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 ~  Abstract
 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 288-292
 

Utility of a multiplex real-time polymerase chain reaction for combined detection and serotyping of dengue virus in paediatric patients hospitalised with severe dengue: A report from Chennai


1 Department of Pediatrics, Kanchi Kamakoti CHILDS Trust Hospital, Chennai, Tamil Nadu, India
2 Department of Pediatrics, The CHILDS Trust Medical Research Foundation, Chennai, Tamil Nadu, India

Date of Submission11-Jun-2020
Date of Decision15-Aug-2020
Date of Acceptance06-Sep-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Anand Manoharan
The CHILDS Trust Medical Research Foundation, Kanchi Kamakoti CHILDS Trust Hospital, 12-A, Nageswara Road, Nungambakkam, Chennai - 600 034, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_249

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 ~ Abstract 


Objective: Molecular detection and serotyping are rapid, sensitive and accurate techniques for early diagnosis of paediatric dengue. The present study evaluates multiplex real-time polymerase chain reaction (PCR) for diagnosis of dengue virus in children hospitalised with severe dengue (SD) and attempts to establish an association of clinical severity with specific serotypes. Methods: Four hundred and eighty-five samples were received from hospitalised paediatric patients with suspected dengue from March 2019 to February 2020. Multiplex real time PCR was employed for diagnosis. An in-house real-time PCR that combined diagnosis and serotyping was established. Non-structural protein 1 (NS1) assay and real-time PCR were assessed for their accuracy in diagnosing severe paediatric dengue. Results: Three hundred and twenty-five (67%) patients were positive for dengue RNA by real-time PCR. All four serotypes were identified throughout the year; dengue serotype 2 (DEN-2) was predominant (61%) followed by DEN-3, 20%. Compared to the commonly used NS1 testing, multiplex real-time PCR showed greater sensitivity in diagnosing SD. Conclusions: Compared to NS1, multiplex real-time PCR is a rapid and accurate diagnostic test for children hospitalised with SD. DEN-2 was the predominant serotype in severe cases. Continued surveillance of serotypes should be carried out year-round in endemic areas.


Keywords: Dengue serotype 2, multiplex real-time polymerase chain reaction, severe paediatric dengue, Tamil Nadu


How to cite this article:
Balasubramanian S, Chandy S, Peter R, Nachiyar GS, Sudhakar A, Sumanth A, Manoharan A. Utility of a multiplex real-time polymerase chain reaction for combined detection and serotyping of dengue virus in paediatric patients hospitalised with severe dengue: A report from Chennai. Indian J Med Microbiol 2020;38:288-92

How to cite this URL:
Balasubramanian S, Chandy S, Peter R, Nachiyar GS, Sudhakar A, Sumanth A, Manoharan A. Utility of a multiplex real-time polymerase chain reaction for combined detection and serotyping of dengue virus in paediatric patients hospitalised with severe dengue: A report from Chennai. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 24];38:288-92. Available from: https://www.ijmm.org/text.asp?2020/38/3/288/299819





 ~ Introduction Top


Dengue is a major public health problem in India. All serotypes, dengue serotype 1 (DEN-1), DEN-2, DEN-3 and DEN-4 co-circulate in urban and semi urban areas making it hyperendemic for dengue.[1],[2] The diagnosis of dengue during the acute phase is important for clinical management, surveillance and implementation of control measures. The non-structural protein 1 (NS1) dengue viral antigen is a sensitive and early marker, routinely being used for diagnosis in the ELISA format, however, its sensitivity is reportedly less in secondary dengue. Serological immunoglobulin M (IgM) and IgG testing are retrospective and cannot provide timely diagnosis nor help in clinical decision-making.[3]

These antigen/antibody assays cannot provide information on the infecting serotype. Reverse transcriptase-based polymerase chain reaction (RT-PCR) is increasingly being used for detection and serotyping of dengue virus. Multiplex real-time RT-PCR has a good sensitivity (80%–100% and specificity (99%–100%). However, cross contamination leading to false positives can be a limiting factor of PCR.[3],[4],[5] Detection followed by serotyping is the format used by commercial real-time PCR kits. In-house assays can be developed to combine detection and serotyping thereby improving turnaround time, diagnosis and lowering testing costs.

The predominant serotypes reported from different regions of the country during 1982–2015 are: DEN-2 and DEN-1 in the northern region, DEN-2 and DEN-3 in the southern region, and DEN-1 and DEN-2 in the eastern and the western regions, respectively. Dominant serotypes in a region get displaced over time.[6] Hyperendemicity and new serotypes in a region can contribute to dengue severity. The association between dengue serotypes and disease outcomes has been explored but no definite link has been established. Temperature and rainfall together with intricate interactions of ecology, environment, vector and virus serotypes are key factors driving dengue outbreaks.[7]

This study was undertaken at Kanchi Kamakoti Childs Trust Hospital, a paediatric hospital in Chennai. Study objectives were to assess disease burden, serotype diversity in children hospitalised with suspected dengue and the utility of a rapid real-time PCR for combined diagnosis and serotyping of severe cases.


