Research ArticleA Cross-Sectional Comparative Study of the Performance of theWidal Test and the Typhidot Immunoassay for Typhoid FeverDiagnosis in the West Region of Cameroon

Karimo Ousenu,1 Innocent Mbulli Ali ,1,2 Leonard Fonkeng Sama ,1,3

Marcel Nsangou Ndam,4 Thibau Florant Tchouangueu ,1

and Christopher Bonglavnyuy Tume1,5

1Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, Faculty of Science,University of Dschang, Dschang, Cameroon2MARCAD Program, "e Biotechnology Centre, University of Yaounde 1, Yaounde, Cameroon3Higher Institute of Applied Science, Gulf of Guinea University Institute, Douala, Cameroon4Dschang District Hospital, Dschang Health District, Dschang, West Region, Cameroon5Department of Biochemistry, Faculty of Science, University of Bamenda, Bamenda, Cameroon

Correspondence should be addressed to Innocent Mbulli Ali; dr.alinn@gmail.com

Received 19 April 2021; Accepted 19 July 2021; Published 9 August 2021

Academic Editor: Maria De Francesco

Copyright © 2021 Karimo Ousenu et al. (is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Background. (e diagnosis of typhoid fever based on the Widal slide agglutination test remains a major hurdle in developingcountries due to varied perceptions of the value of the Widal test in determining clinical decision-making. We undertook a studyto evaluate the diagnostic performance of the Widal test and the Typhidot immunoassay in patients suspected of having typhoidfever in the Menoua division, West Region of Cameroon.Methods. Blood and stool samples were collected from 558 consentingfebrile patients on the basis of suspicion of typhoid fever. (ese patients attended three district health services of the Menouadivision between April 2018 and September 2019. (ese patients had clinical symptoms suggestive of typhoid fever as determinedby their consultant. Serum was used for the Widal slide agglutination test and for the Typhidot rapid immunoassay test based onmanufacturer’s guidelines. A composite reference of fever plus positive coproculture for Salmonella typhi and Salmonellaparatyphi was used as the reference. (e sensitivity, specificity, and predictive values of the positive and negative tests werecalculated as well as Cohen’s kappa for agreement between the two tests. Results. Of 558 patients, 12.90% tested positive for thereference method, 57.17% tested positive for the Widal slide agglutination test, while 15.59% were positive for Typhidot-IgM.(eoverall sensitivity, specificity, and predictive values of the positive and negative tests were 80.56%, 94.03%, 66.6%, and 97.03% forTyphidot-IgM and 94.44%, 48.35%, 21.32%, and 98.33% for the Widal slide agglutination test, respectively. Cohen’s kappaestimates were 0.1660 (0.121–0.211) and 0.386 (0.312–0.460) for the Widal test and Typhidot immunoassay for 53.6% and 76.16%agreements of all observations, respectively. Conclusion. (e Widal test was found to have a lower predictive value for thediagnosis of typhoid fever in our setting. However, the Typhidot test, although better, was not ideal. Diagnosis of typhoid fevershould therefore rely on adequate clinical suspicion and a positive Typhidot test to improve the clinical management of typhoidfever in our setting.

1. Background

Enteric fever caused by Salmonella typhi and Salmonellaparatyphi remains a major burden in developing countries

due to varied perceptions of the value of the Widal test indetermining clinical decision-making. (e burden of ty-phoid fever, worldwide, shows that it causes 16.6 millionnew infections and about 600,000 deaths each year [1].

