Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource, hyper-endemic setting with varied microgeographic variation in reactivity.

The microscopic agglutination test (MAT) is the standard serological reference test for the diagnosis of leptospirosis, despite being a technically demanding and laborious procedure. The use of a locally optimised MAT panel is considered essential for proper performance and interpretation of results. This paper describes the procedure of selecting such an optimised panel for Sri Lanka, a country hyper-endemic for leptospirosis. MAT was performed using 24 strains on 1132 serum samples collected from patients presenting with acute undifferentiated fever. Of 24 strains, 15 were selected as the optimised panel, while only 11% of serum samples showed positivity. A geographical variation in predominantly reactive serovars was observed, whereas reactivity was low with the saprophytic strain Patoc. Testing with paired sera yielded a higher sensitivity but provided only a retrospective diagnosis. Serological tests based on ELISA with complementary molecular diagnosis using PCR are a feasible and robust alternative approach to diagnose leptospirosis in countries having a higher burden of the disease.

Introduction

Leptospirosis, caused by a group of spirochetes in the family Leptospiraceae, is considered to be the most common zoonotic disease worldwide; with many mammalian species, mainly rodents, acting as reservoir hosts[1-3].Genomic classification of Leptospira has identified 64 species to date, with 17 in the type 1 pathogenic group which are responsible for the majority of disease cases[4]. Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri are the predominant circulating species worldwide[5-7]. The serologic classification divides the strains into serovars according to the lipopolysaccharide structure of the outer cell wall; with more than 300 serovars grouped into 25 serogroups[3,8,9]. The importance of serologic classification is appreciated in part due to the established links with particular serovars and reservoir animals; for example, the serovar Icterohaemorrhagiae and Ballum in rodents and serovar Hardjo and Pomona in cattle[10,11].

The lack of a rapid and accurate diagnostic test in the acute stage of the disease is a major challenge for the management of leptospirosis. A further complication is the similar clinical manifestations of dengue fever, rickettsial infections, and malaria; which are common in leptospirosis endemic regions[12,13]. However, to diagnose leptospirosis clinicians mainly depend on clinical features combined with a history suggestive of exposure to a susceptible event. Unlike many other bacterial infections, culture isolation is not an option in the acute setting due to the fastidious nature of the organism, which needs special culture media and might take a minimum of 3 weeks to give a positive result[7,14].

The microscopic agglutination test (MAT) has long been in use as the serological reference field test of leptospirosis[15-19]. MAT depends on the immune response of the patient with the production of antibodies which are specific to the infecting serogroup. According to the criteria made by the “Leptospirosis Reference Epidemiology Group” (LERG), a confirmed case of leptospirosis should have an acute MAT titre of ≥1 in 400 or a four-fold rise of titre between acute and convalescent samples. A probable case is defined when the MAT titre is ≥1 in 100 in a non-endemic area [20]. Although MAT results mainly provide a retrospective diagnosis, the test serves to diagnose cases mainly for epidemiological purposes. This is especially important in estimating the true disease burden, and some studies have used MAT data to predict circulating serogroups within possible reservoir hosts[21-24].

The World Health Organisation (WHO) recommends to use a locally optimised MAT panel that represents the currently circulating strains in a particular region; or to use a broad panel of serogroups in the absence of such knowledge[11]. The basis for this is to improve the sensitivity of the test, as patient sera are likely to react well with local strains. However, knowledge on currently circulating strains is scarce in many high endemic settings. This is particularly a challenge given the laborious, low sensitive and resource intensive procedure involved in culture isolation of locally prevalent strains. Even the use of strains representative of a broad panel of serogroups for MAT is not feasible given the resource intensity and expense of the procedure.

The lack of a simple diagnostic test with higher sensitivity and specificity during the acute phase of the illness is a major challenge in managing patients with leptospirosis; and this could have an impact on optimal patient care. Currently, molecular based diagnostic tests are rapidly replacing culture and serology based assays in diagnosing leptospirosis. Several qPCR assays utilizing pathogen specific primers have been assessed and found to have a higher sensitivity during the acute phase; a time point at which the serological response is lagging [25,26]. These molecular tests have the added advantage of being able to detect positive samples with non-viable organisms, such as when culture techniques fail following antibiotic therapy. In addition, there are published reports of several molecular based platforms which have been developed to determine the infecting species directly from the blood sample, without the need of culture[27,28].

ELISA and lateral flow based serological tests have largely replaced the conventional MAT test due to their ease of performance with comparable sensitivity and specificity, particularly during the acute stage of the disease[15,18,29-31]. Some of these serological tests have gained the status of point of care rapid screening tests[19,32-35].

Sri Lanka has a high incidence of leptospirosis with several recent outbreaks [1,36-38], and still faces challenges related to diagnostics to be used in the acute stage as well as selecting a locally optimised MAT panel. The knowledge on currently circulating strains is low, with a single recent report on two isolates[39]. However, MAT is used as the serological standard test in Sri Lanka and optimisation of the panel is an essential prerequisite to improve the sensitivity of the test. The purpose of the present study was to describe the procedure for selecting a sensitive as well as cost effective MAT panel in Sri Lanka using a cohort of patients with acute undifferentiated fever. This study might serve as an example for settings where there are knowledge gaps on circulating Leptospira serogroups.

