Masters Theses

Development of RT-PCR for rapid detection of dengue virus type 1-4 from clinical specimens and a preliminary phylogenetic study of dengue virus isolates from Sri Lanka

Tue, 18 Oct 2016 06:26:23 GMT

Development of RT-PCR for rapid detection of dengue virus type 1-4 from clinical specimens and a preliminary phylogenetic study of dengue virus isolates from Sri Lanka Velathanthiri, V.G.N.S. Attached; Dengue virus is the causative agent of dengue fever (DF) and its complications; dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). It has four serotypes (Dengue 1-4) and is a member of the family flaviridae. It is one of the important causes of inorbidity and mortality through out the subtropical and tropical regions, including Sri Lanka. 1. Patients with dengue need to be closely monitored for evidence of haemorrhage and shock. In order to do this, it is necessary to differentiate patients with dengue from non dengue patients. This however, is a challenge since dengue often presents with non specific symptoms such as fever, headache and body aches. Therefore, it requires an aetiological diagnosis based on laboratory confirmation of disease. In this regard, an ideal diagnostic tool should be sensitive, specific, reliable, rapid, cheap, technically less demanding and it should also be able to detect dengue in the early stages of the disease. Detection of the circulating serotype IS very important In epidemiological surveillance of the disease. Laboratory diagnosis of dengue infection is normally based on detection of dengue virus specific antibodies and isolation of dengue virus from patient's serum. Serological diagnosis has been proven to be of less value in the early stages of illness. Current technologies for isolation and identification of the dengue virus are based on complicated systems such as suckling mouse brain or mosquito inoculation or cell culture technique. These methods are technically demanding, expensive and time consuming. The availability of molecular diagnostics such as the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique has made it possible to perform rapid detection of the viral RNA. In this study, I present an alternative design of the RT-PCR technique that can detect and simultaneously identify the sero type ofthe dengue virus. I designed sets of primers and developed an RT-PCR for rapid detection and simultaneous identification of dengue virus and its serotypes in cell culture supernatant and in serum samples. It was evaluated for sensitivity, specificity and compared with other standard laboratory diagnostic methods. The test is based on 5 sets of primer pairs specific -for dengue viruses within the nonstructural (NS) 5 region of the dengue virus genome. A universal primer set that would bind to target sequence shared by all the four serotypes of the virus within the NS5 region, are used. The resulting PCR products are detected by gel electrophoresis and staining with ethidium bromide. The RT-PCR was developed with serotype specific primers, which were also designed within the NS5 region of each serotype for the identification of dengue serotypes. The amplified products of different sizes were obtained with different dengue serotypes and were detected by gel electrophoresis. The RT-PCR technique is simple and rapid, capable of not only detecting the dengue virus but also identifying its serotype in clinical specimens. The RT-PCR protocol developed by me was shown to more sensitive than virus isolation in cell culture and equally sensitive in detecting dengue virus and its serotypes in serum specimens. It was also shown not to cross react with other flaviviruses. In the preliminary phylogenetic study, I compared the nucleotide sequence homology of the Sri Lankan dengue virus isolates of the present study with dengue viruses isolated form other parts of the world. Nucleotide sequence analysis was performed by an automated nucleic acid sequencer on 14 dengue virus isolates (13 dengue type 2 and one dengue type 3). The dengue 2 viruses were most closely related to dengue 2 virus isolated in Sri Lanka and Seychelle in 1990 and 1976 respectively. The dengue 3 virus was most closely related to dengue 3 viruses recovered in Sri Lanka in 1991, 1985 and 1981. Our results suggested that these dengue virus serotypes are evolving locally and any introduced strains are failing to become established.

THE STUDY OF CONVENTIONAL OSTEOLOGY & DNA ON HUMAN SKELETAL REMAINS AT POTHANA & COBRA HOOD CAVE IN SIGIRIYA

Thu, 02 Jan 2014 07:49:59 GMT

THE STUDY OF CONVENTIONAL OSTEOLOGY & DNA ON HUMAN SKELETAL REMAINS AT POTHANA & COBRA HOOD CAVE IN SIGIRIYA

LOCALIZATION OF CHOLINERGIC NERVES AND THEIR COGNATE RECEPTORS IN TISSUES OF THE IMMUNE SYSTEM- AN ANIMAL STUDY

Thu, 02 Jan 2014 07:49:22 GMT

LOCALIZATION OF CHOLINERGIC NERVES AND THEIR COGNATE RECEPTORS IN TISSUES OF THE IMMUNE SYSTEM- AN ANIMAL STUDY

Epidemiology, Geographical Distribution and Investigation of possible Hydrological Relationship of Chronic Renal Failure Patients in North Central Province of Sri Lanka

