|Year : 2015 | Volume
| Issue : 2 | Page : 237-242
Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever
KJ Prasad, JK Oberoi, N Goel, C Wattal
Department of Clinical Microbiology and Immunology, Sir Ganga Ram Hospital, New Rajinder Nagar, New Delhi, India
|Date of Submission||02-Jan-2014|
|Date of Acceptance||13-Nov-2014|
|Date of Web Publication||10-Apr-2015|
Department of Clinical Microbiology and Immunology, Sir Ganga Ram Hospital, New Rajinder Nagar, New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: Enteric fever is a major public health problem in developing countries like India. An early and accurate diagnosis is necessary for a prompt and effective treatment. We have evaluated the diagnostic accuracy of two Rapid Salmonella-IgM tests (Typhidot-IgM and Enteroscreen-IgM) as compared to blood culture in rapid and early diagnosis of enteric fever. Materials and Methods: A total of 2,699 patients' serum samples were tested by Rapid Salmonella-IgM tests and blood culture. Patients were divided into two groups. Test group - patients with enteric fever and blood culture positives for Salmonella Typhi; and three types of Controls, i.e. patients with non-enteric fever illnesses, normal healthy controls and patients positive for S. Paratyphi- A. In addition to this we have also evaluated the significance of positive Salmonella-IgM tests among blood culture-negative cases. Results: The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Typhidot-IgM test and Enteroscreen-IgM test considering blood culture as gold standard were 97.29% and 88.13%, 97.40% and 87.83%, 98.18% and 92.03%, 96.15% and 82.27%, respectively. Typhidot-IgM test was found to be significantly more sensitive and specific as compared to Enteroscreen-IgM. Among blood culture-negative patients, Rapid Salmonella-IgM tests detected 72.25% additional cases of enteric fever. Although the Rapid Salmonella-IgM tests are meant to diagnose S. Typhi only, but these tests detect S. Paratyphi- A also. Thirty-eight patients who were blood culture-positive for S. Paratyphi- A were also positive by Rapid Salmonella-IgM tests. Conclusion: Rapid Salmonella-IgM tests offer an advantage of increased sensitivity, rapidity, early diagnosis and simplicity over blood culture.
Keywords: Blood culture, Enteroscreen-IgM, enteric fever, rapid salmonella-IgM tests, typhidot-IgM
|How to cite this article:|
Prasad K J, Oberoi J K, Goel N, Wattal C. Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever. Indian J Med Microbiol 2015;33:237-42
|How to cite this URL:|
Prasad K J, Oberoi J K, Goel N, Wattal C. Comparative evaluation of two rapid Salmonella-IgM tests and blood culture in the diagnosis of enteric fever. Indian J Med Microbiol [serial online] 2015 [cited 2020 May 29];33:237-42. Available from: http://www.ijmm.org/text.asp?2015/33/2/237/154861
| ~ Introduction|| |
Enteric fever caused by Salmonella More Details Typhi or S. Paratyphi- A is a major health problem in India. Most of the salmonella infections are diagnosed primarily on clinical grounds and treated presumptively leading to delayed diagnosis and emergence of drug resistance. Moreover during the first week of fever, enteric fever cannot be easily distinguished from other illnesses, like malaria, dengue, chikungunia, infectious mononucleosis, leptospirosis, rickettsial diseases, acute gastroenteritis, etc. Therefore, reliable laboratory tests are essential to establish aetiologic diagnosis for appropriate treatment.
Gold standard for diagnosing enteric fever is the blood culture.  However, its positivity rate is only 50-70%.  In most of the developing countries, irrational and widespread use of antibiotics is the prime reason for the low sensitivity of blood cultures.  The sensitivity of stool and urine cultures is much lower and they become positive after the first week of infection. Bone marrow cultures are more sensitive, but have a little use in public health settings.