Methodology

Ethics statement

Written informed consent was obtained from all patients prior to sample collection. For minors, written informed consent was obtained from the parents/ guardians. This study is approved by the Ethics Review Committee of the Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka. Protocol No.ERC/2015/18.

Study design

This study is part of a large multi-centre study to characterise the clinical, epidemiological, and aetiological aspects of leptospirosis in Sri Lanka. The study protocol is published elsewhere[40].

Study setting

The study sites include a wide range of geographic areas differing in temperature, altitude, rainfall, ecology, human behaviour, and leptospirosis endemicity. Data were collected from June 2016 to January 2019.

The main data collection sites for the serological study were the Teaching Hospital Anuradhapura (THA) and Teaching Hospital Peradeniya (THP). A short-term data collection was done at the Base Hospital Awissawella (BHA) and Provincial General Hospital Rathnapura (PGHR), following flooding in 2017. These study sites represent four districts within four provinces of the country. In addition, the geographical distribution represents dry and wet zones, and low and high altitudes.

Sample collection

Blood was collected from two types of patients. Firstly, acute undifferentiated febrile (AUF) patients (temperature >38°C, fever <15 days) from outpatient departments and hospital wards. Patients from paediatric wards (age less than 12 years) were not included in this set of patients. Also excluded were physician-diagnosed cases of fever due to other causes, such as probable or definite acute bacterial meningitis, lower respiratory tract infections (e.g., consolidated lobar pneumonia), traumatic, post-operative, or fever due to nosocomial infections, and any patient confirmed with other diagnosis as a cause for the fever. The second type of patients were probable cases of clinical leptospirosis from any ward, without a restriction of age or fever duration. Most of the probable cases were actually detected as acute undifferentiated febrile patients for the first group. However, there were patient who were missed during the initial recruitment and or developed those symptoms later and the physicians wanted to exclude leptospirosis as an alternative diagnosis.

Patient recruitment

The first category of patients was recruited on admission, by visiting all selected hospitals on a daily basis. A clinical data collector (graduate nurse or physician) screened the patients using the eligibility criteria and patients were recruited. Sample and data collection was done on the first day of admission. The second group of patients were recruited as reported.

Sample processing and MAT procedure

From the eligible patients, 4 ml of blood was collected into plain tubes, allowed to coagulate for 30 minutes, and then centrifuged at 1300 rpm for 10 minutes. The serum was separated and 500 μl aliquots were prepared and stored at -20°C and -80°C for short term and long-term storage, respectively. The procedure of serum separation and storage was carried out within 2 hours of sample collection. A follow up serum sample was collected three weeks from the onset of fever and similarly prepared and stored.

The MAT panel selected for the study consists of WHO recommended serovars and is used by the Centre for Disease Control (CDC)[11]. Five additional CDC strains were included, which were isolated from the wet zone of Sri Lanka during the period 1950–1970 [36,41]. All strains were maintained in EMJH liquid media and sub-cultured weekly to maintain the live antigen panel. Cultures which were 4–5 days old and showed a growth equivalent to a 0.5 McFarland solution were selected for the study. For the MAT testing, two strains from the original panel were not included due to inadequate growth in the EMJH liquid media. These were L. borgpetersenii serovar Javanica str. Veldrat Bataviae and L. interrogans serovar Cynopteri str. 3522C. The final MAT panel consisted of 24 strains representing 17 serogroups which included a saprophytic species Leptospira biflexa serogroup Semaranga serovar Patoc.

MAT was run in two steps; screening and run out tests. In the first step serum samples were screened in 1 in 50 dilution by diluting with phosphate buffered saline (PBS) and screened in 96 well flat bottom microtitre plates with the panel of 24 live antigens. The first row of each plate contained antigen controls which were loaded in 50 μl of PBS and 50 μl of a live antigen strain making a final volume of 100 μl. The remaining rows were dedicated for a single serum sample and each column for a single strain. These wells were loaded with 50 μl of diluted serum (dilution of 1 in 25) and 50 μl of a specific live antigen making a final dilution of 1 in 50. An internal quality control step was carried out daily with each test run. Ideally, standard antisera should be available for each test strain for this purpose. However, it was available only for the L. interrogans serovar Weerasinghe str. Weerasinghe (purchased from the reference laboratory Amsterdam, Netherland). The standard antisera was mixed with the corresponding serovar plus a distinct serovar in the panel to make sure that only specific agglutination reactions are occurring with appropriate test conditions.

After loading of samples and test strains, the plates were put into an orbital plate shaker for 5 minutes allowing optimal mixing of sera and live antigens. This was followed by incubation for two hours in a 30°C incubator.

Agglutination reactions were read by adding 5 μl drops onto a clean glass slide from each well under dark field microscopy with 200x magnification. Positive samples were selected as those which gave 50% reduction of free, motile Leptospira compared to the antigen control. Reading was done column-wise where reaction to a single serovar was tested at a time. When paired sera were available, both samples were tested in the same plate.

