Dengue (DEN) is a neglected tropical viral disease. This arthropod-borne viral infection is estimated to cause 390 million infections annually and about 2.5 billion people in more than 120 countries are at risk of infection around the world. Every year about 500,000 DEN patients require hospitalization for dengue haemorrhagic fever (DHF). India is considered as endemic for the disease and DEN is expanding its peripheries in rural and tribal areas in the country. Dengue Virus (DENV) belongs to the genus flavivirus in the Flaviviridae family. The virus comprises lipopolysaccharide envelope and single stranded positive sense RNA. The DENV complex is primarily comprised of four different serotypes DENV1, DENV2, DENV3 and DENV4 which are antigenically distinct.
An accurate and timely diagnosis is very important to tackle the critical situation posed by DEN. DEN initially shows symptoms similar to other diseases such as Chikungunya, malaria and influenza[4,5]. Distinguishing DEN from these disease conditions can help healthcare providers for correct treatment and public health officials to adopt preventive strategies. There are many well established serological and molecular tools available for diagnosis and serotyping of DENV; serological testing such as Non-structural Protein 1 (NS 1) ELISA, DENV specific IgM antibody captures ELISA are used. The nucleic acid amplification tests such as nested RT-PCR or a one-step multiplex RT-PCR assay, Real time RT-PCR are the most reliable and standard tests for DENV diagnosis in the early phase of infection. These molecular tests require intact RNA and for this the samples need to be transported within time maintaining cold chain which require cooling boxes with ice packs, which is a challenging task. Further, transporting wet samples has chances of spillage and leakage leading to Biosafety hazards and wastage of samples. Moreover, it is not cost effective. To overcome these hurdles, this study was planned with the objective to develop and evaluate a method to use the serum sample mixed with RNA protecting solution blotted and dried on Whatman filter paper for detecting, serotyping and genotyping of DENVs.
MATERIAL & METHODS
The State Level Virus Research and Diagnostic Laboratory (SL-VRDL) at ICMR-NIRTH, Jabalpur, India is a designated Apex Referral Laboratory (ARL) by National Vector Borne Disease Control Programme (NVBDCP). Blood and serum samples collected from suspected patients from districts of central India are referred to this laboratory for DEN and Chikungunya diagnosis maintaining a cold chain. The samples collected in the acute phase of illness <5 days of onset are subjected to NS 1 ELISA (Abbott Panbio Dengue Early ELISA, Cat. No. 01PE40) and samples collected 6 days onwards are tested by DENV IgM capture ELISA (ICMR-NIV, Pune). The remaining part of serum is stored -70 °C for future use. Forty samples collected in the acute phase of illness (20 positives and 20 negatives) were selected for this study [Figure 1].
The stored serum sample (100 µl) was mixed with RNA stabilization solution (RNAlater™) (Cat no: AM7021, Thermo Fisher Scientific, USA)" at 1:1 ratio in a micro centrifuge tube. Then, the solution was blotted in previously prepared 10 mm × 10 mm Whatman filter paper Grade 3 (Cat. No. WHA1003185, Meark India) made of cellulose. The blots were kept at room temperature till they were completely dried. The dried filter paper was warped in a zip lock polethene pouch, these pouches were kept at different temperatures. Two sets were prepared, first (Set-I) kept at 4°C and second (Set-II) at 28°C for 7 days. Nuclease free water mixed with RNAlater was included as a negative control in each set.
Viral RNA extraction
After seven days of incubation, the filter paper was taken out, allowed to come to room temperature and then cut into small pieces. The RNA was extracted using QIAmp® Viral RNA mini kit (QIAGEN, Cat No. 160053700) with slight modification. The Whatman filter paper pieces were transferred to a 1.5 ml microcentrifuge tube containing 600 µl AVL Lysis buffer with carrier RNA. These tubes were incubated for two hours at room temperature. The tubes were centrifuged at 3000 RPM for 10 min and the supernatant (about 560 µl) was further used for RNA extraction following the manufacturer’s protocol. Simultaneously, RNA was extracted from serum of all forty samples (100 µl /each) following the manufacturer’s protocol.
CDC Trioplex RT-PCR Assay
The RNA extracted from serum samples was subjected to CDC Trioplex RT-PCR (Real-time PCR) assay for detection of DENV, Chikungunya and Zika virus present in serum following the manufactures guidelines.
Multiplex RT-PCR assay
The positive samples (n=20) were further stereotyped using CDCs DENV1-4 real time RT-PCR multiplex. The SuperScript III Platinum One-Step qPCR kit (Invitrogen, Cat. No. 12574-035) and 10µl of extract RNA was used.
Further, in order to identify the circulating genotype/s the RNA, dry blot samples were subjected to RT-PCR with high fidelity platinum Taq polymerase (Invetrogen, Cat. No.11708-013) and primer sets for C-preM region of DENV. The resulting PCR products of 511 bases were sequenced as described earlier and submitted to National Centre for Biotechnology Information (NCBI) GenBank. The genotypes were identified based on sequence similarity in Basic Local Alignment Search Tool (BLAST) and constructing phylogenetic tree using MEGA 5.
Sensitivity and specificity were calculated by the detection method using the 4 × 4 table and Microsoft Excel was used for calculations.
The study participants gave informed written consent. The study was done under the project “Establishment of SL-VRDL” at ICMR-NIRTH, Jabalpur (approved by institutional ethical committee (NIRTH/IEC/2229/2018 dated Nov 1/11/2018)).
Out of 40 selected samples, 24 were from male and 16 were from female with age ranging from 01 to 55 years. Twenty (M/F: 10/10) were positive for DENV by NS1 ELISA. The results of Trioplex RT PCR on 20 samples showed that those were positive for DENV and negative for Chikungunya and Zika virus infections. The Ct value for DENV was ranging from 18 to 34.4. The RNA extracted from dried blot from Set-1 and Set-II showed similar results. In Set-I Ct values ranging from 18.7 to 30.9, whereas in Set II the rage was 19.36 to 34. The 20 subjects found negative for DENV by NS 1 ELISA were (M/F: 14/6) were negative to all three infections by Trioplex RT PCR [Figure 1].
When results of Whatman filter paper kept at 4°C for seven days were compared with the serum extracted RNA and subjected to RT-PCR, the sensitivity and specificity was 100% at 95% confidence level with Ct value ranging from 14.1 to 35.0. Similarly, the samples kept at 28°C for seven days were compared with serum extracted RNA RT PCR result, 100% sensitivity and specificity were found 95% with, confidence level with Ct value 22–36.
The serotyping results on serum extracted 20 positive samples and dried blot paper extracted 40 (Set I =20 and Set II =20) showed that 5 samples were DENV-1, 9 were DENV-2, 1 was, DENV-3 and 3 were DENV-4. Mixed infection of DENV-1 and DENV-2 was found in 2 samples. The Ct value of positive samples ranged from 15–36.5. The results of serum samples corroborated with Set-I and Set-II serotyping results.
When we subjected the 20 positive samples amplified by conventional RT-PCR for sequencing 13 gave satisfactory results. The 13 sequences were submitted to NCBI and GenBank accession number(s): OL597875 to OL597887. The 13 sequences were further analyzed using NCBI blast for genotyping and it was found that three (OL597876, OL597879, OL597887) of DENV 1 were belonging to Genotype III, whereas eight DENV 2 sequences showed that those belonged to Genotype-IV (OL597875, OL597878, OL597880 to OL597885) and two DENV-4 sequences showed a close relationship with Genotype-1 (OL597877, OL597886) [Figure 1]. Two samples showing mixed infections were not subjected to sequencing. The DENV-3 sequences, although confirmed, the sequence quality was not up to mark and hence were not considered for genotype analysis.
This pilot study demonstrates that DENV can not only be diagnosed from dry serum blot extracted RNA but also can be subjected to serotyping and genotyping using real rime RT-PCR and conventional RT-PCR-sequencing techniques. Accurate and efficient detection of DEN is important. The initial clinical picture of DEN overlaps with several other diseases like Chikungunya, Zika, influenza, COVID-19, and malaria.. In the recent past DEN and other vector borne diseases are frequently reported from rural and tribal areas. Different serotypes and genotypes of serotypes are known to differ in their virulence and transmission capacities, thus molecular diagnosis identifying serotype and preferably genotype helps in clinical care management and is important epidemiologically and for outbreak mitigation. However, in limited resource settings it becomes a challenge. Further, transporting samples from remote areas in cold chains taking all Biosafety precautions is a difficult task.
There are many studies that suggest that filter paper-based sample transportation can be used to diagnose several viral diseases such as HIV, hepatitis B and C infection and DEN. The World Health Organization (WHO) has already adopted filter paper-based blood collection and transport in low-income settings to improve diagnostics and care of HIV, hepatitis B and C infection in remote populations. Dried serum blots are also used for seroprevalence and diagnosis of HIV in newborns as perinatal HIV infection. Prado et al. has also demonstrated that DENV 2 live virus can be stored in filter paper for up to 72 h at 4 or -70°C and after that can be stored at room temperature for 48 h. Studies have shown that using dry blood spot DENV-specific antibodies, viral RNA, and NS1 antigen can be detected. Studies in the past have demonstrated that blood spotted onto filter paper extracted RNA have 78% to 100% sensitivity and up 93% specific for detection of DENV using RT-PCR[18,19,20].
Using viral RNA stabilizer (RNAlater®) to protect viral RNA gave the advantage over earlier reported methods and we could not only store the samples for longer period (up to seven days) at room temperature (28°C) but could also detect viral RNA and serotype and genotype DENVs with higher specificity and sensitivity though with small sample size. Our study also documents for the first time that the dry blots can be used for genotyping of DENVs. We could genotype only 13 of the 20 samples those were positive, probably because of low amount of RNA in rest of 7 samples.
All three serotypes i.e., DENV 1, DENV 2 and DENV 4, detected in the study belonged to same genotype reported earlier from central India. Though there are many advantages owing to simple, cost-effective sample storage and long-distance transportation in ambient conditions; our study has few limitations like testing limited number of samples, not including DENV negative serum sample in dot blot experiment, failure to sequence and genotype DENV 3, not attempting virus isolation from dry blots and not actually testing it in field conditions; so we suggest to test this method with larger sample size in the field conditions. On the other hand, strength of the study is that it demonstrates that the serum mixed with RNA protecting solution blotted on Whatman filter paper and dried, can be stored for seven days and used for diagnosis with great accuracy, serotyping and genotyping of DENVs and will be very beneficial for molecular surveillance.
Conflict of interest:
The authors sincerely thank the Secretary to Government of India, Department of Health Research and Director General, Indian Council of Medical Research for financial support under the State VRDL Project (Ref. No.DHR/VDL/04/2018). We also thank NVBDCP for funding for ARL. The Trioplex RT PCR and DEN 1-4 RT PCR kits were provided by CDC, USA free of cost. The authors thank Dr. Aparup Das, Director, National Institute of Research in Tribal Health, Jabalpur for his kind support and encouragement during the study. We also thank the technical support by student (Ms. Miss. Anjana Marko) and staff at the Division of Virology and Zoonosis at IC-MR-NIRTH, Jabalpur. Help in sequencing by Mrs. Nazia Anwar Ali at the institute is also acknowledged. This MS was approved by institutional publication committee (Ref No: ICMR-NIRTH/PSC/06/2022).
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