Development and Validation of a simple score for diagnosis of Leptospirosis at outpatient departments.

BACKGROUND: Leptospirosis is an important zoonotic disease within the tropics. Diagnosing leptospirosis is a clinical obstacle, as clinical presentations are similar to other tropical infectious diseases. Available serological tests are often insensitive and not cost-effective. Many clinical diagnostic scorings had been developed but most were based on hospitalized patients, and wound not be suitable for use in suspected patients in setting of ambulatory care.
OBJECTIVES: To develop and internal validate multivariable diagnostic prediction score of leptospirosis in patients suspicious of leptospirosis at out-patient clinics of community hospitals.
MATERIALS AND METHODS: We performed a prospective, multisite diagnostic prediction research with development of a diagnostic score. The development cohort was based on patients suspicious of leptospirosis who visited five community hospitals in Si Sa Ket province, Thailand during December 2017 to November 2018. Leptospirosis confirmed cases were defined when one of the three standard confirmatory tests was positive. Multivariable logistic regression was used for score derivation. Test of AuROC equality was done to compare diagnostic performance of the newly derived score and conventional WHO score.
RESULTS: A total of 262 leptospirosis suspicious patients were enrolled. Eighty-two patients (31.5%) were leptospirosis confirmed cases. Five final predictors remained within the reduced logistic model which were history of exposure to wet ground at workplace, history of contact water reservoir used by animal, urine protein and urine blood positive from dipstick test, and neutrophil count from CBC ≥80%. The OPD score diagnostic performance was AuROC 0.72 (95%CI 0.65-0.79). Test of equality revealed significant differences of AuROC between the OPD and WHO score (0.72 vs 0.62, p-value 0.014). Patients were categorized into low and high probability of having leptospirosis at score point of 3.5 with sensitivity 72.4% and specificity 61.7%.
CONCLUSIONS: This study developed and internal validated the OPD score. This clinical risk score could be one of the important tools for diagnosis of leptospirosis at the outpatient clinic.

Introduction

Leptospirosis is an emerging tropical zoonotic disease caused by a genus of spiral-shaped bacteria, Leptospira. Humans usually acquired the disease from environmental exposure to the organism shed from the urine of mammal hosts.[1-3]. In Thailand, the prevalence of leptospirosis was estimated at 5 to 9 cases per 100,000 population with mortality rate at 5 to 10 percent [4]. Si Sa Ket province, a northeastern province with 70 percent of population at risk from occupational exposure [5], reported the highest incidence of 18.7 to 28.2 per 100,000 populations from 2011 to 2015 and also carried three times higher case fatality rate than national averages [6-8].

Diagnosing leptospirosis is a clinical challenge, as the initial presentations are usually difficult to distinguish from other tropical infectious diseases [9-11]. Spectrum of the disease varies from subclinical case, mild case to severe case which could be fatal. Therefore, early detection and early treatment in suspicious groups of patients are vital to attenuate disease progression [1, 11–13]. At present, several options of laboratory investigations are available but each carries its own limitation, especially in countries with limited resources. Bacterial isolations from specimen culture are less sensitive and the results are delayed [11, 14]. Serological testing such as microagglutination test (MAT) needs two sets of blood samples for interpretation of the result and only few laboratories are accessible [15-17]. Rapid antibody test is proved to be non-sensitive and non-specific especially in the early phase of the infection [14, 18–20]. Polymerase chain reaction (PCR) requires specialized laboratories and technicians which is not applicable and not cost-effective [1, 11, 14].

Primary care physician relies primarily on clinical characteristics, signs and symptoms of the patients for diagnosing leptospirosis. Faine’s criteria or WHO score and modified version in 2004 and 2012, were developed as diagnostic guide for clinicians to make presumptive diagnosis of leptospirosis [21-24]. As they were developed from hospitalized patients cohort, the criteria seems to be appropriate for patients with higher degree of severity which require hospitalizationand hospitals where serological investigation are available. However, whether the use of such criteria is suitable for implementation in primary care setting, where patients usually presented in early phase with vague clinical syndrome, is still questionable.

This study aimed to develop a practical diagnostic tool for leptospirosis that incorporates clinical signs and symptoms, history of exposure to possible risks, and routinely available laboratory data to aid physicians and associated health care workers in community hospitals within an endemic area for early detection and early treatment of the disease.

Methods

Ethics statement

The study protocol was approved by the ethical review committee for human research, of The Faculty of Public Health, Mahidol University (MUPH 2017–204), and the ethical committee for research in human subject of Si Sa Ket Hospital (COA No.004 REC No. 071/2560). All study participants were requested for informed consents prior to study inclusion.

Study design and setting

A diagnostic prediction research and clinical diagnostic score development was performed. The data was prospectively collected from five community hospitals with top highest prevalence of leptospirosis within Si Sa Ket Province (Khukhan, Khun Han, Phu Sing, Phrai Bueng, and Prang Ku Hospitals).

Study participants

Patients suspicious of leptospirosis by physicians from initial clinical presentation who visited out-patient clinic in each study site during December 1, 2017 to November 30, 2018 were asked for consents and subsequently included into study. Clinicians suspected the diagnosis of Leptospirosis based on typical syndrome stated in the classic WHO clinical criteria such as the presence of acute febrile illness (onset of fever less than 14 days), headache, myalgia with history of exposure to animal water reservoirs or flooded environments either at home or at work [11]. Patients with unstable vital signs requiring resuscitation at first visit, and patients who were unable to communicate with Thai or local language were excluded and were not interviewed. Patients whose disease progressed and did not survive for the second blood sample collection were excluded from analysis.

