Nghiên cứu đặc điểm nhiễm orientia tsutsugamushi, ấu trùng mò tại khu vực tây bắc và chế tao bộ sinh phẩm phát hiện tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF MALARIOLOGY - PARASITOLOGYENTOMOLOGY -*** - TRAN QUANG PHUC STUDY OF CHARACTERISTICS OF Orientia tsutsugamushi INFECTION, TROMBICULID MITE LARVA IN THE NORTHWEST REGION, AND DEVELOPMENT OF A DIAGNOSTIC KIT Major: Parasitology Code: 62 72 01 16 SUMMARY OF DOCTORAL DISSERTATION OF PHILOSOPHY IN MEDICINE Ha Noi - 2019 The dissertation is completed at the NATIONAL INSTITUTE OF MALARIOLOGY-PARASITOLOGYENTOMOLOGY Scientific supervisors: Assoc Prof Dr Nguyen Van Ba Assoc Prof Dr Le Thanh Dong Reviewer 1: Reviewer 2: The dissertation is defended against University-level Examination Board at National Institute of Malariology-Parasitology- Entomology at……, date ……month…….year……… This dissertation can be found at: - The National Library of Vietnam - The Library of National Institute of Malariology - Parasitology -Entomology INTRODUCTION Scrub typhus (also known as Tsutsugamushi disease) is an acute infectious disease caused by Orientia tsutsugamushi The disease has natural source of infection, transmitted to humans by the bite of the larva of trombiculid mite The disease mainly occurs in rural, mountainous areas The symptoms of scrub typhus may be mistaken with many other diseases of which the treatment regimens are different (such as dengue hemorrhagic fever, leptospirosis) The mortality rate is high if not diagnosed promptly and used specific antibiotics Therefore, the early detection of the disease plays a decisive role in the treatment of scrub typhus [5], [42], [133] Currently, there are few technical tools for diagnosis of scrub typhus in Vietnam, especially in the remote areas where medical resources are limited With the strong development of molecular biology, many modern tools have been applied to determine and diagnose the pathogens In particular, the isothermal nucleic acid amplification method Recombinase Polymerase Amplification (RPA) proves many outstanding advantages compared to the traditional molecular biological techniques, especially allowing low-temperature amplification (37 - 42°C), the reaction duration for determination of the samples is very short (5 - 20 minutes) The RPA method allows the amplification of nucleic acid without using an expensive, bulky Thermal Cycler and unstable in field conditions Meanwhile, the sensitivity of the RPA method is evaluated to be equivalent or better than the PCR method The equipment is easy to use and transport to the place where specimens are taken [64], [97], [140] The Northwestern region is an area with difficult socio-economic conditions where many ethnic minorities are living Its natural conditions are favorable for the development of many natural nidus, including scrub typhus In recent years, there have been a number of studies on the vectors of the disease in the Northeast provinces [8], [12], [13] but no research has been conducted in the Northwest region Therefore, it is necessary to study the characteristics of natural nidus and the proportion of people who have antibodies against Orientia tsutsugamushi to enhance the effectiveness of preventing and controlling scrub typhus in the Northwest region In consideration of the above issues, "Study of characteristics of Orientia tsutsugamushi infection, trombiculid mite larva in the Northwest region and development of a diagnostic kit" is carried out for the objectives: 1) To describe some characteristics of Orientia tsutsugamushi infection, trombiculid mite larva and the distribution of scrub typhus in the Northwest region in 2016, 2017 2) To develop an Orientia tsutsugamushi diagnostic kit by using isothermal amplification technique Recombinase Polymerase Amplification in laboratory NOVELTY, SCIENTIFICITY AND PRACTICALITY OF STUDY Novelty This is the first time the isothermal amplification technology Recombinase polymerase amplification has been used to produce a diagnosis kit of Scrub typhus in Vietnam This is a test method of which the sensitivity and specificity is high, with quick result (after only 20 minutes), it is not required to extract samples or use thermocycler or complex equipment, so the deployment in field is very convenient and suitable The study has assessed the curent situation of people exposed to O tsutsugamushi, scrub typhus and O tsutsugamushi infection in vectors in provinces of Northwest region: Hoa Binh, Son La, Dien Bien and Lai Chau Scientificity The study uses standard scientific methods such as: - Descriptive epidemiological study with analysis by cross-sectional surveys to study characteristics of Orientia tsutsugamushi infection, host and infectious vectors; Retrospective study analyzing the distribution characteristics of Scrub typhus from records of patients who had scrub typhus and treated in provincial hospitals - Laboratory study: Develop procedures of and produce O tsutsugamushi diagnostic kit by using isothermal amplification technique Recombinase Polymerase Amplification in laboratory Research, evaluate, and analyze the sensitivity, specificity, stability of the product These are modern and standard research methods that are applied in Vietnam and worldwide, so its reliability is high Besides, the research uses modern techniques such as gene transformation technique through plasmids, polemerase chain reactiongene technique, these are modern techniques with high reliability Practicality The study results provide a fast, accurate tool for diagnosis of scrub typhus and suitable for local health center, contributing to reduce the burden of the disease in the community On the other hand, the study is also