1 FOREWORDS Gnathostomiasis is a food-borne parasitic disease of public health concern caused by Gnathostoma spp infection The parasite is coiled up in the stomach walls cats, dogs, tigers, lions, and weasels Human infection occurs accidentally in which the parasite fails to reach the sexual maturity (called dead-end parasite), while remaining in the forms of larvae or immature worms The Gnathostoma genus has five species including G spinigerum, G hispidum, G doloresi, G nipponicum, and G Binucleatum Among which, the first species (G spinigerum, detected by Owen in 1936) is predominantly infecting humans in coutries of the Southeast Asia In Vietnam, the first case of human Gnathostoma infection was reported in 1965 in a four-year old girl living in Tay Ninh province In 1992, three more cases were detected In 1997, a case of lung infection owith G spinigerum was recorded in Ha Noi, with the patient having cough up with blood and adult worms During 19992003, over 600 cases of Gnathostoma were detected in Ho Chi Minh City So far, least have been studied on Gnathostoma in Viet Nam and, if any, they are merely investigations on animals, intermediate hosts, and sporadic case reports In order to further explore the scientific evidence for the effective diagnosis and treatment of gnathostomiasis in Vietnam, the study “Clinical and para-clinical characteristics, treatment outcomes of Gnathostoma spp, and species identification of the parasite on humans and intermediate hosts in Southern Vietnam (2016-2017)” was conducted with the following objectives: To describe the clinical and paraclinical characteristics of human Gnathostoma spp infection in Southern Vietnam (2016-2017) To evaluate the treatment outcomes of ivermectin on Gnathostoma spp at the study sites To provide species identification of Gnathostoma spp on humans and ontermediated hosts using morphological and molecularbiological techniques 2 SCIENTIFIC SIGNICICANCE, PRACTICALITY, AND INNOVATION OF THE THESIS Innovation While other studies on Gnathostoma spp mainly focuses on case reports, this study was conducted on the description and analyses of clinical and para-clinical characteristics of the infected patients The study strived to combine the conventional method (morphological species identification) with modern approach on the basis of identification and functional analysis of Cox-1 as molecular target, and species identification by 5.8S rRNA-ITS2 sequencing for Gnathostoma spp Scientific significance The study inherited existing methods applied on a wide scale in Vietnam and on around the world as well Techniques and procedures performed in this study are regularly applied in the health sector This was among the studies to apply the 5.8S rRNA-ITS2 sequencing in Gnathostoma spp species identification Practicality Results of the study played a source of reference for scientific and teaching, and good foundation for upcoming studies STRUCTURE OF THESIS The thesis has totally 120 pages with foreword (2 pages), medical literature review (29 pages), study subjects and methods (23 pages), results (31 pages), discusions (34 pages), conclusions (2 pages), and recommendations (1 page) Total figures (19 images and figures), 55 tables The references included 112 (38 Vietnamese and 74 English references), and other annexes Chapter GENERAL MEDICAL LITERATURE REVIEW 1.1 Introduction of the Gnathostoma spp 3 On the scientific classification, the Gnathostoma spp is beloging to kingdom of animalia, phylum of Nematoda, class Secernentea, order of Spirurida, suborder Spirurina, family of Gnathostomatidae, genus of Gnathostoma, and many of different species, in which G doloresi, G spinigerum, G nipponicum, G hispidum , G malaysiae, and G binucleatum may causes human gnathostomiasis, and as food-borne trematode (FBTs) zoonosis due to raw or uncooked freshwater food consumption In human body, larva can not develop into mature form, but alive larva migrant to other organs and tissues Most of human cases has mild symptoms, but in case of visceral larva migrans (VLMs) to central nervous system with serious comlications, even death 1.2 