The widely used serological test, i.e. Widal test for the diagnosis of typhoid fever lacks sensitivity and specificity and reliance on it alone in areas where enteric fever is endemic leads to errors in diagnosis. , A paired serum sample with a fourfold rise in titer is needed for a meaningful result. Moreover, it takes more than 1 week for the significant titers of antibodies to appear.  Accurate diagnosis of enteric fever at an early stage also helps to identify individuals who may serve as potential carriers, who may be responsible for acute enteric fever outbreaks. 
Newer serological tests like Typhidot, Enteroscreen, Typhipoint, Tubex, IgM-dipstick, etc., which directly detect IgM or IgG antibodies against specific S. Typhi antigens have been developed. Detectable levels of IgM antibodies against S. Typhi can be detected as early as within 4-5 days of fever.  Initial studies done in Asian countries like Malaysia, Indonesia, Philippines, Pakistan, Bangladesh and India have shown variable sensitivity (73-95%) and specificity (68-95%) of these tests. ,,,,
We at a tertiary care centre in North India, have been using Rapid Salmonella-IgM tests, like Typhidot-IgM and Enteroscreen-IgM since September 2008. However, the diagnostic accuracy of these tests has not been evaluated. We therefore have analyzed our data retrospectively, to evaluate the diagnostic accuracy of Typhidot-IgM and Enteroscreen-IgM in the early diagnosis of enteric fever vis-a-vis blood culture, at our hospital. To the best of our knowledge, there are no studies in the literature about the significance of positive Salmonella-IgM tests in the blood culture negative cases. Here an attempt has also been made to evaluate the utility of these Rapid IgM tests in blood culture-negative patients.
| ~ Materials and Methods|| |
This retrospective study was conducted at Dept. of Clinical Microbiology and Immunology of Sir Ganga Ram Hospital, New Delhi. The patients admitted in the hospital or attending Out Patient Department (OPD)/Emergency Department from September 2008-June-2012, with clinical symptoms of enteric fever and for whom both, blood culture and Rapid Salmonella-IgM tests (Typhidot-IgM/Enteroscreen-IgM) were done, were included (n = 2699). The distribution of patients for two different types of Rapid Salmonella-IgM tests (Typhidot-IgM/Enteroscreen-IgM) have been summarized in [Figure 1].
|Figure 1: The distribution of patients for two different types of Rapid Salmonella-IgM tests (Typhidot-IgM/ Enteroscreen-IgM)|
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To evaluate the diagnostic accuracy of two commercially available Rapid Salmonella-IgM tests (Typhidot-IgM and Enteroscreen-IgM) in the diagnosis of enteric fever taking blood culture as gold standard the data was evaluated retrospectively. The following groups of patients were included.
(Patients with blood culture positive for S. Typhi, n = 229). This group comprised all the enteric fever patients confirmed by positive blood culture (n = 111 for Typhidot-IgM group and n = 118 for Enteroscreen-IgM).
Three types of controls were included.
Control group I
(Patients with fever other than Salmonella infection). A total of 111 patients diagnosed with infection other than enteric fever (dengue, Hepatitis-A, malaria, amoebic liver abscess, tuberculosis, isolation of organisms other than Salmonella spp, in blood cultures and Urinary Tract Infections, etc.) were included as controls. Out of these patients, 57 were tested by Typhidot-IgM and 54 by Enteroscreen-IgM.
Control group II
(Normal healthy controls). Blood samples from 40 normal healthy individuals not having any current history of infection (healthy blood donors) were also included as negative controls. Among them, 20 each were tested either by Typhdot-IgM or Enteroscreen-IgM test.
Control group III
(Patients who were blood culture positive for S. Paratyphi A). This group comprised 66 patients (n = 20 for Typhidot-IgM group and n = 46 for Enteroscreen-IgM group). This group was included to find out an additional advantage of these tests (recommended only for S. Typhi) in detecting S. Paratyphi A also.
In addition to the above (blood culture positive cases), we have also evaluated the significance of positive Salmonella-IgM tests among blood culture-negative cases. There were in all 2,404 patients who were blood culture-negative. However, there were 463 patients who were positive by Salmonella-IgM tests (n = 303 by Typhidot-IgM and n = 160 by Enteroscreen-IgM) and the rest 1,941 patients were negative by both Rapid-Salmonella -IgM tests and blood culture. (n = 744 by Typhidot-IgM and n = 1197 by Enteroscreen-IgM, [Figure 1]).
