Detection of Dengue Virus-Specific IgM and IgG Antibodies through Peptide Sequences of Envelope and NS1 Proteins for Serological Identification

Dengue is an acute febrile illness caused by positive-sense single-stranded RNA virus, belonging to the family Flaviviridae and genus Flavivirus. Transmission of virus among the individuals occurred by blood-feeding Aedes mosquitoes. This virus has four serotypes differentiated on the basis of antibody neutralization assay. At present, there is no particular treatment or vaccine candidate available for dengue infection. Approximately 3.9 billion human populations are at risk of dengue virus (DENV) infection. Thus, precise diagnosis of dengue at the early stage is very essential for disease control and effective therapy in order to treat or prevent severe complications. Indeed, the accurate diagnosis of DENV remains a problem because of low detection accuracy along with high testing price. Sensitivity and specificity of available kits vary from test to test, and cross-reactivity with other Flavivirus is a challenging issue for diagnosis. In this study, linear epitopes of envelope (E) and NS1 proteins were identified to diagnose the DENV. Whole protein sequences of E and NS1 of DENV were obtained from UniProtKB database. On the basis of algorithm prediction from DNASTAR, BCEPRED, and IEDB data resources, twelve peptides of E (EP1 to EP12) and eight peptides of NS1 (NS1-1 to NS1-8) were selected, which were common in all serotypes. Sequence homologies of peptides with other Flavivirus were checked by Multiple Sequence Alignment Tool ClustalX2. Peptide sequences were synthesized chemically by solid-phase peptide synthesis technique. Dengue-specific IgM and IgG (secondary response) antibodies in the patient’s antisera were tested with the peptides using ELISA protocol. Peptides EP1, EP2, EP4, EP7, EP10, and EP12 of E protein and NS1-1, NS1-3, NS1-4, NS1-7, and NS1-8 of NS1 protein were considered the best immunoreactive peptides with the sensitivity (73.33-96.66%) and specificity (82.14-100%). Such peptides together can be used to construct the multiple antigen peptides (MAP) or multiplexed microbeads for designing a precise, cost-effective, and easy-to-make peptide-based immunodiagnostic kit for DENV detection

Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria

Joint Involvement Can Predict Chikungunya in a Dengue Syndemic Setting in India

Abstract Dengue and chikungunya have been endemic in India but have the tendency to cause periodic epidemics, often together, wherein they are termed ‘syndemic’. Such a syndemic was observed in 2016 in India which resulted in a further scarcity of already resource-poor specific diagnostic infrastructure even in many urban conglomerates. A cross-sectional study was thus conducted, on 978 fever patients that consulted the ICMR-NIMR fever clinic, New Delhi, in September 2016, with an objective to identify symptom/s that could predict chikungunya with certainty. The overall aim was to rationally channelize the most clinically suitable patients for the required specific diagnosis of chikungunya. Based on their clinical profile, febrile patients attending NIMR’s clinic, appropriate laboratory tests and their association analyses were performed. Bivariate analysis on 34 clinical parameters revealed that joint pain, joint swelling, rashes, red spots, weakness, itching, loss of taste, red eyes, and bleeding gums were found to be statistically significantly associated predictors of chikungunya as compared to dengue. While, in multivariate analysis, only four symptoms (joint pain in elbows, joint swelling, itching and bleeding gums) were found in statistically significant association with chikungunya. Hence, based on the results, a clinician may preferably channelize febrile patients with one or more of these four symptoms for chikungunya-specific diagnosis and divert the rest for dengue lab diagnosis in a dengue–chikungunya syndemic setting

Evaluation of SYBR green I based visual loop-mediated isothermal amplification (LAMP) assay for genus and species-specific diagnosis of malaria in P. vivax and P. falciparum endemic regions

Background & objectives: Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid based diagnostic approach that is easily adaptable to the field settings with limited technical resources. This study was aimed to evaluate the LAMP assay for the detection and identification of Plasmodium falciparum and P. vivax infection in malaria suspected cases using genus and species-specific assay. Methods: The 18S rRNA-based LAMP assay was evaluated for diagnosis of genus Plasmodium, and species-specific diagnosis of P. falciparum and P. vivax, infection employing 317 malaria suspected cases, and the results were compared with those obtained by 18S nested PCR (n-PCR). All the samples were confirmed by microscopy for the presence of Plasmodium parasite. Results: The n-PCR was positive in all Plasmodium-infected cases (n=257; P. falciparum=133; P. vivax=124) and negative in microscopy negative cases (n=58) except for two cases which were positive for P. vivax, giving a sensitivity of 100% (95% CI: 97.04-100%) and a specificity of 100% (95% CI: 88.45-99.5%). Genus-specific LAMP assay missed 11 (3.2%) microscopy and n-PCR confirmed vivax malaria cases. Considering PCR results as a reference, LAMP was 100% sensitive and specific for P. falciparum, whereas it exhibited 95.16% sensitivity and 96.7% specificity for P. vivax. The n-PCR assay detected 10 mixed infection cases while species-specific LAMP detected five mixed infection cases of P. vivax and P. falciparum, which were not detected by microscopy. Interpretation & conclusion: Genus-specific LAMP assay displayed low sensitivity. Falciparum specific LAMP assay displayed high sensitivity whereas vivax specific LAMP assay displayed low sensitivity. Failed detection of vivax cases otherwise confirmed by the n-PCR assay indicates exploitation of new targets and improved detection methods to attain 100% results for P. vivax detection

An epidemiological study of dengue and its coinfections in Delhi

Objectives: To study the epidemiology of dengue with reference to serological, demographic profile, spatio-temporal distribution, vectors, circulating serotypes and coinfections. Methods: Demographic data and presenting symptoms of fever cases reporting to the clinic were recorded. Suspected patients were tested for dengue, chikungunya and malaria. Dengue specific RT-PCR was performed to detect circulating DENV serotypes. Vector surveys were carried out to detect Aedes breeding. Results: Of the 5536 fever patients tested during 2012 to 2015, 1536 (27.7%) had confirmed dengue. The peak in dengue positivity was observed during September and October. Of the 60 samples analysed, 10 (16.7%) had concurrent infection with multiple dengue serotypes; one of them had all the four serotypes. Coinfection of dengue with malaria and chikungunya was also observed. The occurrence of dengue and malaria was inversely related. Seven percent of the dengue patients required hospitalization. Vector surveys in the draining area revealed Aedes breeding with a high house index. Conclusion: Delhi being hyperendemic, the occurrence of concurrent infections with multiple DENV serotypes has become a frequent finding. The study emphasizes the need of epidemiological and entomological surveillance to monitor trends in dengue distribution, seasonal patterns and circulating serotypes to guide dengue control activities. Keywords: Dengue, Epidemiology, India, Serotype

Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results

MOESM1 of Comparison of the safety and efficacy of fixed-dose combination of arterolane maleate and piperaquine phosphate with chloroquine in acute, uncomplicated Plasmodium vivax malaria: a phase III, multicentric, open-label study

Additional file 1: Table S1. Adverse events