Data collection

Enrolled patients were interviewed with standardized questionnaire by trained research personnel. Data collected consists of 5 components which are (1) baseline characteristics and demographic: gender, age, occupation, comorbidities, drinking habit, duration of living within the area, (2) clinical presentations: chief complaint (e.g. fever, myalgia, headache, calf pain), symptoms (e.g. cough, rhinorrhea, red eye, nausea, vomiting, diarrhea, dyspnea, oliguria, jaundice and hemoptysis), onset and duration of chief complaint, prior treatment or visit to other health care providers, prior antibiotics prescribed, (3) physical examination: initial vital signs (body temperature, pulse rate, blood pressure, respiratory rate), calf tenderness, conjunctival suffusion, jaundice, and presence of wounds in hand, foot or leg, (4) exposure to possible environmental risk factors within the past month: flooded house compound, contact with animal, contaminated water and soil around workplace, presence of wound on dependent parts, contact with animal water reservoir, and features of work. (5) hematologic and biochemical laboratory findings: complete blood count, urine dipstick test, and renal function test.

Blood sample and specimen collection

The first set of blood samples were sent for blood culture, real time polymerase chain reaction (PCR) and microscopic agglutination test (MAT). Patients were also scheduled for another visit for the second blood samples collection, which would be used as a paired serum to evaluate rising of MAT.

On the first day of enrollment, 12 ml of blood and 30 ml of urine sample were collected. Blood samples for laboratory investigations other than culture were collected in EDTA tubes and clotted blood tubes. Both plasma and urine sample were centrifuged at 3000g for 10 minutes and were subsequently frozen at -20 degree Celsius until being transferred to the central laboratory. Both samples were stored at -80 degree Celsius until they were taken for analysis. For direct culture of Leptospira, a drop of blood and urine was separately inoculated into 4 mL of liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) at 29 degree Celsius for 14 days. Detection for Leptospira was done with direct observation via dark field microscopy [26]. We confirmed all isolation of leptospira by performing 16S rRNA sequencing. For real time polymerase chain reaction (PCR), 200 μL of whole blood was sampled from EDTA tube. A high Pure PCR template preparation kit (Roche Diagnostics, Germany) was used for DNA extraction with 50 μL elution buffer. The amplification primers for LipL32 gene were LipL32-45F (5’-AAG CAT TAC CGC TTG TGG TG-3’) and LipL32-286R (5’-GAA CTC CCA TTT CAG CGA TT-3’). The fluorescent probe was LipL32-189P (FAM-5’-AA AGC CAG GAC AAG CGC CG-3’-TAMRA). The PCR reactions of the samples were performed in a final volume of 20 μL which contained 5 μL of genomic DNA and 15 μL of reaction mix (10 μL of 2X TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 1 microliter of each 10 μM primer, 0.4 μL of 10 μM probe under 2.6 μL distilled water). The real time PCR program consisted of 45 cycles, each consisting of 95 degree Celsius for 15 seconds and 60 degree Celsius for one minute. Positive and negative controls were included in every experiment done. Results were read by threshold cycle (Ct) value [27]. Microscopic agglutination test or MAT was performed as described in the standard protocol of the World Health Organization (WHO) guideline [11]. A positive MAT was defined as a single serum cut-point of ≥1:800 based on confirmed laboratory diagnosis by CDC definition 2013 [28]. For all urine dipstick test, the reported results of trace or more (1+, 2+, 3+, and 4+) were considered positive.

Confirmation of cases [28]

Clinically suspected patients were defined as “Leptospirosis confirmed cases” if one of the following laboratory criteria were met: (1) isolation of Leptospira from clinical specimen with confirmation by performing 16S rRNA sequencing (2) Leptospira agglutination titer of ≥800by microscopic agglutination test (MAT) in one or more specimens, or four-fold rising of Leptospira agglutination titer between acute and convalescent phase (3) detection of pathogenic Leptospira DNA by polymerase chain reaction from a clinical specimen. Patients who did not fulfil any of the criteria were classified as “non-cases”. The confirmation of diagnosis other than Leptospirosis, in non-cases patients was not done. We defined patients as severe leptospirosis cases if they required any dialysis support, or required mechanical ventilation support or manifested with clinical jaundice. All laboratory confirmation results were blinded to study site physicians, investigators and research assistances.

Statistical analysis and study size estimation

Continuous variables were checked for normality and presented with mean and standard deviation for normally distributed data. Median and interquartile range was used for non-normally-distributed data. The differences of means between the two contrast groups were compared using independent t-test or rank-sum test based on normality test. Categorical variables were presented with frequency and percentage. The comparisons of two independent proportions were done with exact probability test or chi-square as appropriate. Univariable logistic regression analysis was done for each potential predictor to explore for its diagnostic performance. The diagnostic odds ratios (dOR) and area under the receiver operating characteristics curves were presented. A statistical significance was declared if two-sided p-values fall below 0.05. Stata statistical software version 15 was used for all analyses. For development of clinical prediction rules, there is currently no standard approach for estimation of study size. The authors reviewed the unpublished data and patient records comparing the clinical characteristics of leptospirosis confirmed cases and non-cases at Si Sa Ket hospital during 2015. The proportion of patients reported exposure to contaminated water was 0.73 and 0.25 for confirmed cases and non-cases of leptospirosis, respectively. Using the comparison of two proportions approach, 12 confirmed cases and 47 non-cases were needed to achieve 80% statistical power and a two-sided alpha error of 0.05. A 10-events-per-variable rule of thumb was suggested by many literatures including the TRIPOD statements for reporting of clinical prediction rules development [29]. For our study, as we planned to include at least 5 potential predictors within the final model, at least 50 confirmed cases were required for model derivation. At confirmed cases: non-cases ratio of 1:4 [30], this study planned to recruit at least 250 patients (50:200).