Out of the above 463 blood culture negative but Rapid Salmonella-IgM-positive patients, only 173 hospitalized patients whose medical records were available could be re-evaluated retrospectively. Their clinical history, signs and symptoms, laboratory findings, provisional and final diagnosis and treatment, etc., were studied.
Blood culture was done according to the standard procedures using the BacT\ALERT 3D automated system (bioMerieux, France). This system utilizes colorimetric sensor and reflects light to monitor the presence and production of carbon dioxide released by bacterial growth, which gets dissolved in the culture medium. Any isolated bacteria were identified with the help of Vitek-II (bioMerieux, France), identification system and confirmed by slide agglutination with specific antisera (Remel Europe Ltd., U.K.).
Typhidot-IgM (Salmonella Typhi specific IgM antibody assay)
It is a qualitative antibody detection test based on dot enzyme immunoassay technique, to detect the presence of IgM antibodies against a specific antigen on the outer membrane of S. Typhi. The test was carried out as per the manufacturer's instructions (Malaysian Biodiagnostics Research Sdn. Bhd, Selangor Darul Ehsan, Malaysia). Positive and negative controls supplied in the kit were also included with each test run. Serum samples were diluted to 1:100 with the supplied diluents. The positive control formed duplicate dark spots of 2 mm diameter each. Any test sample showing similar or darker spots was defined as positive. The absence of any visible spot indicated a negative test result.
It is a rapid, qualitative, sandwich immunoassay based on the principle of immunochromatography for the detection and differentiation of IgM and IgG antibodies to S. Typhi in human serum/plasma or whole blood specimen (Zephyr Biomedicals, Verna, Goa, India). The control band acts as a procedural control and serves to validate the results. For this study, we have only included the findings of IgM antibodies and have therefore used the term Enteroscreen-IgM.
To evaluate the diagnostic accuracy of Typhidot-IgM and Enteroscreen, the various parameters, like sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and accuracy of the test were calculated. The statistical analysis of results was also done to establish the test of significance for clear and confirmatory statements. Nominal scale variables were described using relative and absolute frequencies, and the Chi-square (χ2 ) test was used to assess differences between the groups. Fisher's exact test was used if expected frequencies were < 5. For all statistical tests, a P value less than 0.05 was taken to indicate a significant difference.
Ethics clearance was obtained from Hospital Ethics committee, Ethics No.EC/07/12/381, dated 9 th August 2012. A written consent was obtained from healthy blood bank donors for testing their left over blood samples for enteric fever.
| ~ Results|| |
Out of 1,178 patients tested for both blood culture and Typhidot-IgM, 111 (9.42%) were positive for S. Typhi. by blood culture and 427 (36.24%) were positive by Typhidot-IgM test. Among blood culture-positive cases (Test Group), out of 111, 108 (97.29%) were also positive by Typhidot-IgM antibody test [Table 1].
Among 57 non-enteric fever cases (Control Group-I), Typhidot-IgM test was positive in 2 (3.5%) cases, whereas none of the healthy controls (Control Group-II) were positive by Typhidot-IgM test [Table 1].
The overall sensitivity, specificity, PPV and NPV of the Typhidot-IgM test, considering blood culture positive for S. Typhi as gold standard, were 97.29%, 97.40%, 98.18% and 96.15%, respectively [Table 2]. The diagnostic accuracy of Typhidot-IgM for diagnosing S. Typhi infections was as high as 97.34%.
|Table 2: Diagnostic accuracy of Rapid Salmonella-IgM Tests-Typhidot-IgM and Enteroscreen-IgM |
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Although Typhidot-IgM is recommended only for S. Typhi, we used it on S. Paratyphi- A blood culture-positive patients also; out of 20 S. Paratyphi- A patients, Typhidot-IgM was positive in 16 (80%) cases [Table 1].