In the second run out test, reactive sera via screening were tested against respective serovars with a dilution series from 1 in 5o to 1 in 3200 to detect the maximum dilution which yielded a 50% reduction of free, motile Leptospira. A positive MAT test was defined as seroconversion, a four-fold increase in titre between acute and convalescent samples or an acute titre of >1 in 400.

Results

Patient characteristics

From 2016–2018, we received 1132 samples from 982 patients for MAT. These samples were from four hospitals representing four districts (Table 1) and the majority were from inward patients (n = 904, 92%). Of these, 832 were received as single samples mostly during the acute illness and 150 as paired samples in the acute and convalescent periods. Patient ages ranged from 12 to 87 years. The median duration of fever was 4 days (range 2–5 days).

Table 1

Characteristics of the patient samples.

	n	%
Age
<20	65	7
20–29	139	15
30–39	179	20
40–49	202	23
50–59	175	20
60–69	90	10
≥70	27	3
Sex
Male	722	80
Female	181	20
Ethnicity
Sinhala	842	93
Sri Lankan Moor/ Malay	27	3
Sri Lankan Tamil	32	3
Other	3	0.3
Hospital/District
ABH	149	15
RGH	72	7
THA	513	52
THP	248	25
Patient presentation
Outpatient	71	7
Hospitalized	904	92
Clinical presentation
Three classical* only	382	50
Three classical and jaundice or conjunctival suffusion	156	20
Three classical and jaundice and conjunctival suffusion	28	3
Any two classical features out of three and jaundice/conjunctival suffusion	32	4
Other	62	8
No clinical details	95	12

*Three classical features: headache, myalgia, and fever

Summary of the MAT results is given in Table 2. Of the 150 paired samples, 19 (13%) had titres of ≥1 in 400 during the acute stage and another 11 (7%) samples had titres ranging from 1 in 50 to 1 in 200. Paired sample analysis showed 49 (33%) confirmed cases; with 22 (15%) seroconversions(from non reactive to 1/50–2, to 1/200-2 and to ≥1/400-18), 12 (8%) with four-fold rise of titres, and another 15 (10%) with titres ≥1 in 400 in the acute stage but didn’t show a four fold rise in the convalescent sample. Of the 832 single samples, 58 (7%) had titres ≥1 in 400.

Table 2

Final diagnosis for 982 patients based on the MAT results.

		n	%
Confirmed 107 (11%)	Seroconversion	22	2
	Fourfold rise	12	1
	Single titre ≥1 in 400	73	8
Reactive/Probable	Reactive (≥50 titre <1 in 400)	50	5
Negative 825 (84%)	Non-reactive paired sample	98	10
	Non-reactive single sample	727	74
Total		982	100

To understand the MAT reactivity with all Leptospira strains used in the study, patient sample reactivity for individual antigens/strains in the MAT panel (irrespective of a single sample or paired samples) were screened. As shown in Table 3, all 24 Leptospira strains reacted with the MAT panel.

Table 3

Highest titres of 1132 serum samples to individual antigens used in the MAT panel.

Strain	1 in 50	1 in 100	1 in 200	1 in 400	1 in 800	1 in 1600	1 in 3200	>1 in 3200	Total reactive samples
L. interrogans serogroup Australis serovar Bratislava str. Jez-Bratislava	13	8	12	17	8	8	15	3	84
L. interrogans serogroup Canicola serovar Canicola str. Ruebush	10	6	13	14	10	3	8	3	67
L. interrogans serogroup Autumnalis serovar Weerasinghe str. Weerasinghe	9	14	12	13	4	1	0	5	58
L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae str. RGA	2	4	11	8	9	7	6	3	50
L. santarosai serogroup Mini serovar Georgia str. LT 117	6	13	13	9	2	0	0	0	43
L. interrogans serogroup Bataviae serovar Bataviae str. Van Tienen	0	3	5	8	10	5	5	0	36
L. interrogans serogroup Icterohaemorrhagiae serovar Mankarso str. Mankarso	2	5	5	4	4	5	1	2	28
L. biflexa serogroup Semaranga serovar Patoc strain Patoc1	6	2	4	7	4	1	1	0	25
L. interrogans serogroup Pyrogenes serovar Alexi str. 616	6	7	8	1	0	0	1	0	23
L. santarosai serogroup Pyrogenes serovar Pyrogenes str. Salinem	0	4	2	4	7	3	0	1	21
L. borgpetersenii serogroup Javanica serovar Ceylonica str. Piyasena	4	1	4	6	2	2	0	0	19
L. interrogans serogroup Australis serovar Australis str. Ballico	7	2	5	1	2	1	1	0	19
L. weilii serogroup Celledoni serovar Celledoni str. Celledoni	7	2	0	3	2	1	0	0	15
L. interrogans serogroup Sejroe serovar wolfii str. 3705	0	3	4	4	0	1	0	0	12
L. kirschneri serogroup Grippotyphosa serovar Ratnapura str. Wumalasena	4	3	4	0	1	0	0	0	12
L. interrogans serogroup Autumnalis serovar Autumnalis str. Akiyami A	2	0	3	3	1	1	0	0	10
L. interrogans serogroup Djasiman serovar Djasiman str. Djasiman	1	2	2	2	0	2	0	0	9
L. borgpetersenii serogroup Ballum serovar Ballum str. Mus 127	1	4	3	0	0	0	0	0	8
L. borgpetersenii serogroup Tarassovi serovar tarassovi str. Perepelitsyn	3	4	1	0	0	0	0	0	8
L. santarosai serogroup Autumnalis serovar Alice str. Alice	1	1	1	0	2	0	0	0	5
L. interrogans serogroup Grippotyphosa serovar Grippotyphosa	0	1	0	2	1	0	0	0	4
L. interrogans serogroup Sejroe serovar Geyaweera str. Geyaweera	0	0	0	0	1	0	0	1	2
L. santarosai serogroup Hebdomadis serovar Borincana str. HS 622	2	0	0	0	0	0	0	0	2
L. interrogans serogroup Pomona serovar Pomona str. Pomona	0	0	0	0	0	1	0	0	1

