Clinical spectrum of endemic leptospirosis in relation to cytokine response.

OBJECTIVES: To describe the clinical spectrum and the cytokine response of leptospirosis patients in an endemic setting of Sri Lanka.
METHODS: Patients presenting to the university teaching hospital, Anuradhapura, Sri Lanka with a leptospirosis-compatible illness were recruited over a period of 12 months starting from June 2012. Daily clinical and biochemical parameters of the patients were prospectively assessed with a follow-up of 14 days after discharge. A magnetic bead-based multiplex cytokine kit was used to detect 17 cytokines.
RESULTS: Of the 142 clinically suspected leptospirosis patients recruited, 47 were confirmed and, 29 cases were labeled as "probable." Thrombocytopenia and leukocytosis were observed at least once during the hospital stay among 76(54%) and 39(28%) patients, respectively. Acute kidney injury was observed in 31 patients (22%) and it was significantly higher among confirmed and probable cases. Hu TNF-α and IL-1β were detected only in patients without complications. Hu MIP-1b levels were significantly higher among patients with complications. During the convalescence period, all tested serum cytokine levels were lower compared to the acute sample, except for IL-8. The cytokine response during the acute phase clustered in four different groups. High serum creatinine was associated GM-CSF, high IL-5 and IL-6 level were correlates with lung involvement and saturation drop. The patients with high billirubin (direct)>7 mmol/l had high IL-13 levels.
CONCLUSIONS: Results of this study confirms that the knowledge on cytokine response in leptospirosis could be more complex than other similar tropical disease, and biosignatures that provide diagnostic and prognostic information for human leptospirosis remain to be discovered.

Introduction

Globally, 2.9 million disability-adjusted life years per annum are estimated to be lost owing to leptospirosis [1]. This burden results in more than 1 million estimated cases and 58,900 deaths [2]. Global studies in recent years have promoted awareness of leptospirosis and at the same time increased the demand for high-quality data on which to design better interventions to prevent and better treat this disease. Despite the accumulating evidence on various aspects of leptospirosis, its control, prevention and clinical management remain as major challenges for clinicians and public health professionals in tropical countries where the disease burden is highest.

Clinical studies on leptospirosis have revealed a wide variation of morbidity and mortality. A recent systematic review showed that, among untreated patients, median mortality ranges from 2% to 37% among diagnosed cases [3]. With appropriate treatment—requiring prompt diagnosis mortality was much lower and outcomes better. Based on the study setting, country, selection criteria, and infecting strains, the clinical syndrome presented by most patients varies between typical Weil’s disease and self-resolving undifferentiated fever. Understanding the typical disease pattern is often difficult in terms of disease burden estimates, owing to sampling biases [3].

Leptospirosis is hyperendemic in Sri Lanka. Since 2008 [4], incidence based on routine notification exceeds 4,000 cases per year. Moreover, since 2003, prospective studies from Kandy [5], Colombo [6,7], Gampaha [6], Galle [8], Peradeniya [9,10], Kegalle [11], and Anuradhapura [12] have reported different rates of severe disease, varying case fatality rates, and a wide range of clinical manifestations. These differences have been attributed, at least in part to micro-geographical differences [12]; molecular characterization has demonstrated that infecting Leptospira different in different ecological and socio-demographic contexts [7,9,11,12].

The different disease outcomes in leptospirosis could be attributed to various factors such as; presence of virulence factors in certain serovar [13] or difference in host immune response [14] such as patterns of cytokine production in early phase of the infection. IL-6, IL-8, IL-10 TNF-α and long pentraxin-3 (PTX3) are shown to be closely related to disease severity and mortality [14-17]. However, Mikulski et al showed that TNF-α, IL-β, IL-1ɤa, PTX3, IL-6 and IL-10 levels between mild and severe cases are not significantly differ [18]. Contradictory results are available for IL-10/TNF-α ratio [17,18]. Bandara et al shows an involvement of Th17 cells in the immunopathogenesis of leptospirosis with an increase in IL-17A, IL-21 and IL-23 levels [19]. However, the cytokine response reported in different studies were shown to be not conclusive and more data are required to have conclusive evidence [20].

To better understand the various aspects of clinical leptospirosis, robust data based on prospective studies with minimal sampling bias need to be carried out. The present study aimed to describe the full clinical spectrum and laboratory findings (including cytokine response) of a prospectively studied, systematically sampled cohort of patients from a single clinical research unit over a 1-year period in Anuradhapura, Sri Lanka.

Materials and methods

Study setting

This prospective, longitudinal cohort study included subjects based on presenting with a leptospirosis-compatible illness, beginning at the time of each patient’s hospital admission and continuing through outpatient follow-up after discharge up to 14 days. The study was carried out in the Faculty of Medicine and Allied Sciences teaching unit of the teaching hospital, Anuradhapura (THA) affiliated with Rajarata University of Sri Lanka. Anuradhapura district typically reports 100–150 cases of leptospirosis annually, with the majority of cases reported from THA. THA, the only sentinel site in Anuradhapura district for leptospirosis surveillance, has three medical units at the time of present study, and patient admission is equally distributed among these units.