A total of 1,521 patients were tested by both blood culture and Enteroscreen-IgM test. Out of which only 118 (7.75%) were positive for S. Typhi by blood culture [Table 3] and 286 (18.80%) were positive for Enterscreen-IgM. Among test group patients of 118 culture-positive patients, 104 (88.13%) were also positive by Enteroscreen-IgM.
|Table 3: Results of entertoscreen-IgM test in test group and control groups |
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In control Group-I patients (54, non-enteric fever patients), Enteroscreen-IgM gave false positive results in 9 (16.66%) patients. However, none of the control Group-II patients (Healthy Controls) were positive with Entertoscreen-IgM indicating thereby high specificity of the test [Table 3].
The overall sensitivity, specificity, PPV and NPV of the Enteroscreen-IgM test for S. Typhi were 88.13%, 87.83%, 92.03% and 82.27%, respectively [Table 2]. The diagnostic accuracy was 95.31%.
When tested on S. Paratyphi- A blood culture-positive patients, out of 46 patients, Enteroscreen-IgM was positive for 22 (47.83%) patients, only [Table 1].
Typhidot-IgM vs. Enteroscreen-IgM
The comparative evaluation of diagnostic accuracy of both the tests for the diagnosis of enteric fever has been summarized in [Table 2]. Typhidot-IgM was definitely a more sensitive and specific test and the PPV and NPV were also very high, whereas Enteroscreen-IgM had significantly low sensitivity (88.13%) as compared to Typhidot-IgM (97.29%). Similarly, the diagnostic accuracy of Typhidot-IgM was also significantly higher than Enteroscreen-IgM (97.34% vs. 88.02%; P = 0.0005). Although the PPV of Entertoscreen-IgM test was high (92.03%), it was lower than Typhidot-IgM (98.18%). The negative predictive value of Enteroscreen-IgM was quite low as compared to Typhidot-IgM (82.27% vs. 96.15%; P = 0.005). The sensitivity of Typhidot-IgM for S. Paratyphi- A was significantly higher (80%) than that of Enteroscreen-IgM (47.83%).
Blood culture negative but rapid Salmonella-IgM positives
Out of a total of 2,699 patients tested for enteric fever, 2,404 were blood culture negative. Among these blood culture-negative patients, there were 463 patients (Typhidot-IgM, n = 303 and Enterscreen-IgM, n = 160) who were positive by Salmonella-IgM tests. Medical records of only 173 patients, (Typhidot-IgM, n = 84 and Enterscreen-IgM, n = 89) who were admitted in the hospital could be obtained. Among these 173 patients, 125 patients (72.25%), inspite of being blood culture negative, were diagnosed clinically as enteric fever cases, on the basis of clinical symptoms and positive Salmonella-IgM tests and other laboratory findings, i.e. total leukocyte count, differential leukocyte count, C-reactive protein. All these patients were treated successfully with intravenous (IV) Ceftriaxone. The rest 48 patients, who had a history of fever associated with underlying features, like laryngitis, thrombocytopenia, pneumonia, septic shock, gastroenteritis, trigeminal neuralgia, etc., although not clinically pre-diagnosed as enteric fever cases, responded well to IV Ceftriaxone. These patients were discharged when afebrile. The concomitant infection with salmonella could not be ruled out.
| ~ Discussion|| |
We have evaluated two commercially available Salmonella-IgM tests-Typhidot-IgM and Enteroscreen-IgM, for early diagnosis of enteric fever. The results of these two serological tests and blood culture as gold standard have been analyzed retrospectively. During September 2008 to June 2012, a total of 3,946 patients' serum samples were tested for Salmonella-IgM. Out of which, only 2,699 samples, for whom request for both blood culture and serological test were received, were included in this study.