 ~ Methods Top


Paediatric patients hospitalised with suspected dengue were included. Clinical diagnosis was based on the WHO guidelines; cases were classified as dengue without warning signs (DW) (D), DW and severe dengue (SD).[5],[8] Those with clinical evidence of urinary tract infection (UTI), pneumonia, abscess or any other apparent cause of fever were excluded. Written consent was obtained from the parents/guardians. The study was reviewed by the Institution Review Board (IRB) for its scientific content. (IRB No: IEC-31/Feb 2019). Clinical data was recorded in a case report form. Two patients were excluded, as sample volumes were inadequate. Results of NS1 testing, done in the Department of Microbiology (ELISA format) and in laboratories outside the institution, were available from the hospital records.

Total nucleic acids were extracted from serum samples using the Versant 1.0 Reagent kit (Siemens, Belgium). Automated extraction (VERSANT kPCR, Siemens) was done if load was more 10 patient samples.

The Fast Track Diagnostics (FTD) Dengue/Chik kit (FTD, Junglinster, Luxembourg) was used for dengue RNA screening. An internal control provided in the kit was added to the samples during extraction. Real-time PCR was performed on Quant Studio 5 Dx (Thermo Fisher Scientific®). Screening positives were serotyped with FTD dengue differentiation kit.[9],[10] The presence of dengue RNA was reported as cycle threshold (Ct). Ct cut-off for detection and serotyping was ≤35 as per the manufacturer's instructions.

In-house real-time PCR was standardised in multiplex (for DEN-1, DEN-2 and DEN-3) and uniplex (DEN-4) format employing Superscript™ III Platinum™ One Step quantitative RT (qRT)-PCR kit (Invitrogen, Cat No: 11732-020).[11] Primers and probes were procured from Macrogen Inc., (Seoul, Korea) [Table 1]. Amplification curves were assessed and thresholds set manually when needed. Ct cut-off of in-house assays was based on duplicates tested on the commercial FTD kit and NS1 results.
Table 1: In house real time polymerase chain reaction for dengue serotype-1, dengue serotype-2, dengue serotype-3 and dengue serotype-4

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Statistical analysis

All categorical variables were summarised as percentages. The association between categorical variables was studied by Pearson's Chi-square test. For data analysis, statistical software Stata 11.0 was used. Concordance between the tests was calculated using the formula:




 ~ Results Top


Between March 2019 and February 2020, 485 patients were recruited. Individuals were from Tamil Nadu (TN, n = 364), Andhra Pradesh (AP, n = 119), Telangana (n = 1) and Andaman (n = 1) and were clinically classified as (D, n = 93), (DW, n = 213) and (SD, n = 70). Patients with undifferentiated febrile illness (n = 109) were also included; 24 of these were diagnosed as non-dengue (enteric fever, bacterial UTI and viral acute respiratory tract infection). About three-fourth of recruited subjects presented at the hospital within day 5 (D5) of illness.

The median age of patients was 7.8 years and ranged from 3 months to 17 years and 9 months. Male:female ratio was 1.3:1. Real-time PCR was performed on 485 subjects, NS1 results were available for 362. Ct cut-off of in-house real-time PCR was ≤37.5. Overall, 325 (67%) patients were positive for Dengue RNA [Table 2]. Of 362 samples tested for Dengue NS1 antigen, 230 (63.5%) were reactive. Overall concordance between PCR and NS1 was 77.07%. Concordance between real-time PCR and NS1 decreased from 81.8% on D0–D2-76% on D3–D5. All serotypes were seen throughout the year, DEN-2 was predominant (n = 197, 61%) followed by DEN-3 (n = 65, 20%) [Table 2]. There was no significant association between infecting serotypes and age (P = 0.752). DEN-2 was the predominant serotype circulating in TN. Seventy-three per cent of cases from AP were confirmed as dengue. Serotypes diversity in AP was DEN-3 (46%), followed by DEN-2 (27.5%) and DEN-1 (25%). Seventy-five per cent of the cases (n = 366) occurred during the monsoon months. There was a significant association between the season and dengue cases (P < 0.001).
Table 2: Dengue burden and serotype distribution: 2019-2020

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Dengue without warning signs (D, n = 93), dengue with warning signs (DW, n = 213) and severe dengue (n = 70)

Average age of D, DW and SD patients was 7.8, 8.5 and 7.3 years, respectively. Real-time PCR diagnosed 60%, 82% and 91% of D, DW and SD cases, respectively. NS1 testing confirmed dengue in 74.5%, 77% and 78% of D, DW and SD cases, respectively.

