|Year : 2018 | Volume
| Issue : 1 | Page : 5-13
Laboratory tests and clinical management of dengue in 26 healthcare institutions in India
Sanjay Bhattacharya1, Gaurav Goel1, Kalpana George2, Ranjani Shamsundar3, J Lancy4, J Sudarsana5, S Rema Devi6, S Sangeetha7, Kamla Kant8, KA Sathiavathy9, KL Sarada Devi10, K Rejitha11, TS Shailaja12, Geetha Raveendran2, Anu P John13, T Sohanlal14, George Varghese15, Anitha Madhavan16, Barsha Sen17, KV Suseela18, Syed Mustaq Ahmed19, Renu Mathew20, Shoba Kurian Pulikkottil21, J Beena Philomina22, Jily P Chinnan23, Soumya Rani24, Ranganathan Iyer25, Sourav Maiti26, Swapna R Nath27, Shabina M Balakrishnan22, Kavita Raja28
1 Department of Microbiology, Tata Medical Centre, Kolkata, West Bengal, Kerala, India
2 Department of Microbiology, Government Medical College, Kollam, Kerala, India
3 Department of Microbiology, St. John's Medical College Hospital, Bangalore, Karnataka, India
4 Department of Microbiology, Government Medical College, Kochi, Kerala, India
5 Department of Microbiology, Baby Memorial Hospital, Kozhikode, Kerala, India
6 Department of Microbiology, Dr. Somervell Memorial CSI Medical College, Karakonam, Kerala, India
7 Department of Microbiology, Rajarajeswari Medical College and Hospital, Bangalore, Karnataka, India
8 Department of Microbiology, General Hospital JIPMER, Karaikal, Puducherry, India
9 Department of Microbiology, Jubilee Mission Medical College and Research Institute, Thrissur, Kerala, India
10 Department of Microbiology, Government Medical College, Thiruvananthapuram, Kerala, India
11 Department of Microbiology, Government Medical College, Manjeri, Kerala, India
12 Department of Microbiology, PK Das Institute of Medical Sciences, Ottappalam, Kerala, India
13 Department of Microbiology, Government Medical College, Thrissur, Kerala, India
14 Department of Microbiology, Aster MIMS, Kozhikode, Kerala, India
15 Department of Microbiology, Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla, Kerala, India
16 Department of Microbiology, Government Tirumala Devaswam Medical College, Alappuzha, Kerala, India
17 Department of Microbiology, Sri Aurobindo Seva Kendra, Kolkata, West Bengal, Kerala, India
18 Department of Microbiology, Amala Institute of Medical Sciences, Thrissur, Kerala, India
19 Department of Microbiology, Muslim Educational Society Medical College, Perinthalmanna, Kerala, India
20 Department of Microbiology, Believer's Church Medical College Hospital, Thiruvalla, Kerala, India
21 Department of Microbiology, Government Medical College, Kottayam, Kerala, India
22 Department of Microbiology, Government Medical College, Kozhikode, Kerala, India
23 Department of Microbiology, VPS Lakeshore Hospital and Research Center, Ernakulam, Kerala, India
24 Department of Microbiology, SR Medical College and Research Center, Varkala, Kerala, India
25 Department of Microbiology, Global Hospital, Hyderabad, Telangana, India
26 Department of Microbiology, Institute of Neurosciences, Kolkata, West Bengal, Kerala, India
27 Department of Microbiology, Regional Cancer Centre, Thiruvananthapuram, Kerala, India
28 Department of Microbiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
|Date of Web Publication||4-Jul-2018|
Dr. Kalpana George
Department of Microbiology, Government Medical College, Kollam, Kerala
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Bhattacharya S, Goel G, George K, Shamsundar R, Lancy J, Sudarsana J, Devi S R, Sangeetha S, Kant K, Sathiavathy K A, Sarada Devi K L, Rejitha K, Shailaja T S, Raveendran G, John AP, Sohanlal T, Varghese G, Madhavan A, Sen B, Suseela K V, Ahmed SM, Mathew R, Pulikkottil SK, Philomina J B, Chinnan JP, Rani S, Iyer R, Maiti S, Nath SR, Balakrishnan SM, Raja K. Laboratory tests and clinical management of dengue in 26 healthcare institutions in India. J Acad Clin Microbiol 2018;20:5-13
|How to cite this URL:|
