a study by Bandara et al., L. interrogans was reported in 78% of leptospirosis patients presenting with complications such as AKI, liver insufficiency, myocarditis and pulmonary haemorrhage [11]. Further, L. borgpetersni was found in 14% of cases and was associated with liver insufficiency. L. kirshneri was identified in 7% and was associated with AKI. Jayasundara et al. reported that L. interrogans infections were more frequently linked to acute kidney injury and cardiovascular complications, while both L. interrogans and L. borgpetersenii were associated with thrombocytopaenia and liver dysfunction [31]. This pattern suggests that L. interrogans may be inherently more virulent in the Sri Lankan context, driving more severe disease. Furthermore, these evidences suggest that specific Leptospira species, genotypes, or serogroups contribute disproportionately to severe clinical outcomes. Elevated levels of IL-10 and TNF-α have been strongly linked to severe leptospirosis. In a Sri Lankan study, a significantly higher IL-10 was found in those with severe disease and fatal outcomes, with an elevated IL-10/TNF-α ratio predicting severity and mortality [32]. Meanwhile, a larger case–control study showed that IL-10, along with other cytokines such as IL-6, was markedly raised in fatal cases, suggesting a dysregulated “cytokine storm” response. In parallel, Kidney Injury Molecule-1 (KIM-1) has emerged as a sensitive biomarker for renal injury in leptospirosis [33].

This study highlights the importance of early clinical recognition and routine laboratory markers in identifying patients at risk of severe leptospirosis, particularly in Sri Lanka where access to pathogen-specific diagnostics remains limited. Collectively, these findings underscore the need for integrated diagnostic strategies that combine clinical presentation, basic laboratory tests, and where available, pathogen and host biomarkers to improve early risk stratification and patient outcomes.

Conclusion

The most frequent serological response was observed against L. interrogans serovar Bakeri. Fever, myalgia, and anuria/oliguria were key clinical features, while abnormalities such as thrombocytopaenia, elevated creatinine, serum urea liver enzymes, and CRP effectively differentiated leptospirosis from other febrile illnesses. Severe leptospirosis occurred in over one-third of patients, with acute kidney injury as the most common complication, resulting in a case fatality rate of 21.4%. Early haematological and biochemical derangements within the first 3–5 days of illness were strongly associated with progression to severe disease. Thus, the study emphasizes the potential clinical impact of routine haematological and biochemical tests in predicting disease severity of leptospirosis in tropical, resource limited settings.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AKI

Acute kidney injury

ALT

Alanine transaminase

AST

Aspartate aminotransferase

CDC

Centers for disease control and prevention

CRP

C reactive protein

DNA

Deoxyribonucleic acid,

ECG

Electrocardiogram

EDTA

Ethylenediaminetetraacetic acid

EMJH

Ellinghausen–McCullough–Johnson–Harris

GGT

Gamma-glutamyl transferase

ICU

Intensive care unit

IgM

Immunoglobulin M

MAT

Microscopic agglutination test

qPCR

Quantitative polymerase chain reaction

WBC

White blood cell

WHO

World health organization

Author contributions

TN involved in sample and data collection, laboratory work, data analysis and interpretation and writing the manuscript. KB involved in preparing the manuscript. CG involved in proposal writing, research question, data analysis, writing the manuscript. MW designed the study, involved in proposal writing, data analysis and writing the manuscript. NR and CM selected and recruited the patients for the study and involved in drafting the article. CDG involved in laboratory diagnosis and writing the manuscript. NF involved in proposal writing, research question, data analysis and writing the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by grants awarded by University of Sri Jayewardenepura, Sri Lanka. [ASP/01/RE/MED/2015/37, ASP/01/RE/MED/2016/48 and ASP/01/RE/MED/2017/29].

Data availability

The datasets generated and/or analysed during the current study are available as NCBI GenBank accession number SLUSJ\_182-MH243436 and SLUS\_\_183 MH243437.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Review Committee of University of Sri Jayewardenepura (Application number: 2/17). Institutional clearance was obtained from Regional Director of Health Services Colombo/Kaluthara and Director, Colombo South Teaching Hospital Sri Lanka (Application number: 559). Samples were obtained from patients after obtaining the informed written consent of the patients. Thumb print was taken from illiterate people in front of a witness.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.