Dissertation on Schistosoma Mansoni Antibody Detection

Developing a Schistosoma mansoni antibody detection with recombinant proteins

Table of Contents

1. Introduction

1.1. Burden of disease

1.2. Disease control and our goal

1.3. Current tests for schistosomiasis

1.4. Next generation tests for Schistosoma mansoni

2. Materials and methods

2.1. Study population and sample preparation

2.2. Polymerase chain reaction and agarose gel electrophoresis

2.3. Spectrophotometry and protein measurement by the Bradford method

2.4. Enzyme linked immunosorbent assay with recombinant proteins

2.5. Ethics and regulations

3. Results

4. Discussion

5. References

6. Appendices

1. Introduction

1.1. Burden of disease

Every year, 700 million people are at risk of schistosomiasis, 240 million get infected, and it contributes towards 4.5 million disability adjusted life years globally (Munisi et al., 2017). schistosomiasis is a water borne infection Caused by five Schistosoma species (Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Schistosoma intercalatum, and Schistosoma mekongi). Sub Saharan Africa shoulders 93% of this burden (Beltrame et al., 2017, Munisi et al., 2017, Ferreira et al., 2017) most of which is  attributable to  Schistosoma mansoni and Schistosoma haematobium including a combined mortality of 300,000 cases per annum (Kinkel et al., 2012). A recent outbreak in Corsica (Beltrame et al., 2017)secondary to a West African Schistosoma strain which was a mixture of Schistosoma haematobium and hybrids has highlighted the possibility of long distance and local transmission in Europe and perhaps the importance of migration in schistosomiasis transmission (Kinkel et al., 2012).

The infective stage of schistosomiasis, cercarie is shed by fresh water snails and it penetrates the skin when an individual is exposed to the water where it is present(Hinz et al., 2017).  Schistosoma mansoni, is responsible for intestinal schistosomiasis and patients can remain asymptomatic during the acute phase(Munisi et al., 2017, Ferreira et al., 2017). At an advanced stage Schistosoma mansoni can present with abdominal pain, ascites , hepatomegaly and liver failure(Ferreira et al., 2017).

1.2. Disease control and our goal

The good thing with schistosomiasis is that effective treatment which also acts as prevention exists. Studies have demonstrated morbidity reduction and sustained transmission interruption with use of praziquantel, a relatively inexpensive drug that is readily available(Munisi et al., 2017)  Central to the success of anti-schistosomiasis control efforts are good screening and diagnostic tests(da Frota et al., 2011). A good test which is affordable, acceptable, can help with generation of prevalence data informing mass drug administration (MDA) programs. On the other hand, a good diagnostic test with good  specificity will help sustain control efforts post MDA. The goal of our work is to evaluate new tests with potential to revolutionise how we detect Schistosoma mansoni.

1.3. Current tests for schistosomiasis

Over the years, the Kato Katz stool microscopy test which provides proof of the presence of the parasite has been widely used as the gold standard test for the diagnosis of Schistosoma mansoni in endemic settings (Ferreira et al., 2017). This method is dependent on the variation of the infection intensity, number of specimens collected in a day, day to day variation in the egg counts in stool and decay of eggs in the stool which can be affected by storage and transportation(Bärenbold et al., 2017). These factors make the diagnosis with Kato Katz method have a low sensitivity especially in low endemic areas(Bärenbold et al., 2017). (Bärenbold et al., 2017, Hinz et al., 2017).

Schistosoma mansoni worms produce Circulating Cathodic Antigen (CCA) which can be detected in urine before the parasite’s eggs which can only be detected with the Kato Katz test from six to eight weeks after infection, this gives an alternative way to test for Schistosoma mansoni. Meta-analysis of CCA test in studies conducted in endemic areas, the CCA test, showed an average sensitivity and specificity of 89% and 55%, respectively for Schistosoma mansoni compared to the Kato Katz technique (Ochodo et al., 2015). However, sensitivity and specificity of CCA also varies with prevalence and intensity of infection(Ferreira et al., 2017).