Bhattacharya S, Goel G, George K, Shamsundar R, Lancy J, Sudarsana J, Devi S R, Sangeetha S, Kant K, Sathiavathy K A, Sarada Devi K L, Rejitha K, Shailaja T S, Raveendran G, John AP, Sohanlal T, Varghese G, Madhavan A, Sen B, Suseela K V, Ahmed SM, Mathew R, Pulikkottil SK, Philomina J B, Chinnan JP, Rani S, Iyer R, Maiti S, Nath SR, Balakrishnan SM, Raja K. Laboratory tests and clinical management of dengue in 26 healthcare institutions in India. J Acad Clin Microbiol [serial online] 2018 [cited 2019 Jul 16];20:5-13. Available from: http://www.jacmjournal.org/text.asp?2018/20/1/5/235916
| Introduction|| |
Dengue is a mosquito-borne (Aedes aegypti) acute viral infection caused by a single-stranded (positive sense) ribonucleic acid virus belonging to the Flaviviridae family. The disease is transmitted generally by the bite of infected mosquito but can also be transmitted rarely from blood products or donated organs of donors incubating the virus., In India, the National Vector Borne Diseases Control Program (Directorate General of Health Services, Ministry of Health and Family Welfare) gives comprehensive information about cases and deaths from dengue from all states and union territories. For example, in the last four years: 2014, 2015, 2016 and 2017, the number of cases reported from the whole of India was 40,571, 99,913, 129,166 and 153,635, respectively. The deaths from dengue reported from the same years were 137 (0.33%), 220 (0.22%), 245 (0.19%) and 226 (0.15%), respectively. There is obviously an upward trend in the number of dengue cases reported from India and also a noticeable decline in the mortality rate from dengue. The reasons for these trends may be due to various factors such as increased case detection, greater awareness amongst general population and willingness to seek early medical intervention (for testing and management), better testing and case management strategies.
Despite the positive news about decreasing mortality rate in India from dengue, a lot of clinical, epidemiological and technical information about the disease and its detection remains to be known. There are also many challenges with regard to optimal diagnosis, timely case detection, better treatment and appropriate follow-up. In most healthcare institutions, the microbiology laboratory is responsible for laboratory diagnosis of dengue. A variety of different dengue test kits and diagnostic strategies are available globally and in India. However, large-scale surveys are not common with regard to the dengue diagnostic tests used in microbiology laboratories. The denominator data (number of samples tested for dengue) are also not readily available. This current survey done in 26 microbiology laboratories mostly from Kerala (n = 20), but also a few from West Bengal (n = 2), Karnataka (n = 2), Telangana (n = 1) and Puducherry (n = 1) attempts to understand the laboratory diagnostic practices and results from various hospitals in India.
| Materials and Methods|| |
A pre-designed questionnaire was used to collect data of dengue tests done and infection rates for the year 2017 from different microbiology laboratories attached to Medical Colleges or private institutions in India [please refer to the Appendix 1 at the end of the paper on 'A questionnaire on laboratory tests and clinical management of dengue in India']. The questions were set after mutual consultation by members of the Editorial Board of the Journal of the Academy of Clinical Microbiologists (JACM) and subsequently circulated to microbiology peer groups through known e-mail contacts. The responses were subsequently collated and analysed (using Excel) by one of the editorial board members of JACM. The Editorial Board subsequently reviewed the data and finalised the formal report. Although e-mail or telephonic reminders were given to survey participants, no other incentive (apart from authorship in publication) was given to survey participants in the invitation e-mail [please refer to 'Invitation Letter' in the Appendix 1].