Model development

The model was based on complete-case analysis, no data imputation was done. All clinically relevant parameters were included in multivariable logistic regression model to explore for significant predictors of leptospirosis. Backward elimination was done based on both statistical significance from p-value of each predictor and total predictive performance of the model via area under the receiver operating characteristic curve (AuROC). Non-contributing factors with large p-values and lowest magnitude of effect (odds ratio closest to 1.00) were initially eliminated from the regression model. After each predictor was removed, we checked for model diagnostic performance via the AuROC. The predictor was re-entered into the model, if its removal caused substantial decreases in AuROC. The steps were done consecutively until all of the remaining predictors within the model had a p-value of lower than 0.10 on condition that the AuROC of the reduced model must be well preserved. Measure of discrimination and calibration of the final reduced model was done with the use of AuROC curves and Hosmer-Lemeshow goodness of fit test.

Score derivation and validation

The score was assigned for each of the predictor within the final model based on its logit coefficients. In score transformation, the lowest coefficient of all predictors was used as a denominator, while others were used as numerators. After division of coefficients, the products were rounded up to non-decimal figures. Score was calculated for each patient within the development cohort. Measure of discrimination and calibration were similarly done for score based logistic regression model. The score was further categorized into two risk groups (low and high probability of having leptospirosis) at an appropriate cut-point. Sensitivity, specificity, positive likelihood ratio and AuROC of each risk category were be displayed. The diagnostic ability of developed score was compared with that of the standard WHO score (conventional Faine’s Criteria) with 10 predictors by AuROC equality test and visualized with comparative AuROC curves. We performed internal validation with non-parametric ROC regression with 1,000 bootstrapped sampling.

Results

Participants

During study period, a total of 262 patients suspected with leptospirosis were enrolled. Two patients were excluded due to incomplete data, 260 patients were included in score development and internal validation cohort. Of these patients, 82 (31.5%) were confirmed leptospirosis cases (Fig 1). Forty-three cases (52.4%) were treated as outpatient treatment and appointed for subsequent follow-up visit. Thirty cases (40.2%) were admitted for in-patient hospital care. Six cases (7.3%) were defined as severe leptospirosis and were transferred to higher level hospital. However, none of the patient had lethal complication. The monthly epidemiologic curve showed the distribution of suspected leptospirosis cases during study period and the proportion of confirmed cases and non-cases within each month (Fig 2). The number of suspected cases was highest in October and lowest in April. Most of the suspected patients were male (72.3%) without medical comorbidities (84.2%). The mean age of the cohort was 47.0±16.6 years. The mean onset of fever in leptospirosis confirmed cases and non-leptospirosis cases were 3.0±2.3 and 3.2±3.3 days, respectively. Around two third of the patients were farmers. Most patients presented with fever (64.1% vs 72.1%, p = 0.233), myalgia (12.7% vs 18.0%, p = 0.328) and headache (10.3% vs 12.7%, p = 0.675). Most of clinical presentations between both groups were similar. The symptom which showed statistical significance was fatigue (75.6% vs 60.7%, p = 0.024). From physical examination, confirmed cases compared to non-cases had lower systolic blood pressure (115.7±20.2 vs 120.7±15.8 mmHg, p = 0.033), higher pulse rate (100.4±17.6 vs 94.9±16.2, p = 0.011), higher respiratory rate (21.4±2.6 vs 20.7±2.0, p = 0.015), higher proportion of jaundice (6.1% vs 1.1%, p = 0.035) (Table 1).

Fig 1

Study flow.

Fig 2

Epidemiologic curve visualizing distribution and frequency of leptospirosis suspected patients during study period and proportions of cases and non-cases within each month.

Table 1

Baseline demographic data, presenting symptoms, physical examinations and initial laboratory investigations of the derivation cohort, comparison of confirmed-cases and non-cases of Leptospirosis.

