Nghiên cứu thực trạng, một số yếu tố liên quan đến nhiễm giun lươn strongyloides spp và kết quả điều trị bằng ivermectin tại huyện đức hoà, tỉnh long an (2017 – 2018) tt tếng

According to the study results in Cu Chi district, HCM city previously, the prevalence of Strongyloides spp infection in the community was quite high.. Stemming from this reality, we con

INTRODUCTION Strongyloides spp is a chronic causative infectious agent This agent is considered to be one of the neglected tropicals infectious pathogens, but it is an important public health problem due to the characteristic of the autoimmune cycle, leading to hyper infection and death in some patients

People are acquired Strongyloides spp agent through contact with contaminated soil sources directly such as agricultural cultivation, recreation activities, etc The adult worms usually located in the intestine wall, causing abdominal pain, prolonged diarrhea, or colitis, In addition to disease at the gastrointestinal tract, the stage when infective larvae of Strongyloides spp enter the human body, can move to many organs, causing very diverse clinical diseases The problem of accurate diagnosis of the case therefore also faces many difficulties

Treatment strongyloidiasis cases is more difficult than other intestinal helminths, especially with hyper infection The treatment of cases has still been inconsistent with the timing of treatment and drug selection

Although strongyloidiasis was discovered in Southern Vietnam at first time, but there has not been many studies on this pathogen recently According to the study results in Cu Chi district, HCM city previously, the prevalence of Strongyloides spp infection in the community was quite high While Duc Hoa district of Long An province was located bordering with Cu Chi district, HCM city, there still has not been any research on these pathogens

Stemming from this reality, we conducted the thesis: Studying the actual situation, various factors related to human Strongyloides spp infection and ivermectin effectiveness in Duc Hoa district, Long An province (2017 – 2018).”with the objective:

1 Determine the actual situation and factors related to human Strongyloides spp infection in Duc Hoa district, Long An province in 2017-2018

2 Determine component species of Strongyloides in human strongyloidiasis disease by morphological characterization and molecular biology

3 Describe the clinical symptoms, para-clinical and evaluate results of treatment for strongyloidiasis by ivermectin single dose

NOVELTY, SCIENTIFIC AND PRACTICAL SIGNIFICANCE OF THE

THESIS The thesis provides details of valuable scientific data on the status of Strongyloides spp infection and related factors in many research sites of Duc Hoa district, Long An province

Provide details of the presence of symptoms, signs and value of para-clinical test in cases of gastrointestinal strongyloidiasis

Determine the clinical efficacy of ivermectin and the effectiveness of larval clearance, which is the basis for developing an appropriate intervention program For the first time, a new species of Strongyloides spp was identified from patient in Duc Hoa district that derived from animal origin, by using molecular biology techniques

THESIS STRUCTURE The thesis consists of 136 pages divided into the following sections: Introduction (02 pages), Chapter 1: Literature review (31 pages), Chapter 2: Study subjects and methods (29 pages), Chapter 3: Study results (34 pages), Chapter 4: Discussions (36 pages), Conclusions (02 pages), and Recommendations (1 page) There are 36 tables, 13 figures, and 115 references (12 pages, including 28 Vietnamese documents and 87 English documents)

Chapter 1 LITERATURE REVIEW 1.1 History of discovering strongyloidiasis

In July 1876, Louis Normand found the parasite in the stool samples of French diarrhea patients with a history of coming to the Southern Vietnam He named this agent Anguillula stercoralis and the corresponding disease has been called Cochin -China diarrhea Coming to 1915, the council named scientific unification named this pathogen Strongyloides stercoralis

1.2 Pathogen

There are about 104 species of Strongyloides spp, including 52 common species, some of which cause disease in domestic pets and other animals The main pathogen cause human disease is S stercoralis, and less is S fuelleborni Some other species such as S procyonic (host is raccoons), S myopotami and S ratti (hosts are rats and rodents) are considered to be parasitic diseases transmitted from animals to humans

1.2.1 Morphology

The development stages of Strongyloides spp including: parasitic female living worm, free-living female worm, free-living male worm, the first stage larvae (rhabditiform), the second stage larvae (filariform) and eggs Parasitic male worm has still been not found

1.2.2 Biological development cycle of Strongyloides spp

Strongyloides spp have two stages of the pathogenesis cycle: the parasitic cycle and the free living cycle Free living cycle often happen in the tropics due

to adaptive with conditions of external environment In addition, strongyloidiasis also has autoinfection cycle The autoinfection cycle occurs when all or some of rhabditiform larvae reside in the intestinal wall, rapidly molt to the infected stage, establishing a parasitic development stage inside the host and this phenomenon may remain maintenance of host life This phenomenon also often occurs in patients who have immunosuppression status This autoinfection process leads to two severe status of strongyloidiasis: hyperinfection syndrome and disseminated strongyloidiasis