The overall sensitivity, specificity, PPV and NPV of the Typhidot-IgM test were 97.29%, 97.40%, 98.18%, and 96.15%, respectively. Similar results were reported by a study carried out by Jesudason and Shivakumar,  at CMC, Vellore, i.e. sensitivity = 92.3% and specificity = 98.8%. Similarly, a sensitivity of 90.3% and specificity of 91.9% have been reported by Choo et al.  from Malaysia. Sherwal et al. at Lady Harding Medical College, Delhi have also shown high sensitivity (92%) but comparatively low specificity (87.5%). It may be attributed to the use of combined Typhidot (IgM + IgG) kit. Our results are also comparable to that of other studies carried out in India and abroad ,,,,,,,, , who have recommended Typhidot-IgM to be a useful test in early diagnosis of enteric fever and an alternate to Widal. However, we could not compare the paired serum samples by Widal.
The overall sensitivity, specificity, PPV and NPV of the Enteroscreen-IgM test were 88.13%, 87.83%, 92.03% and 82.27%, respectively. Typhidot-IgM appears to be a more sensitive and specific test with high PPV and NPV. However, in case of emergency especially during odd hours when Typhidot-IgM cannot be performed, as it takes at least 3 hours and especially in rural set-ups, one can rely on rapid point of care tests like Enteroscreen-IgM, considering its high PPV (92.03%) and a reasonable diagnostic accuracy, i.e. 88.02%.
Although the available rapid Salmonella-IgM tests are meant to diagnose S. Typhi only, but these tests detected S. Paratyphi- A also. It may be due to cross-reactivity between outer membrane protein (OMP) antigen of S. Typhi and S. Paratyphi A. Typhidot-IgM test was positive in 16 such patients who were blood culture positive, for S. Paratyphi- A and gave a sensitivity of 80%. Similar results have been reported from Vellore, India with 50% sensitivity and 98.8% specificity.  Enteroscreen-IgM showed comparatively low sensitivity for S. Paratyphi- A (47.83%). Out of 46, S. Paratyphi- A blood culture positive cases, only 22 were found positive.
Among blood culture-negative patients, Rapid Salmonella-IgM tests (both tests combined) were positive in additional 463 patients, of which 173 could be followed up. Out of these 173 blood culture negatives, 125 were clinically diagnosed and treated as enteric fever cases with clinical cure. Thus, additional 72.25% such cases of enteric fever were treated successfully with IV ceftriaxone. Similarly, Baig et al. picked up 63% more cases of true typhoid fever by Typhidot-IgM as compared to blood culture. This can be explained by the fact that sensitivity of blood culture is low, i.e. 50-70%  and the sensitivity of blood culture decreases after the first week of illness and antibiotic therapy.  Moreover, rapid tests can detect IgM antibodies as early as two days of fever and also up to second week of fever.
The strength of our study is that for the first time we have compared the diagnostic accuracy of Typhidot-IgM-an Enzyme Immuno Assay (EIA) with a rapid immunochromatographic, test (ICT), i.e. Enteroscreen-IgM. The significant feature of our study is that we have also evaluated significance of the positive Salmonella-IgM test in blood culture negative cases. An additional 68 (72.25%) of blood culture negative and Salmonella-IgM positive patients could clinically be diagnosed as having enteric fever. However, further investigation needs to be done by carrying out a planned study to evaluate the role of Salmonella-IgM tests in diagnosing the enteric fever in blood culture negative cases where molecular test could act as a gold standard.
| ~ Conclusion|| |
Rapid Salmonella-IgM tests offer increased sensitivity, rapidity, early diagnosis and simplicity over blood culture and can replace the Widal test, the most commonly used serological test. Positive Salmonella-IgM tests among blood culture negative patients should always be correlated with clinical picture of the patient. Many a times, blood culture may be negative due to other reasons like prior intake of antibiotics, etc., However, culture isolation of Salmonella remains essential, especially for antibiotic susceptibility testing and these serological tests should be used in conjunction with culture for the early diagnosis of enteric fever.
| ~ Acknowledgement|| |
Authors are thankful to Ms. Parul Takkar for her help in statistical analysis.
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[Table 1], [Table 2], [Table 3]