This strain level analysis shows a large number of reactive antigens which could be used for the diagnosis of leptospirosis in Sri Lanka. However, the cross reactions were high, especially in the samples with high titres (Fig 1), and some antigens seemed to yield consistently low titres. In samples with a titre of >1:3200, around 50% of the samples had cross reactivity across more than 10 antigens.

Fig 1

Percentage distribution of cross reactivity of patient samples by the highest MAT titre.

The colour legends 1–18 shows the number of strains reacting with a single serum sample.

We characterized the antigens providing the highest titres for particular samples and titres ≥ 1:400 (Table 4) to select the best panel of Leptospira for the Sri Lankan MAT panel.

Table 4

Number of samples with diagnostic (≥ 1 in 400) and highest tires with individual antigens for 1132 patient sera.

Strain	Titres ≥1 in 400	Highest titre for the sample	Diagnostic and highest titre	Cumulative percentage of diagnosed samples
L. interrogans serovar Bratislava str. Jez-Bratislava	51	70	46	34%
L. interrogans serovar Canicola str. Ruebush	38	35	20	49%
L. interrogans serovar Icterohaemorrhagiae str. RGA	33	17	15	60%
L.interrogans serovar Weerasinghe str. Weerasinghe	23	21	15	71%
L. interrogans serovar Bataviae str. Van Tienen	28	12	9	78%
L. santarosai serovar Pyrogenes str. Salinem	15	5	5	82%
L. interrogans serovar wolfii str. 3705	5	6	5	85%
L. interrogans serovar Mankarso str. Mankarso	16	4	4	88%
L. borgpetersenii serovar Ceylonica str. Piyasena	10	8	4	91%
L. santarosai serovar Georgia str. LT 117	11	8	3	94%
L. biflexa serovar Patoc strain Patoc1	13	5	2	95%
L. interrogans serovar Australis str. Ballico	5	2	2	97%
L. interrogans serovar Geyaweera str. Geyaweera	2	2	2	98%
L. interrogans serovar Djasiman str. Djasiman	4	3	1	99%
L. interrogans serovar Pomona str. Pomona	1	1	1	100%
L. weilii serovar Celledoni str. Celledoni	6	0	0
L. interrogans serovar Autumnalis str. Akiyami A	5	1	0
L.interrogans serovar Grippotyphosa	3	0	0
L. interrogans serovar Alexi str. 616	2	1	0
L. santarosai serovar Alice str. Alice	2	0	0
L. kirschneri serovar Ratnapura str. Wumalasena	1	0	0
L. borgpetersenii serovar Ballum str. Mus 127	0	1	0
L. borgpetersenii serovar tarassovi str. Perepelitsyn	0	1	0
L. santarosai serovar Borincana str. HS 622	0	0	0

This analysis shows that, although the reactivity is high, for diagnostic purposes more than 95% sensitivity could be achieved using 11 antigens and 100% with 15 antigens, compared with the full 24 antigen panel. The selected panel included three Sri Lankan isolates plus the genus specific saprophyte Patoc strain.

Varying highest titre based on day of sampling

Of the 19 paired samples with positive results, 15 had a titre of ≥1 in 400 in the acute samples with a higher titre in the convalescent period. We examined these samples to determine the serovar/serogroup prediction using the highest titre. Of the 15 samples, 12 (80%) had highest titres for different antigens in the convalescent samples, showing that the routine diagnostic MAT based serovar or serogroup prediction is unreliable and may depend heavily on the day of sampling. Fig 2 and Table 5 show reactivity of patient sera in different geographical settings.

Fig 2

Reactivity of patient sera from different geographical settings in Sri Lanka.

(Maps used for the baselayer in Fig 2 are freely available from The United Nations Office for the Coordination of Humanitarian Affairs ). Serological reactions differed across the four hospitals used in the study. We looked at only those strains with high titres (Fig 2). Peradeniya (wet zone high lands) showed high titres to at least five strains, while Anuradhapura patients were predominantly reacting against only one strain. The observed geographical differences of L. interrogans serovar Bratislava str. Jez-Bratislava, L. interrogans serovar Icterohaemorrhagiae str. RGA and L. interrogans serovar Bataviae str. Van Tienen in reactivity were statistically significant (chi-square 34.1, p = .04).

