JCM Accepted Manuscript Posted Online 3 February 2016 J. Clin. Microbiol. doi:10.1128/JCM.02620-15 Copyright © 2016 Vallur et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Accurate sero-detection of asymptomatic Leishmania donovani infection using defined
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antigens
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Aarthy C Vallur1, Caroline Reinhart1, Raodoh Mohamath1, Yasuyuki Goto1,*, Prakash Ghosh2,
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Dinesh Mondal2, Malcolm S Duthie1 and Steven G Reed1, #
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Diseases Research, Laboratory Sciences Division, Dhaka, Bangladesh
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Running title: Novel antigens for detecting L. donovani infection
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*
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Life Sciences, University of Tokyo, Tokyo, Japan
Infectious Disease Research Institute, Seattle, USA,
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International Center for Diarrhoeal
Present address: Laboratory of Molecular Immunology, Graduate School of Agricultural and
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#
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Steven G Reed
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e-mail:
[email protected]
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Key words: Leishmania, kala azar, antigen, asymptomatic, protein, diagnosis, antibody,
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surveillance, elimination
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Abbreviations: VL: visceral leishmaniasis, Ld/ L. donovani: Leishmania donovani, DAT: Direct
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Agglutination Test, EC: endemic control, ELISA: enzyme-linked immunosorbent assay, NEC:
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non-endemic control, OD: other diseases, PBS: phosphate-buffered saline, PBS-T: PBS-Tween,
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PCR: Polymerase Chain Reaction, PKDL: Post-Kala azar dermal leishmaniasis, RDT: Rapid
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Diagnostic Test, TR: tandem repeat, SLA: Soluble lysate antigen
Address correspondence and reprint requests to
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ABSTRACT
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Infection with Leishmania donovani is typically asymptomatic, but a significant number of
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individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200
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million people in endemic areas. While diagnosis of acute VL has been simplified by the use of
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cost-effective confirmatory serological tests, similar standardized tools are not widely available
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for detecting asymptomatic infection which can be 4-20 times more prevalent than active
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disease. A simple and accurate serological test capable of detecting asymptomatic L. donovani
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infection will be useful for surveillance programs targeting VL control and elimination. To
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address this unmet need, we evaluated recombinant antigens for their ability to detect serum
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antibodies in 104 asymptomatic L. donovani infected individuals (qualified as positive for L.
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donovani-specific antibodies by direct agglutination test; DAT) from the VL hyperendemic
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Mymensingh district of Bangladesh. The novel proteins rKR95 and rTR18 possessed the
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greatest potential and detected 69% of DAT positive individuals, with rKR95 being more robust
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in reactivity. Agreement in results with individuals with high DAT responses, who are more
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likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard
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antigen used to confirm clinical diagnosis of VL but which is now becoming widely used for
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surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined
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estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests
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amenable to rapid or high-throughput screening could enable simple and accurate detection of
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asymptomatic infection. Such tests will be important tools to measure L. donovani infection
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rates, a primary goal in surveillance and a critical measurement with which to assess elimination
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programs.
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Introduction
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Visceral Leishmaniasis (VL or Kala azar) is one of the deadliest and most neglected
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tropical diseases in the world. It affects the poorest among mostly rural endemic populations,
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with around 300,000 new cases every year (1, 2). Of these, about 90% occur in the Indian sub-
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continent, Brazil and East Africa, while VL is an emerging threat in the Mediterranean basin (1).
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In the Indian sub-continent, VL is caused by infection with Leishmania donovani.
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Importantly, VL in the Indian subcontinent has some unique features that appear to make it a
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candidate for elimination (3, 4). The presence of a single vector, anthroponotic transmission and
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a defined, rural area of endemicity have empowered the governments of India, Nepal and
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Bangladesh to propose the Kala-azar elimination program, which aims to reduce the incidence
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of VL to less than 1 case per 10,000 individuals by 2015 (5). Early case detection, treatment
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and integrated vector control are three of six pillars of the program (5-7). Of these, the practical
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difficulties plaguing early case detection have been a roadblock in advancing toward the
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elimination program objectives (8, 9). Recognizing the potential of the rk39-based rapid
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diagnostic tests (RDT), the elimination program has made it readily available to clinics and
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health care workers in endemic regions, thereby enhancing detection of acute VL cases. A
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major challenge for the elimination campaign is the large population of asymptomatic L.
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donovani infected individuals residing in endemic regions. Such individuals harbor low levels of
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infection but display no symptoms of VL. Although clearance of the parasites appears to be the
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typical outcome, these individuals can potentially transmit parasites to others and are at an
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elevated risk of developing VL (10, 11). Early case detection hinges on identifying
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asymptomatics and monitoring them periodically. Thus, surveillance, detection, and monitoring
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of asymptomatic L. donovani infection are absolutely necessary to realize Kala-azar elimination.
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Existing diagnostic tools are not entirely suitable for detection of asymptomatic infection.
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Diagnostic methods such as microscopy of splenic aspirates are unethical in asymptomatic
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individuals and unsuitable for large population surveillance. The rk39 RDT recommended for
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confirming VL disease in the Indian sub-continent is not fully reliable to screen for asymptomatic
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L. donovani infection (12). At present, ELISA against rK39 and the Direct Agglutination Test
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(DAT) are commonly used in large surveillance studies for asymptomatic infection in the Indian
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sub-continent (13-16). Although DAT is effective for detecting L. donovani infection because it
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offers a broader antigen panel, the use of freeze-dried promastigotes as the detecting antigen
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can render it susceptible to lot-to-lot variations (13, 17). In addition, DAT is labor intensive,
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markedly lower in throughput than a standard ELISA and most importantly, being a visual test, it
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is extremely difficult to set uniform standards for widespread use.
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Detection of asymptomatic L. donovani infection is an urgent need within VL control
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programs. Given that asymptomatic L. donovani infections are more common than VL, there is a
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need for simple and standardized tools to provide sensitive, specific and quantitative results
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whilst also facilitating high-throughput screening within endemic regions. In an attempt to
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develop a recombinant antigen-based serological test with these properties for use in
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surveillance for asymptomatic infection, we evaluated several Leishmania antigens in an ELISA
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on sera from likely asymptomatic L. donovani infected individuals in Bangladesh. Through this
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process we selected two recombinant antigens that complemented rk39 ELISA and were
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comparable to DAT in detecting asymptomatic L. donovani infection. We discuss our results in
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terms of a dedicated serological tool to screen endemic areas for asymptomatic L. donovani
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infections in a conventional ELISA or a rapid test format.
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Methods and Materials:
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Samples
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All samples were collected following approval from the respective Ethics Committee and after obtaining individual consent forms. Blood was obtained and sera/DNA prepared from individuals with no past history of VL
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or PKDL residing in the VL hyper-endemic region in Harirampur Union, Trishal sub-district,
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Mymensingh district, Bangladesh as described before (10). Initial consent was obtained from
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the head of household to screen household members, then individual written consent was
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obtained from participants prior to study enrollment. Sera from clinically confirmed VL patients
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were included as positive controls. Sera from 46 healthy individuals in the US who had no
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history of travel outside the US (purchased from Equitech, TX), were used as non-endemic
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controls (NEC) to establish cut-offs for sensitivity. In addition to the NEC, sera from healthy
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endemic controls (EC) from the Mymensingh district were used. To measure cross-reactivity
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with other diseases (OD), sera from patients with non-VL febrile illnesses from a non-endemic
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region (the Philippines) were used. These individuals were defined with no previous history of
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VL due to negative responses in DAT and rK39 RDT.
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Initial sample characterization
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Initial serum characterization was conducted using the Direct Agglutination Test (DAT)
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(KIT Biomedical, Amsterdam) performed according to manufacturer’s instructions at icddr,b,
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Dhaka, Bangladesh. Based on a DAT titer >1600 in these evaluations, 104 sera samples were
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designated as being DAT positive and are referred to as asymptomatic L. donovani infected
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individuals in this study. The 104 individuals were also tested on finger prick blood for positivity
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with the rK39-RDT (KalaAzar DetectTM, InBios International, Seattle) by investigators from
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icddr,b, Dhaka, Bangladesh. Healthy endemic controls were defined as DAT and rK39-RDT
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negative individuals from the same area as the asymptomatic individuals. Aliquots of the sera
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samples were shipped to IDRI and archived.
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Recombinant proteins
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Several tandem repeat (TR) proteins previously identified in a bioinformatics screen and
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validated as being reactive with VL patient sera were evaluated (18). Kinesin-like protein was
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identified by a LC-MS/MS analysis of sera and urine samples from VL patients performed using
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standard procedures at ITSI Biosciences (Johnstown, PA). Peptide hits were then screened for
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Leishmania specificity against L. donovani and L. infantum databases using Proteome
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Discoverer 1.2 and the SEQUEST algorithm. Peptides specific to VL patient samples but not
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healthy US samples were considered as Leishmania-specific peptides. L. donovani kinesin-
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related protein, henceforth referred to as rKR95 (GI.112293604) was identified by 5 peptide hits
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in 4 VL sera and 1 peptide hit in 1 VL urine sample and was chosen for further validation after
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confirming lack of homology to human counterparts (Table 1). The construct encoding the 742
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amino acids sequence that contains all six peptides was synthesized and cloned into pET17a
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with an N-terminal 6XHis tag (Gene Dynamics, Tigard, OR). The sequence verified expression
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construct was expressed from E. coli strain BL21 (DE3) pLys (Invitrogen) and purified as
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described previously (18).The purity and integrity of each protein was confirmed by SDS-PAGE.
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rK39 antigen was made as described previously(19).
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Ld SLA was made
by lysing 1×107 cultured L. donovani/MHOM/80/IN/DD8
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promastigotes in the presence of 1X protease inhibitors (HALT protease inhibitor cocktail,
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Thermofisher) and 5mM EDTA by repeated freeze thawing followed by sonication and
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separating insoluble material by centrifuging for 45 minutes at 15,000 rpm.
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Antibody reactivity by ELISA
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All ELISAs were done at IDRI. High binding 384-well plates (Corning, NY) were coated
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with 1µg/mL recombinant protein or 2.5µg/mL Ld SLA in the ELISA Coating buffer
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(eBioSciences, Inc., San Diego, CA) at room temperature for 4 hours and washed with PBS6
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Tween (PBS-T) before being blocked overnight at 4oC with PBS-T with 1% BSA. Plates were
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washed as before. Serum diluted 1:200 in PBS-T with 0.1% BSA was then added to each well
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and plates were incubated at room temperature for 2 hours on a shaking platform. After washing
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as before, horseradish peroxidase-conjugated anti-human IgG (heavy and light chains)
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(Invitrogen) diluted 1:8000 in PBS-T with 0.1% BSA was added to each well and incubated for
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1h at room temperature. Plates were washed again and developed with SureBlue™ TMB
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Microwell Peroxidase Substrate (KPL, Inc., Gaithersburg, MD). 1N H2SO4 was then added
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to each well to stop the reaction. The optical density of each well was immediately read at both
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450nm and 570nm. The final, background-adjusted signal for each sample was calculated by
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subtracting the 570nm reading from the 450nm reading. Cut-off for sero-positivity on each plate
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was defined as the mean signal of normal sera added 2 standard deviations. Signal to noise
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ratios were calculated as ELISA signal over cut-off for any antigen. Ratios over 1 were
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considered positive.
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Sequence alignments
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The BLASTn suite (NCBI) using the cloned sequences as queries against Leishmania
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and non-Leishmania genomes or cDNA sequences when genomes were not available.
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Statistical analysis
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One-way ANOVA was used to determine the statistical significance between non-
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parametric data sets to assess the performance of antigens, with p