a reference material for scientific research and teaching at universities, and is a base for further research THESIS STRUCTURE The thesis includes 121 pages: Introduction (2 pages), chapter Literature Review (35 pages), chapter Study Subjects and Methods (23 pages), chapter Study results (34 page), chapter Discussion (24 pages), Conclusion (2 pages), Recommendations (1 page), 33 tables, 17 figures, 150 references Chapter LITERATURE REVIEW 1.1 Characteristics of Scrub typhus The disease caused by larva of trombiculid mites (Scrub typhus /Tsutsugamushi disease), is an acute infectious disease belonging to Group C in the Law on Prevention and Control of Infectious Diseases Its causative agent is O tsutsugamushi [2], [4], [141; the vector is the larvae of trombiculid mite Leptotrombidium The disease’s clinical features are fever lasting for 2-3 weeks, accompanied by skin ulcers, lymph nodes on body and rash If left untreated, there will be serious complications, causing damage to many organs and leading to death If not promptly diagnosed and treated by specific antibiotic, the mortality rate may range from 7-10% [141], even up to 70% depending on different epidemiological regions [40], [136] 1.1.1 Symptoms of scrub typhus Clinical case: Incubation period: 8-12 days on average (6 to 21 days); At first, the area bitten by the larva of trombiculid mite has a nodule of about 5mm, painless [3], the patient often does not pay attention; After the incubation period, there will be following symptoms: Fever ≥ 38 - 40°C, persistent, lasting for 15 - 20 days, for some cases the fever may last 27 days if left untreated; sometimes chills in the first 1-2 days, with severe headache, muscle aches [4], [19] Specific ulcers (eschar, the typical symptom of Scrub typhus): The rate of ulcers is common in some disease outbreaks in Vietnam, at about 80%, the others may be spots which are small or difficult to find (in ear canal, pussy ) Ulcers occur mainly in soft and moist areas such as the genitals area, anal area, groin, armpits, neck, and other areas such as thighs, abdomen, chest, and back Sometimes the ulcer located in the ear ring, navel, eyelid (easily mistaken for the stye) The ulcers are usually painless, not itchy; the patient usually has only one round or oval nodule with the diameter of mm to 20 mm; the initial nodule may develop gradually into cloudy fluid on a reddish area, after to days they will be broken into a spot with light brown or black scales depending on soft or hard skin area and premature or old skin of the ulcer; the scales is flaked and revealing a shallow concave ulcer, light pink, with no pus or fluid, red or dark edge depending on the disease is developing or stopping; the ulcer will be gradually healed when the fever stops[4], [19] Nodes and maculapapular rash: The nodes in ulcer area is usually swollen and painful, not red, still moves, occurs with fever or after 2-3 days, is the area of the ulcers; The nodes in body is less painful, except for severe cases The maculopapular rash appears at the end of the first week and the begin of the second week, in most area of the body, except for the palm of the foot, lasts for a few hours to a week, with low frequency than the classic Dengue fever, about 35-70% of the patients current, depending on when the patient is examined; sometimes with petechiae (less than 10%) In the early days, skin and mucous membranes were congested in most cases (about 88%), different from malaria and typhoid [5], [71], [115] For severe patients: heart sound is faint, blood pressure is low, pulse is weak compared to temperature, nosebleeds, gastrointestinal bleeding, bronchitis, a typical pneumonia [37], [146] In addition, scrub typhus may also be hidden and atypical (no ulcers) If treated by appropriate antibiotics, the fever will be reduced quickly If the intervention is late or ineffective, there may be complications such as myocarditis, septic shock, pneumonia, respiratory distress, meningitis meningitis leading to death [39], [102] The mortality rate caused by O tsutsugamushi varies from country to country, depending on the species circulating in the local The mortality rate in Vietnam, Indonesia and Taiwan, Malaysia, Japan is about 1%; 5-20%, 15-20% and 20-60%, respectively [5], [134] 1.1.2 Diagnosis of scrub typus The diagnosis of scrub typus is based on clinical and laboratory standards Criteria for determining the disease [5]: - A clinical standard only: + Fever, specific ulcers, there may be swollen lymph nodes, maculopapular rash, white blood cells 4,000 - 12,000, normal or increased lymphocytes, increased blood flow + Required criteria: specific ulcers - If there is no specific ulcers, one positive test of the following is required: The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA); or Indirect Immunofluorescence Assay (IFA test); or indirect immunoperoxidase test (IIP test) - Weil-Felix reaction with the antigen OX-K of Proteus mirabilis, the sensitivity and specificity is not high but cheap, suitable for district level - Trial treatment: For early treatment, the patient should try tetracycline or chlorocid, only applied for suspected diagnosis [5], [119], [137] Differential diagnosis: Disease caused by spirochete; Typhoid; Dengue fever; marsh fever Treatment of scrub typhus Specific antibiotics for treatment of scrub typhus are chloramphenicol, doxycycline and tetracycline However, because these antibiotics have no effect on killing but only controlling the bacteria, O tsutsugamushi bacteria still alive and exist in the lymph nodes, in the reticuloendothelial system for many days, months and easy to relapse [5], [26], [30] 1.1.3 Prevention of scrub typhus Implement some measures to propagate health education combined with hygiene for prevention of disease Carry basis investigation to detect outbreaks in areas which are suspectd and inhabited to have resolution (catch rodentia, trombiculidae, classify, subdivide R.orientalis, find antibodies, detect patients) At an identified or suspected outbreak: Measures to prevent from being bitten by trombiculidae: Removal of trombiculidae in the environment: residual spraying into moist soil, bushes of grass under 20 cm high around the house, diazinon, fenthion, malathion, lindane, dieldrin, chlordan Kill rats seasonally, paying attention to sprinkle the substance for killing trombiculidae first Clearing the vegetation around the house selects clusters of many larvae plants (trombiculidae source) Preventive treatment: This measure is limited because it is not easy for infection in outbreaks; Doxycycline 200 mg / week [4], [5], [19] can be used when a military team has to pass the outbreak for their mission 1.2 Molecular Biology Technology for disgnosis of scrub typhus Serological tests for detecting Orientia tsutsugamushi antibodies are not suitable to diagnose the disease in the acute stage because antibody level may be below the detection threshold during the disease onset stage Therefore, it is important to detect antigen/germ before the specific antibody level increases Currently, most of antigen/germ detection tests are based on PCR, quantitative realtime PRC or nested PCR targeting different target genes, including 56 kDa, 47 kDa, groEL of Orientia tsutsugamushi [47], [74], [94] However, all of these tests require training to operate the PCR thermal cycler, and in the condition of limited resources, it is difficult to ensure the equipment, maintenance and adjustment of the thermal cycler operate stably New technologies of nucleic acid amplication Nucleic acid analysis test is hoped to provide a fast, sensitive, and specific diagnostic tool for parasitic diseases New generation of diagnostic equipment allow detection of parasitic germs in the field, allowing clinicians to quickly provide reliable diagnostics for effective treatment The complex biochemical nature of clinical specimens, low levels of nucleic acid of most current samples and biosensor technologies requires us to use some technologies for amplification of nucleic acid to improve the sensitivity, meet clinical requirements when analyzing small samples, apply in field analysis [97], [141] Most nucleic acid analysis technologies are now very expensive and require trained testers, so it is only suitable for central labs that are heavily invested with abundant resources in the big cities and urban areas This makes the benefits of nucleic acid analysis tests unavailable to residents living in under-resourced areas and in isolated geographic areas Therefore, molecular diagnosis technology with reasonable price, not too complicated is essential to protect the health of each individual and the community Recent scientific and technological advances have shown that nucleic acid analysis devices can be scaled down and easily transported to the field To overcome the limitations of traditional PCR in the nucleic acid amplification used in molecular diagnostics in the field, recent studies tend to change to isothermal nucleic acid amplification methods Isothermal amplification method uses enzymes to separate the fiber, while the traditional PCR must use high temperature Isothermal amplification techniques are a very useful tool for developing field-based diagnostic models which is capable to amplify nucleic acids without the need for thermal steps and related control mechanisms [109], [111] Isothermal Amplication Technology Recombinase polymerase amplification Recombinase polymerase amplification (RPA) is a technology that uses a mixture of recombinase of progenitor to help primers attach substrate to the target gene Fiber-substituted DNA polymerase enzyme (large fraction of Bacillus subtilis PolI, Bsu) catalyzes the extended primer reaction to synthesize new added DNA fibers[49] The important thing in RPA is the use of single-stranded DNA-binding protein (SSB) and strand-displacing polymerase to replace the modified step in traditional PCR The RPA technique maximizes the advantages of simplicity, cost savings and short reaction time To determine the target sequence, the RPA requires two simple designed oligonucleotide primers (an additional probe may be required) for occurrence of pairing reaction This method has a specificity in high sequence in a nucleic acid mixture without extracting the sample This technology allows amplify the target gene by continuously synthesizing DNA without the denaturation of the double-stranded DNA at high temperatures The test can be carried out at temperatures between 24°C and 45°C with very high efficiency, so the signal from the target molecule can be detected after only to 20 minutes which is short compared to traditional PCR (20 - 180 minutes), Reltime PCR (3-4 hours) and LAMP (45 - 60 minutes) RPA technology was developed to detect HIV, Rift Valley Fever virus, Ebola virus, Sudan and Marburg, MERS-CoV, virus causing hand-foot-mouth disease and Coronavirus in cow The technique has also been developed for detection of Chlamydia trachomatis in urine samples, diagnosis of Cryptosporidiosis in animals and human samples and detection of Neisseria gonorrhoeae, Salmonella enterica, Staphylococcus aureus against methicillin (MRSA), Francisella tularensis, Streptococci group B [81], [93], [105] The sensitivity and specificity of RPA technique is also very high Studies showed that RPA technology can detect a copy of the target DNA sample (or