Table 5

Reactivity of patient sera from different geographical settings.

	ABH		RGH		THA		THP
	n	%	n	%	n	%	n	%
L. interrogans serovar Bratislava str. Jez-Bratislava	13	38	8	38	42	51	9	19
L. interrogans serovar Canicola str. Ruebush	8	23	3	14	14	17	10	21
L. interrogans serovar Weerasinghe	6	17	3	14	5	6	7	15
L. santarosai serovar Georgia str. LT 117	3	8	2	9	1	1	2	4
L. interrogans serovar Icterohaemorrhagiae str. RGA	1	2	2	9	5	6	9	19
L. interrogans serovar Bataviae str. Van Tienen	1	2	1	4	2	2	8	17

Discussion

Although MAT is considered to be the serological reference test for the diagnosis of leptospirosis, it has many inherent issues in performance. The inability to standardize the test conditions may lead to interpersonal variations in the interpretation of results. The necessity to maintain live cultures for the MAT panel is a laborious procedure due to the fastidious nature of the species, and imposes a risk of laboratory acquired infections. Apart from these technical problems, the sensitivity of MAT in the acute stage has been questioned. Patients might not elicit detectable immune responses in the acute stage and sensitivity can be significantly low in such cases [32,42-44]. Therefore, for a proper interpretation, paired sera should be used[11][32]. However, relatively higher specificity of MAT has been reported in both acute and paired sera for the diagnosis [32,33,42].

The findings of this study have several important implications. This study used a broad panel of 24 strains representing 17 serogroups per WHO recommendation, in the absence of recent published data on widely circulating local strains. This is the recommended practice for settings where knowledge on current circulating serovars is missing. Our results show that in Sri Lankan settings, 100% sensitivity of MAT could be achieved using 15 rather than the recommended 24 strains. These 15 strains compose a locally optimised panel since the reactive sera were collected from patients residing in different geographical regions, including dry and wet zones and high and low altitudes. Although the hospitals were from four provinces, the actual patient population was from eight of nine provinces. Furthermore, the samples were collected over a 2.5 year period where both endemic and epidemic cases were included in the study.

In this study, differential patterns of sera reactivity were observed in the diverse geographical settings. Predominant reactivity only with serogroup Australis serovar Bratislava in the dry zone contrasts with the shared reactivity with several serovars in the wet zone. This might reflect the diversity of reservoir hosts in different geographical contexts. Some studies have used MAT data to predict infecting serogroups[45]; however, this has been shown to be inaccurate due to cross reactions with other serovars in the acute stage and is not generally recommended[11,46,47]. This study similarly revealed such multiple cross reactions, particularly with sera at high titres.

In comparison, published MAT data using an almost similar representative serogroup panel in a 2008 leptospirosis outbreak in Sri Lanka revealed L. interrogans serogroup Pyrogenes serovar Pyrogenes to be the predominant reactive serovar[48]. The shift in the reactive serovar from Pyrogenes to Bratislava in this study may also be indirect evidence of changes in predominant reservoir hosts over time. It may reflect the predominant role of one serovar in an outbreak setting and geographical difference in circulating serovars. The 2008 study was focussed in a wet zone whereas this study included both dry and wet zones, with predominant reactivity in samples from the dry zone. However, support for this would be to simultaneously recover local isolates from humans and veterinary animals where serotyping can be used to establish important and potential reservoir hosts for human leptospirosis.

We did not observe an anticipated high reactivity with the local isolates in the MAT panel. Only three isolates were among the 15 serovars of the selected final panel: serovars Weerasinghe, Ceylonica, and Geyaweera. These three isolates were recovered from the wet zone of Sri Lanka during 1964–1965[41]. The analysis of agglutination reactions with local serovars shows that only serovar Weerasinghe was reactive with 37% of the total reactive samples whereas that of serovar Ceylonica and serovar Geyaweera showed only 12% and 1% reactivity, respectively. These observations follow a similar pattern to MAT data published in the 2008 Sri Lanka study[48]. Changes in antigenic structures or emergence of new serovars over time might have contributed to this reaction pattern. Similar results have been found in other international studies where local isolates have failed to give better agglutination reactions[49]. Inclusion of an adequate number of currently circulating Leptospira isolates in the MAT panel and a re-evaluation procedure is required before committing to the use of new isolates. The on-going studies in Sri Lanka with recently recovered isolates will provide adequate new strains for this purpose[50]

A relatively low reactivity was observed with the saprophytic non-pathogenic serovar Patoc (16%). Usually the saprophytic species L. biflexa is considered to have a broader reactivity and is therefore recommended to be included in MAT panels [11]. However, our results were consistent with an ELISA-based study in Sri Lanka which showed reduced sensitivity of saprophyte L. biflexa in serological assays compared to a pathogenic local isolate [51]. Similar results have been observed in studies done in other countries with the Patoc strain[52]. Based on these observations, MAT should be performed with a panel of serovars; and testing only with saprophytic L. biflexa, assuming a broader range of reactivity, could lead to gross underestimation of leptospirosis cases.