Study enrollment

All clinically suspected cases of leptospirosis at THA were consecutively recruited for this study. The study was a component of a larger fever-surveillance study, and systematic assessment and investigations were carried out among all febrile patients admitted to the THA teaching unit from June 2012 to May 2013. Dengue, typhus, and leptospirosis were the primary focuses, and after excluding all other causes of fever, all remaining undifferentiated fever patients were assessed clinically for possible cases of leptospirosis. We previously showed that such a case definition may have low sensitivity—especially to detect anicteric leptospirosis and mild cases [11]; therefore, we did not use the surveillance case definition proposed by the WHO and adopted by the epidemiology unit of Sri Lanka for possible cases. Rather, we used a less stringent case definition as proposed earlier to enhance inclusivity [21]. All clinically suspected cases were considered as “possible” cases of leptospirosis.

Patient recruitment and data collection

During the study period, the first author, a physician in the THA teaching unit assessed all febrile patients daily to consider each patient as a “possible” case for enrollment. All febrile cases were assessed using a symptom and sign checklist given upon admission, at which time a serum sample was also collected by a registered nurse. Data were collected using an interviewer-administered questionnaire. A standardized protocol was used to systematically record clinical and laboratory data. All patients were assessed daily while hospitalized. From day 1 of hospital admission, routine measurements included complete blood counts, serum creatinine, blood urea nitrogen, transaminases, alkaline phosphatase, bilirubin level, serum electrolytes, and urine biochemistry. In addition, a routine X-ray was done for all patients who exhibited at least one sign of respiratory involvement. All patients with probable cardiac involvement had electrocardiograms performed. All patients were given an appointment for follow-up assessment, and the examinations and routine investigations were carried out during the follow-up visit at 14 days post-discharge from the hospital. All samples were stored in -80°C until further analysis.

All cases were confirmed according to the WHO Collaborating Leptospirosis Reference Laboratory in France using the microscopic agglutination test (MAT) with a broad panel of serovars (Table 1). We also used a lateral flow immunoassay (Immunemed Leptospira, Korea) as a bedside/point-of-care diagnostic test. (Dahanayaka et al., 2017) Final diagnosis was established based on a combination of results from these tests. In MAT titre, single high titre>1/400, seroconversion of fourfold increase were considered as disease confirmation. Positive MAT titre without falling in to those categories or positive LFIA was considered as probable cases. All other clinically suspected cases were considered as possible cases.

Table 1

Serovar used in the MAT panel.

Serial	Species	Serogroup	Serovar	Strain
1	L. interrogans	Australis	Australis	Ballico
2	L. interrogans	Autumnalis	Autumnalis	Akiyami A
3	L. interrogans	Bataviae	Bataviae	Van Tienen
4	L. interrogans	Canicola	Canicola	Hond Utrecht IV
5	L. borgpetersenii	Ballum	Castellonis	Castellon 3
6	L. kirschneri	Cynopteri	Cynopteri	3522 C
7	L. kirschneri	Grippotyphosa	Grippotyphosa	Moskva V
8	L. interrogans	Sejroe	Hardjobovis	Sponselee
9	L. interrogans	Hebdomadis	Hebdomadis	Hebdomadis
10	L. interrogans	Icterohaemorrhagiae	Copenhageni	Wijnberg
11	L. noguchii	Panama	Panama	CZ 214 K
12	L. biflexa	Semaranga	Patoc	Patoc 1
13	L. interrogans	Pomona	Pomona	Pomona
14	L. interrogans	Pyrogenes	Pyrogenes	Salinem
15	L. borgpetersenii	Sejroë	Sejroë	M 84
16	L. borgpetersenii	Tarassovi	Tarassovi	Mitis Johnson
17	L. interrogans	Icterohaemorrhagiae	Icterohaemorrhagiae	Verdun
18	L. weilii	Celledoni	ND	2011/01963
19	L. interrogans	Djasiman	Djasiman	Djasiman
20	L. borgpetersenii	Mini	ND	2008/01925
21	L. weilii	Sarmin	Sarmin	Sarmin
22	L. santarosai	Shermani	Shermani	1342 K
23	L. borgpetersenii	Javanica	Javanica	Poi
24	L. noguchii	Louisiana	Louisiana	LUC1945

Multiplex cytokine assay

Multiplex cytokine assay was performed at Scripps Research Institute, La Jolla, Sand Diego. A magnetic bead–based multiplex cytokine kit (Bio-Plex Pro Human Cytokine, [Bio-Rad, Hercules, CA) was used to detect 17 cytokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, Interferon-γ (IFN-γ), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1b), and tumor necrosis factor (TNF-α). Briefly, antigens supplied with the kit were used to generate a standard eight-point dilution series. Samples were diluted 1:100 using the Bio-Plex standard diluent. The coupled beads (50 μL) followed by standard or test sample were added to the wells of a pre-wetted 96-well microtiter plate. After the recommended washing, plates were incubated for 30 min in a shaker (850 rpm) at 25°C. Detection antibody mixture (25 μL) was added, and the samples were incubated on ice for 30 min and then washed. Finally, 50 μL of streptavidin-phycoerythrinwas added, and after the recommended incubation [on ice for 10 min] and washing cycles, the beads were resuspended in 125 μL assay buffer and analyzed using the BioPlex suspension array system (Bio-Rad Laboratories) and Bio-Plex manager software. A curve fit was applied to each standard curve according to the manufacturer’s manual, and sample concentrations were interpolated from the standard curves. Each sample was assessed in triplicate, and the mean cytokine concentration was taken as the final value.

Levels of individual cytokines were statistically aggregated using principal component analysis with Varimax rotation and Kaiser Normalization. This was done to understand the complex relationship between cytokines and the clinical outcomes.