HindawiCanadian Journal of Infectious Diseases and Medical MicrobiologyVolume 2021, Article ID 8279122, 9 pageshttps://doi.org/10.1155/2021/8279122

However, the incidence of typhoid fever has dropped toabout 10/100,000 population/year in developed countriesdue to improved living standard, proper hygiene and san-itation, and better healthcare systems, but the incidence isstill higher, 100/100,000 population/year, in less developedcountries [2]. A key challenge to the effective control oftyphoid fever is related to poor diagnosis. Diagnosis oftyphoid fever in clinical settings is complicated because ofoverlapping symptoms with other common infectionssuch as malaria, dengue, and viral enteritis [3–5]. Forproper diagnosis, a test with a good diagnostic perfor-mance, especially in children with febrile diseases [6, 7], isof great importance. In addition, the misuse of antibioticsvia automedication makes diagnosis difficult on a clinicalbasis [8]. (e gold standard for the diagnosis of entericfever is blood culture, but this test not only has a poorsensitivity in clinical settings but also is time consumingand expensive for patients and clinics in remote settingswhere culture facilities may not be always available andthe population is poor [9]. (e main diagnostic in suchsettings is based on the Widal slide agglutination testwhich is difficult to interpret for several reasons: cross-reactivities, time lag between infection and production ofantibodies, and persistence of target antibodies long aftertreatment with very low correlation with active disease[10, 11]. (e tube dilution technique enables a quantifi-cation of specific antibodies, and a change in titer canindicate active disease, but it is not very accessible. Manystudies suggest that the Typhidot test, a plausible alter-native based on the detection of antibody productionagainst the outer membrane preparation common toSalmonella typhi and Salmonella paratyphi, has betterperformance characteristics. However, variations insensitivity and specificity in the diagnosis of enteric feveramong adults and children have also been noted [12–14].(e assay gives good results during early infections with asensitivity of 68–95% and a specificity of 75–95% [15, 16].An increase in the negative predictive value is importantin endemic areas [17]. Recommendations from the WorldHealth Organization for typhoid rapid antibody testing[18] and some studies that have evaluated the Typhidotability to detect antibodies have shown variations insensitivity and specificity [15] in different settings. Al-though comparative studies have relied on the use ofimperfect gold standard tests, a composite reference hasbeen suggested to improve diagnostic values, but noagreement has been reached on which combinations canform a good composite reference standard, Storey et al.[19]. In this study, we undertook to evaluate the diagnosticperformance of the Widal and Typhidot tests against acomposite reference made of a combination of fever(≥37.50 C), 3–7 days or more, and a positive stool culturetest for Salmonella typhi and/or paratyphi in combinationwith one or more of the following clinical symptoms:persistent headache, abdominal discomfort, vomiting,and nausea. (e choice of this combination was based onthe feasibility and independence of the tests conditional tothe disease, and although less specific, measures weretaken to exclude other common febrile conditions such as

malaria and respiratory tract infections.(is was done in abid to find a local strategy to better diagnose typhoid fever.

2. Methods

2.1. Study Area and Period. (eMenoua division, one of thesix divisions of the West Region of Cameroon, covers asurface area of 1380 km2; it is divided into six subdivisions(Figure 1) as follows: Dschang, Santchou, Nkong-Ni, Penka-Michel, Fokoue, and Fongo-Tongo.(e altitude ranges from600 to 800m in Santchou through Dschang at 1500m andDjuititsa at an altitude of 2200m. (e division has an av-erage rainfall of about 1717.7mm, and temperature rangesfrom 13.6°C to 25.35°C. About four in every five indigenespractice subsistence and/or smallholder farming, and themost important food stuffs grown include cabbage, carrot,onion, maize, banana, tomato, plantain, and beans. As of2005, the division had a total population of 285,764. (ecapital of this division is Dschang.

(is study was conducted from April 2018 to September2019.

2.2. Study Design and Population. (is was a hospital-based,cross-sectional study among febrile patients suspected ofhaving typhoid fever. (ese patients had fever (temperature>37.5°C at inclusion) or reported febrile episodes in the pastthree days. Eligible patients who consented to study pro-cedures were recruited and attributed a unique study code.(is code was repeated instead of the patients’ names orinitials in all samples collected in the context of this study.(e enrolment form was password secured and maintainedby a study staff who was not associated with subsequentstudy procedures. A questionnaire containing demographicand clinical data and known risk factors was administered toeach participant.

2.3. Assay Methods

2.3.1. Blood Sample Collection. With the help of a sterilesyringe and needle, about 2-3ml of blood was collected fromeach participant as they arrived the laboratory. (is wasdone by well-trained laboratory technicians independent ofthe study team.

