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Table of Content - Volume 21 Issue 1 - January 2022
Deepali Vagdalkar1*, B Swetha2
1Associate Professor, 2Post Graduate, Department of Microbiology, Malla Reddy Institute of Medical Sciences, Suraram, Hyderabad, Telangana, INDIA.
Abstract Background: Early and confirmatory diagnosis of dengue is important to prevent morbidity and mortality associated with dengue. There are many diagnostic tests. Some are gold standard like viral isolation and culture, RT-PCR, Antibody based and rapid tests, which detects NS1 antigen using kit. Rapid tests are cheaper, easily available and gives results very fast, but often people doubt its diagnostic accuracy. Objective: To study diagnostic accuracy of Immunochromatography based RDT kit compared to Dengue NS1 Ag Microlisa and Dengue IgM Microlisa in the diagnosis of Dengue infection Methods: Diagnostic evaluation study was carried out using 84 blood samples. In all the samples, Immunochromatography based RDT kit results were compared with Dengue NS1 Ag Microlisa and Dengue IgM Microlisa (gold standard). The sensitivity, specificity, positive predictive value and negative predictive value were calculated for the rapid test. Results: Males were more than females (55.9% vs. 44.1%). Most common affected age group was 20-39 years (55.9%). Majority (59.5%) were having platelet count >1lakh/cumm and they were classified as having no risk. 46.4% were having mild disease.Rapid test was able to identify 86.5% correctly as having disease with a sensitivity of 86.5%. 80% cases, which were diagnosed as negative by rapid test, were also negative on ELISA giving a specificity of 80%. Positive predictive value of rapid test was 96.9% while negative predictive value was 44.4% compared to ELISA Conclusion: Rapid test was found to have good diagnostic accuracy in the diagnosis of dengue compared to ELISA and can be used in the low resource settings. Key words: diagnostic accuracy, Immunochromatography, ELISA, diagnosis, Dengue
INTRODUCTION Dengue is a public health problem. It is a vector borne disease. 10% of all dengue infections constitute the severe and very severe form of the disease where the patient can even be admitted to the intensive care unit and mortality in such cases is very high. The clinical features of dengue resemble to those other febrile illnesses prevalent in those areas. Laboratory findings also match to a certain extent. Hence, it is difficult to have early diagnosis of the dengue fever.1 Hence, early diagnosis is the key to prevent the morbidity and mortality associated with the dengue fever. For this, a reliable, easy to use, cost effective and accessible diagnostic test is much needed. At present viral isolation and culture is one test that is costly, time consuming (takes 6-10 days to give the results) and not available everywhere. Detection of dengue viral RNA is another test which is also costly and may not be available everywhere. Third, one is using the paired sera of the antibodies for specific IgM/IgG antibodies. The gold standard considered for the diagnosis of the dengue is the combination of these three tests. RT-PCR based tests give results in 24 hours. Paired sera of the antibodies for specific IgM/IgG antibodies gives results in few hours. However, the antibodies take 4-5 days to develop and hence the antibody-based tests done during this period do not yield results. One, 2 NS1 (Non-structural Protein 1) based diagnostic tests for dengue are available now a days. NS1 is a glycoprotein found in the viruses like yellow fever virus, Japanese encephalitis virus, tick borne encephalitis virus and the dengue virus. 3 It is highly specific ranging from 86.1% to 100%. 4 But the sensitivity is highly variable ranging from 37% to 98.9% from different studies. 5-12 The range in the variability of the sensitivity of the NS1 test is because it decreases over time and affected by the presence of the secondary infections. 7, 10, 11 Hence, it has been suggested that IgM and IgG specific antibodies should be added to improve the results and improve the sensitivity and specificity to NS1 bases tests as a single kit. 13 With this background in mind, to find the diagnostic accuracy of the Immunochromatography based RDT kit compared to Dengue NS1 Ag Microlisa and Dengue IgM Microlisa in the diagnosis of Dengue infection, present study was carried out.