1.3 Epidemiological characteristics

The infection prevalence is usually less than 1% in temperate zone, but may

be above 25% in many parts of the tropics

1.3.1 The situation of Strongyloides spp infection in the world

Strongyloidiasis is an infectious disease in many countries, especially in West Africa, the Caribbean, Southeast Asia, tropical regions of Brazil, Cambodia and Spain Southeast Asia area has the highest prevalence of the disease

1.3.2 The situation of Strongyloides spp infection in Vietnam

According to a Galliard survey in 1940, in the northern Vietnam, the prevalence of Strongyloides spp infection was from 0.2 to 2.5% of the population Recent studies using ELISA technique have shown that the infection rate is higher than 7.6-10.9%

Strongyloides spp are widely distributed in the southern provinces of Vietnam, provinces such as Long An, Binh Duong, Tay Ninh, Dong Nai and Ho Chi Minh City (Cu Chi, Thu Duc and Hoc Mon districts) and some provinces in the Central region and Highlands

1.4 Strongyloidiasis pathology

The strongyloidiasis has an incubation period approximately 1 month The majority of cases in endemic areas often have no symptoms or vague symptoms Strongyloidiasis is divided into two forms:

1.4.1 Chronic, uncomplicated Strongyloidiasis: happen in normal individuals, without immunodeficiency, possible symptoms include:

Skin manifestations: The cutaneous larvae migrans, skin bruises, nonspecific urticaria,

Gastrointestinal manifestations: Abdominal pain, diarrhea, weight loss, anal itching

Other manifestations: Patient coughing, pneumonia, signs of cachexia, 1.4.2 Severe disease, complications

This form of the disease is common in immunocompromised individuals who use corticosteroids, immunosuppressing or accompanied by other chronic diseases such as chronic obstructive pulmonary disease, chronic kidney failure, malignancy, diabetes, alcoholism, malnutrition

1.4.3 Hyperinfection syndrome and disseminated strongyloidiasis

Hyperinfection syndrome is manifestted by a phenomenon in which the rapid increasing the number of pathogens leads to an excessive burden without the spread of external larvae Disseminated strongyloidiasis is the ultimate consequence of hyperinfection syndrome Affected organs include the lungs, liver, heart, kidneys, endocrine organs and central nervous system Strongyloidiasis is severe and lead to death

1.5.2 Indirect diagnosis

Currently, serological testing methods are popular and widely used due to their high sensitivity and general applicability The most widely used technique is the ELISA test to detect immunoglobulin G (IgG) antibodies

1.5.3 Molecular biology diagnosis

The PCR test detects parasitic DNA in feces, especially Strongyloides spp, which has the advantage of high sensitivity and specificity PCR techniques have been used include as follows: real-time PCR, nested PCR, multiplex real-time PCR and multiplex PCR

1.6 Treatment

Treatment human strongyloidiasis was based on using current drugs: Ivermectin 150-200 µg/kg/ day single dose, albendazole 10-15 mg/kg/day and thiabendazole 50 mg/kg/day The dose of each drug is determined but no consistent guide about the number of treatment days

CHAPTER 2 STUDY SUBJECTS AND METHODS 2.1 Study subjects, sites and duration

2.1.1Study subjects

People residing in Duc Hoa district, Long An province meet the selection criteria for the sample

Patients infected with gastrointestinal strongyloidiasis

Strongyloides larvae were collected from a patient's stool sample test Ivermectin pills single dose

2.1.2 Study sites (location)

In the field works: 4 communes Duc Lap Thuong, My Hanh Nam, Hiep Hoa,

An Ninh Tay and 1 Duc Hoa town of Duc Hoa district, Long An province In each commune or town, the location is people's residence and health station

- Laboratory of medical parasitology, Pham Ngoc Thach university of medicine, Laboratory of Hematology and Immunization, Hospital for Tropical Diseases in Ho Chi Minh city

- Genome sequencing at First BASE Laboratories-Axil Scientific, Malaysia 2.1.3 Study duration

The study was conducted from July 2017 to November 2018

Determining the prevalence of intestinal strongyloidiasis in each commune / town of Duc Hoa district, Long An province

Description and analysis: A number of demographic factors related to the prevalence of Strongyloides spp infection in people such as sex, age group, economic status and education level Some of the related behaviors such as agriculture job, toilet use and contact soil directly in daily life

Techniques used in the study

Interview technique of data collection

Technique for diagnosis of Strongyloides spp infection

 Modified Harada Mori culture (Sasa, 1986)