2.3.2. Test Procedure for the Widal Slide Agglutination Test.(e qualitative slide agglutination test was performed usingfebrile patient antigen kits for Salmonella typhi and Sal-monella paratyphi (TYDAL Widal Antigens for Slide andTube tests, Spain). (is test was used to determine thepresence of anti-TO and TH antibodies in patients’ sera.Collected blood was centrifuged using a bacteriologicallaboratory centrifuge (Andreas Hettich GmbH & Co. KG,78532 Tuttlingen, Germany) at 5000 turns for 5 minutes. Forthe determination of Salmonella antibodies by slide agglu-tination, a drop of Salmonella typhi O and H antigens wasadded on a drop of serum on the slide card, rotated at100 rpm for one minute, and recorded as either reactive ornonreactive.

2 Canadian Journal of Infectious Diseases and Medical Microbiology

2.3.3. Test Procedure for the Typhidot (Immunoassay) Test(Typhidot®Malaysian Biodiagnostic). (e Typhidot IgG andIgM rapid test device detects IgG and IgM antibodies toS. typhi and S. paratyphi through visual color development.Recombinant O and H antigens and antihuman IgG andantihuman IgM antibodies are used to detect the specificantibodies in human whole blood, serum, or plasma sam-ples. (e patient’s serum sample was added to the samplewell on the test panel, and specific IgG and/or IgM anti-bodies, if present, bind to the recombinant O and H antigensconjugated to colored particles on the sample pad. As thespecimen migrates along the strip by capillary action andinteracts with reagents on the membrane, the complex willbe captured by antihuman IgG and/or antihuman IgMimmobilized at the detection zone.

Venous blood was collected from patients suspected tohave Salmonella infections (about 2-3ml). (e bloodspecimens were then centrifuged at 5000 rpm for 5minusing a laboratory centrifuge (Andreas Hettich GmbH&Co.KG, 78532 Tuttlingen, Germany). Using the provided pi-pette, one drop of serumwas added to the test well on the testcassette followed by one drop of buffer. (e setup wasallowed for 15 minutes for migration across the membraneand color development in the result windows.

2.3.4. Results’ Interpretation. After 15 minutes, the testoutcome was read as follows. A result was consideredpositive for IgG and IgM if one red band appeared on the

control window (C) and two other red bands in both IgG andIgM windows, respectively. (e shade of color may varyfrom pink to purple but indicates a positive result even witha faint line based on manufacturer’s recommendations. IgMwas considered to be positive when one red band appearedon the control window (C) and on the IgM window, whilepositive IgG was considered when one red band appeared onthe control region (C) and on the IgG window. A negativeresult was indicated by the appearance of red color only onthe control window. A test assay was considered invalid ifthe control line did not appear.

2.3.5. Stool Sample Collection and Coproculture.Approximately 3 to 4 g of fresh stool was collected by febrilepatients for the coproculture test after a brief training onhow to collect the sample using a sterile wide-mouthed andtransparent container. Assessors were provided to each ofthe participants. After collection, the stool was mixed in 3mlnormal saline, and 1ml of this inoculum was diluted with9ml of freshly prepared Selenite F Broth (Oxoid CM0395Band LP0121A) onto prelabelled tubes and incubated at 37°Cfor 24 hours aerobically in a bacteriological incubator(Laboratory Incubator, DNP-9052-1, Midfield Equipment &Scientific, England) for amplification of bacterial growth. Aloop full of inoculum from 3ml normal saline was streakedon SSA agar (L: S-Biotech, USA) and MacConkey agar(Microxpress®, India) and incubated for 24 hours for thegrowth of colonies. After 24 hours, a loop full of bacterial

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Localisation of different sites of the study in the Menoua division

Date: 19/12/2020Coordinate system : WGS 1984-UTM-zone 32N

Figure 1: Sample collection location sites (A: Cameroon, B: West Region of Cameroon, and C: the six subdivisions of the Menoua division;turquoise circles represent study sites).