Situation of human gnathostomiasis 1.2.1 In the globe In 1889, the first case was reported in Thailand, afterward human gnathostomiasis recorded in many countries as Malaysia, India, China, Vietnam, Indonesia, Thailand, Japan, Korea, Philippines, Laos, Taiwan, Bangladesh, Pakistan, and Israel However, highest prevalence and predominant in Thailand, Japan At the present, in the world have at least six gnathostomiasis induced Gnathostoma species in human being, composed of G binucleatum, G doloresi, G hispidum, G malaysiae, G nipponicum, G Spinigerum, and G spinigerum is most common in Southeast Asia 1.2.2 In Vietnam Vietnam is the third country with high case numbers in Asia, most of them come from local residence and tourist Four Gnathostoma spp that infected in human and animal in Vietnam of G spinigerum, G hispidum, G Doloresi, and G vietnamicum 1.3 Clincal manifestations, and laboratory findings 1.3.1 Clinical symptoms After penetrating gastric wall, larva may migrans to other organs or tissues as namely larva migrans The most common clinical manifestations of the infection in the skin higher than internal organs, but if yes, pernicious viceral form Disease forms are based on larva migratory to tissues, such as skin, soft tissues and neural form are most common Rarely, in the ears, eyes, lungs, or gastrointestinal, hepatobiliary, and urinogenital forms) 4 1.3.2 Laboratory parameters Haematology: Eosinophil is an important sign as early warning point in diagnosis and evaluation of treatment effectiveness Immunodiagnosis of ELISA Enzyme-linked immunosorbent assay (ELISA) is a popular analysis tool in detection of serum IgG anti-Gnathostoma spp., and ELISA kit sensitivity and specificity varied from different products (56-100%) Molecular identification and diagnosis Gnathostoma spp mitochondrial gene analysis was wide-applying in Gnathostoma spp identification, population genetic structure analysis, and as molecular marker in Gnathostoma spp phylogenetic tree (Gu et al., 2014) 1.4 Diagnosis Confirmed diagnosis when we collected Gnathostoma spp larva or young worm from lesions in mucocutaneous tissue, ocular or viceral location, but rarely occured Hence, most of clinician usually based on diagnosis criteria as follow: o Uncooked or raw freshwater foods eating in the past history, or travelling to popular endemic area o Cutaneous or visceral larva migrans symdrome, such as itching, urticaria, red rash, or creeping eruption o Blood eosinophil up to 500 cells/ ml o Positive serum immunogdiagnosis of IgG antibody antiGnathostoma spp or positive Gnathostoma spp antigen 1.5 Treatment 5.1 Internal treatment Many of wide-spectrum effective antiparasitic drugs can be used in human gnathostomiasis treatment Albendazole for 3-4 weeks has been shown to result in cure in several trials > 90% Oral thiabendazole dose of 50 mg/kg/day for 1-2 days or 2-7 days (belonging to clinical forms) with cure rate range from 91.37-96.55% However, ivermectin has good point in single dose 200 microgam per kg and cure rate > 80% while long-course of three weeks in albendazole regimen 1.5.2 Surgical treatment The best treatment option is surgical excision of the larvae remained the only in case of confirmed ocular or cutaneous larva migrans 5 Chapter SUBJECTS AND METHODS 2.1 Objective and 2: To descriptive of clinical manifestations and laboratory findings of human Gnathostoma spp infection in Sounthern and Central region Vietnam (2016-2017) Evaluation of the human gnathostomiasis treatment outcome by oral ivermectine regimen 2.1.1 Study subjects Total of 112 patients who has gnathostomiasis met following criteria: - Inclusion criteria: The patients at parasite-specific clinic met inclusion criteria will be enrolled in the study:  Patient has just positive ELISA immunodiagnosis with IgG antiGnathostoma spp antibody + Four following criteria: o Uncooked or raw freshwater foods eating in the past history, or travelling to popular endemic area o Cutaneous or visceral larva migrans symdrome, such as itching, urticaria, red rash, or creeping eruption o Blood eosinophil is higher than 500 cells/ ml o Positive