| Results|| |
Twenty-six laboratories responded to the questionnaire. Of these, 19 laboratories were attached to medical colleges and seven to private hospitals. All laboratories tested for dengue in their own laboratory except one which outsourced the tests to external commercial laboratories [Figure 1], [Figure 2], [Figure 3]. Dengue tests were part of routine External Quality Assurance Scheme, or Inter Laboratory Comparison in 15 laboratories. A total of 56859 (NS1 antigen), 48557 (immunoglobulin M [IgM] antibody) and 20727 (immunoglobulin G [IgG] antibody) tests were done in the institutions participated. Number of tests done and positives are shown in [Figure 4].
|Figure 3: Number of laboratories using various platforms to detect dengue markers|
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Dengue antigen and antibody detection: enzyme-linked immunosorbent assay and rapid tests
Kits from >10 manufacturers were used by various laboratories. [Figure 5] shows the number of institutions using kits by various manufacturers. All questionnaire responders were satisfied with respect to the performance of various enzyme-linked immunosorbent assay (ELISA) kits used for detecting NS1 antigen, IgM and IgG antibodies irrespective of the manufacturer except one laboratory which rated ELISA kits by two manufacturers as fair. These laboratories reported the observation that the cut-off values of negative controls of these kits tended to decrease with time and they encountered grey zoning for one kit frequently. Rapid tests for detection of dengue markers were reported to be 'satisfactory' by majority of users. None of the participating institutions did in-house tests for NS1 antigen, IgM or IgG antibodies. NS1 antigen was the most common test done (with about 27% of the samples being reactive), followed by IgM serology (about 25% reactive). IgG serology was done in less than half of the cases (with about 14% testing reactive).
Dengue polymerase chain reaction and genotyping
Dengue polymerase chain reaction (PCR) was done only by Pushpagiri Institute of Medical Sciences and Research Center, Thiruvalla. They used in-house kit for the PCR by conventional method. None of the participating institutions carried out genotyping of dengue virus.
Turn-around time for dengue test results
Out of the 19 (73%) laboratories performing NS1 antigen ELISA tests, 18 issued the reports on the same day. IgM ELISA was done by 21 (80.5%) laboratories, of which 16 (76.1%) issued the results within 24 h. One laboratory reported the results in one week and the rest in 2–3 days. IgG ELISA was done by 13 (50%) laboratories who participated in the survey either onsite or outsourced, of which eight issued reports on the same day. Laboratories which did rapid tests for any of the markers gave out reports on the same day. Various laboratories used time cutoffs for issuing same day results. The laboratory which did PCR, issued the result, on the third day.
Epidemiology, clinical management of dengue cases and pest control
We noted from the survey that majority of the clinical management of dengue cases was done by physicians from general medicine (internal medicine) and by paediatricians, followed by multidisciplinary care, clinical microbiologists, departmental physicians, infectious disease physicians and other physicians (nephrologists and intensive care specialist were part of team that managed dengue patients in two hospitals) [Figure 4]. In an oncology hospital, dengue patients requiring inpatient care but without an active oncology problem at the time of presentation were referred to other hospitals for specific care. Only seven (26.9%) hospitals had a policy to take care of dengue patients in isolation rooms during in-patient stay. Majority of the centres relied on both haematocrit values and platelet count for monitoring of dengue cases. Few centres used liver function test, renal function tests, urine and serum albumin and international normalised ratio for monitoring dengue cases [Figure 5]. In dengue negative case or in cases not responding adequately to management, other investigations for infection screen included blood cultures, tests for malaria, typhoid, chikungunya, leptospirosis, rickettsial infections, viral hepatitis, Brucella More Details and Kyasanur Forest Disease [Figure 6]. All laboratories observed a seasonal variation in the occurrence and distribution of dengue with a maximum number of cases occurring between the months of May and November [Figure 7]. Twenty-one (80.7%) hospitals which took part in the survey had blood bank facility and 19 (73%) hospitals had pest control practices in place.