Clinical Characteristics	Confirmed cases(n = 82)		Non-cases(n = 178)		Crude OR (95%CI)	P-value
	n	(%)	n	(%)
Demographic
Male	59	(72.0)	129	(72.5)	0.97 (0.54–1.75)	1.000
Age, years (mean±SD)	45.9	±14.6	47.6	±17.5	0.99 (0.98–1.01)	0.432
Symptoms
Myalgia	71	(86.6)	141	(79.2)	1.69 (0.82–3.52)	0.172
Jaundice	7	(8.5)	6	(3.4)	2.68 (0.87–8.23)	0.121
Fatigue	62	(75.6)	108	(60.7)	2.01 (1.12–3.61)	0.024
Vomiting	19	(23.2)	27	(15.2)	1.69 (0.87–3.25)	0.120
Breathing difficulty	14	(17.1)	16	(9.0)	2.07 (0.96–4.48)	0.093
Physical examinations
Body temperature,°C (mean±SD)	38.1	±1.1	37.9	±1.1	1.20 (0.94–1.54)	0.141
Pulse rate, per mins (mean±SD)	100.4	±17.6	94.6	±16.2	1.02 (1.00–1.04)	0.011
SBP, mmHg (mean±SD)	115.7	±20.2	120.7	±15.8	0.98 (0.97–0.99)	0.033
DBP, mmHg (mean±SD)	69.5	±12.0	71.4	±10.5	0.98(0.96–1.01)	0.184
Respiratory rate, per mins (mean±SD)	21.4	±2.6	20.7	±2.0	1.15 (1.02–1.28)	0.015
Jaundice	5	(6.1)	2	(1.1)	5.71 (1.08–30.10)	0.035
Risk factors
Flood or wet ground at home	38	(47.5)	57	(32.0)	1.92 (1.23–3.30)	0.025
Flood or wet ground at workplace	71	(87.7)	133	(75.6)	2.30 (1.09–4.84)	0.031
Contact animal water reservoir	34	(42.0)	51	(28.7)	1.80 (1.04–3.12)	0.045
Laboratory findings
WBC, /μL (median, IQR)	10,000	6,600, 12,200	7,900	5,700, 11,800	1.00 (0.99–1.00)	0.049
Neutrophil count ≥80%	37	(45.7)	41	(23.3)	2.77 (1.58–4.85)	<0.001
Neutrophil, % (mean±SD)	73.3	±14.1	68.3	±13.0	1.03 (1.01–1.05)	0.006
Lymphocyte, %,(median, IQR)	13.7	9.2, 23.6	19.9	13.1,27.3	0.97 (0.94–0.99)	0.003
Monocyte, %(median, IQR)	5.1	4, 8	7.1	5.6, 9.1	0.86 (0.78–0.94)	<0.001
Platelet, /μL(median, IQR)	194,000	141,000, 271,000	214,500	181,500, 259,000	1.00 (0.99–1.00)	0.105
eGFR, mL/min/1.73 m2 (mean±SD)	85.8	±23.7	90.0	±22.9	0.99 (0.98–1.00)	0.187
Urine glucose positive	8	(10.4)	9	(5.3)	2.07 (0.77–5.60)	0.175
Urine protein positive	44	(57.1)	65	(32.2)	2.15 (1.25–3.72)	0.008
Urine blood positive	38	(49.4)	46	(27.2)	2.61 (1.49–4.56)	0.001
Urine bilirubin positive	7	(9.2)	7	(4.1)	2.36 (0.80–6.99)	0.137
Urine ketone positive	19	(24.7)	29	(17.1)	1.59 (0.83–3.06)	0.169

Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cell count; eGFR, estimated glomerular filtration rate; OR, odds ratio; CI, confidence interval; SD, standard deviation; IQR, interquartile range.

For history of exposure to environmental risk factors, confirmed leptospirosis cases had higher proportion of flooded house compound (47.5% vs 32.0%, p = 0.025), flooded or wet ground at workplace (87.7% vs 75.6%, p = 0.031) and contact with water reservoir used by animal (42.0% vs 28.7%, p = 0.045). Other factors such as animal contact, presence of wound or skin abrasion and features of work did not reveal significant differences (S1 Table).

Confirmed leptospirosis cases had significantly higher percentage of neutrophil (73.3±14.1 vs 68.3±13.0, p = 0.006), lower percentage of lymphocyte (13.7(IQR 9.2, 23.6) vs 19.9 (IQR 13.1, 27.3), p = 0.003) and monocyte (5.1 (IQR 4,8) vs 7.1 (IQR 5.6,9.1), p<0.001) than non-cases. The result of urine dipstick test showed significant differences between groups in presence of urine protein (57.1% vs 32.2%, p = 0.008), urine blood (49.4% vs 27.2%, p = 0.001), urine bilirubin (9.2% vs 4.1%, p = 0.137), and urine ketone (24.7% vs 17.1%, p = 0.169). (Table 1). The remaining diagnostic predictors without statistical significant differences from univariable comparison were shown in S1 Table.

Model development and validation

All potential predictors with p-value from univariable analysis less than 0.2 were included in multivariable logistic regression for model development and score derivation (Table 2). Sequential elimination was carried out based on both statistical significance and model diagnostic performance. Five final predictors remained within the reduced logistic model which were history of exposure to wet ground at workplace, history of contact water reservoir used by animal, urine protein and urine blood positive from dipstick test and neutrophil count from CBC ≥80% (Table 3).

Table 2

Multivariable logistic regression analysis.

	Full modelmOR	95% CI	P-value	Reduced model mOR	95% CI	P-value
Demographic
Male	0.83	0.38–1.79	0.630
Age, years	1.00	0.97–1.02	0.750
Symptoms
Myalgia	1.15	0.45–2.94	0.773
Jaundice	0	0	0.991
Fatigue	1.48	0.70–3.13	0.303
Vomiting	1.20	0.51–2.83	0.672
Breathing difficulty	1.53	0.55–4.27	0.418
Physical examinations
Body temperature,°C	0.94	0.65–1.35	0.722
Pulse rate, per mins	1.02	0.99–1.05	0.094
SBP, mmHg	1.00	0.97–1.02	0.876
DBP, mmHg	0.98	0.94–1.02	0.257
Respiratory rate, per mins	1.08	0.94–1.25	0.291
Jaundice	0	0	0.990
Risk factors
Flood or wet ground at home	1.46	0.72–2.95	0.290
Flood or wet ground at workplace	1.82	0.67–4.92	0.238	2.65	1.07–6.58	0.035
Contact animal water reservoir	1.82	0.89–3.72	0.101	1.64	0.89–3.03	0.111
Laboratory findings
WBC, /μL	1.00	0.99–1.00	0.510
Neutrophil, %	0.95	0.87–1.04	0.298	1.02	0.99–1.05	0.052
Lymphocyte, %	0.94	0.85–1.05	0.283
Monocyte, %	0.86	0.75–0.99	0.036
Platelet, /μL	1.00	0.99–1.00	0.978
eGFR, mL/min/1.73 m2	1.00	0.98–1.02	0.744
Urine glucose positive	1.12	0.30–4.16	0.860
Urine protein positive	1.23	0.59–2.56	0.582	1.71	0.94–3.10	0.079
Urine blood positive	2.02	1.01–4.05	0.048	1.99	1.09–3.62	0.026
Urine bilirubin positive	1.77	0.44–7.09	0.419
Urine ketone	0.85	0.35–2.06	0.713
Constant (intercept)	116.16			0.02

Abbreviations: SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cell count; eGFR, estimated glomerular filtration rate; mOR, multivariable odds ratio; CI, confidence interval; SD, standard deviation; IQR, interquartile range.