Concordance between PCR and NS1 amongst SD cases was 74.5%. Thirty-one per cent of SD cases developed complications; NS1 was reactive for 88% while PCR diagnosed them all. DEN-2 was the predominant serotype in the study and the causative serotype in 38%, 48% and 53% of D, DW and SD cases, respectively. Complications included hepatomegaly, hepatosplenomegaly, myopathy, encephalopathy, respiratory distress and acute liver failure. In our study, 53% of dengue related complications could be attributed to DEN-2. Two males, aged 8 and 16 years died. DEN-2 was the causative serotype in both [Table 3] and [Table 4]. None of the non-dengue like cases was reactive by real-time PCR. A case of suspected malaria was positive by NS1 and PCR.
Table 3: Utility of polymerase chain reaction versus non-structural 1 in diagnosis of suspected dengue

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Table 4: Serotype distribution based on the clinical classification of cases

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D

Paediatric dengue is difficult to diagnose clinically in the early stages. In hyperendemic areas, children develop SD due to secondary infection. Multiplex real-time PCR can provide rapid and accurate results with additional serotype information to predict disease severity.

The mean age of recruited patients in the study was 7.8; those with confirmed dengue were 8.2 years. Infants were the least affected. Similar results have been documented in studies from Orissa, Puducherry and Chennai.[12],[13] Dengue outbreaks recur in South India every year with increasing severity and a shift in circulating serotypes. Enrolment of subjects with a high degree of clinical suspicion could have yielded a high positivity (67%) seen in the study. DEN-2 (61%) was the predominant serotype in 2019. During the year 2013–2015, DEN-2 and DEN-3 were the predominant serotypes seen in Chennai.[14] Overall, multiplex real-time PCR and NS1 detected 67% and 63.5% of the cases. The mean number of days of detection of dengue RNA postonset, was 6. Dengue RNA was detected in 58 samples after 8 days' postonset. Theoretically, dengue RNA is most often detected during the viremic phase (5–7 days after onset), however, there are published reports of dengue RNA being detected even after 14 days after onset.[3],[15] Multiplex real-time PCR detected 91% of SD cases compared to NS1 test (78%).

Multiplex real-time PCR has enabled early diagnosis of paediatric dengue with a single blood sample. Diagnosis and serotyping can be combined in a single sensitive assay as demonstrated in the methods section. We multiplexed DEN-1, DEN-2 and DEN-3 as they are the commoner serotypes in the region. DEN-4 is rare and was standardised as a uniplex real-time PCR which was performed for samples nonreactive by the multiplex real-time PCR. The in-house real time PCR assay had shorter turnaround time (TAT), performed marginally better and was cost-effective compared to the commercial multiplex real-time PCR kit The TAT of the in-house method was 40% less than when the commercial kits were employed for diagnosis and serotyping. The cost of diagnosis and serotyping by the commercial kit was around Indian Rupee (INR) 3000 while that of the in-house assay was in the range INR 1000–1200.

All four serotypes were detected throughout the study with a predominance of DEN-2. Data from dengue outbreaks worldwide have shown the association of DENV-2 and DENV-3 with increased hospitalisation and greater severity. DEN-1 is associated with less severe disease. DENV-1 and DENV-3 have been associated with primary infections while DENV-2 and DENV-4 with secondary infections 5. Our study provides preliminary data on the severity of DEN-2 infections in children. However, larger studies have to be done to show significant association of serotypes with severity in paediatric cases.

Serotypes circulating in TN and AP were very diverse in proportions. In TN, DEN-2 was predominant, while DEN-3, DEN-2 and DEN-1 caused infections in AP. In 2017, DENV-2 (41%) was the predominant serotype in AP followed by DENV-4 (37%). In TN, DEN-3 followed by DEN-1 was dominant in 2012 and DEN-2 followed by DEN-3 during the year 2013–2015.[16],[17],[18] This shift of serotypes along with hyperendemicity may result in severe forms of dengue specially in paediatric population. Sequential infections with multiple dengue serotypes increase disease severity so rapid and accurate diagnosis is important in improving patient outcomes and effective dengue control.

Acknowledgement

The authors would like to thank Drs. Julius Scott, T. Vasanthi, Lalitha Janakiraman, V.V. Vardarajan, Sivabalan S, Janani Sankar and Ravikumar K for contributing clinical cases. Mr Abdul Hameed and Mr Subramani are thanked for technical assistance and data entry.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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