|Figure 6: Other infections for which screening is done in dengue negative cases|
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|Figure 7: Number of labs that recorded a seasonal distribution (month wise distribution)|
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Notification to public health authority
In 11 (42.3%) and 14 (53.8%) laboratories, notification to the public health authority was done within 24 h and seven days, respectively, of the test coming positive. Four of the laboratories also reported the total number of samples tested (including those which tested negative). In one instance, the laboratory was not directly involved in the notification process (and the hospital administration did the notification). Eleven (42.3%) institutions notified dengue to public health authorities on the basis of rapid tests only. The laboratory which did PCR notified dengue to health authority based on PCR results alone.
| Discussion|| |
The current study highlights some important practical points regarding the diagnosis, prevention and management of dengue in various tertiary care settings within India. NS1 antigen and IgM for dengue are the most common tests used in diagnosis [Figure 1] and not all centres are testing or having the opportunity to test for dengue. This may be because presentation of cases to healthcare facilities happen during the acute phases of illness and also due to the fact that not all patients come back for follow-up. We also note that most of the laboratories within this survey do not outsource the dengue serology test [Figure 2]. This is useful as outsourcing can lead to increase in turn-around time and delay the diagnosis. Dengue is an acute illness and rapid diagnosis is required. A delay in diagnosis can be costly both for the patient as well as for the institution. Although majority of the centres use ELISA, there are significant numbers who use rapid tests based on immunochromatography or both [Figure 3]. This is because although ELISA has superior sensitivity and specificity than rapid tests, manual ELISAs are operationally difficult because of the need of focussed human resources and significant periods needed in test procedures (sample/reagent addition to ELISA plates, incubation, washing, etc.). It is also not cost-effective for laboratories whose workload is not significant. ELISAs available in dengue serology are mostly manual and not automated in most centres increasing the operational requirements. We noted that the most common serological marker to come reactive in dengue serology was NS1 antigen (27.2% of the samples tested), closely followed by IgM (24.9%) of the samples tested. IgG for dengue had a reactivity rate of 14.3% only [Table 1]. We also observed that dengue serology was being done using a variety of kits from different manufacturers [Table 2]. It is important that when kits are selected due attention is given to not only the cost of performing the test but also sensitivity, specificity, markings (CE-Conformité Européenne, IVD approval: forin vitro diagnostics only, or FDA approved-US Food and Drug Administration), ease of performance, technical support, availability and quality control and quality assurance parameters. We noticed through the survey that physicians of general medicine and paediatrics are the most common healthcare service providers along with ID physicians and clinical microbiologists. This implies that, for better management of dengue, there has to be a close clinical liaison between the concerned departments to optimise diagnosis, management and follow-up. Platelet count and haematocrit are the common haematological parameters used along with clinical parameters (pulse, respiration, blood pressure, orientation/consciousness, jaundice and bleeding) to monitor cases of dengue [Figure 5]. The platelet count indicates chances of bleeding (especially when it is <10,000/cu. mm) and the need of platelet transfusion. Haematocrit is an indicator of the degree of haemoconcentration arising because of fluid loss from increased capillary permeability and the need for hydration. Since many of the clinical signs and symptoms of dengue are nonspecific, it is important that other investigations that are clinically and epidemiologically relevant are done. This may involve tests for excluding other infections which are part of the differential diagnosis (e.g., chikungunya, typhoid, etc.) [Figure 6]. Our survey confirms that dengue continues to be a disease with significant seasonal bias with majority of the cases occurring in the rainy season and the post-monsoon months [Figure 7]. This information is important for vector control programme and healthcare infrastructure logistics development and reinforcement during the high incidence period.