Table 3

Multivariable logistic model with score transformation via weighing of logit coefficients.

Predictors	mOR	95% CI	P-value	Coefficient	Score
Wet ground at workplace	2.66	1.06–6.66	0.037	0.976	2
Contact animal water reservoir	1.73	0.94–3.20	0.079	0.550	1
Urine protein positive	1.99	1.09–3.64	0.026	0.529	1
Urine blood positive	1.70	0.93–3.09	0.084	0.686	1.5
Neutrophil count ≥80%	2.27	1.24–4.15	0.008	0.818	1.5
Constant	0.07			-2.611

Abbreviation: mOR, multivariable odds ratio.

The score was derived by division of larger coefficients with the smallest coefficient resulting in a total score of 7 points. The newly derived diagnostic scoring scheme was named the “OPD Lepto Score”. Patients with history of exposure to wet ground at work were assigned 2 points, history of contact with water reservoir used by animal were assigned 1 point, urine protein positive were assigned 1 point, urine blood positive were assigned 1.5 points, and neutrophil count ≥80% were assigned 1.5 points (Table 3). Three pre-specified cut-off points for dichotomization of the score were compared based on sensitivity, specificity, positive likelihood ratio and AuROC (Table 4). The score of 3.5 was chosen based on its AuROC of 0.67 (95%CI 0.61–0.73) and higher sensitivity at 72.4% (95%CI 60.9–82.0) than that of score ≥4. Consequently, suspected patients with OPD score less than 3.5 would be defined as low risk while patient with OPD score ≥3.5 would be defined as high risk of having leptospirosis (Table 5).

Table 4

Selection of score cut point, sensitivity, specificity, LHR+, AuROC.

Score cut point	Sensitivity (%)	Specificity (%)	LHR+	AuROC(95%CI)
<3	86.8 (77.1–93.5)	39.5 (32.1–47.4)	1.44(1.24–1.67)	0.63 (0.58–0.69)
≥3.5	72.4 (60.9–82.0)	61.7 (54.8–69.1)	1.89 (1.49–2.39)	0.67 (0.61–0.73)
≥4	64.5 (52.7–75.1)	71.3 (63.8–78.0)	2.24 (1.68–3.00)	0.68 (0.61–0.74)

AuROC, area under the receiving operating curve

Table 5

Score categorization and likelihood ratio of positive (LHR+) in OPD Lepto Score and Faine’s score.

Probabilitycategories	Score	Confirmed cases(n = 75)		Non-cases(n = 167)		LHR+	(95%CI)	P-value
		n	%	n	%
OPD Lepto score
Low	<3.5	21	28.0	103	61.7	0.45	(0.31–0.67)	<0.001
High	≥3.5	54	72.0	64	38.3	1.88	(1.48–2.39)	<0.001
Mean±SD	-	4.2	±1.5	3.0	±1.6	-		<0.001
Faine’s’ score
Low	<26*	97.3	76.0	165	98.8	0.99	(0.95–1.03)	0.407
High	≥26*	2	2.7	2	1.2	2.23	(0.32–15.5)	0.407
Mean±SD	-	19.55	±3.2	17.7	±4.01	-		0.002

*Cut point of the conventional Faine’s’ score is definition of a presumptive diagnosis of leptospirosis from Part A, or Parts A and B score [21]

The OPD score diagnostic performance was considered acceptable at the AuROC of 0.72 (95%CI 0.65–0.79) (Fig 3). The measure of calibration was done with Hosmer-Lemeshow goodness-of-fit test which yielded non-significant p of 0.637. The diagnostic ability of OPD score with 5 predictors was then compared with that of WHO score with 10 predictors, the test of equality revealed statistically significant differences of AuROC between the two scores (0.72 vs 0.62, p = 0.014). The bootstrapped ROC from internal validation of OPD score was 0.67 (95%CI 0.59–0.74). In a clinical setting where complete blood count was unavailable, the OPD score still could maintain its diagnostic performance at the AuROC of 0.70 for the remaining 4 predictor variables.

Fig 3

Receiver operating characteristic curve in diagnostic prediction of leptospirosis, comparison of OPD score and standard WHO score within development cohort.

Discussion

In early stage of leptospirosis infection, the clinical signs and symptoms were found to be indifferent and did not provide significant diagnostic value. In this study, we explored for potential predictors for diagnosis of early leptospirosis infection in terms of initial clinical characteristics, history of environmental exposure to the organism and basic laboratory investigations which could be done routinely. We identified five final predictor variables which includes mainly the environmental exposure risk and basic routinely-available laboratory investigation. A history of wet ground at workplace, contact with animal water reservoir, positive urine protein and urine blood from dipstick test, and a neutrophil count of more than 80% from complete blood count were included in the multivariable model for diagnosis of early leptospirosis patients.

The original Faine’s criteria which was widely endorsed since 1982 consisted of three parts which were clinical data, epidemiological factors and bacteriological and laboratory findings [21]. Nine predictor variables were needed from patient’s clinical presentation such as headache, fever, body temperature 39 ≥degree Celsius, conjunctival suffusion and meningism. For epidemiological factors, the patients were asked for history of contact with animals or contaminated water. Two types of laboratory investigation, culture and MAT, were necessary to fulfil the criteria. In 2004, the criteria were revised to improve applicability in many endemic areas [22-24]. The Modified Faine’s 2004 added rainfall as another important epidemiological factors and split animal contact and contact with contaminated environment into two independent factors. ELISA test for IgM and slide agglutination test or SAT were appended as serology options. Eight years later, the latest version of the criteria was launched as Modified Faine’s criteria 2012 [25]. The criteria added hemoptysis and dyspnea in clinical data and polymerase chain reaction as another option for laboratory investigation. All the criterion were based on acutely-ill patients with typical clinical syndrome and they also required advanced laboratory testing which was usually unavailable especially in health resource limitted areas.

After a widespread leptospirosis endemic in Thailand during 1997–1998, the Faine’s criteria was implemented for use in high risk areas specifically in the northeastern and southern region of the country. The bacteriological and laboratory findings component of the score was not completed owing to financial and logistic limitations. Only few specimens were sent to the central laboratory investigation for epidemiologic purposes. The diagnostic performance varied and was unsatisfied with 68% sensitivity and 58% specificity [31]. For hospitalized patients, who usually presented with severe clinical symptoms, an on-admission diagnostic tool (THAI-LEPTO Score) was developed based on in-patient data from local hospitals within the northeastern and southern part of Thailand [32]. The score contains 8 clinical parameters which would be present in leptospirosis patients with end-organ damage. Although the predictive performance was good, the test might not be sensitive for use in out-patient settings where early phase leptospirosis patients usually present with mild and vague clinical syndromes.

In this study, clinical presentations, both signs and symptoms, did not reveal any significant differences and thus might not be efficient to consider as potential predictors. As patients with acute febrile illness in the tropical regions usually presented in the early phase with fever without any distinctive features to distinguish leptospirosis from other types of infections [9, 33–34]. Since leptospirosis infection required environmental exposure to leptospira, history of exposure to potential risk factors were crucial. It was concordantly found that history of flooded or wet ground at workplace was a good predictor [35-37]. Patients with flooded workplace had high probability of exposure to infective organism for longer duration than other places. Patients with flooded house compound also carried some risk but not as high as at workplace because people tend to reside within dry area at home. Contact or utilize animal water reservoir was another predictor within the OPD Lepto Score. In agricultural area within the northeastern, animal water reservoirs were usually natural sources without steep curb. Animals and livestock can drink and bath within this basin. Patients who contacted or utilized the water within same natural water reservoirs had higher risk of exposure to leptospira.

Presence of protein and blood in the urine of suspected patients was due to renal involvement of leptospirosis, which could be one of the most distinguish features from other tropical diseases. Leptospira directly invades the vessels causing vascular damages and stimulate a chain of inflammatory processes. Kidneys are the most commonly involved organs [1, 38], thus, even in the early stage of the disease, urine abnormalities from dip stick test can be positive. The last predictor was percentage of neutrophil count ≥80% [39-40]. Infectious disease with higher percentage of neutrophil could indicate bacterial origin, while other tropical diseases were caused by either viral or atypical organism such as rickettsia.

Five potential predictors constituted a total score of seven with acceptable diagnostic performance according to Hosmer-Lemeshow categorization of AuROC. Comparing to standard WHO score or conventional Faine’s Criteria, the OPD Score had higher diagnostic ability with fewer number of predictors (Table 6).

Table 6

Comparison of study characteristics and diagnostic indices between previous diagnostic criteria of leptospirosis and newly derived OPD Lepto Score.

	Chifou W. et al.,1997[31]	Bhatia M. et al.,2015[23]	Bandara K et al., 2016[25]	Linda Rose Jose. et al., 2016[24]	Temeiam N. et al.
Criteria or score	Faines’ criteria 1982	Modified Faine’s criteria (2004)	Modified Faine’s criteria (with amendment) 2012	Modified Faine’s criteria (2004)	OPD Lepto Score
Country	Thailand	India	Sri Lanka	India	Thailand
Reference standard	MAT titer ≥ 400 or 4 fold raising of MAT paired serum	MAT titer ≥ 1:80	MAT1≥400 or PCR	IgM ELISA(PanBio,Brisbane, Australia) and MAT1>1:100	Culture or PCR or MAT1 ≥ 800 or 4-fold raising MAT2
Patient domain	Hospitalized 54.1% and non-hospitalized 45.9%	Hospitalized	Hospitalized	Hospitalized	Outpatient clinic
Sample size(suspected/confirmed)	74 /24	100/49	168/66	332 /115	260/82
Sensitivity (%)	68	N/A	89.39	N/A	72.4
Specificity (%)	58	N/A	58.82	N/A	61.7
PPV (%)	64	21	58.42	N/A	46.2
NPV (%)	59	N/A	89.55	N/A	83.1
Recommendations	Urine protein≥1+ plus fever, headache, and myalgia increase specificity to 80%* cutoff point ≥ 20	Further evaluation of the diagnostic utility ofmodified Faine’s criteria is needed of the hour.	Utilized only immunochromatographic assay (Leptocheck WB, India) in Part C be useful in the presumptive diagnosis of leptospirosis.	All persons with fever for >5 daysshould be screened for leptospirosis utilizing modified Faine’s criteria	Two risk factors, protein and blood urine dipstick, and neutrophil count ≥ 80%

Abbreviations: MAT, microscopic agglutination test; PCR, polymerase chain reaction; N/A, not applicable.

In the OPD Lepto Score implementation, we chose the cut-off at ≥ 3.5 points because the sensitivity and specificity were in acceptable margin and not too low to cause error in diagnosis. Other factors should be considered together with the use of the score in real practice. The score was designed to be used in undifferentiated fever patients intended to be diagnosed of leptospirosis, so the patients’ signs and symptoms must be relevant. Timing and season of visit should also be taken into account prior to antibiotic prescription. Patients who scored low risk during low prevalence season should be closely monitored by village health volunteers or local health care personnel for disease progression, while patients who scored high risk could be offer a choice of antibiotics such as doxycycline. Although a portion of high-risk patients might receive unnecessary antibiotics, the benefit certainly outweighed the risk for this group of patients [41]. After treatment initiation, it is mandatory to schedule the patient for evaluation.

Another obstacle that delay patients visit to the hospital occurred during harvesting season, where outbreaks usually took place. Large portions of high-risk patients, the farmers, avoided distant traveling to hospital by visiting primary care units within their villages. The primary care units did not have attending physician and were not equipped with laboratory investigation other than urine drip stick test. Local health care workers could exploit the remaining 4 predictors of OPD score as a guide in initiation of early antibiotic treatment by leaving out neutrophil percent count and still preserved a diagnostic performance of 0.70. This could help alleviate disease progression and probably decrease fatality rate of leptospirosis.

Our study has several strengths. First, this is a prospective study with data collected from multiple sites within the leptospirosis endemic regions in the northeastern parts of Thailand. The study was primarily designed for patients who presented with undifferentiated fever and physicians suspected of having leptospirosis. Second, the domain of patients was shifted from clear clinical syndromes as in other derived score (e.g. Faine’s Criteria or WHO Score, and THAI-LEPTO Score) to patients with vague symptoms for this purpose. We believed that early diagnosis and early treatment in suspected group of patients could impede disease progression and decrease mortality. Third, the included predictors used in OPD Lepto Score were based on routinely available laboratory investigations and exposure to risk factors could be simply asked within short time.

However, there were some limitations need to be addressed. First, the study protocol planned to schedule all enrolled patients for 2nd visit for blood samples for MAT to observe and interpret four-fold rising of convalescent titers, but only 48.9% of patients came for the blood tests. Thus, the number of confirmed cases could be underestimated in some degree. Second, distinguishing a group of patients at their early phase of the disease was troublesome and resulting in only fair discriminative performance of the model. However, we believed that as the model was derived entirely from the specific study domain intended to be used in outpatient practice, its performance should overcome previously developed criterion. Third, in terms of generalizability, the development cohort was based on highly endemic region of leptospirosis, so the application of OPD Lepto Score to non-endemic area might not be beneficial. Fourth, an external validation study is needed for evaluation of generalizability before the OPD Lepto Score being endorsed for real clinical use. Fifth, the confirmation of diagnosis other than leptospirosis in non-cases was not done. These some non-cases may be actual cases but remained undiagnosed because of various reasons including lesser severity which lowers diagnostic techniques sensitivity, higher antibiotic prescription (even non-significantly). These might increase the risk of false negatives.

In conclusion, this study developed and internally validated the OPD Lepto Score, a practical clinical risk score for the diagnosis of leptospirosis in suspected patients with acute undifferentiated fever who presented to the outpatient clinics in high endemic areas. With 5 predictors, the score was more practical for outpatient setting than the conventional WHO score with 10 predictors.

Univariable analysis of baseline demographic data, presenting symptoms, physical examinations and initial laboratory investigations of confirmed-cases and non-cases of Leptospirosis.

(DOCX)

Click here for additional data file.

14 Oct 2019

Dear Dr. Srisawat:

Thank you very much for submitting your manuscript "DEVELOPMENT and VALIDATION of a SIMPLE SCORE for DIAGNOSIS of LEPTOSPIROSIS at OUTPATIENT DEPARTMENTS" (#PNTD-D-19-01397) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

When you are ready to resubmit, please be prepared to upload the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt.

(5) To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

We hope to receive your revised manuscript by Dec 13 2019 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there.

Sincerely,

Nicholas P. Day

Associate Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Authors have developed a score used for as a diagnosis tool to identify mild or early phase symptoms of human leptospirosis in rural healthcare units with limited laboratory resources based on data from "cases" and "non-cases" patients in Si Sa Ket, a province in Northeast Thailand, between December 2017 and November 2018.

Most methods were lacking of reference citations for example, all laboratory tests used in the study such as culture, polymerase chain reaction, and MAT methods; cases and non-cases definitions; the method used for sample size estimation; and the modified WHO score used in the study.

Section "Blood sample and specimen collection" is not well described. For example, which kinds of blood tubes were used for culture, PCR and MAT, which parts of blood (or urine) samples were collected after centrifuge and for what tests, centrifugation force was not specified. It is confusing that frozen samples were sent to culture (this definitely need the step-by-step-protocol or reference citation.

For the definition of leptospirosis cases, the first criteria "isolation of Leptospira from clinical specimen" might not accurate. How much do authors be certain if pathogenic Leptospira species isolated from urine samples collected from patients who having 2 days of fever? Or how much author be certain the species or group (pathogens or non-pathogens) of Leptospira isolated from blood or urine specimens.

Clinical outcomes (at least after 7 days, the second visit) and treatments are important for evaluation the success of non-intervention routine practices. However, these data were not collected in this study.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The results of Leptospira isolations (both blood and urine specimens), PCR and MAT (paired serum) were not presented. These results are keys results for proper "cases" and "non-cases" definitions. Only final number of cases and non-cases were described.

The results of WHO score in each "cases" and "non-cases" groups are important, however, they were not shown.

Table 1 and 3 shows combination of both category parameters and numeric parameters. However, the heading are not specify well.

Is it accurate to use mean (in stead of median and IQR) to compare the OPD score of "cases" and "non-cases" group as shown in Table 6?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The data shown in the manuscript supported that "the OPD score" is better than the WHO score in the study endemic setting of Si Sa Ket province. However, the discussion on why the WHO score is not successful when used in this setting is not mention (even Laboratory data was available). Is the OPD score is suitable for human leptospirosis with other complication such as respiratory or heart diseases (not only kidney complication). Or is the OPD score is suitable for other endemic area in the Southern of Thailand or Malaysia, where less cattle farms and rice paddy fields but more rubber gardens, forest trekking and stray dogs.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: 1. Revision the "Methods" with all possible reference citations and laboratory brief protocols. And also specify the following details; PCR format (conventional or real-time PCR), target gene (rrs, lipL32 or others), Laboratory institute where all tests performed.

2. Adding PCR, culture and MAT results; and the WHO score results.

3. Improving heading and legend in Table 1 and 3.

4. Using median and IQR instead of mean and SD when it is appropriate.

5. Improving discussion on the limitation of the "OPD score".

6. Checking spelling and grammar for the whole manuscript again. Please correct the "drip stick". It should be "dipstick".

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: Manuscript Review: Development and Validation of a Simple Score for Diagnosis of Leptospirosis at Outpatient Departments

Journal: PLoS NTD

Manuscript ID: PNTD-D-19-01397

This study proposes a scoring system for identifying patients with early leptospirosis. The rationale for the study is that identification of early leptospirosis using a simple set of questions and tests would be broadly applicable and could enable therapy at a stage that could prevent or reduce more severe complications. Although cases of leptospirosis were confirmed by MAT, serologic testing and other leptospirosis-specific tests were not considered as these are not available in many settings.

An OPD score was developed based on five relatively novel and potentially useful criteria. The “wet ground at workplace” criterion is certainly more specific than the Modified Faine’s criterion of “rainfall”. Likewise, contact with an “animal water reservoir” is more specific than “animal contact” or contact with “contaminated environment”. The three laboratory criteria are readily obtainable in many outpatient settings. Although the proposed OPD scoring system could be potentially useful, I have a number of concerns that should be addressed.

Comments:

1. The authors should summarize Faine’s original and modified criteria for readers not familiar with these diagnostic scoring systems.

2. The authors should summarize the sensitivity and specificity of Modified Faine’s Criteria as reported by others (references 24-27) and compare these published results with those reported here.

3. The crude odds ratios for “wet ground at workplace” of 2.83 is not supported by the percentages in confirmed (27.4%) and non-cases (20.2%).

4. How much protein or blood in the urine is required for a positive score for those criteria?

5. The data for patients with neutrophil percentage >80% among confirmed and non-cases is missing.

6. The data for some of the modified Faine’s criteria (such as rainfall) among confirmed and non-cases is missing.

7. What percentage of non-cases were diagnosed with infections other than leptospirosis? What were those alternative diagnoses and how often did they occur?

8. Did any of the leptospirosis patients in this study develop more severe complications including death? How many of the leptospirosis cases in this study were later admitted to the hospital or treated for leptospirosis?

9. What was the average time from onset of symptoms to evaluation?

Reviewer #3: The OPD score is an alternative tool for screening human leptospirosis for rural healthcare units with limited laboratory resources. However, further sensitivity and specificity validations in other endemic (various prevalence) and non-endemic regions should be emphasized.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: Review PLoS NTD-PNTD-D-19-01397_TA.docx

Click here for additional data file.

20 Nov 2019

Submitted filename: Response to reviewers_7Nov2019_NS.docx

Click here for additional data file.

8 Dec 2019

Dear Dr. Srisawat,

We are pleased to inform you that your manuscript, "DEVELOPMENT and VALIDATION of a SIMPLE SCORE for DIAGNOSIS of LEPTOSPIROSIS at OUTPATIENT DEPARTMENTS", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please note: your manuscript will not be scheduled for publication until you have made the required changes.

IMPORTANT NOTES

* Copyediting and Author Proofs: To ensure prompt publication, your manuscript will NOT be subject to detailed copyediting and you will NOT receive a typeset proof for review. The corresponding author will have one final opportunity to correct any errors when sent the requests mentioned above. Please review this version of your manuscript for any errors.

* If you or your institution will be preparing press materials for this manuscript, please inform our press team in advance at plosntds@plos.org. If you need to know your paper's publication date for media purposes, you must coordinate with our press team, and your manuscript will remain under a strict press embargo until the publication date and time. PLOS NTDs may choose to issue a press release for your article. If there is anything that the journal should know, please get in touch.

*Now that your manuscript has been provisionally accepted, please log into EM and update your profile. Go to http://www.editorialmanager.com/pntd, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

*Note to LaTeX users only - Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines [http://www.plosntds.org/static/latexGuidelines.action] in the meantime.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Nicholas P. Day

Associate Editor

PLOS Neglected Tropical Diseases

Mathieu Picardeau

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

2 Jan 2020

Dear Dr. Srisawat,

We are delighted to inform you that your manuscript, "DEVELOPMENT and VALIDATION of a SIMPLE SCORE for DIAGNOSIS of LEPTOSPIROSIS at OUTPATIENT DEPARTMENTS," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases