TYPHOIDYNE : Microspot Multi-Antigen Arrays Enzyme Immunoassay - The synergistic effects of species-specific and genus conserved antigens towards an improved definitive and differential diagnosis of typhoid fever

doi:10.21203/rs.3.rs-1754131/v1

Typhoid fever is a neglected re-emerging and potentially fatal infectious disease caused by S.Typhi. The disease is characterized by illness of multi-organ system and acquired through faecal-oral route by consumption of contaminated food and water. The disease is endemic in developing countries but alarming number of cases linked to domestic sources have been reported in industrialised countries. The clinical presentations of typhoid fever is highly variable and often overlap with other acute undifferentiated febrile illness (AUFI). Culture isolation is the gold standard for the diagnosis of typhoid. However, culture isolation is time-consuming, laborious with variable sensitivity and often limited to secondary or tertiary laboratory facilities. On the other hand, serological interference has been recognized as a major drawback in the reported diagnostic kits for typhoid fever. Hence, accurate, rapid and cost-effective diagnostic test are required for early life saving treatment and effective epidemiological intervention. This present study report the development of TYPHOIDYNE EIA using arrays of multi-antigen for definitive and differential diagnosis of typhoid fever. Multiple antigens were comprised of whole cell protein (WCP), cell surface protein(CSP) and surface depleted-whole cell protein(sdWCP) derived from differentially extracted whole cell bacterial proteins of S.Typhi and S.spp . Sensitivity and specificity of TYPHOIDYNE EIA were evaluated using panel of sera consisting of typhoid patient, typhoid vaccinated subject, healthy subject and subject with other diseases. The performance of the test was encouraging with sensitivity of 97.3% and specificity of 100%. The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 100% and 97%, respectively. Multi-antigens derived from species-specific and genus conserved play an important role in profiling heterogenicity in typhoid immune response that lead to excellent synergistic effect in differential and definitive diagnosis of typhoid fever. This preliminary report showed that TYPHOIDYNE EIA successfully detected and differentiated typhoid cases, typhoid vaccinated individuals, typhoid carriers and healthy individuals. To our knowledge, this is the first report to describe the successful use of multi-antigens based microspot arrays enzyme immunoassay to characterize the heterogenicity of immune response and their application in diagnosis of typhoid fever.

Typhoid fever is an infectious disease caused by Salmonella enteretica serotype Typhi which is also known as typhoidal Salmonella. Salmonella Typhi is a gram-negative bacterium from the family of Enterobacteriaceae. Human are the definitive host for Salmonella Typhi, and the disease is transmitted through fecal-oral route by consumption of contaminated food or water¹. Typhoid fever is known to be highly endemic in developing countries throughout the world. Typhoid scenario in developed countries is usually associated with travelers returning from endemic countries. However, recent report confirmed alarming typhoid cases in developed countries associated with domestic sources and not linked with visit to endemic counties^2–4. Typhoid fever is one of the etiologies for acute undifferentiated fever⁵. Therefore, diagnosis based on clinical presentation is not always possible because the overlapping clinical symptoms of typhoid fever with other acute undifferentiated febrile illness.

Culture isolation is the gold standard for laboratory diagnosis of typhoid fever. Specimens from patient which include blood, stool, urine and bone marrow aspirate (BMA) are collected to isolate and detect presence of S.Typhi^6–9. Bacterial isolation is time consuming, laborious and often not available in resource limited facilities¹⁰. Sensitivity and specificity of the culture isolation varies and depend on type of specimen and time of collection. These are the major drawback in culture isolation.

Serology is another alternative indirect method for the diagnosis of typhoid fever complimenting the culture isolation. The classic widal test has several limitations including the need for acute and convalescent sera, moderate sensitivity and specificity and not widely used in serodiagnosis of typhoid fever¹¹. Various serology test has been developed and reported over the years using various antigen preparations which includes flagellar antigen, hemolysin E (HlyE), 50 kD outer membrane protein, O9 lipopolysaccharide antigen and some undisclosed antigen. However, the reported serology tests have several drawbacks which include insufficient sensitivity and specificity and not all the reported serological test are available for laboratory use.^9,12−18. Another major drawback is link to the serological interference in the reported tests caused by individuals with other disease, vaccinated individual against typhoid, typhoid carrier and invasive non-typhoidal salmonella infection¹⁹. The pitfall in reported serological tests for typhoid fever indicated a strong requirement for an improved and effective serodiagnosis tool that able to differentiate active typhoid cases, typhoid carrier, individuals vaccinated against typhoid and patient in the category of acute undifferentiated febrile illness(AUFI).