serum immunogdiagnosis of IgG antibody antiGnathostoma spp or positive Gnathostoma spp antigen  To be of sound mind, ability of hearing, understanding, and answer by Vietnamese language  Willingness to comply with the study protocol for the duration of the study and to comply with the study visit schedule;  No limit of age and gender - Exclusion criteria  Patients has positive ELISA immunodiagnosis with IgG antibody with other parasite, except G spinigerum  Unwilling and unvolunteering with study  Presence of acute or chronic seriously illness as asthma, bronchitis  Presence of psychological disorders and fatigues  History of ocular disorders such as cataract, and retinal degeneration  Người có tiền sử bệnh dày tá tràng  A positive pregnancy test or breastfeeding women 6  History of hypersensitivity reactions to fungus, foods, or any of the medicine(s) being tested 2.1.2 Location and timeframe - Location  Case record forms and data collection from 66 gnathostomiasis patients at parasite-specific clinic of the Institute of Malariology, Parasitology, and Entomology (IMPE) Quy Nhon, and 46 cases of gnathostomiasis patients from the general clinic of Trong Nghia, Ho Chi Minh city  Parasite-specific or microbiology laboratories at the IMPE Quy Nhon, the general clinic of Trong Nghia, and the microbio-parasitology department of the medicine and pharmacy university in Ho Chi Minh city - Timing: from May 2016 to April 2017 2.1.3 Methodology Study design - The descriptive prospective study design on all selected patients These patients enrolled in interventional treatment group list and evaluation of treatment outcome via non-randomized controlled clinical trial - Experimental study in laboratory with collected Gnathostoma spp larva from gnathostomiasis by morphological and bio-molecular analysis Sample size - In the case of ivermectine drug with an expected failure rate of 20%, a confidence interval of 95% and a precision level of 10%, at meantime a minimum of 61 patients should be enrolled - To avoid of number of withdraw or loss of follow during long course follow-up, plus 20%, hence final sample size is 73 cases, but in practical samples of this study up to 112 cases Sampling - Patients who met inclusion criteria at two parasite-specific clinic will be enrol, serum sample storage and case record forms (CRFs), infromed consent froms (ICFs) until enough required sample size 2.1.4 Study process and procedures Description of the clinical and sub-clinical characteristics of human Gnathostoma spp in the Southern Vietnam (2016-2017) - Clinical characteristics + Mucocutaneous system: Itching, urticaria, rash or erythema, intermittent swelling, cutaneous larva migrans or creeping eruption syndrome  Digestive tract: Epigastric pain, digestive disorder (watery or semiliquid) stool, anorexia, nausea  Respiratory system: Persistent cough (dry cough, no mucus), chest pain, shortness of breath, wheeze  Vision: visual impairment (blurred vision), orbicularis oculi muscular pain, diplopia  Nervous system: Headache, vertigo, sleep disorder (insomnia) - Sub-clinical characteristics + Complete blood count: white blood cell, eosinophil count + Liver function test: SGOT, SGPT + ELISA with anti-Gnathostoma IgG: S/Co cut-off level (S/Co≥1.0 positive and S/Co < 1.0 negative) Evaluation of Gnathostoma spp outcome treatment with ivermectin  Evaluation of treatment for Gnathostoma patient at the study site with 0.2mg/kg single dose ivermectin through the reduce of clinical and subclinical symptoms at months and months post-treatment  Ivermectin (Pizar®) 3mg or 6mg, log No 18003, Mafg date 22.9.2015 Exp.date 22.9.2018, and manufactured by DAVI Pharm JSC  Evaluation of some unexpected events after use of ivermectine 2.1.5 Techniques used in the study - Interview technique, clinical examination, taking notes and copying original CRFs based on the information provided in the designed CRFs - Doctors and lab technicians received Good Clinical Practices (GCPs) training before conducting the study - Giving prescription to patients; explain and persuade the patients to comply with the treatment regimen and