This study has few technical shortcomings. Ideally, for the internal quality control step, antisera should be available for each strain in the test panel and should be run with each test. In addition to ensuring quality control of the test, this step will also enable identifying the mislabelling of live antigens which might happen during long-term maintenance. However, due to financial issues the purchasing of antisera for the whole panel wasn’t possible, and hence only one antiserum was used as a quality control.

We observed that a large number of “clinical leptospirosis” patients were negative. This low sensitivity could be due to several possibilities. Ideally, before concluding a negative result, paired sera should be available to look for seroconversion or a four-fold rise in titre. However, the lack of paired sera for a majority of samples (available only for 15%) in this study might contribute to a low level of detection. The findings of this study also highlight the poor utility of MAT as a diagnostic tool in the acute stage of the disease which is the critical period in patient management.

Another possibility for the low positivity could be due to other aetiologies in these patients such as simple viral fever, dengue fever or rickettsiosis as they are from a cohort of acute undifferentiated fever patients. Of the patients we tested, 51% had only fever, headache, and myalgia—a set of symptoms which may appear due to many tropical fevers.

On other other hand, some of the patients confirmed as having a single high titre may probably be affected by high background tires. There are no Sri Lankan studies estimating background titres and it may slightly overestimate the positivity. However, it will not affect the main focus of the study.

MAT is used as the serological reference test despite having low sensitivity, low value as a clinical diagnostic tool, and involving labour intense procedures. Hence determining the optimum number of strains to be included into the test panel is a crucial step in many settings where the resources are limited. For Sri Lanka, further improvement of the proposed panel is required with the addition of newly isolated strains.

Considering the laborious nature of the test procedure and low sensitivity in the acute stage, the utility of MAT as a reference test seems to be imprudent. Rapid bed side diagnostics are a reasonable alternative to overcome the inherent issues with MAT. These diagnostics include lateral flow immune assays as screening tests, supported with ELISA-based tests as confirmatory assays. However ELISA based serological studies also need to be validated as there can be many cross reactions with other infections. Furthermore the antigen should be regionally optimised to increase the sensitivity of the test as has been shown in studies done in the local setting[51]. A highly sensitive and specific serological tests like immunofluroscence assays haven’t been extensively studied limiting its’ use in the resource poor endemic regions[20] Although a PCR-based molecular diagnostic is expensive and technically demanding, it would be a good complimentary test to be used in the early stage of the disease before a serological response develops. Ideally the use of MAT should be preserved for epidemiological purposes, especially in endemic countries.

5 May 2021

Dear Dr. Suneth Agampodi,

Thank you very much for submitting your manuscript "Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource, hyper-endemic setting with varied microgeographic variation in reactivity." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Sincerely,

Vasantha kumari Neela

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Bastos

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: General Observations:

The microscopic agglutination test (MAT) is the basis or cornerstone test for serological classification and diagnosis. However, in the recent past MAT is used for the sero- epidemiological studies than diagnosis. MAT antibodies appear at the end of the first week of the onset of the disease, reaches to peak during the second week or early third week. Hence detecting MAT antibodies using MAT may not much useful for the clinical management of patients . More so single MAT may not provide conclusive information on current clinical infection or confirmatory diagnosis and technically cumbersome. Molecular genetic-based techniques with a conjunction of IgM ELISA (for early and late reporting cases respectively ) are in use to provide the highest patient care. Authors themselves expressed the similar opinion. Nevertheless, MAT is the reference test and the choice for serological classification and tracking of animal vectors. Optimizing the panel of serovars / strains for the microscopic agglutination test (MAT) for the diagnosis of Leptospirosis (use of representative strains/ serovars reported (one or two strains from each serogroup ) and inclusion local isolates is well known and not a new . More so geographic genomics - gene acquisition and gene loss on evolutionary time scale is a continues process in leptospires and it is evidence from classification – about 300 serovars and several species (sensu stricto ). In view of the above phenomenon , optimizing MAT panel at particular point of time may not be ideal or optimized panel at another point of time.

Specific points:

Introduction- Line 92 – 94: No single MAT titre can be regarded as diagnostic of acute or current clinical infection , in a proportion of confirmed patients may have low MAT titres or past infection may have a high titre ( it is well known that microscopic agglutinating antibodies are long-lasof ting and persist several months to several years ). More so , prevalence of microscopic agglutinating antibodies are commonly observed among the community , use of single MAT may increase the false positive results . Since the central dogma of serology is defined as four-fold rise in titre or greater rise in titres, demonstration of rising titres by using paired MAT only confirm the current infection . More so it is observed that the dilutions are mentioned as 1: 400 or 1: 800 or so on. Please be noted here that In the diagnostic serology, the dilutions should be expressed as 1 in 400 or 1in 800 or so on as 1 in 400 is different from 1: 400 . (1 part of serum and 399 parts diluent and 1 part of serum and 400 parts diluent respectively) . Can be please rewritten for better understanding.

Methodology : Line 183 -186 : Please see general observations - MAT panel for the diagnosis of leptospirosis and please clarify .

Reviewer #3: The theme was well contextualized in the introduction subsidizing the development of the manuscript. The importance this work has it’s from the optimization of MAT with the proposal to change the panel of serovars used mainly in the countries with limited resources, reduction of the number of strains tested without loss of reactivity and epidemiological assessment of the disease. The etiological agent, phenotypic and genotypic classification, diversity, occurrence and it interaction with different hosts were referenced; difficulty in early diagnosis (acute phase) of leptospirosis. The definition of cases for non-endemic areas was considered as proposed by “Leptospirosis Reference Epidemiology Group”. Also, complexity of its implementation, limitations and importance of MAT, as well as the advantages and application of the techniques available for the diagnosis of leptospirosis.

The purpose of the study is clear. The study design was appropriate to achieve the objectives of the study. The authors will use samples from a cohort of patients with undifferentiated fever. These were divided into two types/groups: the first group of patients was with undifferentiated acute fever treated in outpatient department, hospital wards and paediatrics wards with ≥ 12 years old; the second group were patients with suspect symptoms of leptospirosis from any ward, without age restriction. The cohort study was from June 2016 to January 2019 in which the samples came from geographic areas with different characteristics (temperature, altitude, ecology, rainfall, human behavior and endemicity). The study observed the ethical requirements.

Considering that Sri Lanka is a hyperendemic country for leptospirosis, is the cut-off point 1:100 or 1:50? Why was the cut-off point 1:100 used in the screening test while in titration it was used 1:50?

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

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: Results :

Lines 235-240: It is stated that 19 of the 150 patients with paired samples had a titre of 1 in 400 or more in the acute sample. Subsequently it is stated that 15 patients had a titre of 1 in 400 or more, but did not meet the other criteria for a positive diagnosis. I assume that 4 patients had an initial titre of 1 in 400 or more, but showed 4 fold rise in titre in the convalescent sample. If this is so, it may not be clear to the reader. Please state this clearly.

Lines 247 – 251: ‘We predicted the number of positives, if paired samples were available for all patients…’. Does this mean that the authors applied the proportion of positive cases among the 150 patients (33%) to the entire cohort of 982 patients to estimate the number of cases among them? If so, please clarify this and state the estimated number.

Lines 249-251: Did the authors calculate the 95% confidence interval of the estimated number? What is 29%? Is it the width of the confidence interval of the estimated prevalence of leptospirosis among the 150 patients whose paired sera was available? Please state the confidence limits as lower and upper bounds.

Line 250-251: It is not clear what ‘difference’ was statistically significant. How McNemar test, which is for paired data, was performed? . Please clarify .

Line 253 , table 2 : A total of 107 patients confirmed as positives and out which 73 based on single MAT . Since the study was conducted in hyperendemic area out 73 confirmed patients , there is a probability that the proportion these 73 cases may be false positives (please see Specific points mentioned – Introduction -line 92 – 94 ) . The base line MAT titres in the community may vary from one geographical area to other . Is there any community based study conducted at the study area to estimate the base line titre and to draw cut -off titre for single MAT . Please clarify .

Reviewer #3: The characterization of patients does not refer to the two types (groups) of patients initially presented (lines 157 to 166). The clinical characteristics and the disease course are extremely important for results interpretation of MAT as well as other methodologies.

I recommend adding stratify the samples evaluated by MAT according to the disease course (days or weeks after the onset of symptoms).

The authors used samples with less then 15 days. MAT shows reactivity from the 7th or 8th day of onset symptom and there is a considerable difference in sensitivity before and after the first week of illness. The analysis of the results considering the first and second week of illness could provide different information. This method is the gold standard recommended by the WHO as well as by other institutions and entities such as CDC and ILS. Was other methodology performed (PCR, ELISA) in parallel to MAT of the two types/groups of patients studied? Table 1 shows data from 982 samples from 1132 patients. Of the total samples, 105 patients did not report age, 79 not report sex, 78 ethnicity, 7 patients were lost without information about they were hospitalized or were on an outpatient departments and about the clinical presentation, 227 patients not even were mentioned if they presented other symptoms.

I recommend completing the information even when it has not been properly reported.

Under the table 1, line 237, the three classic characteristics (symptoms) are referenced: headache, myalgia and fever. I recommend that, in the same way, the two classic characteristics (symptoms) be referenced (myalgia and fever?).

The line 239 mention of the 150 paired samples, 19 (13%) had titres of ≥1:400 during the acute stage and lines 240-241 mention another 11 (7%) samples had titres ranging from 1:50 to 1:200. Paired sample analysis showed 49 (33%) confirmed cases. I can conclude that, of the 49 paired samples 19 it were from the convalescent phase?

I recommend conceptualize the acute phase by defining the period in days and rewriting the text from lines 239 to 244 with the exception of the last sentence to improve understanding.

The writing of the line 243 is not clear “and another 15 (10%) with titres ≥1: 400 but not falling into the above two categories”.

In Table 2, which type of patients according to the group mentioned in lines157 to 166 does the 107 leptospirosis confirmed patients belong to?

Table 3 show that were used serovars of 17 serogroups recommended by the WHO, but there are different serovars and strains from the recommended panel. If were used the exactly recommended serovars and strains, could it generate a different reactivity profile?

Please review the wording for lines 239 and 290. The line 239 mention that is 19 positive paired samples while the line 290 mention 15 positive paired samples in the acute phase? Which is the correct number of sample?

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

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: Despite the shortcomings of the MAT, well described here, also ELISA tests do have shortcomings (ie cross reactions). This also should be adressed in the discussion, to avoid that readers use any ELISA which tests for antileptospiral antibodies.

Reviewer #2: A total of 107 patients confirmed as positives and out which 73 based on single MAT . Since the study was conducted in hyperendemic area out 73 confirmed patients , there is a probability that the proportion these 73 cases may be false positives (please see Specific points mentioned – Introduction -line 92 – 94 ) . The base line MAT titres in the community may vary from one geographical area to other . Is there any community based study conducted at the study area to estimate the base line titre and to draw cut -off titre for single MAT . Please clarify .

Reviewer #3: Manuscript discussion emphasizes the difficulties in performing the MAT technique. Considering that, the objective of the article was “The purpose of the present study was to describe the procedure for selecting a sensitive as well as cost effective MAT panel in Sri Lanka using a cohort of patients with acute undifferentiated fever”.

I recommend including in the discussion the limitations of the other diagnostic methods, mainly the serological tests.

In this study, were the samples submitted to analysis by another methodology? If so, it would be important to present these data.

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

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: 1. When reading the procedure of the MAT, it seems that the authors did not include the volume of the leptospiral antigen in the titers. They describe the serum sample is diluted 1:50. They also describe their dilution series ranging from 1:50 to 1:3200. In the results titres of 1:50 are described. To my knowledge most of the laboratories performing MAT report titers including the added antigen. I would suggest the authors to state cleary that they report the serum dilutions, without the antigen, so that this is clear to all readers.

2. The sentence from line 257 to 258 needs to be reformulated.It seems the authors mean that from the panel of leptospiral strains, there was with each serovar a MAT titer with 1 or more of the 982 serum samples. Or they should say that by MAT panel they mean the sera tested by MAT. But earlier in the methods (line 183) they state that the MAT panel consists of leptospiral serovars.

3. strain Van Tienan should be spelled as Van Tienen.

Reviewer #2: As Suggested under methodology and results

Reviewer #3: (No Response)

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

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: This is a clear study, which gives a good example about how to set up a MAT panel for a certain geographical area. Most important is to realize that a situation is not static and new serovar might appear in a region.

Reviewer #2: The microscopic agglutination test (MAT) is the basis or cornerstone test for serological classification and diagnosis. However, in the recent past MAT is used for the sero- epidemiological studies than diagnosis. MAT antibodies appear at the end of the first week of the onset of the disease, reaches to peak during the second week or early third week. Hence detecting MAT antibodies using MAT may not much useful for the clinical management of patients . More so single MAT may not provide conclusive information on current clinical infection or confirmatory diagnosis and technically cumbersome. Molecular genetic-based techniques with a conjunction of IgM ELISA (for early and late reporting cases respectively ) are in use to provide the highest patient care. Authors themselves expressed the similar opinion. Nevertheless, MAT is the reference test and the choice for serological classification and tracking of animal vectors. Optimizing the panel of serovars / strains for the microscopic agglutination test (MAT) for the diagnosis of Leptospirosis (use of representative strains/ serovars reported (one or two strains from each serogroup ) and inclusion local isolates is well known and not a new . More so geographic genomics - gene acquisition and gene loss on evolutionary time scale is a continues process in leptospires and it is evidence from classification – about 300 serovars and several species (sensu stricto ). In view of the above phenomenon , optimizing MAT panel at particular point of time may not be ideal or optimized panel at another point of time.

Reviewer #3: I congratulate the authors for the research developed. The article is relevant for the improvement of leptospirosis diagnosis. The leptospirosis is neglected in many countries because they are unable to diagnose it in a timely manner.

The MAT method is extremely labor and there is a need for the development of new methodologies that allow early diagnosis as well as the identification of the different serovars circulating in different countries.

Currently, MAT is important both for the diagnosis of leptospirosis and to verify the endemicity of the disease.

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Submitted filename: MAT -less resorce settings Sri lanka.docx

Click here for additional data file.

21 May 2021

Submitted filename: Author_Responce.docx

Click here for additional data file.

15 Jun 2021

Dear Dr. Suneth Agampodi,

We are pleased to inform you that your manuscript 'Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource, hyper-endemic setting with varied microgeographic variation in reactivity.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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

Best regards,

Vasantha kumari Neela

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Bastos

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)

**********

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)

**********

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)

**********

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: I just noticed a small typo: In line 387 "On other rother hand"

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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

28 Jun 2021

Dear Dr Agampodi,

We are delighted to inform you that your manuscript, "Optimizing the microscopic agglutination test (MAT) panel for the diagnosis of Leptospirosis in a low resource, hyper-endemic setting with varied microgeographic variation in reactivity.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases