Development of a Novel Enzyme-Linked Immunosorbent Assay and Lateral Flow Test System for Improved Serodiagnosis of Visceral Leishmaniasis in Different Areas of Endemicity

ABSTRACT Visceral leishmaniasis (VL) is caused by protozoan parasites of the Leishmania donovani complex and is one of the most prominent vector-borne infectious diseases with epidemic and mortality potential if not correctly diagnosed and treated. East African countries suffer from a very high incidence of VL, and although several diagnostic tests are available for VL, diagnosis continues to represent a big challenge in these countries due to the lack of sensitivity and specificity of current serological tools. Based on bioinformatic analysis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed. The diagnostic performance of rKLi8.3 was evaluated by enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) on a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other diseases, including tuberculosis, malaria, and trypanosomiasis. The diagnostic accuracy of rKLi8.3 was compared with rK39 and rKLO8 antigens. The VL-specific sensitivity of rK39, rKLO8, and rKLi8.3 ranged from 91.2% over 92.4% to 97.1% and specificity ranged from 93.6% over 97.6% to 99.2%, respectively. In India, all tests showed a comparable specificity of 90.9%, while the sensitivity ranged from 94.7% to 100% (rKLi8.3). In contrast to commercial serodiagnostic tests, rKLi8.3-based ELISA and LFT showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer improved VL serodiagnostic efficiency in East Africa and other areas of endemicity. IMPORTANCE Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases. Both prototype rKLi8.3-based enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other areas of endemicity, compared to currently commercially available serodiagnostic tests.

leishmaniasis. The aim of the study presented herein was to develop two serological tests, ELISA and a lateral flow test, both of which show a higher sensitivity and specificity compared to current available in vitro diagnostic tests. The work and presentation are overall well written and should be of importance to researchers and medical technicians interested in the diagnostic of visceral leishmaniasis.
Major points While this reviewer agrees with the authors conclusions that the sensitivity of the ELISA and the lateral flow test was increased compared to antigens previously used for the diagnosis of visceral leishmaniasis, there are some areas of improvement, in particular in the discussion of the data. But what this reviewer consider the weakness -not way a fatal flaw -is the small sample size of serum samples examined from patients with other infectious diseases used as controls. For almost all tests conducted, only a few serum samples from patients with malaria, schistosomiasis, Chagas disease, amoebiasis, and echinococcosis were employed. Also, the sample size of sera collected from India was very small (n= 21) as well. Below are comments for the authors to consider.
1. As highlighted in the general comments above, the number of serum samples from patients with other bacterial or parasitic infections used as further controls is very small while the number of control serum samples from healthy donors is quite large (n= 85). Despite the acquisition of serum samples is very difficult, it would be of importance to add more serum samples to evaluate the data collected. If collection of more serum samples is for any reason impossible, the authors have to point out this obvious limitation of the study, maybe in the discussion.
2. On page 6, the authors mentioned that kinesins have been used as antigens for the sero-diagnosis of visceral leishmaniasis caused by species of the L. donovani complex. In their previous work, they used a recombinant antigen, rKLO3 from an L. donavani isolate. Here, a novel antigen from L. infantum has been used for evaluation and optimization of the test. It is not entirely clear, why the authors decided to use a kinesin from L. infantum. Also, the rationale why a comparative sequence analyses of kinesins of L. infantum strains were conducted is not clear as well (see page 6, lines 191-196). Why they end up with the three kinesins described in the following paragraph? This should be clarified in the result.
3. To show the differences between the three selected antigens (rKLi6.3, rKLi7.3, and rKLi8.3) and to make the manuscript more reader-friendly, a separate figure showing the structural differences of these three antigens would be strongly recommend.
4. In figure 1, the authors presented data from the ELISA performed with three different antigens. It seems that the identical data collected with the rKLi8.3 antigen were also used for figure 2C and 3C. The authors should clearly indicate whether these data were used for the calculations of the data presented in figure 2D and 3D. To avoid any misunderstandings, the data obtained with rKLi8.3 should be only presented in figure 1. 5. On page 7, second paragraph, To evaluate the performance of the ELISA, two additional antigens, rK39 and rKLO8, were investigated. The origin or source of these antigens are unclear. Should the antigens genetically engineered by the authors, a full description including construction (amplification, cloning, etc.) and purification have to be included in the Materials and Methods. Should these antigens are commercially available, the source of these proteins have to be included.
6. The discussion is very short and needs improvement. A comprehensive discussion of the strengths and limitation (e.g. sample size of the controls are very small) of the study are strongly recommended as well as the future perspectives how to increase the sensitivity of the novel test systems. Also, the benefit of the developed tests described herein compared to in vitro diagnostics currently used have to be explored in a better way.