Canadian Journal of Infectious Diseases and Medical Microbiology 3

culture from incubated tubes containing the selenite culturewas restreaked into the Salmonella-Shigella agar (SSA)plates.(e plates were examined carefully for the presence ofcharacteristic colonies of Proteus spp., E. coli, Salmonellaspp., Citrobacter spp., Shigella spp., Klebsiella spp., andSerratia spp. Suspected colonies were restreaked on Sal-monella-Shigella agar for further identification and confir-mation using API 20E gallery (bioMerieux, France). (etechnicians performing coproculture were blinded to theprocedure and results of the Widal and Typhidot tests.

2.3.6. Test Outcome Classification. (e Widal test outcomewas classified as positive or negative based on the slide re-activity of patient’s serum. If, after the procedure, the patient’sserum was reactive, it was noted as a positive test outcome. Ifthe serum was nonreactive, it was noted as a negative testoutcome. (e procedure and classification were strictly fol-lowed for the total number of participants in the study.

(e Typhidot immunoassay test outcome was determinedby IgG and IgM positivity based on the manufacturer’sguideline. Based on this guideline, positive IgM and/orIgM+IgG were considered definite diagnosis of typhoid fever.For the purpose of this study, positive IgG “only” was notconsidered positive for the reference test as it may reflect onlypast exposure and also because the test has no quantitative value.

(e reference test was coproculture positive for Sal-monella typhi/paratyphi and a history of fever (≥37.50C)within 3–7 days or more including the hospital visit day.Other nontyphoidal Salmonella, although identified, did notconstitute a positive test. (ey were therefore considerednegative by the reference method. A positive Salmonellatyphi/paratyphi coproculture and fever as determined byaxillary temperature >37.5 C or reported fever within 3–7days or more were considered the referent. We includedthese criteria to avoid spectrum bias as much as possible,given the spectrum of disease severity that may be present atthe health facility and the likelihood of patients exposed toantibiotics before visiting the hospital.

2.3.7. Statistical Analysis. We calculated the sample sizebased on the nomogram published by Carley et al., in 2003.Based on the nomogram for the sensitivity plot atalpha� 0.05, we estimated the sample size by using thefollowing criteria: an estimated prevalence of typhoid feverof 15%, a confidence limit of 95%, and a type 1 error of 0.05.We obtained an estimated sample of 558 patients.

(e primary outcome of this study was the test accuracyand the predictive values of the positive and the negativetests of the Widal slide agglutination test and the Typhidotimmunoassay. Secondary outcomes were a comparison ofthe performance characteristics such as sensitivity, speci-ficity, and likelihood ratios of positive and negative testsbetween the Widal and the Typhidot test in our setting. (earea under the receiver operating curve was used to estimatethe diagnostic accuracy of the Widal and Typhidot tests,while the sensitivity, specificity, and predictive values werecalculated based on standard formulae. Regression analysiswas used to assess risk factors of typhoid fever in the study

population. All data were recorded in a predesigned ques-tionnaire designed on Epi Info before transferring toGraphPad Prism 8.0.2 (GraphPad software 2019, California)for further processing. A P value <0.05 was consideredsignificant for all comparisons.

3. Results

3.1. Sociodemographic Characteristics of Respondents. Weenrolled a total of 558 patients presenting signs andsymptoms suggestive of typhoid fever at the study sites(Table 1). (e majority of the participants were female,accounting for 67.38% (376/558) of the total sample. (emajority of participants were in the age range [11–20] years(219/558, 39.27%) and were students. According to occu-pation, respondents that were students were more repre-sented (47.67% (n= 266)), followed by participants workingin the private sectors (25.80% (n= 144)). (en, we hadhousewives accounting for 21.68% (n= 121) of the sampleand finally civil servants representing only 4.83% (n= 27) ofthe total study population.