Serological tests make use of the presence of antibodies in the host that are produced by the different stages of the parasite(Kinkel et al., 2012). These are very useful in people with acute infections and widely used in nonendemic areas(Kinkel et al., 2012). Other serological tests include; enzyme-linked immunosorbent assays (ELISAs) using different antigens, such as crude or purified adult worm antigen (AWA), soluble egg antigen (SEA), Schistosoma mansoni IgG and Western Blot containing Schistosoma mansoni soluble antigen(Kinkel et al., 2012). However, some serological tests have reported low specificity because of their inability to differentiate between Schistosoma mansoni and other helmith infections(Hinz et al., 2017).  Another short fall of these tests is the tests inability to discriminate between present and past infections by the presence of antibodies(Hinz et al., 2017, Kinkel et al., 2012). These tests are currently not widely used in some settings due to the need for sophisticated equipment in addition to the need of well trained personnel to conduct the test and interpret results(Kinkel et al., 2012).

1.4. Next generation tests for Schistosoma mansoni

The objective of our work was to assess an antibody detection method using ELISA with recombinant proteins for investigating the exposure to the parasite Schistosoma mansoni.  ELISA with recombinant protein is promising to improve the sensitivity and specificity of Schistosomiasis detection. A study by Lv et al (2016) evaluated the diagnosis of goat schistosomiasis with the use of recombinant multi-epitope proteins.  Hence the use of purified and recombinant proteins may overcome the low sensitivity resulting from cross reactivity with other helminths that occurs when crude antigens are used(Carvalho et al., 2017).

The aim of the experiment was to conduct ELISA on participants sera to assess recombinant Protein A and Protein B for its potential in assessing exposure to Schistosoma mansoni. The coding sequence of two genes A and B from the parasite were cloned, then the recombinant plasmids containing these inserted genes were isolated. The genes were then expressed and purified as recombinant proteins for use in the ELISA plates

2. Materials and methods

2.1. Study population and sample preparation

The laboratory experiment was conducted at Liverpool School of Tropical Medicine (LSTM) MSc laboratory. In this experiment, stool, urine and sera samples were obtained from 40 participants from a UK military battalion. Samples were from 20 participants who had a travel history to a Schistosoma mansoni endemic area and the other 20 from participants who did not have travel history to Schistosoma mansoni endemic areas. The following diagnostic tests were performed on all the 40 participant samples following the LSTM Standard Operating Procedure (SOP) and the manufacturers recommendation; Kato Katz faecal egg count (eggs per gram), Circulating Cathodic Antigen test and serum antibody test using three commercial assays as well as ELISA with the recombinant proteins. Recombinant proteins used for the ELISA were analysed with Polymerase Chain Reaction (PCR) for the band sizes and protein concentration with the Bradford assay.

2.2. Polymerase chain reaction and agarose gel electrophoresis

PCR was performed followed by agarose gel electrophoresis to visualise the band sizes of the gene inserts (MSc lab SOP, 2017). Master Mix with the required components (Appendix A) was prepared for the reactions. Primers were designed for the inserted Gene A and Gene B with a final concentration of 100 nm for both primers. The dNTPS in the master mix had a concentration of 200 μM. PCR was set up with 40 cycles for the 4 tubes that were prepared for; gene A, gene B and two negative controls. The PCR temperatures were; 95 o C for the initial denaturation, and melting, Primer annealing was set at 55 o C and 72 o C extension and the final extension.

The PCR products were run on 2% Agarose gel (according to manufacturer’s instructions). Ladder was loaded on the first lane, negative controls on lane three and five, with gene B and gene A on lane two and four respectively.  The gel was run for 40 minutes, then placed on a trans illuminator to visualise the band sizes of the amplicons.

2.3. Spectrophotometry and protein measurement by the Bradford method

To determine concentration of the expressed and purified recombinant proteins, spectrophotometry and protein measurement with the Bradford method (Bradford, 1976) was performed. Bovine serum albumin (BSA) standard solution at 2mg/ml with standard protein concentration was used to set up a standard curve with the absorbance readings at 595 nm. From the initial total volume of 15 ml that was expressed and purified, five different dilutions of recombinant Schistosoma mansoni proteins A and B were prepared with reference to the BSA concentrations. Absorbance readings at 595 nm were recorded with each protein sample run in triplicate. The equation from the standard curve and dilution factors of the proteins was used to calculate the concentrations of the recombinant proteins.