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This paper describes the development of a novel more specific and sensitive ELISA and lateral flow test for the diagnosis of visceral Leishmaniasis in several countries to newly described antigen. The group compared previous antigens on a range of positive and control patients to understand the specificity and sensitivity.

Major Comments
It would be interesting to determine if the false positive seen in endemic controls in Fig 1 was positive in all of the tests/antigens or where these different FPs?
Do you see varying antibody response based on region of Sudan ?   Fig 1, 2, 3, and 4.A -What statistic measure was used to designate your cut off or was this arbitrarily designated?
Does this have an effect on the sensitivity and specificity, what if you changed this to be the same for all antigens and compared values across the board for example all at 0.106?  All tables; if you had the same cut offs, would this impact sensitivity and specificity?
Please find the names of the various "anonymous" authors in the references.

Minor Comments
Line 45 -"L" in linked should be capitalized.
Line 59 -Cross reactivity should be two words or hyphenated.
Line 97 -remove "here" Line 101 -please add references to where the previous ethics were approved for obtaining these samples from patients.
Line 163 -please name the control protein used.
Line 199 -add spaces between "L. and species".
SF3 -Again, was the same false positive the same in all 3 antigen ELISA tests?

Figure 4: In (A) add a color key on the image to indicate the three types of tests as in (B). Or it
can be mentioned in the caption to cover both figures. Table 1-4: Mention the unit of cut-off.  Fig S1: Add the full name for the abbreviations: N, C, T, C, AB, S Figure S3: Change 'toxoplasmose' to 'Toxoplasma gondii' Change 'malaria' to 'Plasmodium spp.'

Point by point reply
We thank both reviewers for taking time and their constructive comments which improved our manuscript. Thank you very much! Please find below our point by point reply:

Reviewer #3
First, we want to thank for the appreciation of our work!

1) the number of serum samples from patients with other bacterial or parasitic infections used as further controls is very small…
Reply: We agree that the number of sera with other infections is limited. However, it is extremely difficult to get enough control sera from patients with other infections from the same (VL) endemic regions. Despite these limitations, no cross-reactivity was observed with 11 different pathogens. In the revised manuscript, we mentioned the difficulties to obtain control sera (line 279-282).

2) In their previous work, they used a recombinant antigen, rKLO3 from an L. donavani isolate.
Here, a novel antigen from L. infantum has been used for evaluation and optimization of the test. It is not entirely clear, why the authors decided to use a kinesin from L. infantum. Also, the rationale why a comparative sequence analyses of kinesins of L. infantum strains were conducted is not clear as well (see page 6, lines 191-196). Why they end up with the three kinesins described in the following paragraph? This should be clarified in the result.
Reply: The aim of the project was to identify and develop an antigen that provides better diagnostic performance in East-Africa than rKLO8 or the rK39 from L.donovani. Our selection was based on the following rationale: Antigens that contain charged AA give better antibody responses than antigens with uncharged AA, i.e. the substitution of charged for uncharged AA reduces the antigenicity. We thus compared multiple recombinant kinesin proteins from different strains (L.donovani, L. archibaldi and L.infantum). We identified only one kinesin protein (rKLi8.3 from L.infantum) where charged AA were not substituted (Supplemental Table S2). Besides the sequence, the structure of the kinesin antigen (number of tandem repeats) might also influence the antigenicity, as predicted by BepiPred and shown in supplemental figure S3. Since these analyses were only in silico-predictions, we therefore tested the influence of kinesin sequence (Fig 1) and structure (Fig 2)  3) To show the differences between the three selected antigens (rKLi6.3,rKLi7.3,and rKLi8.3) and to make the manuscript more reader-friendly, a separate figure showing the structural differences of these three antigens would be strongly recommend.

Reply:
In the revised version, we have added a scheme (new supplemental figure S2), which illustrates the structures of rKLi6.3, rKLi7.3 and rKLi8.3.

4)
In figure 1, the authors presented data from the ELISA performed with three different antigens. It seems that the identical data collected with the rKLi8.3 antigen were also used for figure 2C and 3C. The authors should clearly indicate whether these data were used for the calculations of the data presented in figure 2D and 3D. To avoid any misunderstandings, the data obtained with rKLi8.3 should be only presented in figure 1.