Based on the level of education, about half of our studyparticipants had a secondary level of education (54.48%)followed by those who attained a tertiary level of education(31.18%) and then by those who had only a primary edu-cational level (13.97%).(e least represented were those whonever went to school (0.17%). Married people were morerepresented (56.27%), followed by those who were single(37.27%), whereas widows were the least represented(6.45%) (Table 1).

3.2.Distributionof thePrevalenceofTyphoidFever in theStudyPopulation. (e distribution of patients according to gendershows a significant difference (P � 0.0148) between the maleand female populations, with the male population beingmore infected (18.1%) compared to the female population(10.4%). Marital status also shows a significant difference(P � 0.0025), and the prevalence of typhoid fever among theunmarried population was higher (19.2%). Neither of thefollowing factors: age group, occupation, or level of edu-cation were associated with typhoid fever (Table 2).

3.3.Clinical SignsandSymptomsofStudyParticipants. In thisstudy, we recorded clinical signs and symptoms expressed bystudy participants.We estimated the frequency of these signsand symptoms, calculated the relative risk of acquiring ty-phoid fever given a symptom and presented the data inTable 3. As observed, febrile patients with abdominal painhad more than 2.5x the risk of typhoid fever compared tobeing febrile only. Other symptoms did not increased therisk of typhoid fever among the febrile patients.

3.4. Performance of Widal and Typhidot Tests for TyphoidFever Diagnosis. We found interesting comparisons be-tween the Widal and Typhidot tests in their ability to di-agnose typhoid relative to the reference test (Table 4). (eoverall sensitivity, specificity, and predictive values of the

4 Canadian Journal of Infectious Diseases and Medical Microbiology

positive and negative tests were 80.56%, 94.03%, 66.6%, and97.03%, respectively, for Typhidot-IgM and 94.44%, 48.35%,21.32%, and 98.33%, for the Widal slide agglutination test.Cohen’s kappa estimates were 0.386 (0.312–0.460) and0.1660 (0.121–0.211) for the Typhidot immunoassay andWidal test, respectively. Of all observations, the percentageagreementsfor Typhidot immunoassay was 76.16% and53.76% for the Widal test (Table 4).

3.5. Prevalence of Typhoid Fever in the Study Population.Out of the 558 participants in this study, 319 were positivefor typhoid fever based on the Widal test with an antibodytiter of 1 : 80 for both “O” and “H” antigens being taken ascutoff point values indicative of a recent typhoid infectiongiving a prevalence of 57.17%. On the contrary, 42.83%(n= 239) were negative for typhoid fever when Widal wasused as the diagnostic tool. Interestingly, the prevalence oftyphoid fever was 15.59% (n = 87) based on Typhidot-IgMand close to 12.90% (n = 72) based on the reference test. InTable 5, we summarise the differences in prevalence esti-mates by the two idea tests and the reference test.

As shown, the difference in estimating the prevalence oftyphoid fever was significant (P � 0.0001) comparing thethree diagnostic methods.(emost likely estimate of thetrue

prevalence estimate is that obtained with the referencemethod..

4. Discussion

Typhoid fever constitutes one of the major causes of mor-bidity and mortality in developing countries due to variedperceptions of the value of the Widal test in determiningclinical decision-making. (e clinical diagnosis of this dis-ease remains difficult because its symptoms are similar tothose of malaria and many other diseases [3, 21]. Laboratoryconfirmation of infection has relied on cheap slide agglu-tination tests which have their drawbacks leading to theappearance of false positive test results, the magnitude ofwhich depends on the endemicity of the disease. Other testssuch as the Typhidot immunoassay and the Tubex Widaldilution test do exist but have not been studied to under-stand their value in the diagnosis of typhoid fever in oursetting. In the current study, we set out to compare theperformance of the Widal slide agglutination test and theTyphidot test in patients suspected of typhoid fever in theWestern Region of Cameroon.