Written informed consent was obtained from all eligible patients prior to enrollment in the study. Ethics clearance for this study was obtained from the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka.

Results

Case confirmation

From June 2012 to May 2013, 142 clinically suspected leptospirosis patients were recruited to the study. The cohort included 121 males (85%) and 21 females (15%). Median age of the patients was 41 years (interquartile range 32–52 years) with approximately equal number of men and women. Paired serum samples were available for analysis among 79 (55.6%) patients. Of the study sample, 36 men, and 11 women were confirmed to have leptospirosis (Fig 1). In addition, 29 cases were labeled as “probable” based on the pre-set case definitions used. The rest (66) were considered as possible cases.

Fig 1

Leptospirosis case confirmation among febrile patients admitted to the professorial medical unit, teaching hospital Anuradhapura from June 2012 to May 2013.

Temporal distribution of cases

There was a clear peak of leptospirosis cases from December to March, which was compatible with the case notifications received by the regional epidemiologist for leptospirosis from Anuradhapura district (Fig 2).

Fig 2

Comparison of trends of leptospirosis cases admitted to the professorial medical unit, teaching hospital Anuradhapura from June 2012 to May 2013 and routinely reported data from Anuradhapura district, Sri Lanka during the same period.

Exposure history

Of the 76 cases (confirmed and probable), an exposure history was available for 74. The most common exposure type was working in rice paddy fields (n = 63, 85%), proximity to marshy land (n = 32, 43%), having contact with stagnant water (n = 19, 26%), and having contact with surface waters (n = 16, 22%). All confirmed cases were able to provide a typical history of one of these types of, whereas 3 probable cases and 12 possible cases were unable to recall any likely leptospiral exposure.

Clinical profile of patients

Median duration of fever on admission was 4 days (IQ range 2–5 days), with no difference among the confirmed, probable, and possible cases. Assessment of symptoms and signs on admission revealed that myalgia and arthralgia were the commonest clinical presentation associated with fever (Table 2). Classical features of leptospirosis, such as conjunctival suffusion and icterus, were present in ~25% of study participants. There was no significant difference (Chi square test, p < .05) between possible probable or confirmed cases and even after amalgamating confirmed and probable cases, results were same.

Table 2

Clinical features of leptospirosis presented by patients admitted to the teaching hospital Anuradhapura, Sri Lanka from June 2012 to May 2013.

Clinical feature	Confirmed n %		Probable %		Possible n %		Total n %
Myalgia	46	97.9	22	75.9	60	90.9	128	90.1
Arthralgia	42	89.4	25	86.2	59	89.4	126	88.7
Retroorbital pain	23	48.9	11	37.9	22	33.3	56	39.4
Abdominal pain	24	51.1	11	37.9	20	30.3	55	38.7
Vomiting	24	51.1	8	27.6	22	33.3	54	38.0
Calf muscle tenderness	16	34.0	6	20.7	23	34.8	45	31.7
Conjunct injection	18	38.3	6	20.7	14	21.2	38	26.8
Hepatomegaly	17	36.2	8	27.6	10	15.2	35	24.6
Reduced urine output	14	29.8	9	31.0	9	13.6	32	22.5
Thigh muscle tenderness	10	21.3	5	17.2	17	25.8	32	22.5
Chest pain	9	19.1	5	17.2	16	24.2	30	21.1
Icterus	13	27.7	5	17.2	11	16.7	29	20.4
Diarrhea	12	25.5	3	10.3	12	18.2	27	19.0
RH tenderness	10	21.3	2	6.9	6	9.1	18	12.7
Epigastric tenderness	4	8.5	4	13.8	2	3.0	10	7.0
Palpitation	3	6.4	2	6.9	4	6.1	9	6.3
Flushed appearance	2	4.3	1	3.4	6	9.1	9	6.3

None of these differences were significant (Chi square test, p>.05).

Clinical evolution of leptospirosis among the cohort

All hospitalized study participants were clinically assessed daily by one of the investigators. For all study participants, routine laboratory tests were carried out daily, including white cell and neutrophil counts. Total white cell count, neutrophil count and platelet count was not significantly differ between confirmed/probable and possible cases (S1 Dataset). Further analysis was done only for confirmed and probable cases (Fig 3). Thrombocytopenia was common at the time of hospital admission. Total white cell and neutrophil counts dropped steadily until day 8, whereas the platelet count remained below the normal range throughout. Total white cell, neutrophil, and platelet counts returned to normal at 2 weeks post-discharge from the hospital among all cases. A slight increase of packed cell volume (PCV) on day 2–4 was observed among all confirmed cases (S1 Dataset). Of the 47 confirmed cases, 13 (27.6%) had PCV increase more than 20% of the baseline value.

Fig 3

Changes in total white cell, neutrophil, and platelet counts during the course of illness among probable and confirmed cases of leptospirosis admitted to the professorial medical unit, teaching hospital Anuradhapura, Sri Lanka from June 2012 to May 2013.

Complications and sequelae

Clinically significant hemorrhage was not observed in confirmed leptospirosis patients, despite thrombocytopenia. Acute kidney injury, defined as an increase in serum creatinine of >1.5-fold from baseline, was observed in 31 possible, probable and confirmed patients (22%). Among these, 15, 10, and 6 patients had risk, injury, or kidney failure, respectively, according to the RIFLE classification [22]. Confirmed/probable patients were having significantly higher percentage (31.6% vs 10.6%) of AKI compared to possible cases. Only AKI was significantly different between confirmed/probable vs possible cases (Table 3).