METHODS The present study was diagnostic evaluation study where we took Dengue NS1 Ag Microlisa and Dengue IgM Microlisa (J. Mitra and Co. Pvt. Ltd, India) as our gold standard for the present study (henceforward will be called as ELISA in the remaining part of this article). The results of the Immunochromatography based RDT kit (henceforward will be called as RDT i.e. rapid diagnostic kit in the remaining part of this article) (J. Mitra and Co. Pvt. Ltd, India) were compared against the results of the ELISA to see the sensitivity, specificity, positive predictive value and negative predictive value. Present study was conducted from July 2021 to November 2021 in the Department of Microbiology, Malla Reddy Institute of Medical Sciences, Hyderabad, India. 84 Blood samples of clinically suspected Dengue patients were collected from OPD and IPD in the Central laboratory of the institution Inclusion criteria: All patients whose blood samples were available in the laboratory during the study period. All age groups of both sexes Exclusion criteria: Inadequate blood samples data were excluded from the present study. Those with doubtful reports were excluded. Institution Ethics Committee permission was not taken as this study did not identify the patients and do not include the identifying information in any form. Secondly, sending samples of the suspected dengue cases is a routine procedure and we were not involved in the sample collection. Hence, we did not take Institution Ethics Committee permission. The blood samples of the patients from OPD and IPD who were clinically suspected to have dengue infection were sent to the Microbiology department of Malla Reddy Institute of Medical Sciences, Hyderabad for confirmation of the diagnosis as a routine protocol. Under this study, we carried out both rapid and ELISA test for each sample received by us during the study period. All the blood samples were collected under aseptic precautions routinely. After the sample was received, it was entered in the receipt register of the laboratory. First, the serum was separated andstored for further analysis. All serum samples were tested for dengue dentification by Immunochromatography based RDT kit (J. Mitra and Co. Pvt. Ltd, India) which detects NS1 antigen, IgM and IgG antibodies. Dengue NS1 Ag Microlisa (J. Mitra and Co. Pvt. Ltd, India) and Dengue IgM Microlisa (J. Mitra and Co. Pvt. Ltd, India) were carried out as second confirmatory test. All blood samples were subjected to complete blood picture with special emphasis on platelet count using standard recommended methods. Statistical analysis: The data was expressed as proportions. Sensitivity, specificity, positive predictive value and the negative predictive value was calculated for rapid test when compared with the gold standard i.e. ELISA.
RESULTS Table 1: Age and sex wise distribution of study subjects
Males were slightly more than females (55.9% vs. 44.1%). Most common affected age group was 20-39 years (55.9%) followed by the 1-19 years of age (26.2%) (Table 1) Table 2: Distribution of study subjects as per their platelet counts
Majority of the study subjects (59.5%) were having platelet count of more than one lakh per cumm and they were classified as having no risk. 35.7% of the cases had platelet count of 40,000 to less than one lakh per cumm and they were classified as having low risk. Only one case was having high-risk disease with platelet count less than 21,000. (Table 2)
Table 3: Distribution of study subjects as per the disease severity based on clinical presentation
On clinical examination, 46.4% were found to have mild disease followed by moderate disease in 39.3% of the cases. 14.3% were found to have severe disease (Table 3)
Table 4: Sensitivity and specificity of NS-1 rapid antigen test compared to ELISA
The rapid test was able to identify 86.5% of the cases correctly as having disease with a sensitivity of 86.5%. The rapid test which identified two cases (20%) as positive were actually negative. 80% of the cases which were diagnosed as negative by rapid test were also negative on ELISA giving a specificity of 80% for the rapid test. The positive predictive value of the rapid test was 96.9% while the negative predictive value was 44.4% compared to ELISA (Table 4)
DISCUSSION Males were slightly more than females (55.9% vs. 44.1%). Most common affected age group was 20-39 years (55.9%) followed by the 1-19 years of age. Majority of the study subjects (59.5%) were having platelet count of more than one lakh per cumm and they were classified as having no risk. 35.7% of the cases had platelet count of 40,000 to less than one lakh per cumm and they were classified as having low risk. Only one case was having high-risk disease with platelet count less than 21,000. On clinical examination, 46.4% were found to have mild disease followed by moderate disease in 39.3% of the cases. 14.3% were found to have severe disease. The rapid test was able to identify 86.5% of the cases correctly as having disease with a sensitivity of 86.5%. The rapid test which identified two cases (20%) as positive were actually negative. 80% of the cases which were diagnosed as negative by rapid test were also negative on ELISA giving a specificity of 80% for the rapid test. The positive predictive value of the rapid test was 96.9% while the negative predictive value was 44.4% compared to ELISA. Osorio L et al.14 carried out paired analysis of 310 samples out of which 218 were positive and 92 were negative. They used “Platelia™ Dengue NS1 Ag, second generation Pan-E™ Dengue Early ELISA, SD Dengue NS1 Ag ELISA, Dengue NS1 Ag STRIP™, and SD BIOLINE™ Dengue Duo (NS1/IgM/IgG)”. They observed that highest sensitivity of 80.7% was seen for “SD BIOLINE™ NS1/IgM/IgG”. The sensitivity for ELISA format test was less than 75%. The sensitivity for “STRIP™ and SD NS1” was less than 65%. As the duration of taking sample from the onset of the fever increased, the sensitivity decreased. Fry SR et al. 15evaluated efficacy of “Dengue Early Rapid test for NS1” and conducted a study to determine its efficacy in combination with “commercial IgM/IgG rapid test.” They included 198 positive and 100 negative samples. They found that the sensitivity was 69.2% and the specificity was 96%. Tricou V et al.16 calculated the specificity and sensitivity of “Bio-Rad NS1 Ag Strip and SD Dengue Duo (NS1/IgM/IgG) lateral flow rapid tests” among 245 patients. They noted that the sensitivity of the rapid tests was around 60% but had very good specificity of 100%. After including IgM/IgG results from the SD Dengue Duo, the sensitivity increased to 75.5%. Pal S et al.17evaluated the tests like “Dengue NS1 Ag STRIP”, “Platelia Dengue NS1 Ag ELISA” (Bio-Rad, France), “Dengue NS1 Detect Rapid Test (1st Generation)” and “DENV Detect NS1 ELISA” (InBios International, United States), “PanBio Dengue Early Rapid (1st generation)” “PanBio Dengue Early ELISA (2nd generation)” and “SD Bioline Dengue NS1 Ag Rapid Test” (Alere, United States). They reported that the sensitivity of the rapid tests was 71.9% to 79.1% and the specificity was 85.6% to 95.9%. They used the virus isolation as the gold standard. Garg A et al.18 estimated the sensitivity and specificity in 80 samples for “Dengue Cassette (PanBio, Australia)”, “Bioline Dengue Duo (SD Diagnostics, Korea)”, “Dengue Day 1 test (J Mitra and Co., India)”, and “Dengucheck Duo (Tulip Diagnostics, India)”. They noted that all the rapid tests had low sensitivity but good specificity. Sathish N et al.19 compared “An IgM capture ELISA (National Institute of Virology, Pune, India)” with two commercial tests, “PanBio Rapid Immunochromatographic Card Test (Brisbane, Australia)” and “PanBio Microwell IgM ELISA”. They found that the positivity rate with NIV IgM capture ELISA was 38.9% compared to 22.7% of PanBio rapid and 20.7% of the PanBio IgM. The sensitivity of the NIV MAC-ELISA was 96% while it was 73% for the PanBio rapid and 72% for the PanBio IgM ELISA.
CONCLUSION The sensitivity, specificity, positive predictive value and the negative predictive value of the rapid test was good and comparable. Thus, the diagnostic accuracy of the rapid test was comparable. Hence, we recommend using the rapid tests for the diagnosis of the dengue in the low resource settings. This will help to go for the early diagnosis and this early diagnosis will help in prevention of the morbidity and mortality associated with the dengue.
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