Use of dry blots for serotyping and genotyping of dengue viruses: A pilot study

Background & objectives: Dengue (DEN) is a result of infection by one or multiple types of four dengue viruses known as Dengue virus (DENV) 1-4. Identifying circulating serotype and genotype is epidemiologically important, however, it is challenging in resource limited areas. Moreover, transporting samples from the collation site to the laboratory in appropriate condition is an exigent task. To overcome this, we evaluated the usefulness of dry blots of serum for DENV diagnosis, serotyping and genotyping. Methods: Serum samples received for diagnosis were divided into parts; one was used for providing the diagnosis. Remaining sample was distributed in three parts (100 µl each), one part was used for molecular testing and two parts were mixed with RNAlater reagent® in equal volumes and was blotted on Whatman filter paper no 3. The blots were dried and stored at 4°C and 28°C and tested for presence of dengue RNA, serotypes and genotypes after 7 days of incubation. Results: The diagnosis and serotyping results of serum sample and dry serum blots were in concordance. Out of 20 positive samples, 13 (65%) gave satisfactory sequencing results. Genotype III of DENV-1, Genotype IV of DENV 2 and Genotype I of DENV-4 were detected. Interpretation & conclusion: The results demonstrate that serum mixed with RNA protective solution and blotted on Whatman filter paper no 3 can be effectively used for diagnosis, serotyping and genotyping of DENVs. This will help in easy transportation, diagnosis and effective data generation in resource limited settings.

Identification of hepatitis B virus genotypes detected in Lahaul & Spiti district of Himachal Pradesh, India

Background & objectives: Infection by hepatitis B virus (HBV) results in acute or chronic hepatitis. Based on sequence differences of eight per cent or more, HBV is divided into 10 genotypes (A to J) and 35 sub-genotypes. Molecular characterization of the circulating HBV genome has helped in understanding the epidemiology and its clinical importance. Spiti valley in Himachal Pradesh, which shares its border with Tibet, is one of the most HBV prevalent areas in India. Since information about the circulating genotype/s of HBV in this area is limited, this study was conducted to identify the circulating HBV genotypes. Methods: The surface and partial reverse transcriptase gene regions were sequenced using 14 hepatitis B surface antigen-positive samples. Results: Out of the 14 hepatitis B surface antigen-positive samples 11 sample gave quality sequence for further analysis. All the 11 samples belonged to subtype ayw2. The phylogenetic and recombination analysis revealed that five out of 11 samples were of genotype CD1 and the rest six were of genotype D3. Interpretation & conclusions: The CD1 recombinant sub-genotype might have immigrated during past or present transcontinental migration between the adjacent countries. Further studies using full-genome sequencing and high sample size will be helpful to understand this epidemiology and to combat the high prevalence of HBV in the area.

Co-circulation of dengue virus serotypes with chikungunya virus in Madhya Pradesh, central India.

Background: Dengue and chikungunya present with very similar signs and symptoms in the initial stage of illness and so it is difficult to distinguish them clinically. Both are transmitted by Aedes aegypti and Aedes albopictus mosquitoes. This study was conducted with the aim to explore the co-circulation of dengue and chikungunya viruses in central India. Materials and methods: Samples from suspected dengue cases were subjected to dengue immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) and dengue-negative samples were tested with chikungunya-specific IgM ELISA. The samples collected in acute phase of illness were tested by nested reverse transcription polymerase chain reaction (nRT-PCR). Chikungunya virus (CHIKV) sequences were analysed to determine their genotype. Results: Of 138 samples screened for dengue, 21 (15.2%) were positive, and of 119 samples screened for chikungunya, 13 (10.9%) were positive. Dengue viruses 1 and 4 were found co-circulating with chikungunya virus in Jabalpur, central India. The chikungunya virus detected belonged to the East Central South African genotype. Conclusion: Accurate and timely diagnosis would help in patient management and use of resources. It is advocated to simultaneously test samples for these two diseases in endemic areas. This will also aid in understanding the epidemiology of chikungunya.

Vector-borne diseases in central India, with reference to malaria, filaria, dengue and chikungunya.

Background: Vector-borne diseases (VBDs) caused by parasites and viruses are a major cause of morbidity and mortality in Madhya Pradesh (MP), central India. These diseases are malaria, lymphatic filariasis, dengue and chikungunya. Epidemiological information is lacking on different VBDs that are commonly prevalent in rural-tribal areas of MP, except on malaria. Methods: The studies were carried out at the request of Government of Madhya Pradesh, in three locations where many VBDs are endemic. Data on malaria/filaria prevalence were collected by repeatedly undertaking cross-sectional parasitological surveys in the same areas for 3 years. For dengue and chikungunya, suspected cases were referred to the research centre. Results: Monitoring of results revealed that all the diseases are commonly prevalent in the region, and show year-to-year variation. Malaria slide positivity (the number of malaria parasitaemic cases, divided by the total number of blood smears made) was 18.7% (190/1018), 16.4% (372/2266) and 20.4% (104/509) respectively in the years 2011, 2012 and 2013. There was a strong age pattern in both Plasmodium vivax and P. falciparum. The slide vivax rate was highest among infants, at 5% (odds ratio [OR] = 3.8; 95% confidence interval [CI] = 1.5 to 9.4; P<0.05) and the highest slide falciparum rate was 20% in children aged 1–4 years (OR = 2.0; 95% CI 1.5 to 2.7; P<0.0001). This age-related pattern was not seen in other VBDs. The microfilaria rate was 7.5%, 7.6% and 7.8% in the years 2010, 2012 and 2013, respectively. Overall, microfilaria rates were higher in males (8.7%) as compared to females 6.4% (OR = 1.5; 95% CI = 1.1 to 2.0; P < 0.01). The prevalence of dengue was 48% (dengue viruses 1 and 4 – DENV-1 and DENV-4), 59% (DENV-1) and 34% (DENV-3) respectively, in the years 2011, 2012 and 2013 among referred samples, while for chikungunya very few samples were found to be positive. Conclusion: Despite recent advances in potential vaccines and new therapeutic schemes, the control of VBDs remains difficult. Therefore, interruption of transmission still relies on vector-control measures. A coordinated, consistent, integrated vector-management approach is needed to control malaria, filaria, dengue and chikungunya.

New method of electrode placement for determination of cardiac output using impedance cardiography.

In thirty eight young healthy adult subjects, cardiac output was determined non-invasively by using two methods of electrode placement viz. vertical (uses silver braided wires in a band shape) and horizontal (conventional stick-on type surface ECG electrodes), using the technique of Impedance Cardiography. The recordings were taken in supine position on the same day in two separate sets with 30.0 min interval between two sets. In each set of recording, five successive recordings, each at an interval of five minutes were taken. The mean values of cardiac out put by two methods were compared. For the set I, the respective values (Mean +/- SD) of cardiac output by horizontal and vertical methods for the each of the five recordings were 4.87 +/- 0.77 and 5.03 +/- 0.64 for the first, 4.87 +/- 0.71 and 4.91 +/- 0.66 for the second, 4.99 +/- 0.67 and 5.00 +/- 0.70 for the third, 4.78 +/- 0.69 and 4.98 +/- 0.61 for the fourth, 4.84 +/- 0.69 and 4.98 +/- 0.62 for the fifth recording in supine position. The respective P values for these pairs for between the group comparisons were 0.33, 0.50, 0.96, 0.17, and 0.36. In addition, to see the repeatability for each method, within the group comparison was done, the P values were 0.71 and 0.91 for the horizontal and vertical methods, respectively. The mean value of cardiac output did not differ significantly between two methods for recordings of set II in supine position. The cardiac output measurement by placing four spot electrodes horizontally, gave consistent result on repeated measurements and their values showed concordance with the cardiac output values obtained by conventional four band electrodes tied around the chest.

Comparison between pre-prandial and post-prandial heart rate variability (HRV).

After food ingestion, peptides are released in GIT, which cause local vasodilatation. Therefore, after meals, redistribution of blood occurs because of shifting of large amounts of blood into GIT. In normal individuals, this is well compensated and does not lead to post-prandial hypotension. The mechanism of post-prandial hypotension is well known. We hypothesized that there may be a decrease in parasympathetic activity (tone) after meals to compensate for the change in blood distribution. We carried out the study to find out the changes in the autonomic tone before and after meals (lunch) in normal individuals, using Heart Rate Variability (HRV). From the series of RR intervals marked, the time domain and frequency domain measures of HRV were obtained using Nevrokard software (version 6.4). Continuous ECG was recorded in 15 healthy adult subjects (mean age 29.06 +/- 6.2; 13 males and 2 females). The ECG was recorded in pre-prandial and post-prandial state for a period of five minutes each as follows: (1) just before the subjects had lunch, (2) 15 minutes after lunch, (3) 1 hour after lunch, and (4) 2 hours after lunch. Time domain and frequency domain measures of HRV were compared between pre-prandial state and rest of post-prandial states. The autonomic tone parameters did not show a significant change between the pre-prandial state and the immediate post-prandial state. [Range, i.e., the difference between the maximum and minimum RR intervals (406 +/- 161.14 vs. 416.66 +/- 125), standard-deviation of normal to normal RR interval (56.33 +/- 22.72 vs. 67.63 +/- 26.50), RMSSD (55.02 +/- 35.85 vs. 63.87 +/- 32.60), NN50 (42.13 +/- 29.43 vs. 51.86 +/- 29.83), PNN50 (12.67 +/- 10.29 vs. 15.27 +/- 9.71), HF (49.53 +/- 15.10 vs. 47.07 +/- 16.88), LF (41.41 +/- 13.18 vs. 46.49 +/- 15.99), LF/HF (0.98 +/- 0.53 vs. 1.26 +/- 0.90), total power (148.27 +/- 37.78 vs. 137.61 +/- 37.10)]. No significant change was seen in the above parameters between the pre-prandial state and the later phases of post-prandial state. Since there is no significant decrease in the time domain measures and the HF value between the pre-prandial and the post-prandial states, we conclude that the parasympathetic tone is not altered. The parameters denoting sympathetic tone, ie, LF and LF/HF, also do not show a significant change. This indicates that the cardiovascular autonomic tone is not affected by ingestion of meals in normal individuals. Thus we refute our hypothesis. In conclusion, the HRV parameters do not alter significantly after meals in normal individuals.