Table 3

Complications observed among patients with leptospirosis admitted to the professorial medical unit from June 2012 to May 2013, and routinely reported data from the Anuradhapura district during the same period.

	Confirmed (47)		Probable (29)		Possible (66)		Total
	n	%	n	%	n	%	n	%
Liver involvement
SGPT>60 ul	17	36.2	4	13.79	23	34.9	44	31.0
Bilirubin (direct)>7 mmol/L	4	8.5	1	3.45	5	7.6	10	7.1
Acute kidney injury	19	42.5	7	36.8	11	14.9	31	21.8
Hypotension or shock
SBP<90mmHg	15	31.9	4	13.79	11	16.7	30	21.1
Required inotropic support	10	21.3	2	6.90	5	7.6	17	12.0
Lung involvement
Saturation drop <92%	1	2.1	1	3.45	6	9.1	8	5.6
Diffuse alveolar shadowing	4	8.5	3	10.3	3	4.6	10	7.0
Myocarditis	1	2.1	0	0.0	3	4.6	4	2.8

The duration of hospitalization ranged from 1 to 12 days with a median of 3 days. During the study period, one death occurred (case fatality rate, 0.7%). This was a 51-year-old male part-time farmer whose fever had been present for 6 days before admission and he died on the 10th day of hospitalization from a syndrome that included oliguric renal failure, pulmonary hemorrhage and refractory shock. Serum creatinine was 140 μmol/L (normal range 90–120 μmol/L) on admission. This patient had a MAT titer of 1/200 and a positive Leptospira IgM in LFIA.

Cytokine analysis of study participants

We prospectively measured serum cytokines in patients suspected to have leptospirosis, with the long-term goal of identifying potential a biosignature of acute leptospirosis. Though acute sample cytokines were done for 43 confirmed/ probable cases, complete clinical and diagnostic data were available from 41patients. The curve fit statistics of the cytokine analysis shows that the generated data are not deviated from the standard curve, confirming the validity of cytokine estimates. The metadata include 30 confirmed and 11 probable cases. Human (Hu) MIP-1b was detected in all samples, and Hu MCP-1 (also called MCAF), Hu IL-8, Hu IL-6, and Hu GM-CSF were detected in more than 75% of the samples. Hu IL-5 and IL-1β were detected in only 2 and 4 samples, respectively, and hence were excluded from the cytokine analysis of the acute samples (Fig 4).

Fig 4

Distribution of cytokines during the acute phase of illness among probable and confirmed cases of leptospirosis admitted to the professorial medical unit, teaching hospital Anuradhapura from June 2012 to May 2013.

We compared the distribution of cytokines among those patients who had complications with those who lacked complications (Table 4). Of the 41 patients studied, 17 (42%) had one or more complications. Impairment of renal function (serum creatinine >1 mg/dL for females, >1.2 mg/dL for males) was observed for 16 patients (39%). Eleven patients (27%) had hypotension (systolic blood pressure <90 mmHg, and diastolic blood pressure <60 mmHg), and 6 patients (15%) required treatment with inotropic agents. Six (15%) patients had pulmonary involvement with abnormal chest X-rays, and 3 patients required intubation and ventilator support.

Table 4

Distribution of cytokines during the acute phase of illness among confirmed and probable leptospirosis patients with or without complications.

	Complications
	Yes (n = 17)		No(n = 24)		p value
	Range	Median	Range	Median
Hu IL-2	0–45.4	0.0	0–394.5	1.0	0.117
Hu IL-4	0–3.5	0.0	0–30.7	0.0	0.898
Hu IL-5	0–90.0	0.0	0–10.0	0.0	0.777
Hu IL-6	0–174.7	30.2	0–99499.4	19.5	0.285
Hu IL-7	0–114.6	0.0	0–12.2	0.0	0.936
Hu IL-8	0–12196.1	50.5	0–6872.1	13.1	0.067
Hu IL-10	0–712.0	12.2	0–846.8	13.7	0.989
Hu IL-12	0–304.5	0.0	0–1773.5	13.5	0.34
Hu IL-13	0–180.5	0.0	0–45.8	0.0	0.869
Hu IL-17	0–28.0	0.0	0–63.0	0.0	0.904
Hu G-CSF	0–14.8	5.7	0–2759.0	5.1	0.58
Hu GM-CSF	0–927.2	215.2	0–2381.2	144.0	0.778
Hu IFN-γ	0–600.3	0.0	0–15066.5	0.0	0.946
Hu MCP-1 (MCAF)	0–481.9	61.8	0–12375.9	42.6	0.853
Hu MIP-1b	207–1436.6	497.5	92–18771.9	300.2	0.028
Hu TNF-α	0–0.0	0.0	0–592.7	0.0	0.028
IL-1β	0–0.0	0.0	0–1808	0.0	0.081

The Mann Whitney U test was used for data analysis. None of the differences were significant.

Hu TNF-α and IL-1β were detected only in patients without complications. Hu MIP-1b was detected in all 41 patients, and the levels were significantly higher among patients with complications. While Hu IL-8 appeared to be elevated among patients with complications, the difference was not statistically significant from uncomplicated cases.