 Stool direct smear technique

2.2.2.2 Objective 2: Determine component species of Strongyloides in human strongyloidiasis disease by morphological characterization and molecular biology

Diagnostic technique: size measurement of morphological structures of Strongyloides spp larvae stage 1,2 or adults

Multiplex real-time PCR technique, 2 steps nested - PCR and gene sequencing 2.2.2.3 Objective 3: Describe the clinical symptoms, paraclinical and evaluate results of treatment for Strongyloides spp by ivermectin single dose

Research content

Describe the presence rate of clinical symptoms in patients who were diagnosed gastrointestinal strongyloidiasis before and after treatment: intermittent loosing diarrhea, abdominal pain, urticaria, weight loss, headache, cutaneous larvae migrans

Describe and analyze the para-clinical parameters of patients who were diagnosed gastrointestinal strongyloidiasis: the number, ratio of eosinophils and serum ELISA diagnosis

Determining the clinical and para-clinical therapeutic effect of single dose ivermectin and the presence rate of side effects

Techniques used in the study

Blood test technique to determine eosinophils

ELISA test for detect specific antibody (IgG) against Strongyloides spp Stool test technique to evaluate treatment results: Apply a combination of 2 direct smear technique and stool culture

2.2.3 Data processing

Analyze the relation among the variables using 2 test, Fisher exact test, t test,

OR with p <0.05 by SPSS 22.0 software in windows

Genetic sequences were obtained after sequencing, will be processed by Bioedit v.2.6 (Tom Hall, 2017) and MEGA 6 (Temura, 2013) sofware

2.3 Ethics in research

Strictly compliance with regulations in biomedical study

CHAPTER 3 STUDY RESULTS 3.1 Determine the actual situation and factors related to human Strongyloides spp infection in Duc Hoa district, Long An province in 2017-2018

3.1.1 Characteristics of study subjects

The total number of samples collected was 1,190 samples, distributed in 5 research sites including 4 communes and 1 town The study site was Duc Lap Thuong commune had the largest sample size corresponding to the largest population (314), followed by Duc Hoa town (233)

The percentage of women participating in the study was 54.1%, more than men People between the ages of 15 and 60 participated in the majority (66.8%) The number of the study participants with education level below high school was 69.1% People with poor and nearby poverty status accounted for 14.3% Farmer accounted for 24.7% and the proportion of unhygienic toilet using was 11.5%

3.1.2 Actual situation of Strongyloides spp in Duc Hoa district

3.1.2.1 The prevalence of Strongyloides spp infection

Table 3.1 Prevalence of Strongyloides spp infection in study site (n = 1,190)

No Commune/town No of Tests No (+)Percentage (%)

The overall prevalence of Strongyloides spp infection in Duc Hoa district is

6.64%

3.1.3 Related factors with Strongyloides spp infection

Table 3.2 Relation between Strongyloides spp infection and sex Strongyloides inf

p < 0.001; OR = 4.06; CI 95%: 2.39 – 6.89 There was a relation between Strongyloides spp infection and sex (p <0.001) Male was at 4.06 times higher risk of infection than women

Table 3.3 Relation between Strongyloides spp infection and age

People over 60 years of age were 2.46 times higher risk about Strongyloides spp

infected than the other groups

Table 3.4 Relation between Strongyloides spp infection and education

Strongyloides inf

p < 0.05; OR = 1.98 ; CI 95%: 1.12 – 3.54

People with education level below high school was 1.98 times risk to be infected

Strongyloides spp

Table 3.5 Relation between Strongyloides spp infection and economic status

Strongyloides inf

Economic status

Inf (+) Non – inf Total

p < 0.001; OR = 10.84; CI 95%: 5.93 – 19.83 People who had poor and nearby poverty status, was 10.84 times more likely

to be infected Strongyloides spp than those in the average economy group or above

Table 3.6 Relation between Strongyloides spp infection and farmer

status Strongyloides inf

p < 0.001; OR = 11.40; CI 95%: 6.99 – 18.59 People who used unhygienic toilets was 11.4 times risk to be infected Strongyloides spp than group used hygienic toilets

Table 3.8 Relation between Strongyloides spp infection and the habit

contact with the soil directly Strongyloides inf

Habit contact with the soil Inf (+) Non – inf Total

p < 0.001; OR = 9.07; CI 95%: 4.48 – 18.33 People who contact direct with soil in daily activities was more 9.07 times risk

to be infected Strongyloides spp than other

Table 3.9 Multivariate analysis of factors related to Strongyloides spp

infection

value correction OR

Educational level (below high school) No > 0.05 1.03

Using toilets (Unhygienic) Yes < 0.01 3.30

Living habits (contact with soil) Yes < 0.05 2.69

Strongyloides spp infection in Duc Hoa district was associated with: male, over 60 of age, poor and nearby poverty economic status, farmer, using unhygienic toilets and contacted soil habits in daily life