2.4. Enzyme linked immunosorbent assay with recombinant proteins

Sandwich ELISA was performed following the LSTM MSc laboratory Practical SOP to assess the recombinant proteins ability to detect Schistosoma mansoni antibodies in the 40 participants sera. The 96 multiwell plates were coated with recombinant Protein A and Protein B and incubated overnight. Blocking with a 5% w/v solution of non-fat skimmed milk powder was done before adding sera which was prepared with a 1:50 dilution in blocking solution and a volume of 100 μl added to the ELISA wells.  A detection antibody was added, after colour development the reactions were stopped by adding 25 μl of sulphuric acid. Optical density was read using a plate spectrophotometer with a 450nM filter.

2.5. Ethics and regulations

The data used in this work was collected in the context of a study protocol approved by the LSTM ethics committee. The specific data used in the analysis did not possess participant identifiable data.

3. Results

Band sizes of the purified recombinant plasmids A and B were visualised on a trans illuminator. The sizes were identified by comparing the bands for gene A and gene B to the ladder which was on lane 1 of the gel. Figure 1 was produced from the Agarose gel.

Figure 1; Agarose gel image from PCR amplification of recombinant Schistosoma mansoni gene inserts. Lane 1; DNA ladder, Lane 2; Schistosoma mansoni gene B, Lane 3; Negative control, Lane 4; Schistosoma mansoni gene A, Lane 5; Negative control

From existing data the recombinant protein products from gene A and B were 100 and 200 amino acids in length respectively. Based on the DNA ladder the band sizes of the two recombinant gene inserts were identified. Gene A had 100 amino acids which corresponds with 300 base pairs as each amino acid codon has three base pairs(Berg et al., 2002). Similarly, for Gene B which has 200 amino acids which corresponded with 600 base pairs.

A standard curve was produced from the absorbance readings of the BSA standard concentrations that were recorded at 595nm (figure 2). As the standards were run in triplicate the average absorbance was used to plot the standard curve. In order to produce a linear graph to use for the calculation of the unknown concentrations, only five standards absorbance readings were used in the final plot to have values that were only falling between 0.1 and 1 which gave a strong R2 value of 0.9988 close to 1. To establish the concentration of the unknown recombinant proteins the absorbance and dilution factors of the unknown proteins were substituted in the standard curve equation y=0.0011x + 0.0114, where y is absorbance. An absorbance that was between 0.1 and 1 was used with the one closest to the middle value for both Protein A and B chosen. Using an average absorbance of 0.462 at 595 nm for protein A and its dilution factor of 10 protein A concentration was found to be 4093.63 μg/ml. Protein B average absorbance was 0.58 at 595 nm, with a 16.67 dilution factor these produced a protein concentration of 8616.87 μg/ml (Appendix B).  Protein A and protein B content in the initial volume of 15ml was 61,404.45 μg and 129,253.05 μg respectively.

Figure 2:  Bovine Serum Albumin protein concentration standard curve using the Bradford reagent method. Protein concentration against absorbance at 595 nm

All participants stool and sera samples were analysed using different diagnostic tests in addition to the Recombinant protein A and B ELISA. 37.5% of the participants had eggs detected in their stool using the Kato Katz faecal egg count method, the mean value was(n=3) 15 out of 20 participants with travel history to endemic countries accounted for all the samples that had eggs present. No eggs were detected in any of the participants with no travel history to an endemic region. CCA test results were the same as those of the Kato Katz test, 37.5% of the participants urine tested positive.

The serum antibody test was performed using three different serological test and results were recorded as positive or negative where two assays were in concordance. The tests showed 50 % of the participants to have Schistosoma mansoni antibodies. All participants sera positive with the Serum Antibody test assay were from the ones with travel history to an endemic region. The Schistosoma mansoni prevalence was 37.5% with the Kato Katz method.