|Table 1: Number of tests done by each laboratory and positives (marker wise)|
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|Table 2: Manufacturers of kits used and the number of institutions using each kit|
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The National Vector Borne Disease Control Programme envisages three-pronged strategies for the control of dengue, which includes early case reporting and management, integrated vector management (for transmission risk reduction) and supporting interventions (including training of trainers, capacity building and legislative support). The early case reporting and management strategy include coordination of sentinel surveillance sites with laboratory support, strengthening of referral services and involvement of private sector in sentinel surveillance of dengue. Despite these objectives, several gaps remain in the actual management of dengue in India – in the field of laboratory diagnosis, clinical management and vector control. Surveys are an important tool to identify gaps as well as strengths of dengue management strategies. A survey can reveal interesting facts about ground level realities in dengue management. Approximately half of participants (of a dengue clinical survey done by CDC Atlanta in Texas, USA) demonstrated knowledge needed to identify dengue cases, fewer than 20% correctly identified all prevention messages, severe dengue warning signs, or circumstances in which a dengue patient should return for care, very few participants correctly identified scenarios when plasma leakage occurred (10%) or a crystalloid solution was indicated (7%). A study from Brazil showed that clinical criteria were not sufficient for distinguishing between dengue and non-dengue febrile illness. The study also showed that dengue PCR had a sensitivity and specificity of 88% and 89%, respectively, compared to NS1 ELISA.
Alere, Australia conducted a retrospective study in two Southeast Asian locations, Vietnam and Malaysia, where they evaluated the efficacy of a Dengue rapid NS1 antigen test and also made an effort to determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. The study reported that, in Vietnam, the sensitivity and specificity of the rapid dengue test were 69.2% and 96%, respectively. In Malaysia, the performance was similar with 68.9% sensitivity and 96.7% specificity compared to real-time polymerase chain reaction. Importantly, when the dengue early rapid test was used in combination with the IgM/IgG test, the sensitivity increased to 93%. A similar study from Thailand reported that the combination of NS1 and IgM tests enhanced diagnosis of dengue. A report from Singapore showed that the WHO classification schemes for bedside diagnosis of dengue had high sensitivity but lacked specificity. The NS1 strip test had high specificity, but its sensitivity was significantly lower in secondary compared with primary dengue infections. Differences in viral serotypes did not affect the performance of any of the three diagnostic approaches. A report from Pakistan showed that acute phase sera of nearly 58% were immunochromatography test were non-reactive, and concluded that in highly suspected cases of dengue infection clinical management should not rely on negative serological results. In India, an ICMR study during a dengue epidemic in Tamil Nadu reported that while dengue-specific IgM responses were predominant amongst infants with dengue fever, IgG and mixed responses (M + G) were seen in 85% of the children with severe forms of dengue.
Guidelines from the National Vector Borne Disease Control Program has stated that, although 'a number of commercial rapid diagnostic test (RDT) kits for anti-dengue IgM and IgG antibodies are at present commercially available, (and) which produces the results within 15–20 min, the sensitivity/specificity of most of these tests is not known since they have not yet been properly validated. The sensitivity and specificity of some RDTs also found to vary from lot to lot. According to WHO guidelines, these kits should not be used in the clinical settings to guide management of DF/DHF cases because many serum samples taken in the first five days after the onset of illness will not have detectable IgM antibodies. Reliance on such tests to guide clinical management could, therefore, result in an increase in case fatality rate'. The current article does not endorse the use of dengue rapid tests but provides information on the prevailing practices.
| Conclusion|| |
The current survey conducted across 26 centres in India is one of the few laboratories and clinical surveys available on dengue in India. It identifies actual practices (from labs) and clinical policies (from wards). It is hoped that the study would be helpful for policymakers in public health to identify gaps and formulate better policies in the future for the control of dengue in India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| Appendix|| |
A questionnaire on laboratory tests and clinical management of Dengue in India
Information for the participants of the questionnaire:
Dengue is an important vector-borne disease and a public health problem in India. Effective management of dengue requires not only good clinical and support services (such as blood bank) but also good quality laboratory infrastructure. Building up dengue control activities would require a better understanding of dengue diagnostic facilities within the country. The following questionnaire has designed after discussion between various members of the editorial board of the Journal of the Academy of Clinical Microbiologists with that objective. Your participation in this questionnaire will provide valuable information for specialists and policymakers involved in the control and management of dengue. Also note that personal details and hospital details would be kept confidential and laboratories/hospital responding to the questionnaire would be anonymised (with code numbers). We look forward to your participation.