Louis Pasteur successfully developed vaccines against diseases such as rabies, chicken cholera, tuberculosis and anthrax based on principal of cross protection and attenuation initiated by Edward Jenner for successful small pox vaccination ²⁰. Pasteur's principle in vaccine development adopted in our research approach to develop diagnostic test for typhoid fever.

In this study, we have determined the synergistic effect of species and genus conserved antigens in the accurate diagnosis of typhoid fever. We included differentially extracted antigens derived from whole cell of S.Typhi and antigens derived from whole cell of closely related species of Salmonella in the development of TYPHOIDYNE Microspot Multi-Antigen Arrays Enzyme Immunoassay (TYPHOIDYNE EIA).

Bacterial collection and maintenance

Two clinical isolates were obtained from stock culture collection diagnostic lab and maintained in Microbiology Laboratory, Advanced Medical and Dental Institute (ADL,AMDI), Universiti Sains Malaysia (USM), Kepala Batas, Pulau Pinang. All isolates were sub-cultured on BSA agar for purity check and maintained in Luria broth at room temperature.

Safety in handling

Salmonella. has been categorized under risk group 2 (RG-2); a pathogen that rarely cause serious human or animal disease and can be prevented by therapeutic interventions ²¹. Therefore, all the experimental procedure involved in culture handling were performed according to the regulation of biosafety level 2 laboratory (BSL2) requirements.

Serum collection and maintenance

The collection of sera was obtained from serum bank at Advanced Medical and Dental Institute (AMDI) with patient consent from previous study and ethical approval was obtained to use the serum collection.

Differential extraction of bacterial proteins derived from S.Typhi and S.spp

Differential extraction of bacterial protein was performed to obtain three cluster of proteins which include WCP, CSP and sdWCP. Three consecutives overnight culture of respective organisms in 1:100 ratio was subjected to protein extraction. WCP extraction was obtained by solubilizing overnight culture in solubilizing buffer(Ph6.8), boiled for 5 minutes and precipitated with ice cold ethanol overnight. Extraction of CSP was performed by solubilizing overnight culture in glycine-HCL (pH2.2), 15 minutes incubation at room temperature and centrifugation at 10000 rpm, 4°C for 10 minutes. The supernatant was calibrated to 7.4, precipitated overnight in ice-cold ethanol. The remaining pellet was solubilized in solubilizing buffer (pH6.8), subject to boiling for 5 minutes and precipitated with ice cold ethanol overnight. All three precipitated cluster of proteins were extracted by centrifugation and dissolved in Tris-PMSF until further use.

Development of TYPHOIDYNE EIA

Titration was done with six different concentrations from 5.0µg/ml to 0.15µg/ml on nitrocellulose membrane to determine the cut off for diagnosis. The dot EIA test for optimization steps are as follows. Serial dilution ranging from 5.0µg/ml, 2.5mg/ml, 1.25mg/ml, 0.6mg/ml and 0.15mg/ml were prepared and dotted on 0.45µm membrane strips (Microfiltration System, CA., USA). Blocking process was done by incubation with Tris buffer saline (TBS) containing 5% skimmed milk for one hour to block unbound sites and reduce non-specific binding. The membrane strips were than incubated with primary antibody serum of typhoid patient and healthy patient diluted in 1:100 TBS for 1 hour. The membrane strips were washed with TBS containing 5% skimmed milk for 5 minutes. This procedure was repeated three times. The membrane strips were than incubated with secondary antibody, IgM, IgG, IgA for 1 hour. IgM were diluted at 1:1000, IgG at 1:2000 and IgA at 1:1000 all in TBS. The membrane strips were than washed again for three times with TBS containing 5% skimmed milk for 5 minutes each. The membrane strips were than incubated for 10 minutes with AP conjugate (NBT-BCIP, Bio-Rad alkaline phosphatase substrate kit, USA) for optimum colour development. The reaction was stopped by washing with distilled water for 10 minutes. Two lowest concentration that produce visible colour intensity that showed difference between healthy and typhoid patient were chosen to develop the assay for serodiagnosis of typhoid fever.