Thu, 02 Jan 2014 07:27:31 GMT

Epidemiology, Geographical Distribution and Investigation of possible Hydrological Relationship of Chronic Renal Failure Patients in North Central Province of Sri Lanka Herath, H.M.J.R.; Dharmagunawardne, P.V.D.S.; Perera, M.A.L.R.; Deheragoda, C.K.M. Despite, Sri Lanka has attained impressive health indices such as low infant mortality, maternal mortality, and high life expectancy. rapidly changing life styles and deterioration of the environment have accelerated the prevalence of Non Communicable Diseases (NCD), which includes Chronic Kidney Disease (CKD). CKD is defined as the presence of sustained abnormalities of renal function and results from different causes of renal injury. End-stage renal disease (ESKD) can be defined by the necessity for life-saving dialysis or kidney transplantation. As recommended by the scientific committee of the National Research Programme of Ministry of Health - Sri Lanka, aetiology of CKD will be considered as "Uncertain or Unknown" if all the criteria stated below are satisfied. i.e. No past History or current treatment for Diabetes Mellitus or Chronic and/or Severe Hypertension, Snake Bite, Urological Disease of unknown aetiology or Glomerulonephritis, Normal HbA1C (>6.5%) and BP < 1601100 mm Hg untreated or < 140/90 mmHg on up to two anti hypertensive agents. CKD among people of North Central Province continues to be an emerging health problem. Chronic Kidney Disease of Unknown Aetiology (CKDU) has reached crisis proportions in the main agricultural region under reservoir based irrigation, in the North Central Province (NCP) of Sri Lanka. Objective of the study is to study the geographical distribution and characteristics and any links to hydrological factors in the aetiology of Chronic Renal Failure patients in NCP and compare these factors with other provinces of Sri Lanka. The study was a Descriptive Cross Sectional one and utilized a structured pre-tested interviewer administered questionnaire for data collection. Sample was selected from diagnosed ESRD patients who were taking treatment in 8 Hospitals in Sri Lanka. Data was collected during the month of September 2009 and all patients who came for Renal Replacement Therapy (RRT) i.e. for Peritonal Dialysis, Haemodialysis and Renal Transplant were included for the study. Most of the CKD patients in NCP are Males (81.7% of the total), Farmers (41.9% of the total), people with poor income level and People of lower socio economic classes, while nationally higher income groups, higher occupational groups and socio economic classes were affected by CKD. Even nationally mostly affected were Males. There were 93 ESRD patients with in the North Central Province with 70 in Anuradhapura and 23 in Polonnaruwa District, (75.3% and 24.7% respectively). Mean Age of these patients were 47.61 +/- SD 11.44 years. Anuradhapura District has 86.53 ESRD patients per million population and for Polonnaruwa District it was 57.5. The prevalence of ESRD patients was 76.92 per million population for NCP. These figures were much higher when considering the national figure of 24.43 per million population. 44 patients in NCP (47.3% of the total) were classified as having CKDU. 75% of them were residing in Anuradhapura. For Sri Lanka, this figure was 25.51%. For NCP prevalence of CKDU was 36.39· patients per million population with 43.26 and 22.5 patients per million population for Anuradhapura and Polonnaruwa districts respectively. This prevalence value was almost six times higher than the National figure of 6.23 patients per million population. Within NCP, Galnewa, Thirappane, Anuradhapura New Town, Mihinthale, Padawiya, Horowpothana, Palugaswewa, Galenbidunuwewa, Kahatagasdigiliya, Hingurakgoda and Thamankaduwa District Secretariat (DS) Divisions had high prevalence of (more than average of NCP) ESRD with unknown aetiology. Except for first four areas, all the DS Divisions were located towards more North East of the province and rest were situated towards South West of the province. Most of the areas of these DS divisions, had higher levels of Fluoride in the soil in excess of 3.0 ppm and lie outside the usual areas of higher prevalence. This indicates that, there is a relationship with higher fluoride levels in soil and higher prevalence of ESRD patients. There was no increase of ESRD with Unknown Aetiology among recently migrant populations (within 25 years) and there was no association with the source of Drinking Water.