The optical density of the recombinant protein ELISA was collected and interpreted as negative or positive. Sensitivity and specificity with a 95% confidence interval of the recombinant protein ELISA was calculated for both protein A and Protein B, based on results of Kato Katz test and the index test (Appendix C and D).  Protein A sensitivity was 93.33% and specificity was 80%. The sensitivity of recombinant protein B was 53.33% while its specificity was 84%. The Positive predictive value (PPV) and negative predictive value (NPV) were also calculated for recombinant protein A and recombinant protein B. PPV for protein A and protein B was 74% and 66.67% respectively, while the NPV for protein A and protein B was 95% and 75% respectively (Table 1A and 1B).

		95% Confidence interval
Sensitivity	93.33%	68.05% to 99.83%
Specificity	80.0%	59.30% to 93.17%
Positive predictive value	73.68%	55.82% to 86.12%
Negative predictive value	95.24%	74.88% to 99.26%

Table 1A: Diagnostic performance of recombinant protein A ELISA based on Kato Katz method as a standard reference test

		95% Confidence interval
Sensitivity	53.33%	26.59% to 78.73%
Specificity	84.0%	63.92% to 95.46%
Positive predictive value	66.67%	42.02% to 84.66%
Negative predictive value	75.0%	62.98% to 84.10%

Table 1B: Diagnostic performance of recombinant protein B ELISA based on Kato Katz method as a standard reference test

4. Discussion

Improved and accurate diagnosis is important in schistosomiasis control and treatment therefore, it is essential for a test to be able to detect all people that are truly infected so that they can receive treatment and prevent any complications that can happen at an advanced stage of Schistosoma mansoni. This will also help to prevent unnecessary treatment of people who are not affected by the parasite. This experiment targeted potential candidate genes that could be used in ELISA to detect Schistosoma mansoni. The genes selected in this experiment, had a known amino acid length which matched with the expected band sizes for both gene A and gene B based on the available literature. This validated the PCR amplification and the gel results in addition to the results from the negative controls that were used. The use of positive controls such as commercial plasmids and plasmids expressing the recombinant proteins should be considered when setting up PCR for the future work to make sure that the reactions worked as well as ensuring quality and accuracy.

The proteins concentrations were detected using the BSA standard curve equation at an absorbance that was between 0.1 and 1 when measured at 595 nm for use in the ELISA plates. A strong R2 value was obtained by only plotting standards that had an absorbance of less than 1 when measured at 595 nm. The different proteins concentrations absorbance increased as the protein concentration in the samples increased. This is shown by the linear relationship between absorbance and concentration, following the Beer Lambert Law. …

This experiment demonstrated that Recombinant protein A and B detected the antibodies that were present in the participants Sera of the population that had the disease hence the two proteins have the potential to be used in diagnostics. The use of recombinant protein in the diagnosis of s. mansoni is expected to improve sensitivity and specificity of ELISA in Schistosoma mansoni detection compared to ELISA which use crude antigens(Carvalho et al., 2017).  Beltrame  (et al., 2017)in their study, described how serological tests are also to detect antibodies from past infections resulting in a  have a low sensitivity. This was similar in this experiment regardless of the use of recombinant proteins.  Protein A had 5 false positives while protein B had 4 false positives in samples that had no eggs using the Kato Katz method, these antibodies in participants could be present due to past infections. It is important for a serological test to have a high sensitivity in order to be able to diagnose asymptomatic and symptomatic individuals with an exposure in areas where schistosomiasis is endemic(Kinkel et al., 2012). ELISA with protein A, was a better test compared to protein B with its 93.33% sensitivity and 80% specificity. The PPV of 74% for ELISA with protein A also confirms the tests ability to detect the antibodies in participants who truly had the disease. ELISA with protein B on the other hand had a sensitivity of 53.33% and specificity of 84%, these make the test inadequate to be used for the diagnosis of Schistosoma mansoni. If protein B was used it would miss a lot of participants with Schistosoma mansoni which will negatively impact the control programme when people with the parasite miss treatment.