3.2 Determine component species of Strongyloides in human strongyloidiasis disease

3.2.1 Survey pathogens by morphology

Table 3.10 Analysis stool tests in human strongyloidiasis (n = 79) Name of technique Number Percentage (%)

The direct smear test alone was only able to detect 58.2% of total cases,

much lower than the culture technique

Table 3.11: Morphology index of larvae stage 1 (n = 79)

Horizontal size (µm) 18.47 ± 0.61 16.5 – 20.0 Length of esophagus (µm) 75.7 ± 5.1 64 – 90.1 Bucal cavity length (µm) 4.4 ± 0.3 3.9 – 5.3 Ratio esophagus length/body length (%) 27.1 ± 2.1 21.0 – 34.0

1st stage larvae: 100% with pointed tail, average length 279m, esophageal length averaged 27.1% compared to body length

Table 3.12: Morphology index of larvae stage 2 (n = 79)

Length of esophagus (µm) 244.7 ± 17.9 210.3 – 132.0 Bucal cavity length (µm) 4.5 ± 0.5 4.0 – 6.0

Ratio esophagus length/ body length (%) 42.5 ± 3.8 36.0 – 53.0 Horizontal size at endpoint of tail (µm) 2.6 ± 0.2 2.2 – 3.4 Endpoint of tail (blunt pointed/split 2) 11/68 (13.9 %/ 86.1 %) When cultured at day 3, 2nd larvae stage has slender shape, the endpoint

of tail has blunt pointed or split 2 in shaped

Table 3.13: Morphology index of free-living male (n = 5)

Body length (µm) 778.8 ± 27.7 740.8 – 812.6 Horizontal size (µm) 45.1 ± 1.7 43.4 – 47.6 Length of esophagus (µm) 131.3 ± 6.9 120.0 – 136.2 Bucal cavity length (µm) 7.1 ± 0.6 6.6 – 8.1

Ratio esophagus length/ body length (%) 17.0 ± 1.0 16.0 – 18.0 Length of genital spines (µm) 33.4 ± 0.9 32.1 – 34.4

Free-living male of Strongyloides spp had 778.8 µm average length, pointed tail

Table 3.14: Morphology index of free-living female (n = 3)

Body length (µm) 916.7 ± 21.6 892.6 – 934.2 Horizontal size (µm) 46.2 ± 1.7 44.2 – 47.5 Length of esophagus (µm) 130.6 ± 4.6 127.4 – 135.9 Bucal cavity length (µm) 6.8 ± 0.4 6.5 – 7.2 Ratio esophagus length/body length (%) 14.3 ± 1.2 14.0 – 15.0 Distance between vulva with head (% of

Free-living female of Strongyloides spp had 916.7µm average length, vulva was located near the middle of the body, slightly forward from 0 to 1% of the body length

3.2.2 Results of real-time PCR in identification of Strongyloides spp

In 79 samples of 2nd stage larvae were collected from 79 patients who infected with Strongyloides spp in Duc Hoa district DNA extraction was conducted according to the manufacturer's procedure, but only 70/79 samples response the requirements (88.6%) A total of 70 samples were included in the real-time PCR test

Perform real-time PCR DNA Strongyloides spp on collected samples to identify genus of Strongyloides based on 28S rRNA gene sequences U3949 The identification of species S stercoralis based on Stro 18S gene sequences AF279916 and identified species S ratti based on the sequence Srat 28S gene location DQ14570

Table 3.15 Components of Strongyloides spp determined

by real-time PCR (n = 70)

Species No Percentage (%)

C: Negative control (H2O); S: DNA sample of Strongyloides spp

All 14 products of 2-step nested PCR included 4 samples with S ratti presence and 10 S stercoralis random samples (obtained from real-time PCR), were sequenced genome

Table 3.16 Analyzing results of sequence of 14 larvae samples in the study

The species components were similarity very high to the isolates that published in the gene bank

Figure 3.2 Phylogenetic tree was built on group 10 S stercoralis larvaes

Figure 3.3 Phylogenetic tree was built on group 4 S stercoralis larvaes 3.3 Describe the clinical symptoms, paraclinical and evaluate results of treatment for strongyloidiasis by ivermectin single dose

The total number of patients tested positive with Strongyloides spp was 79 cases Average age: 52.97 ± 27.64 (min - max = 22 - 84)

3.3.1 Clinical and paraclinical symptoms