Journal of the Academy of Clinical Microbiologists
| Reference|| |
1. National Vector Borne Diseases Control Programme. Director General of Health Services. Ministry of Health and Family Welfare. Available from: http://www.nvbdcp.gov.in/den-cd.html. [Last accessed on 2017 Dec 27].
- Do you test for Dengue in your lab?
- Yes (In-house)
- Outsourced to Commercial labs
- Outsourced to reference centers
- Not done
- Is dengue test part of your routine EQAS, or ILC (Inter-Laboratory Comparison)?
- What is the type of hospital that your laboratory serves?
- Medical college
- Government hospital (non-medical college)
- Private hospital
- Reference Laboratory
- Private standalone laboratory
- Please tick as done in your laboratory:
- If you are doing Dengue tests in your lab what kits are you using for the following: (if not please go to question 4)
- Do you perform Dengue RNA PCR routinely in your lab?
- If Dengue PCR is performed in your lab what platform is used for the same?
- Real-time PCR
- End point (non-real time PCR)
- Others (please specify)
- Not applicable
- Do you perform Dengue genotyping in your lab? Yes/No
- If genotyping is performed in your lab what technique do you use for the genotyping?
- Real-time PCR- multiplex
- End point PCR- multiplex
- Sanger sequencing
- Others (please specify)
- Not applicable
- What is the average turn-around time for Dengue serology in your lab?
- NS1 antigen ELISA (same day/once a week/others [please specify______days])
- IgM ELISA (same day/once a week/others [please specify______days])
- IgG ELISA (same day/once a week/others [please specify______days])
- Rapid immunochromatographic test (same day/done even at night or on holidays/others [please specify______hours])
- Dengue PCR (same day/once a week/others [please specify______days])
- Dengue genotyping (same day/once a week/others [please specify______days])
- Who manages Dengue within your hospital? (you may select more than one)
- Infectious Diseases physicians
- Clinical microbiologists
- General Medicine Physician/Pediatricians
- Respective Departmental consultants
- Others (please specify)
- How often do you notify dengue to your local public health authority? (you may select more than one)
- Within 24 hours of the test coming positive
- Within 7 days of the test coming positive
- Within one month of the test coming positive
- Others (please specify)
- We also notify total number of samples tested (including those which tested negative)
- Do you notify cases of Dengue if they are positive by rapid tests (e.g., non-ELISA immunochromatographic tests) and ELISA was not done or was negative?
- Do you notify cases of Dengue if they are positive by PCR (and ELISA was not done or was negative)?
- Are confirmed dengue patients kept in isolation (in separate rooms) within your hospital?
- In dengue negative cases what other infections do you screen for?
- Blood cultures
- Others (please specify)
- Once positive for Dengue how do you monitor cases of dengue in your hospital?
- By platelet count
- By hematocrit (PCV- packed cell volume)
- By both haematocrit and platelet count
- By other parameters (please specify)
- Do you see a seasonal distribution for the Dengue cases? Yes/No
- Which months or period of the year do you see most cases of Dengue?
- Do you have a blood bank in your hospital which provides platelet components for transfusion?
- Do you have a pest control service within your hospital? Yes/No
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Kabilan L, Velayutham T, Sundaram B, Tewari SC, Natarajan A, Rathnasamy R, et al.
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Guidelines for the Clinical Management of Dengue Fever, Dengue Hemorrhagic Fever and Dengue Shock Syndrome. Guidelines from the National Vector Borne Disease Control Program; 2008. Available from: http://www.nvbdcp.gov.in/Doc/Clinical%20Guidelines.pdf
. [Last accessed on 2018 Jun 05].
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]