appoint a follow-up examination at months and months post-treatment - ELISA immunodiagnosis for detecting of anti-Gnathostoma spp IgG antibody by ELISA kit of Viet Sinh Ltd company, circulating certificate 73/2016/BYT-TBCT in Vietnam, code KST5-GnathoELISA, log 180416, Mafg date 18.04.2016, exp.date 18.04.2019 with sensitivity and specificity of 96.7% and 99.1%, respectively Chapter RESULTS 3.1 Clinical and laboratory findings of human gnathostomiasis in Southern Vietnam (2016-2017) 3.1.1 Manifestations of study patients Table 3.1 Distribution of study patient by resisdent location Resident location (province, city) n(%) Resident location (province, city) n(%) Binh Dinh 19 (16.96) Tra Vinh (2.68) Daklak 12 (10.71) Vinh Long (2.68) Gia Lai 12 (10.71) Bac Lieu (1.79) Quang Ngai (6.25) Ben Tre (1.79) Ho Chi Minh city (6.25) Binh Dương (1.79) An Giang (3.57) Đong Nai (1.79) Long An (3/57) Hau Giang (1.79) Tien Giang (3.57) Kon Tum (1.79) Ca Mau (2.68) Soc Trang (2.79) Can Tho (2.68) Đong Thap (0.89) Dak Nong (2.68) Kien Giang (0.89) Khanh Hoa (2.68) Lam Dong (0.89) Phu Yen (2.68) Ninh Thuan (0.89) Quang Nam (2.68) Tay Ninh (0.89) These patients came from a variety of mountainous, plain, and coastal areas in 28 provincies and cities nationwide, highest proportion in Binh Dinh 19 case (16.96%) Table Error! No text of specified style in document Patient distribution by age and gender (n = 112) Age group Male Female p-value < 15 15 (75%) (25%) < 0.05 ≥ 15 - < 30 (33.33%) 10 (66.67%) > 0.05 ≥ 30 - < 45 (25%) 21 (75%) < 0.05 ≥ 45 15 (30.61%) 34 (69.39%) < 0.05 42 (37.5%) 70 (62.5%) Total Human gnathostomiasis appeared differently by age groups, but predominantly in people aged from 45 years old (41.07%) Table Error! No text of specified style in document Patient distribution by occupation (n = 112) Occupation(s) Pos.(+) rate (%) State staffs 28 25.0 Farmers 20 17.86 Traders 17 15.18 Students 15 13.39 Pupils 13 11.60 Fishers 05 4.46 Others 13 11,60 Study data revealed that state staff represented the highest incidence Table Error! No text of specified style in document Patient distribution by education background (n = 112) Educational background Pos (+) Rate (%) Unalphabetic 03 2.68 Primary school 23 20.54 Secondary school 32 28.57 High school 25 22.32 Upper high school 29 25.89 By education background, the secondary levels represented the highest incidence (28.57%), followed by post-schools (25.89%), the primary schools (20.54%), and the illiterates occupying lowest (2.68%) Table 3.5 Patient distribution by ethnic community (n = 112) Ethnic group Kinh Other (E De Jrai, Tay, Kho Me) Pos.(+) Rate (%) 107 95.54 4.46 The Kinh ethnic represented the predominant incidence of Gnathosoma spp., while other ethnic minority groups occupied the rest 4.46% 10 3.1.2 Risk factors for Gnathostoma spp infestation Table 3.6 Some possible risk factors (n = 112) # Risk factors Pos.(+) Rate (%) Raw fresh-water fish or salad 80 71.43 Uncooked frog meat 73 65.18 vegetables forms of single serves or salads 72 64.29 Steamed or sliced snails mixed with vegetables 69 61.61 Undercooked or rare fresh-water eels 49 43.75 Other aquatic foods salad 43 38.39 Snake salad or snake liquid blood 36 32.14 Sliced raw fish, prawn with wasabi 31 27.68 Raw mussel salads with mustards 23 20.54 15 13.39 10 Drinking of undercooked river and well water Gnathostomiasis is transmitted via digestive system, especially when patients consume at least one of the ten following food groups Study data revealed a variety of food frequently consumed by patients 3.1.3 Clinical manifestations on organs or systems Table 3.7 Time interval prior to study enrollment (n = 112) # Number of symptom days before enrollment Pos.(+) Rate (%) < days 2.68 ≥ - < 15 days 13 11.61 ≥ 15 - < 30 days 35 31.25 ≥ 30 - < 45 days 35 31.25 ≥ 45 days 26 23.21 Data revealed that the proportions of patients having longer interval time (from disease onset to hospitalization) of 15-30 days and 30-45 days were same 31.25%, and those with shorter interval time represented lower proportion Table 3.8 The reasons why patient hospitalized (n = 112) Reasons Pos.(+) Rate (%) 11 Mucocutaneous tissue 92 82.14 Neural system 50 44.64 Digestive system 37 33.04 Ocular organs 13 11.61 Respiratory tract 6.25 Data revealed various reasons for hospitalization and need to treatment Table 3.9 Clinical manifestations on patients (n = 112) Involved organs or tissues Pos (+) Rate (%) Mucocutaneous tissue 91 81.25 Neural system 51 45.53 Digestive system 39 34.82 Ocular organs 13 11.6 Respiratory tract 7.14 Patients with cutaneous and subcutaneous symptoms represented the highest proportion (81.25%), neurological system (45.53%), digestive system (34.82%), occular symptoms (11.6%), and respiratory system as lowest incidence (7.14%) Table 3.10 The clinical symptom on mucocutaneous tissue (n=112) Clinical manifestations Pos (+) Rate (%) Itching, urticaria 84 75.0 Red rash, tunnel traces 38 33.93 Partial rash/erythema 22 19.64 Larva migrans/ Creeping eruption 13 11.61 Regular occurred 47 41.96 Intermittent occured 44 39.28 Lesion location Lesion characteristics Major cutaneous and subcutaneous symtoms included pruritus and urticaria (75%), followed by erythema (33.93%), and localized rash (19.64%), whereas creeping eruption was another less common 12 manifestation (11.61%) The frequencies of clinical features may vary from regular occurrence (41.96%) to intermittence (39.28%) Table 3.11 The clinical manifestations on digestive tract (n=112) Digestive system Pos.(+) Rate(%) Epigastric pain 35 31.25 Digestive disorder (loose stool) 8.04 Anorexia plus nausea 4.46 The epigastric pains represented the highest proportion (31.25%), followed by digestive disorders (8.04%), and poor appetite and nausea (4.46%) Table 3.12 The clinical manifestations on respiratory tract (n = 112) Respiratory system Pos.(+) Rate(%) Chest pain 3.57 Persistent cough (dry without sputum) 1.79 Short breath 1.79 Sweezing 1.79 A small proportion of patients with chest pain (3.57%), other symptome occupied less than 2% Table 3.13 The clinical manifestations on ocular system (n = 112) Ocular system Pos.(+) Rate(%) Periocular myalgia 6.25 Ocular disorder (blurred vision) 5.36 Blurred vision (diplopia) 4.46 The ocular symptoms represented relatively small proportions, including pains of the eyelids (6.25%), vision impairment or blindness (5.36%), and diplopia (4.46%) Table 3.14 The clinical manifestations on neural system (n = 112) Neural system Pos.(+) Rate(%) Headache (+/- dizziness) 40 35.71 Dizziness 31 27.68 Sleep disorder (insomnia) 6.25 13 Major neurological manifestations of the studied patients with gnathostomiasis included headache (possible with diziness) as the highest proportion (35.71%), diziness (27.68%), and disorders (6.25%) 3.1.4 Laboratory findings Table 3.15 Blood eosin proportion before treatment (n = 112) Blood eosinophile (%) Mean ± SD Prior treatment (n = 112) 456.85 ± 419.45 < 100/mm3 (2.68) 100 - 500/ mm3 78 (69.64) > 500 cells/mm3 31 (27.68) Most patients (92%) had the normal range of WBCs, and elevated WBC (>10,000 cells/mm3) was found in the remaining 8% of the patients Eosinophilia (>500 cells/mm3) was present in 27.68% Table 3.16 Liver enzyme SGOT and SGPT before treatment (n = 112) Tested samples SGOT Mean ± SD SGPT 27.94 ± 9.42 Mean ± SD Normal ( 0,05 Respiratory tract WBC > 10.000/mm3 p-value The results indicated that six mos following treatment, 21 cases still had clinical manifestations, including 12 cases with positive ELISA tests This was translated into a cure rate of 92.16%, reduced symptoms of 3.92%, and non-cured of 3.92% Table 3.19 Clinical and laboratory manifestations before and after treatment months post-treatment Before Tx After Tx mos (n = 112) (n = 102) Mucocutaneous 92 (82%) (7.8%) < 0,05 Digestive tract 37 (33%) (1%) < 0,05 Respiratory tract (6,3%) (1%) > 0,05 Ocular vision 13 (11.6%) (1%) < 0,05 Neural system 50 (44.6%) 10 (9.8%) < 0,05 ELISA ≥ 1.0 112 (100%) 12 (11.8%) < 0,05 (8.0%) (4.9%) > 0,05 Organs/Tissues WBC >10.000/ mm3 p-value 15 Eosin > 500 cells/mm3 31 (27,7%) 10 (9,9%) < 0.05 SGOT ≥ 40 U/L 13 (11,6%) 14 (13,7%) > 0,05 SGPT ≥ 40 U/L 10 (8,9%) (5,9%) > 0,05 The results indicated that six mos following treatment, 21 cases still had clinical manifestations, including 12 cases with positive ELISA tests This was translated into a cure rate of 92.16%, reduced symptoms of 3.92%, and non-cured of 3.92% Table Error! No text of specified style in document 20 Evaluation of treatment outcome after months (n=102) Treatment outcome n Rate (%) Cure 94 92.16% Partial recovery, reduction 3.92% Not recovery 3.92% 102 100% Tổng cộng Proportion of recovery (92.16%), reduction (3.92%), non-cure (3.92%) Table 3.21 Several possible ivermectin induced adverse events Adverse events Pos (+) Rate Occurred time after taking ivermectine (min-max) (%) Headache, dizziness 6,25 Early: 1h; Late: 48hs Abdomen pain, nausea 7,14 Early: 2h; Late: 24hs Loose stool or diarrhea 0,89 ca: 24hs Muscle pain 0,89 ca: 48hs Fever Itching, skin rash 5,36 Early: 24hs; Late: 48hs Regarding the unwanted side effects of IVM on patients, our study revealed that 7/112 (6.25%) cases had headace, 8/112 (7.14%) abdominal pains and nausea, 6/112 (5.36%) had urticaria 3.3 Gnathostoma species identification on human and intermediate host by conventional morphological and bio-molecular technique 3.3.1 Species identification of Gnathostoma spp 16 Figure 3.1 Proportion of Gnathostoma larva in the collected eels (2.57%) - Species identification by morphological method Table 3.22 The Gnathostoma spp larva size (n = 81) Number (n) 10 12 18 20 2,4 2,8 Mean (SD) Length (mm) 1,5 1,8 Width (mm) 0,16 0,17 0,2 0,25 0,28 0,3 0,3 0,24 ± 0,05 Size 3,0 4,0 2,50 ± 0,64 The average length (2,50 ± 0,64) and width (0,24 ± 0,05) of larva Table 3.23 Number of spines on Gnathostoma spp larva head bubd Spines row Number of prickles on each row Mean (SD) I 39 39 42 43 43 44 44 42.26 ± 1.71 II 42 47 44 42 45 45 43 44.05 ± 1.65 III 44 48 49 47 49 49 49 48.05 ± 1.41 IV 46 48 50 54 53 52 50 51.28 ± 2.49 No of larva 12 18 20 10 All collected gnathostoma larva had four spines row - Identification of Gnathostoma spp larva by bio-molecular technique Observating the specific Cox-1 gene for Gnathostoma spp larva from the markets and nuclear fragment PCR (250 bp gene Cox-1) on electrophoreisis agarose gel 1.5% 17 cox-1 (250bp) M: ADN 100 bp; Well 1-10: 10 larva samples, well 11: H2O control Figure 3.2 The Cox-1 lane on the Gnathostoma spp larva sample cox-1 (450bp) Figure Error! No text of specified style in document The Cox-1 lane on the Gnathostoma spp larva in collected eels M:DNA 100bp; well 1-10: 10 larva samples; well 11: H2O control well 1,3,7,8,9,10 has band 450bp 450bp Figure Error! No text of specified style in document The gen Cox-1 on Gnathostoma spp larva on the patient 18 M: ADN 100bp; well N1 larva sample from human; well L1 from eels; NC: H2O control - Gene sequencing for Gnathostoma spp identification on specific 5.8S rRNA-ITS2 gene ITS2 (600bp) Figure Error! No text of specified style in document The 5.8S rRNA-ITS2 gene in ten Gnathostoma spp larva M: ADN 100bp, well 1-10: 10 larva samples, well 11: control The result of 5.8S rRNA-ITS2 gene sequencing after nuclear gene polymerase from ten larva sampes of Gnathostoma spp for identify viaBio-edit v.7.2.6, MEGA6, and compare to BLAST on Genbank Data showed that 6/10 samples were identified as G spinigerum, 3/10 samples as G doloresi, and 1/10 sample as G hipidum Heterogenous (%) effect Bảng Error! No text of specified style in document 241 The homogenous effect on nucleotide sequencing between 5.8S rRNA-ITS2 gene of G Doloresi, G hispidum samples and in globe Larva sample Larva sample Homogenous tương đồng (%) 100 0,6 0,9 68,3 99,4 100 0,4 63,8 99,1 99,6 100 66,9 31,7 36,2 33,1 100 effect Mã số gen Ngân hàng gen giới AB181156 G.doloresi AB180100 G.doloresi JN408299 G.doloresi AB181158 G hispidum 19 Figure 3.6 Phylogenetic tree and 5.8S rRNA-ITS2 gene sequencing of six samples of G spinigerum, three G Doloresi, and one G hispidum Chapter DISCUSSION 4.1 Clinical and laboratory characteristics of Gnathostoma spp infection on humans in Southern Vietnam (2016-2017) 4.1.1 Demographic characteristics on the study patients Total 112 cases detected from the Clinic of the IMPE-QN and Trong Nghia Clinic of Ho Chi Minh City were selected for study These patients came from a variety of mountainous, plain, and coastal areas in 28 provincies and cities nationwide This indicated a wide prevalence of the disease, with fluctuated infections by localities Gnathostomiasis appeared differently by age groups, but predominantly in people aged from 45 years old (41.07%) In addition, more women than men (62.5% vs 37.5%), 20 which is in line with a study conducted by Stady et al., (2009) showing the infection rate in women to be 1.6 times as much as in men Across the occupations, our study revealed that government staff represented the highest incidence (25%) of Gnathosoma spp, while fisherman occupied the lowest (4.46%) These results were in agreement with those in the study conducted by Nguyen Van Chuong et al (2013) showing the highest incidence of Gnathostoma spp infection in the governmental staff group (37.21%) and the lowest in the students (16.28%) This might come from the fact that human Gnathostoma transmission occurs via digestive system; and governmetal staff have more eating outs than students, resulting in more infection By education background, no significant differences in Gnathostoma spp incidence were found in the studied groups categorised by education levels The secondary levels represented the highest incidence (28.57%), followed by post-schools (25.89%), the primary schools (20.54%), and the illiterates occupying lowest (2.68%) This indicated gnathosomiasis as a disease transmitted via the digestive system can occur in people with different education backgrounds The Kinh ethnic represented the predominant incidence of Gnathosoma spp., while other ethnic minority groups occupied the rest 4.46% The results were in harmony with the study conducted by Nguyen Van Chuong showing 94.19% of all cases belong to the Kinh ethnic The difference in terms of Gnathostoma incidence might be related to the different eating habits among ethnic groups 4.1.2 Clinical manifestations and risk factors - Risk factors Gnathostomiasis is transmitted via digestive system, especially when patients consume at least one of the ten following food groups Our study revealed a variety of food frequently consumed by patients, ranging from raw fresh-water fish or salad (71.43%); uncooked frog meat (65.18%); vegetables in the forms of single serves or salads (64.29%); steamed or sliced snails mixed with vegetables (61.61%); undercooked or rare freshwater eels (43.75%); other aquatic food salad (38.39%); snake salad or snake liquid blood (32.14%); sliced raw fish and prawn with vegetables or wasabi (27.68%); and raw mussel salads with mustards (20.54%) In addition, the consumption of undercooked river and well water was found in 13.39% of the patients These about-mentioned data revealed 21 Gnathostoma spp incidence was present predominantly in patients having eating habits of raw or undercooked aquatic food (fresh-water fish) This was similar to the etiology of G spinigerum infection following the consumption of raw fresh-water fish containing larvae In 2013, Nguyen Van Chuong reported the connection between the consumption of raw fresh-water fish containing Gnathostoma spp larvae and gnathostomiasis infection In addition, Vailai-B indicated that 90% of the gnathostomiasis patients had history of eating raw or uncooked meat - Interval time from disease onset to hospitalization Data revealed that the proportions of patients having longer interval time (from disease onset to hospitalization) of 15-30 days and 30-45 days were same 31.25%, and those with shorter interval time represented lower proportion The proportion of short interval time (10,000 cells/mm3) was found in the remaining 8% of the patients Eosinophilia (>500 cells/mm3) was present in 27.68%, which was lower than that of the study conducted in 2005 by Le Thi Xuan (75.7%) The difference might be due to the fact that our patients were previously treated with histamine and anti-inflamatory medications following cutaneous manifestations that influenced the immunological responses on patients, hence no eosinophilia The proportion of patients within the normal range of SGOT (