Of the 41 leptospirosis cases with acute sample data, only 14 were followed up as outpatients (8 confirmed and 6 probable), and samples from convalescent patients were available with at least 10 days from the acute phase to convalescence. Of the 14 cases for which paired samples available, 7 (50%) had one or more complications. The distribution of serum cytokine level during the convalescence period was not significantly different between patients with or without complications (Table 5). Analysis of individual cytokines and complications shows that different cytokines are associated with different outcomes (Table 6).

Table 5

Distribution of cytokines during the convalescence phase among confirmed and probable leptospirosis patients with or without complications.

	Complications
	Yes (n = 7)		No (n = 7)		p value
	Range	Median	Range	Median
Hu IL-2	0–45.9	0	0–63.4	14.6	0.535
Hu IL-4	0–5.1	0	0–12.7	0	1.000
Hu IL-5	0–10.7	0	0–0	0	0.710
Hu IL-6	0–2265.5	0	0–20088.4	7.1	0.805
Hu IL-7	0–24.4	0	0–0	0	0.710
Hu IL-8	0–7736.2	53	0–2630.7	34.7	0.902
Hu IL-10	0–260.2	0	0–44.5	0	0.620
Hu IL-12	0–251.8	19.7	0–48.9	0	0.318
Hu IL-13	0–50.1	0	0–4.6	0	0.383
Hu IL-17	0–25.8	0	0–0	0	0.383
Hu G-CSF	0–28.8	0	0–253	0	1.000
Hu GM-CSF	0–781	518.2	126.7–775.6	290.5	1.000
Hu IFN-γ	0–572.9	46.4	0–876.8	0	0.535
Hu MCP-1 (MCAF)	21–2631.7	36.7	0–3387.2	28.3	0.620
Hu MIP-1b	105.2–6199.4	387.6	0–451.7	299.2	0.259
Hu TNF-α	0–51	0	0–424.4	0	1.000
IL-1β	0–58.9	0	0–880.1	0	0.710

The Mann Whitney U test was used for data analysis.

Table 6

Acute phase cytokine levels and selected outcomes among confirmed and probable leptospirosis patients.

Complication	Associated Cytokine	Significance
Thrombocytopenia (Platelet <150,000)	IL-8	p-0.02MVU-100.000
Cardiac involvement requiring Inotropes	MIP-1b	p-0.01MVU-39.000
Billirubin (direct)>7 mmol/l	IL-13	p-0.035MVU -11.000
High serum creatinine (Serum creatinine>100mg/dl for females/120mg/dl per males)	GM-CSF	p-0.03MVU-123.000
Oxygen given	MIP-1b	p-0.06MVU-55.000
Lung involvement	IL-5	p-.045MVU-57.000
Saturation drop	IL-6	p-0.07MVU-10.000
Hypotension	MIP-1b	p-0.08MVU-107.000
	IL-8	p-0.09MVU-109.000

During the convalescence period, all tested serum cytokine levels were lower compared to the acute sample, except for IL-8 (Fig 5). Changes in serum cytokine levels from acute-stage samples to convalescence-stage samples were tested using the paired-sample t test. None of the cytokines showed significant differences in paired samples between patients with and without complications.

Fig 5

Distribution of serum cytokine levels in samples from acute and convalescent patients among probable and confirmed cases of leptospirosis from the professorial medical unit, teaching hospital Anuradhapura from June 2012 to May 2013.

The principal component analysis showed a four factor solution for cytokines which explained 87.6% of the variation (Bartlett’e test of sphericity, chi-square 1298, p < .001). As shown in Table 7, the first component included Hu G-CSF, Hu IL-6, Hu MIP-1b, Hu MCP-1(MCAF), Hu IL-4 and IL-1b. IL-8 loaded as a separate component on its own (Table 6). IL-5, IL-7 and IL-13 were in a separate group with rest in another group. The observed groups were not the typical proinflammatory or anti-inflammatory groups as described in the literature. The cytokines group 1 was positively associated with increased serum creatinine levels (MVU 136, p .087) and the group 4 with Thrombocytopenia.

Table 7

Factor analysis of cytokine response.

Rotated Component Matrixa
	Component
	1	2	3	4
Hu G-CSF	0.989
Hu IL-6	0.988
Hu MIP-1b	0.981
Hu MCP-1(MCAF)	0.974
Hu IL-4	0.907
IL-1b	0.753
Hu IFN-g		0.978
Hu TNF-a		0.967
Hu IL-12(p70)		0.876
Hu IL-2	0.323	0.837
Hu GM-CSF		0.829
Hu IL-10		0.694
Hu IL-5			0.989
Hu IL-7			0.987
Hu IL-13			0.984
Hu IL-8				0.993

Extraction Method: Principal Component Analysis.

Rotation Method: Varimax with Kaiser Normalization.

a Rotation converged in 4 iterations.

Discussion

Predicting the evolution of acute leptospirosis to severe, complicated disease remains a challenge owing to wide variation of clinical disease, complex disease transmission pattern, and variability of local mammalian reservoir hosts, local ecology, climate, host animals and virulence of local, geographically dominant infecting Leptospira. Where leptospirosis is endemic—usually in resource-limited settings—routine diagnostic facilities are not typically available. Hence reliable case reporting is generally not available leading to an underestimation of local as well as global disease burden [23]. Disease burden estimates based on country-specific statistics are not precise because reported data are biased in many ways, particularly based on the identification of typical and severe case presentation for which diagnostic testing was sought.

Despite the prospective nature of the present study, in which sensitive criteria for suspecting cases was adopted and rigorous case definition was employed, we were not able to identify a comprehensive cytokine biosignature of acute or complicated leptospirosis. Routine laboratory testing yield results consistent with previous reports, for example more prominent changes with the same pattern of complete blood counts and serum chemistries, as reported by de Silva et al. (2014) from Colombo, Sri Lanka. This may be attributable to the wide clinical spectrum among patients in our study compared with the study of Silva et al. [24]. Here, we observed that leukocytosis not a feature of leptospirosis in our study region, even though this is expected for bacterial infections. However leukopenia usually differentiates leptospirosis from dengue, which typical is associated with low white blood cell counts. Thrombocytopenia was observed in more than half of our study cohort, which might complicate the clinical diagnosis because of potential confusion with dengue, in places such as Sri Lanka where both infections are transmitted. Hematoconcentration might be a helpful differentiator, a laboratory maker in dengue. However, out data suggested that among confirmed leptospirosis patients, around one fourth were having marked variation in PCV during hospital stay, which may complicate the diagnosis furthermore.

Patterns of leptospirosis complications and clinical features observed in the present study differ from those previously published during a post-flood outbreak of leptospirosis [12]. We observed a high level of renal involvement (55% compared with 22%), but myocarditis was less common (2% compared with 16% among confirmed cases). In addition, we observed that nearly one-third of the confirmed cases developed hypotension with 20% requiring treatment with inotropic agents, which was not observed in our previous studies [11,12]. Observing these markedly different clinical features and outcomes strengthens the hypothesis that endemic and epidemic leptospirosis may differ even in the same regional setting, and this is probably attributable to different infecting Leptospira species or serovars present there [12]. This is a reasonable working hypothesis in countries like Sri Lanka where at least five different species of Leptospira that can cause human leptospirosis are currently circulating, with more than 40 identified serovars [25].

The presently reported cytokine profiles in severe and mild leptospirosis cases, with a subset of such cases including both acute and convalescence phases, resulted in more questions than answers. “Cytokine storm” has been previously reported as a marker of disease severity in leptospirosis [26]. Our prospective study did not confirm such results, but instead results were more complex, with elevated IL-6 and IL-8 among patients with complications. These findings are compatible with previous cross-sectional studies. In contrast we could not confirm a previously reported difference in mean IL-10 levels among severe and mild cases [15,17,27]. IL-10 is known to regulate inflammation by down-regulating monocyte-derived TNF-α and IL-1 [28]. TNF-α was undetectable in most cases, but if detected it was only in patients without complications. Acute-phase MIP-1b level ranged from 92 to 18772 units for patients without complications and from 207 to 1437 for patients with complications. A previous study reported higher levels of MIP-1b (11–2442 units) in severe cases and lower levels (0–765 units) in mild cases [27]. These findings are contradictory but could be attributable to differences in host response to different serovars that might be present in the two study settings. In acute and convalescence phases, we observed marked reduction of almost all cytokines indicated disease resolution. Papa et al. (2015) reported a gradual reduction of IL-6 and IL-8 within the first 5 days of illness and an increase thereafter [27], which differs from what we previously observed [27]. During the convalescence phase, however, the levels of both IL-6 and IL-8 were lower, and there was no significant change in their levels throughout convalescence.

In this study, we were unable to identify a biosignature of leptospirosis using a large panel of cytokine measurements. As reported in several other studies, our study clearly shows the inadequacy of routinely reported data for disease burden estimates even in a known hyperendemic region. The district of Anuradhapura has six other hospitals with more than 100 beds, and another 32 small hospitals with inpatient facilities. Throughout the study period, however, we discovered that more than half of the number of leptospirosis patients in the district were reported by one of the various university teaching units. These data clearly suggest that more than half of the suspected inpatients did not diagnose leptospirosis. In addition, patients seeking outpatient care–which is likely the majority of cases—were not included in these estimates, which biases both reported and understanding the basis of severe leptospirosis disease and complications. This underreporting could be due to a lack of clinical suspicion, as we observed previously [11], lack of diagnostic facilities owing to problems with reporting, or these factors in combination. Current data indicate that approximately 4,000 cases of leptospirosis are reported annually to the central epidemiology unit of the Sri Lanka Ministry of Health. However, the actual number may exceed 10,000 based on observations reported in the present and previous studies [12]. Systematic, population-based studies of patients with undifferentiated fever with efficient diagnostics are to support better public health policies to control this disease on local, regional, national and international levels.

The results of this study have to be interpreted within the study limitations. The analysis is based on confirmed probable and possible cases. On clinical grounds, we have not seen marked differences in probable and possible cases. In a setting where other tropical diseases such as hantavirus infection are common, these probable cases may affect the results. However, these observations, especially the lack of association, needs to be documented in the scientific literature to avoid publication bias. For a better interpretation of results, all data used are provided as supporting information file (S1 Dataset) so that other researchers could use the raw data for further analysis.

The clinical, biochemical and cytokine data of patients.

(SAV)

Click here for additional data file.

2 Nov 2021

PONE-D-21-30348Clinical spectrum of endemic leptospirosis in relation to cytokine responsePLOS ONE

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Reviewer #1: The aim of the study was to describe clinical and laboratory features (including cytokine profile - biosignature) of leptospirosis patients in Sri Lanka.

