| dc.description.abstracteng | Myanmar is a Southeast Asian country with a notoriously high number of cases of snake envenoming. Main concerns for notorious snakebite cases in Myanmar include presence of more than one venomous snake species. Moreover, these species produced similar clinical signs though they are significantly differing in morphological characteristics. In any case, it’s not easy to see which snake has caused the bite if the patients are in panic. People in Myanmar have been living with the threat of snakebites for more than a century. The hypothesis of my first study therefore is to determine whether people who are living in the snake prevalence area have enough knowledge of snakes as well as appropriate first aid measures against snakebites.
The survey area was the Central Dry Zone of Myanmar, renowned for its high amount of snakebite cases in the country. The survey was performed in three townships, each are from three divisions of the Central Dry Zone. A total of nine villages were covered during the period of November 2017 with a total of 434 participants. Villages were classified according to the number of snakebites within one year: HVVG (high number of victims villages), MVVG (moderate number of victims villages) and LVVG (least number of victims villages).
The questionnaires were semi-structural, including open and closed type questions as well as multiple choice questions. The Chi-square test was used for comparing the three village types for their knowledge, attitude and practice and logistic regression for predictors models as statistical analysis. KAP predictors values are based on the socio-economic status of the questionnaire household. Participants who had 70% in each categories considered as good. Among 434 participants, at least 41 % had good knowledge, 57 % had a good attitude and half (50 %) of the participants had good preventive measures against snakebite accidents. Positive correlation was discovered between knowledge and preventive measures. Generally, people with good knowledge showcased better preventive practices. Victims villages, gender, marital status, educational status and occupation were associated with knowledge of the participants.
Our survey uncovered that people need to improve their knowledge of appropriate first aid measures to perform appropriately in cases of snakebites. Gender, educational status and occupation can identify the knowledge of the participants, in which male, higher education and farmers are associated with good knowledge of snakes and preventive measures. Moreover, we can conclude that people who are living in areas with more snakebite victims tend to have better knowledge of first aid measures and snakes in general.
Snakebite envenoming causes devastating consequences such as death, disability and financial difficulties for people in developing countries. Despite existing in all parts of the world except Antarctica, New Zealand and a few smaller islands, snakes have troubled mainly tropical regions and are typically more venomous in those regions. More than that, it is not just one or two species that are venomous and dangerous for people. There are in fact many snake species which are venomous, and which cause serious health threats to populations of tropical regions. Besides, the morphological characteristics and clinical signs of snake bites produced by these species tend to be similar, which makes the diagnostic process challenging. So far, the most effective treatment to snake envenoming is the application of a monovalent antivenom.
Therefore, the second and third part of the study focuses on developing a rapid immunochromatographic assay (also known as lateral flow immunoassay) for the detection of Russell’s viper (Daboia spp.), cobra (Naja spp.) and krait (Bungarus spp.) for victims in the region of South and Southeast Asia.
The immunochromatographic assay is quick, user-friendly, portable and easy to perform by the general public. Several bacteria, viruses, toxins and even hormones can be detected within a few minutes by the immunochromatographic assay. The test has been applied in human and veterinary medicine, as well as by agricultural and environmental sciences. However, less than 5 researches has been performed regarding detection of snake venoms with the immunochromatographic assay.
The second part of the study aims at developing a duplex immunochromatographic assay (duplex immunoassay) for the detection of cobra and krait venoms. The duplex immunoassay uses polyclonal antibodies as capture antibodies and antivenoms as detection antibodies. Polyclonal antibodies are produced from rabbits and used against Naja naja and Bungarus candidus. They were incubated in the nitrocellulose membrane. Detection antibodies used in this study taken from the hyperimmune serum of horses from Queen Saovabha Memorial Institute, Bangkok, were conjugated with gold nanoparticles. Since detection antibodies used in this study are from horses, we used the control line of rabbit anti-horse antibodies in the study. The species of snakes used in the antivenom are Naja kaouthia and B. candidus for cobras and kraits respectively. The detection limit, tested with the in-house venom-spiked PBS-T (phosphate buffer saline-tween) solution has resulted in 1 ng/ml against cobra venom (N. naja, N. kaouthia), 10 ng/ml for B. candidus and 75 µg/ml for B. fasciatus. The antibody combination in the first study was dependent on the polyclonal antibodies and it was intended to capture the whole genus, rather than the specific species. Sensitivity of the krait assay was lower because both capture and detection antibodies used in the study were against B. candidus, whereas in case of the cobra assay, both species of N. naja and N. kaouthia were used as capture and detection antibodies.
The limits of detection were only based on the venom in PBS-T solution, not on the serum of the human. The test still needs further investigation with human serum spike with venoms. The specificity test was performed with different species of snakes including the Russell’s viper (Daboia siamensis), white-lipped pit viper (Trimeresurus albolabris), brown spotted pit viper (Protobothrops mucrosquamatus) and saw-scaled viper (Echis carinatus). No cross-reactivity with duplex immunoassay was observed indicating high specificity. Although the developed duplex immunoassay must evolve to the level of usage in field investigations in South and Southeast Asia, the test is sensitive and specific enough to detect the target venom within 30 min. It would be promising to be applied in developing countries such as Myanmar, where only monovalent antivenoms are available.
Equally, my third study also includes the development of a single plex immunoassay, and a sandwich enzyme linked immunosorbent assay (ELISA) for the detection of Russell’s viper venom. In the study, we were able to find the antibodies combination of the monoclonal antibody (mAb) and polyclonal antibody (pAb) from mice and rabbits respectively. The ideal immunochromatographic assay format is using the monoclonal and polyclonal antibody in the sandwich format. Unlike in previous studies, the antibodies combination of mAb as detection antibodies and pAb as capture antibodies has resulted in higher sensitivity and specificity. The limit of detection for single plex assay was 4 ng/ml, testing the PBST diluted venom. The single plex assay was then taken to test the spike blood with venoms. Due to viscosity, turbidity and colour of blood hinder the visuality of test lines. Hence, it could lower the sensitivity limits to the level of 60 ng/ml. Besides, the experimental time has been delayed due to high molecular weight of the blood cells. Therefore, we developed specific and sensitive sandwich ELISA for the detection of Russell’s viper venom for laboratory settings and for the evaluation of the developed single plex immunochromatographic assay against Russell’s viper. The antibodies used in sandwich ELISA are the pAb and hyper immune serum from the horse against Russell’s viper. Developed sandwich ELISA is highly sensitive and can detect the venom levels at 1 ng/ml. Specificity was performed with different snake species such as the white-lipped pit viper (T. albolabris) , the brown spotted pit viper (P. mucrosquamatus), the saw-scaled viper (E. carinatus), the monocled cobra (N. kaouthia) and the Malayan krait (B. candidus).
Both tests have shown high specificity. These tests were later repeated with serum samples from snakebite victim in Myanmar. Individually, both tests have expressed good sensitivity and specificity. Still, our single plex assay was not available to be used in the field detection yet. However, the sandwich ELISA can use in the laboratory settings in hospitals in tropical developing countries. | de |