Sensitivity and specificity of Kato Katz test can be dependent on the number of samples and dependent on the endemicity in the area(Bärenbold et al., 2017). False negatives with the reference test in such setups affect the sensitivity and specificity of the index test, in this experiment the matching CCA and Kato Katz test may confirm that Kato Katz test detected all the cases that had an active infection as shown by the positive CCA. False negatives with microscopy can be corrected by collecting multiple stool samples on consecutive days(Bärenbold et al., 2017).

Serum antibody tests were not able to differentiate present and past infections(Hinz et al., 2017, Kinkel et al., 2012). In this experiment though it was not used in the sensitivity and specificity calculations, it showed positive in all the participants that had previously been to a Schistosoma mansoni endemic area even if they had been treated for past infections.

The sensitivity, specificity, PPV and NPV calculated shows that recombinant protein A could be a potential candidate that can further be explored for use in Schistosoma mansoni diagnosis with more samples as well as in a Schistosoma mansoni endemic area. However, ELISA with protein A for Schistosoma mansoni diagnosis cannot be used in area with limited resources. This test like other serological test will require specific skills, cannot be used in the fields and sustainability of such a method is questionable in Low and Middle Income countries where schistosomiasis is endemic.

When testing Protein A further, in addition to positive and negative controls samples from the other schistosomiasis species should be included to confirm that the test is not cross reacting with antibodies from other species. Quality control such as using the automated washer should also be ensured to avoid cross contamination that can an occur when using the multiwell and manual washing that was used in the experiment which can also affect the results of the experiment.

5. References

Bärenbold, O., Raso, G., Coulibaly, J. T., N’Goran, E. K., Utzinger, J. and Vounatsou, P. (2017) ‘Estimating sensitivity of the Kato-Katz technique for the diagnosis of Schistosoma mansoni and hookworm in relation to infection intensity’, PLoS Negl Trop Dis, 11(10), pp. e0005953.

Beltrame, A., Guerriero, M., Angheben, A., Gobbi, F., Requena-Mendez, A., Zammarchi, L., Formenti, F., Perandin, F., Buonfrate, D. and Bisoffi, Z. (2017) ‘Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis’, PLoS Negl Trop Dis, 11(6), pp. e0005593.

Berg, J. M., Tymoczko, J. L. and Stryer, L. (2002) ‘Amino Acids Are Encoded by Groups of Three Bases Starting from a Fixed Point’, in 5th (ed.) Biochemistry. New York: W H Freeman.

Bradford, M. M. (1976) ‘A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding’, Anal Biochem, 72, pp. 248-54.

Carvalho, G. B. F., Resende, D. M., Siqueira, L. M. V., Lopes, M. D., Lopes, D. O., Coelho, P. M. Z., Teixeira-Carvalho, A., Ruiz, J. C. and Fonseca, C. T. (2017) ‘Selecting targets for the diagnosis of Schistosoma mansoni infection: An integrative approach using multi-omic and immunoinformatics data’, PLoS One, 12(8), pp. e0182299.

da Frota, S. M., Carneiro, T. R., Queiroz, J. A., Alencar, L. M., Heukelbach, J. and Bezerra, F. S. (2011) ‘Combination of Kato-Katz faecal examinations and ELISA to improve accuracy of diagnosis of intestinal schistosomiasis in a low-endemic setting in Brazil’, Acta Trop, 120 Suppl 1, pp. S138-41.

Ferreira, F. T., Fidelis, T. A., Pereira, T. A., Otoni, A., Queiroz, L. C., Amancio, F. F., Antunes, C. M. and Lambertucci, J. R. (2017) ‘Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil’, Rev Soc Bras Med Trop, 50(3), pp. 358-364.

Hinz, R., Schwarz, N. G., Hahn, A. and Frickmann, H. (2017) ‘Serological approaches for the diagnosis of schistosomiasis – A review’, Mol Cell Probes, 31, pp. 2-21.

Kinkel, H. F., Dittrich, S., Baumer, B. and Weitzel, T. (2012) ‘Evaluation of eight serological tests for diagnosis of imported schistosomiasis’, Clin Vaccine Immunol, 19(6), pp. 948-53.

Lv, C., Hong, Y., Fu, Z., Lu, K., Cao, X., Wang, T., Zhu, C., Li, H., Xu, R., Jia, B., Han, Q., Dou, X., Shen, Y., Zhang, Z., Zai, J., Feng, J. and Lin, J. (2016) ‘Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay’, Parasit Vectors, 9, pp. 135.

Munisi, D. Z., Buza, J., Mpolya, E. A., Angelo, T. and Kinung’hi, S. M. (2017) ‘The Efficacy of Single-Dose versus Double-Dose Praziquantel Treatments on Schistosoma mansoni Infections: Its Implication on Undernutrition and Anaemia among Primary Schoolchildren in Two On-Shore Communities, Northwestern Tanzania’, Biomed Res Int, 2017, pp. 7035025.

Ochodo, E. A., Gopalakrishna, G., Spek, B., Reitsma, J. B., van, L. L., Polman, K., Lamberton, P., Bossuyt, P. M. and Leeflang, M. M. (2015) ‘Circulating antigen tests and urine reagent strips for diagnosis of active schistosomiasis in endemic areas’, Cochrane Database of Systematic Reviews(3), Available: John Wiley & Sons, Ltd. DOI: 10.1002/14651858.CD009579.pub2.

6. Appendices

Appendix A

Components of the PCR master mix

Reagents                                   (1)	Final Conc. (2)	Reaction Vol. (3)	Master Mix Vol.  X 6 samples (4)
Nuclease-free distilled water	–	19.2l	115.2 l
10X Buffer (with loading dye) with MgCl2	1X	2.5l	15 l
dNTP (2.5 mM)	200 M	2l	12 l
Primer 1 or 3 (100 M)	100 nM	0.025l	0.15 l
Primer 2 or 4 (100M)	100 nM	0.025l	0.15 l
Taq DNA polymerase (5 U/l)	1.25 U	0.25l	1.5 l
Plasmid A or B DNA (1 ng/l)	–	1 l	N/A
Total	–	25 l

Appendix B

Spectrophotometry absorbance readings at 595 nm from dilutions of two different recombinant Schistosoma mansoni proteins.

Each protein sample was run in triplicate.  The total starting volume purified of each recombinant protein sample was 15 ml.

Note: Dilution and absorbance used in concentration calculation in red

Recombinant protein sample identification	Volume of undiluted protein sample (µL)	Volume of water (µL)	Absorbance at 595 nm (replicate 1)	Absorbance at 595 nm (replicate 2)	Absorbance at 595 nm (replicate 3)	Average absorbance at 595 nm	standard deviation absorbance at 595 nm	protein concentration per assay (μg/ml)	dilution factor	calculated protein concentration of undiluted sample(μg/ml)
A	20	30	1.756	1.774	1.727	1.752	0.024	1582.363	2.5	3955.908
A	16	34	1.434	1.485	1.414	1.444	0.037	1302.363	3.13	4076.396
A	10	40	0.873	0.881	0.882	0.879	0.005	788.7	5	3943.5
A	5	45	0.459	0.473	0.455	0.462	0.009	409.636	10	4096.36
A	1	49	0.129	0.127	0.128	0.128	0.001	106	50	5300
B	10	40	1.837	1.878	1.882	1.866	0.025	1686	5	8430
B	8	42	1.585	1.577	1.599	1.587	0.011	1432.363	6.25	8952.269
B	5	45	0.982	0.966	0.955	0.968	0.014	869.636	10	8696.36
B	3	47	0.573	0.578	0.588	0.580	0.008	516.909	16.67	8616.873
B	1	49	0.137	0.135	0.131	0.134	0.003	111.454	50	5572.7

Appendix C

Diagnostic performance of recombinant protein A ELISA based on Kato Katz method as a standard reference test

Index test		Positive	Negative
	Positive	14	5
	Negative	1	20

Appendix D

Diagnostic performance of recombinant protein B ELISA based on Kato Katz method as a standard reference test

Index test		Positive	Negative
	Positive	8	4
	Negative	7	21