Our study revealed disparities in the performance of theWidal slide agglutination test and the Typhidot test.Compared to the reference, theWidal test had a sensitivity of0.9444 (0.8657–0.9782) and a specificity of 0.4835(0.4394–0.5279), whereas the Typhidot assay had a sensi-tivity of 0.8056 (0.6997–0.8805) and a specificity of 0.9403(0.9156–0.9581). Our results indicate that the Widal testperforms poorly compared to the Typhidot test. Indeed, theresult indicates that the Widal test correctly identifies abouthalf of patients who are uninfected with the typhoid feverbacteria, while only about 6% of uninfected patients will bewrongly classified as having typhoid fever; meanwhile, theyare uninfected. Also, the Widal test identifies 94% of febrilepatients suspected of typhoid fever as positive, whereas only80% of these patients were positive for the Typhidot im-munoassay. However, only one in five febrile patients sus-pected for typhoid with a positive Widal test result truly hadthe disease (PPV� 21.3%), while two in every three febrilepatients suspected for typhoid fever who have a positiveTyphidot test truly had the disease (PPV� 66.7%). (is lowaccuracy of the Widal test compared to the Typhidot testcould result from exposure to cross-reacting antigens infebrile infections other than Salmonella typhi. Indeed, Sal-monella typhi shares the O and H antigens with othernontyphoidal Salmonella and Enterobacteriaceae, as well assome parasites such as Plasmodium falciparum [22, 23],contributing to an increase in the probability of false positiveresults. Despite disparities in some parameters, we obtainedsimilar estimates of the predictive values of the negative testfor both Widal and Typhidot tests (98.3% and 97.0%, re-spectively). (is indicates that both tests have the ability torule out the disease in a population of suspected febrilepatients.

(e Widal test parameters obtained in the present studyshow some general patterns with some previous studies thatare high sensitivity, low predictive value of the positive test,and high predictive value of the negative test. For example,

Table 1: Sociodemographic characteristics of study participants.

Characteristics Number (n� 558) Frequency (%)GenderFemale 376 67.38Male 182 32.61Age groups (years)≤10 37 6.6311–20 73 13.0821–30 219 39.2431–40 48 8.6041–50 57 10.2151–60 54 9.6761–70 48 8.60≥71 22 3.94ResidenceDschang 237 42.47Fongo-Tongo 20 3.58Nkong-Ni 76 13.62Penka-Michel 12 2.15Santchou 41 7.34OccupationStudent 266 47.67Civil servant 27 4.83Private sector 144 25.80Housewife 121 21.68Level of educationNone 1 0.17Primary 78 13.97Secondary 304 54.48Higher education 174 31.18Marital statusMarried 314 56.27Single 208 37.27Widow 36 6.45

Canadian Journal of Infectious Diseases and Medical Microbiology 5

in the study conducted byMuthaiyan [24] and collaborators,they reported a sensitivity of 88.5% (95% CI: (69.82–97.42)),a specificity of 81.56% (95% CI: (74.16–87.58)), a positivepredictive value of 46.94% (95% CI: (32.54–61.720)), and anegative predictive value of 97.46% (95% CI: 92.74–99.44)for the Widal slide agglutination test. In a similar study inIndia in 2016 [24], the authors reported a sensitivity of80.77% (95% CI: (60.64–97.42)), a specificity of 78.01% (95%

CI: (70.22–84.54)), a positive predictive value of 40.38%(95% CI: (27.01–54.90)), and a negative predictive value of95.65% (95% CI: ()90.14–98.56)). Sherwal et al. [25] alsoreported a sensitivity of 74%, a specificity of 83%, and apositive predictive value of 87.5%. Rahman et al. [26] ob-tained a sensitivity of 81.8%, a specificity of 69.2%, and apositive predictive value of 45.6%. (is pattern generallyshows that the Widal slide agglutination test, although

Table 2: Prevalence of typhoid fever by sociodemographic characteristics in the study population.

Total (N� 558) Salmonella typhi positive (n� 72) P value (chi2)Age groups (years)<10 37 6 (16.2)

0.8010 (3.814)

[11–20] 73 7 (9.6)[21–30] 219 28 (12.8)[31–40] 48 5 (10.4)[41–50] 57 6 (10.5)[51–60] 54 8 (14.8)[61–70] 48 7 (14.6)>71 22 5 (22.7)GenderFemale 376 39 (10.4) 0.0148Male 182 33 (18.1)ResidenceDschang 237 45 (19.0)

<0.0001 (29, 16)Fongo-Tongo 20 1 (5.0)Nkong-Ni 76 10 (13.1)Penka-Michel 12 9 (75.0)Santchou 41 7 (17.1)OccupationStudent 266 33 (12.4)

0.5105 (2.310)Civil servant 27 6 (22.2)Private sector 144 17 (11.8)Housewife 121 16 (13.2)Level of educationNot educated 1 0

0.9459 (0.3724)Primary 78 11 (14.1)Secondary 304 40 (13.15)Higher education 174 21 (12.1)Marital statusMarried 314 28 (8.9)

0.0025 (11.95)Single 208 40 (19.2)Widow/widower 36 4 (11.1)

Table 3: Distribution of clinical symptoms of study participants.

Total (N� 558) Typhoid fever positive P value RR (95% CI)Fever 558 72 0.9999 N/AHeadache 363 48 0.7927 1.074 (0.6854–1.699)Nausea 107 12 0.633 0.843 (0.4691–1.475)Abdominal pain 457 66 0.021 2.431 (1.130–5.401)Fatigue 296 40 0.4444 0.827 (0.5365–1.279)Vomiting 63 8 0.9999 0.9821 (0.4911–1.869)Type of stoolHard 93 4 <0.0001Mucoid 254 23 N/AWatery 170 44 <0.0001Bloody 41 1N: total number of participants; RR: relative risk; N/A: not available.

6 Canadian Journal of Infectious Diseases and Medical Microbiology

highly sensitive, cannot be used to rule out the diseaseamong those with positive results. Furthermore, the highpredictive value of the negative test result indicates that thistest can rule out the disease in patients that have a negativetest outcome. Lastly, the test had a poor agreement indicatedby the low Cohen’s kappa value.

(e prevalence has been shown to vary in differentregions of Cameroon. For example, it was found to be 8.70%in Bamenda (northwest) [27], 21% in Buea, SouthwestRegion of Cameroon [28], and 2.5% obtained by Nsutebuet al. [29] in Tiko, Douala, and Yaounde (Littoral and CentreRegions, respectively). Our estimate was also less than theprevalence obtained in a similar study in Nigeria [30].Several reasons such as differences in sample size, riskfactors, and environmental conditions could account forthese disparities in prevalence. Although disparate, it is notlikely that the pattern of performance parameters of bothtyphoid tests used in the present study may change from oneregion to another, although the magnitude of the perfor-mance parameters may be different. In the present study, fivecases of confirmed typhoid fever by the reference test hadnegative Widal and Typhidot-IgM tests. (ese observations,although few in number, may result from early samplingbefore antibodies become detectable in blood as was re-ported in one study [31]. Furthermore, Andualem et al.found two cases of this category in a study comparing theWidal slide agglutination test against blood culture as thereferent [32]. Although not frequent, this observation mayalso account for false negative results obtained by the indextest in comparative studies of typhoid immunodiagnostics.Delay in antibody production in early disease may be themain determinant of this observation. Similar observationshave been made recently in serology tests used to detectexposure to the COVID-19 virus [33].

Our study has some limitations. Firstly, standard bloodculture has been recognised as the reference test for eval-uating the diagnostic performance of typhoid fever tests.However, there are also problems associated with the test. Itis first of all an invasive procedure [34] and has beendemonstrated to have low sensitivity even when there is noexposure to antibiotics [19]. In addition, obtaining adequateamounts of blood from groups such as children is chal-lenging. (ese make the use of such a test challenging in oursetting. We used a composite reference of a positive stoolculture and fever greater than or equal to 37.5°C or a historyof fever in the past three to seven days. (is is not the idealreference either but represented the optimal combinationunder our local circumstances owing to its practicability.(esearch for a perfect composite reference for diagnosticcomparisons is definitely still a way ahead even withemerging technologies [34]. Secondly, we did not have a wayto check antibiotic exposure history data. We also did notcollect clinical drug prescription data of these patients toquantify overtreatment and therefore the likely impact oftreatment practices on our diagnostic accuracy estimations.(is may likely have a small effect on our results whichshould be interpreted with caution.

5. Conclusion

We compared the diagnostic performance of the Widal slideagglutination test and Typhidot-IgM test against a cop-roculture, and the following results were obtained: thesensitivity of the Typhidot immunoassay was 80.56%,specificity was 94.03%, positive predictive value was 66.67%,and negative predictive value was 97.03%. (e sensitivity oftheWidal slide agglutination test was 94.44%, specificity was48.35%, positive predictive value was 21.32%, and negativepredictive value was 98.33%. Typhidot is a better alternativethan the Widal test in the diagnosis of typhoid fever. (eWidal test only rules out one of five typhoid fever suspectedpatients tested positive, as opposed to two of three whotested positive by the Typhidot immunoassay. Our resultssuggest that the Widal slide agglutination test, in its presentconfiguration, does not have clinical benefits for typhoidfever confirmation in endemic regions such as Cameroon.

Table 4: Performance of the Widal test and Typhidot immunoassay against the reference standard for the diagnosis of typhoid fever.

Stoolculture P value Sen Spec PPV NPV LHR Accu Kappa

(95% CI) Agreement (%)Pos Neg

Widal

67 253 <0.0001 0.938(0.87–0.98)

0.478(0.44–0.53)

0.211(0.17–0.26)

0.980(0.96–0.99) 1.829 0.543 0.167

(0.12–0.21) 53.76

5 233Typhidot-IgM

67 128 <0.0001 0.806(0.70–0.88)

0.940(0.92–0.96)

0.667(0.56–0.76)

0.970(0.95–0.98) 13.5 0.941 0.386 76.16

5 358 (0.31–0.46)PPV: positive predictive value; NPV: negative predictive value; Pos: positive; Neg: negative; LHR: likelihood ratio; Accu: accuracy; Sen: sensitivity; Spec:specificity; IgM: immunoglobulin M.

Table 5: Prevalence of typhoid fever based on the index andreference test methods.

Diagnostic test Positive Prevalence (%) P valueStool culture 72 12.90

<0.0001Widal 319 57.17Typhidot-IgM 87 15.59

Canadian Journal of Infectious Diseases and Medical Microbiology 7

Abbreviations

IgG: Immunoglobulin GIgM: Immunoglobulin MSSA: Salmonella-Shigella agar.

Data Availability

(e datasets and/or analyzed data and materials used duringthe current study are available from the correspondingauthor upon reasonable request.

Ethical Approval

Ethical clearance was obtained from the National EthicalCommittee for Research involving Human Subjects(CNERSH) (no. 2019/11/56/CE/CNERSH/SP), Yaounde,Cameroon. Research authorizations were also obtained fromall heads of districts in the Menoua division and from di-rectors of different hospitals where sampling and datacollection were done. Research authorization was alsogranted by the University of Dschang.

Consent

Information on potential publication of the anonymiseddata was included in the consenting process in the infor-mation sheet. All participants were aware and consented forpublication of the data in this manuscript. (e data andsample were collected only after informed consent wasobtained from each volunteer and guardian. All patientsprovided written informed consent prior to the studyprocedures. (ose who could not write were assisted by astudy-independent individual. Consent forms were trans-lated and back translated in French language. (e infor-mation sheet and consent form used were copies validatedby the ethics committee.

Conflicts of Interest

(e authors declare no conflicts of interest.

Authors’ Contributions

CBT and AIM designed the study. KO, LSF, and MSNperformed sampling and laboratory analysis. KO and TFTperformed statistical analyses. All authors participated in thewrite-up and approved the final version of the manuscript.

Acknowledgments

(e authors wish to thank all participants who sacrificedtheir time and donated blood and stool samples for thisstudy. (ey also particularly thank doctors and laboratorytechnicians of different hospitals for their contributions.

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