Reply.:
We apologize for our mistake in showing the same rKLi8.3 data in Figs 1, 2 and 3. As we have always tested the different antigens simultaneously with rKLi8.3, we have included the corresponding rKLi8.3 data in the revised version for Figs 1, 2 and 3. We also used the new data for the calculation.
5) On page 7, second paragraph, to evaluate the performance of the ELISA, two additional antigens, rK39 and rKLO8, were investigated. The origin or source of these antigens are unclear. Should the antigens genetically engineered by the authors, a full description including construction (amplification, cloning, etc.) and purification have to be included in the Materials and Methods. Should these antigens are commercially available, the source of these proteins have to be included.
Reply: Thank you very much for reminding us to refer to the origin of rK39 and rKLO8.
We inserted a new paragraph in the Material and Method section, explaining the origin and cloning of both proteins, line 148-151. The rK39 protein was purchased from Rekom. Biotech, S.L., Granada Spain and Cloning; expression and purification of rKLO8 has recently been described by us, Plos Neglected tropical disease, 18, 2013.
6) The discussion is very short and needs improvement. A comprehensive discussion of the strengths and limitation (e.g. sample size of the controls are very small) of the study are strongly recommended as well as the future perspectives how to increase the sensitivity of the novel test systems. Also, the benefit of the developed tests described herein compared to in vitro diagnostics currently used have to be explored in a better way.

Reply:
We have improved the discussion by more explicit mentioning the strength and limitations of the newly developed diagnostic tests, line 279-291.

7) It is not clear to what the number of replicates of each serum sample was used in the ELISAs and LFT. This needs clarification
Reply: All ELISAs has been repeated twice with serum samples in duplicates. According to the manufactures protocols, valid LF test (strong internal control band) were performed once. If the control band was weak or absent, LF tests were repeated. This information is now included in the Material and Method section.
8) The definition of the cut off values should be explained more clearly. It is not entirely clear why the authors define the mean absorbance values ´plus three standard deviations´. Please clarify.

Reply:
The cut-off values determine whether a serum sample is considered positive or negative. Determination of the cut-off value is a standard procedure which is calculated as follows: Mean OD value of healthy controls (x) + 3 standard deviation (SD). OD values above x+3SD were considered positive. To achieve a most accurate cut-off value, 50 negative sera were used. Furthermore, the cut-off value for each recombinant protein was confirmed using the receiver operating characteristic (ROC) curve. This has been mentioned in statistical analysis.

9) Minor points
Reply: All minor points are directly addressed in the manuscript.

Reviewer #4
First, we also want to thank the reviewer 4 for very careful reading and commenting our work!
If not explicitly mentioned in this letter, all minor points have been directly addressed in the revised manuscript according to the suggestions of the reviewer.
Line 183: Mention the distribution of the data (Gaussian vs. non-Gaussian) to justify the statistical tools used.
Reply: Comparing defined patient entities (VL vs. endemic controls; VL vs Malaria; VL vs Tuberculosis; etc.) we compare 2 or more independent groups. The data of the antibody response of defined patient group clearly show a parametric (Gaussian) distribution. Therefore we employed student´s t-test for two independent groups or one way ANOVA for several independent groups. In all cases (comparison of two or multiple groups) the significance was P<0.0001. This is now explained in the revised version, section statistical analysis. Reply: The rKLi8.3 prototype ELISA exceeded TECAN/NovaLisa in terms of sensitivity and specificity. Sera from malaria, tuberculosis and Chagas patients have been shown to crossreact with tests using rK39 or whole parasite lysate. This may be due to shared antigenic determinants or a close phylogenetic relationship of e.g. Leishmania with Trypanosomes, which both belong to the family of kinetoplastida. In contrast, the rKLi8.3 ELISA and LFT showed no cross-reactivity with sera from tuberculosis-, malaria-and Chagas patients. The high specificity of rKLi8.3 is most likely due to the combination of a conserved kinesin sequence with high antigenicity (charged AA) and the increased number of TRs. This is now included in the discussion of the revised version, line 281-291. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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Editor, Microbiology Spectrum Editor comments: Line 76: In your Introduction, remind readers which species are associated with visceral leishmaniasis.
Line 180-182: What manufacturer? No manufacturer is mentioned in this section other than the dispense platform manufacturer.
Line 244: Spell out DEV Line 420: Are these variations species-specific? Figure 4: Was there statistical significance between the VL and CL groups, and between the VL and Chagas groups? If this makes the figure too complicated, consider grouping the three CL species into one group.

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