First there is a sample problem. Of the 142 recruited patients, confirmed leptospirosis diagnosis was made only in 47. There were 95 probable or possible cases, based on clinical features (66) and doubtful lab results (29). These harms the analysis. Symptoms suggestive of leptospirosis can be confounded with many other febrile illnesses. Severe cases, more easily diagnosed, were the minority. Clinical features of confirmed, probable and possible cases were the same.

Symptoms suggestive of leptospirosis (calf muscle pain - 31.7%, conjunctival injection - 26.8%, flushed appearance - 6.3%, jaundice - 20.4%) were little frequent. General symptoms, like myalgia, headache, vomiting, etc.) were more frequent.

Second, little more than half of the sample had paired samples for serology. I know it is difficult to get follow-up samples, but it was a clinical study and patients should had been stimulated to come for a follow-up visit and sample collection, to improve the diagnosis yield.

Third, there is no difference between confirmed, probable and possible cases in clinical grounds. This is expected regarding differential diagnosis of leptospirosis in acute febrile illnesses context. So, there is no guarantee that patients with other diseases where included in the analysis, spoiling results.

Figure 3 is quite confusing. What does it means? What is the reaction between duration of fever and cell counts?

Table 3 - confirmed cases had more AKI and shock, but possible cases had more lung involvement and myocarditis. Could it be other disease? Did you considered the possibility of hantavirus?

Does an autopsy was performed on the dead patient? Any attempt to collect tissue for pathological examination and diagnosis?

Cytokine analysis to identify a biosignature of acute leptospirosis - only 41 confirmed (30) and probable cases (11) available for acute analysis. Cytokine profile of complicated cases differed from non complicated cases only on MIP-1b and TNF alpha, but no TNF was detected on complicated cases. This is awkward and unexpected. TNF alpha was expected to be detected in high levels in complicated cases with AKI, shock and lung involvement. By the same way, MIP-1b levels were significantly higher in non complicated cases. These can be observed for the other cytokines levels range. I would like to see the mean and s.d. of cytokines levels instead of the median. MIP-1b broader range on non complicated patients suggests it is not good for case severity discrimination.

Paired cytokine analysis was available in 14 cases (8 confirmed and 6 probable), a very low number os cases. Its impossible to infer a cytokine profile or biosignature with such a low number os patient analysed.

Maybe the choice of statistical method should be reviewed. It is very hard for a medical doctor to deal with obscure statistical techniques. Risk analysis, logistic regression and other more common statistics could be used. Despite this, it is clear to me that no cytokine profile could be inferred from these sample. I consider difficult or even impossible to obtain a cytokine signature for leptospirosis. As you said in discussion, there are many different kinds of leptospirosis, depending on serovar and host. It is a complex relationship. Leptospirosis immune pathogenesis is not yet well understood. We need to study how leptospira express proteins and produces tissue lesion and how host response developes after infection. Epidemiological and social factors may influence response, like nutritional status, previous diseases, etc.

As in bacterial sepsis, probably we will not be able to define a cytokine biosignature for severe leptospirosis.

More efforts must be concentrated in stimulating clinical suspicion and fast laboratory diagnosis, as well as in understanding disease mechanisms.

**********

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Reviewer #1: Yes: Decio Diament

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12 Nov 2021

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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The manuscript file was edited accordingly.

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We have edited the manuscript and included the full data set as a supporting information.

Reply to reviewers

Reviewer #1: The aim of the study was to describe clinical and laboratory features (including cytokine profile - biosignature) of leptospirosis patients in Sri Lanka.

First there is a sample problem. Of the 142 recruited patients, confirmed leptospirosis diagnosis was made only in 47. There were 95 probable or possible cases, based on clinical features (66) and doubtful lab results (29). These harms the analysis. Symptoms suggestive of leptospirosis can be confounded with many other febrile illnesses. Severe cases, more easily diagnosed, were the minority. Clinical features of confirmed, probable and possible cases were the same.

Symptoms suggestive of leptospirosis (calf muscle pain - 31.7%, conjunctival injection - 26.8%, flushed appearance - 6.3%, jaundice - 20.4%) were little frequent. General symptoms, like myalgia, headache, vomiting, etc.) were more frequent.

Second, little more than half of the sample had paired samples for serology. I know it is difficult to get follow-up samples, but it was a clinical study and patients should had been stimulated to come for a follow-up visit and sample collection, to improve the diagnosis yield.

Third, there is no difference between confirmed, probable and possible cases in clinical grounds. This is expected regarding differential diagnosis of leptospirosis in acute febrile illnesses context. So, there is no guarantee that patients with other diseases where included in the analysis, spoiling results.

We humbly accept the point raised. These are common but major issues in clinical research in resource poor settings. We have included these as limitation of our study.

“The results of this study have to be interpreted within the study limitations. The analysis is based on confirmed probable and possible cases. On clinical grounds, we have not seen marked differences in probable and possible cases. In a setting where other tropical diseases such as hantavirus infection are common, these probable cases may affect the results. However, these observations, especially the lack of association, needs to be documented in the scientific literature to avoid publication bias. For a better interpretation of results, all data used are provided as supporting information file (S1 Dataset) so that other researchers could use the raw data for further analysis..”

In addition, the observation that there was no clear difference in probable and possible cases is a major clinical dilemma clinicians are having. Unfortunately, these are not often published in literature, while only the “significant differences” are published leading to a publication bias.

Figure 3 is quite confusing. What does it means? What is the reaction between duration of fever and cell counts?

The figure shows the daily changes of cell counts over the course of illness. This is important for clinicians in these settings where they mostly use clinical features and basic investigations to understand the disease.

Table 3 - confirmed cases had more AKI and shock, but possible cases had more lung involvement and myocarditis. Could it be other disease?

This is actually quite common in leptospirosis. The lung involvement is more common with certain species of leptospirosis, while renal involvement is common in some other species. We have previously reported these microgeographical changes and carried out several studies to confirm this. We have included this point in the discussion.

“Patterns of leptospirosis complications and clinical features observed in the present study differ from those previously published during a post-flood outbreak of leptospirosis. We observed a high level of renal involvement (55 % compared with 22%), but myocarditis was less common (2% compared with 16 % among confirmed cases). In addition, we observed that nearly one-third of the confirmed cases developed hypotension with 20% requiring treatment with inotropic agents, which was not observed in our previous studies. Observing these markedly different clinical features and outcomes strengthens the hypothesis that endemic and epidemic leptospirosis may differ even in the same regional setting, and this is probably attributable to different infecting Leptospira species or serovars present there.”

Did you considered the possibility of hantavirus?

The problem of hantavirus infection is a great concern as suggested. Some of these cases could be hantavirus as shown by a recent study done in the same setting. We have added this as a limitation in the discussion.

Does an autopsy was performed on the dead patient? Any attempt to collect tissue for pathological examination and diagnosis?

No. This could have been a major addition to the study if we could have done. But unfortunately no postmortem samples were taken.

Cytokine analysis to identify a biosignature of acute leptospirosis - only 41 confirmed (30) and probable cases (11) available for acute analysis. Cytokine profile of complicated cases differed from non complicated cases only on MIP-1b and TNF alpha, but no TNF was detected on complicated cases. This is awkward and unexpected. TNF alpha was expected to be detected in high levels in complicated cases with AKI, shock and lung involvement. By the same way, MIP-1b levels were significantly higher in non complicated cases. These can be observed for the other cytokines levels range. I would like to see the mean and s.d. of cytokines levels instead of the median. MIP-1b broader range on non complicated patients suggests it is not good for case severity discrimination. Paired cytokine analysis was available in 14 cases (8 confirmed and 6 probable), a very low number os cases. Its impossible to infer a cytokine profile or biosignature with such a low number os patient analysed.

We agree with this point and the reason for reporting this study is also to have these data in the literature. We firmly believe that publication bias involving “no association” has heavily impacted clinical practice where the journal publication process systematically selecting only “positive associations”. Based on the reviewers comments, we have included all cytokine data with other clinical data as a supporting information file, so that it will be available for readers to download and interpret. As shown in the supplementary file data, the cytokine distribution is extremely skewed and showing the mean and SD will distort the results. This was the reason for using median. However, the supplementary file is provided now with all data.

We have included following points in the discussion to discuss these issues.

“In this study, we were unable to identify a biosignature of leptospirosis using a large panel of cytokine measurements.”

Maybe the choice of statistical method should be reviewed. It is very hard for a medical doctor to deal with obscure statistical techniques. Risk analysis, logistic regression and other more common statistics could be used.

The statistical test used in one of the most simple test (Mann Whitney U test). This is the test that can be used for highly skewed distribution. The suggested statistical tests are not useful for the intended analysis. The factor analysis is required because cytokine profiles could not be analyzed separately. (and the analysis was done by two medical doctors)

Despite this, it is clear to me that no cytokine profile could be inferred from these sample. I consider difficult or even impossible to obtain a cytokine signature for leptospirosis. As you said in discussion, there are many different kinds of leptospirosis, depending on serovar and host. It is a complex relationship. Leptospirosis immune pathogenesis is not yet well understood. We need to study how leptospira express proteins and produces tissue lesion and how host response developes after infection. Epidemiological and social factors may influence response, like nutritional status, previous diseases, etc.

As in bacterial sepsis, probably we will not be able to define a cytokine biosignature for severe leptospirosis.

More efforts must be concentrated in stimulating clinical suspicion and fast laboratory diagnosis, as well as in understanding disease mechanisms.

Agree fully with this comment. This was the reason for us to include the conclusion (highlighted)

“Results of this study confirms that the knowledge on cytokine response in leptospirosis could be more complex than other similar tropical disease, and biosignatures that provide diagnostic and prognostic information for human leptospirosis remain to be discovered.”

We have included these points in the discussion. And as mentioned above It will be important to have these data as evidence to show that cytokine biosignature is not very useful. Thus the data are also included in the manuscript.

“The results of this study have to be interpreted within the study limitations. The analysis is based on confirmed probable and possible cases. On clinical grounds, we have not seen marked differences in probable and possible cases. In a setting where other tropical diseases such as hantavirus infection are common, these probable cases may affect the results. However, these observations, especially the lack of association, needs to be documented in the scientific literature to avoid publication bias. For a better interpretation of results, all data used are provided as supporting information file (S1 Dataset) so that other researchers could use the raw data for further analysis..”

Submitted filename: Responce to reviwers.docx

Click here for additional data file.

23 Nov 2021

Clinical spectrum of endemic leptospirosis in relation to cytokine response

PONE-D-21-30348R1

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: All questions have been addressed adequately. I hope you continue doing research on leptospirosis, regarding differential diagnosis, immune pathogenesis and epidemiology.

**********

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Reviewer #1: Yes: Decio Diament

25 Nov 2021

PONE-D-21-30348R1

Clinical spectrum of endemic leptospirosis in relation to cytokine response

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