Application of TYPHOIDYNE EIA for serodiagnosis of typhoid fever

The optimized concentrations of antigens and conjugates were used to perform this assay to determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) in the serodiagnosis of typhoid fever. Six antigens derived from S.Typhi and S.spp respectively were used in TYPHOIDYNE EIA. The spotted antigens were blocked in TBS containing 5% skimmed milk for 1 hour, conjugated with serum from 4 categories of subjects (typhoid patients, healthy subjects, vaccinated individuals , subjects with other febrile disease) diluted in TBS in 1:100 ratio for 1 hour and followed by secondary antibody (Igm, IgG and IgA) conjugation for 1 hour. The dilution ratio of secondary antibody follows the development method above. The membranes were washed three times with TBS containing 5% skimmed milk after each step above. The membrane strips were than incubated for 10 minutes with AP conjugate (NBT-BCIP, Bio-Rad alkaline phosphatase substrate kit, USA) for optimum colour development and the reaction was stopped by washing with distilled water for 10 minutes. Appropriate cut off controls for IgM, IgG and IgA and negative control were included in each batch of serological test to monitor the validity and sensitivity of the assay. The positive and negative results were interpretated by visual comparison to the positive and negative cut off control. The interpretation of the results was performed while the test strips were still wet. The overall results interpretation was determined based on table below.

Interpretation Criteria for Interpretation of TYPHOIDYNE EIA

Table 1

Interpretation criteria based on IgM, IgG and IgA antibody isotypes
Criteria	Interpretation
Either one or all three antibody isotypes were detected in the sample.	Positive
Absence of detectable level of antibody in patient's serum.	Negative

Table 2

Result interpretation based on synergy of arrays of differentially extracted antigens derived from S.Typhi (species specific) and S.spp (genus conserved)
Criteria		Result
Species specific (S.Typhi)	Genus conserved (S.spp)	Result
positive	positive	Positive typhoid
positive	negative	Carrier
positive (IgG only)	positive (IgG only)	Vaccinated
Negative	Negative	Healthy

Table 3 Overall sensitivity and specificity of TYPHOIDYNE EIA antibody test against WCP of Species specific and Genus conserved antigens preparations

Subject Category	Number of samples	Positive TYPHOIDYNE EIA for WCP antigens		Sensitivity (%)
		Species specific	Genus conserved
Typhoid diseased subject	13	13	13	100

Subject Category	Number of samples	Positive TYPHOIDYNE EIA for WCP antigens		Specificity (%)
Subject Category	Number of samples	Species specific	Genus conserved	Specificity (%)
Healthy subjects	10	6	0	100
Other disease subjects	10	1	0	100
Vaccinated subjects	10	2	2	80

Table 4 Overall sensitivity and specificity of TYPHOIDYNE EIA antibody test against CSP of Species specific and Genus conserved antigens preparations.

Subject Category

Number of samples

Positive TYPHOIDYNE EIA for CSP antigens

Sensitivity

(%)

Species specific

Genus conserved

Typhoid diseased subject

13

12

92

Subject Category	Number of samples	Positive TYPHOIDYNE EIA for CSP antigens		Specificity (%)
Subject Category	Number of samples	Species specific	Genus conserved	Specificity (%)
Healthy subjects	10	0	0	100
Other disease subjects	10	0	0	100
Vaccinated subjects	10	1	1	90

Table 5 Overall sensitivity and specificity of TYPHOIDYNE EIA antibody test against sdWCP of Species specific and Genus conserved antigens preparations

Subject Category

Number of samples

Positive TYPHOIDYNE EIA for sdWCP antigens

Sensitivity

(%)

Species specific

Genus conserved

Typhoid diseased subject

13

100

Subject Category	Number of samples	Positive TYPHOIDYNE EIA for sdWCP antigens		Specificity (%)
Subject Category	Number of samples	Species specific	Genus conserved	Specificity (%)
Healthy subjects	10	6	0	100
Other disease subjects	10	1	0	100
Vaccinated subjects	10	2	2	80

Table 6 Sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) of each differentially extracted antigens of TYPHOIDYNE EIA.

Antigen	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)
WCP	100	100	100	100
CSP	92	100	100	91
sdWCP	100	100	100	100

Table 7 Overall sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) of TYPHOIDYNE EIA for typhoid fever.

Sensitivity	97.3%
Specificity	97%
PPV	100%
NPV	100%

Accurate and rapid diagnosis is essential for early life saving treatment and public health intervention to prevent spread of typhoid fever. A timely treatment along with good prognosis are also of paramount importance towards battling typhoid fever. Various serological tests have been reported in literature using specific antigenic protein for the diagnosis of typhoid fever. However, the performance of the tests was inadequate and the positive predictive value of these tests are the major drawbacks in diagnosis of typhoid fever²². These previous reports have revealed the immunodiagnostic gap which led to under and inaccurate diagnosis of typhoid fever.

Thus, in the present study, we report a newly developed microspot multi-antigen arrays enzyme immunoassay named TYPHOIDYNE EIA, encompassing arrays of antigens for a robust and definitive serodiagnosis of typhoid fever. In this report, we evaluated the effectiveness of genus-conserved and species-specific antigens derived from S.Typhi and S.spp in the serodiagnosis of typhoid fever. TYPHOIDYNE EIA was developed by selecting six optimized arrays of antigen derived from S.Typhi and S.spp respectively. The performance of this assay was evaluated by four categories of serum which includes disease subjects, vaccinated subjects, healthy subjects and other disease subjects. The results of TYPHOIDYNE EIA were interpretated by inclusion and exclusion criteria as described in Table 1 and Table 2. Based on this inclusion and exclusion criteria, we have successfully classified the tested subjects into the following categories which include disease, vaccinated, carrier and healthy subjects. Consecutively, we have also obtained the sensitivity and specificity of the classified tested subjects against each differentially extracted antigens as described in Table 3, 4 and 5.

In our analysis, we achieved an overall of 100% sensitivity for WCP, 92% for CSP and 100% for sdWCP (Table 6). At the same time, when all three differentially extracted repertoire of antigens were combined in a single test, we achieved an overall sensitivity of 97.3% and specificity of 100% (Table 7). We also achieved 100% PPV and NPV of 97% for this assay. Therefore, by inclusion of species specific and genus conserved antigens, the performance of the test has been effectively improved.

Based on our study, we found out that typhoid subjects recognized arrays of antigens derived from S.Typhi and invasive non-typhoidal Salmonella (iNTS). Paradoxically, our study revealed that healthy typhoid carrier recognized antigens derived from S.Typhi in high frequency compared to antigens derived from iNTS (Fig. 1). This is because antibody level produced in healthy carrier is in low frequency due to inadequate infectious dose. On the other hand, we assessed immune response of individuals vaccinated with Ty21a vaccine against the antigen arrays selected for this assay. Interestingly, we observed IgG antibody recognized by antigens derived from S.Typhi and iNTS (Fig. 1). Research from University of Helsinki has reported that the localized immune response expressed by Ty21a vaccine cross react with NTS strain as Ty21a vaccine carries the 09 and 012 antigens that are both present in S.Typhi and iNTS. The difference in immune response reflected in TYPHOIDYNE EIA indicates the different route of pathogenesis and host response towards infection. The outcome of this result showed the variability in magnitude of human response and how it aids in distinguishing categories of infection.

This finding is in line with Felgner et al., indicating the extensive exposure and cross reaction towards closely related species of bacteria warrants for the development of antibody isotypes that reacted with arrays of antigen ²³. Our findings also revealed the presence of cross-reactive antigens in systemic and localized infection which is complementary to Pasteur’s work on closely related species eliciting cross protection. The insightful discovery on the role of specific and cross diagnostic antigens that was adopted by Pasteur's work has led to deeper knowledge in the diversity of microbial species and host interaction. These results also bear marked advantages towards understanding the diversity of humoral response and indicate advantage role of species and cross diagnostic antigens to develop highly predictive assay.

This is a preliminary study with limited number of samples obtained for the evaluation. A larger scale evaluation would be recommended in the future. In addition, we have observed frightening number of iNTS cases documented over the years. Therefore, to complete the diagnosis of Salmonellosis, further experiment is in progress to identify specific and cross diagnostic antigens for diagnosis of iNTS.

In summary, the accurate diagnosis of this assay upholds unequivocal importance for early life saving treatment and reducing the infection rate in low resource settings and developed areas. To our knowledge, this is the first report that demonstrated a versatile and up-scalable assay enriched in arrays of antigens that allows a high throughput test compared to conventional diagnosis. Furthermore, TYPHOIDYNE EIA can be converted readily to point of care test with next-generation technology such as immunochromatography(ICT), label free biosensor(SAW) and ultra-rapid smartphone-based system. Patent for "Typhoidyne EIA" has been filed and is in pending (Application number/MyIPO: PI2021004913).

Author contributions

Z.S.A.K. conceived and design study; K.A.L.K. performed research and wrote the manuscript; Z.S.A.K. supervised project and revised manuscript. All the authors discussed the results and contributed to final manuscript.

Competing interests

The authors declare no competing interests.

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Correspondence and requests for materials should be addressed to Z.S.A.K.

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No competing interests reported.

TYPHOIDYNE : Microspot Multi-Antigen Arrays Enzyme Immunoassay - The synergistic effects of species-specific and genus conserved antigens towards an improved definitive and differential diagnosis of typhoid fever

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Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Additional Declarations

Status:

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