Tóm tắt Luận án Studying the actual situation, various factors related to human Strongyloides spp infection and ivermectin effectiveness in Duc Hoa district, Long An province (2017 – 2018)

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 Strongyloides inf. Age group Inf. (+) Non – inf. Total < 15 0 240 240 15 – 60 55 740 795 Over 60 24 131 155 Total 79 1,111 1,190 p < 0.01 (Fisher test). OR = 2.46; CI 95%: 1.47 – 4.12 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. Education Inf. (+) Non – inf. Total Below High school 64 758 822 High school and more 15 353 368 Total 79 1,111 1,190 p < 0.05; OR = 1.98 ; CI 95%: 1.12 – 3.54 8 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 Poor and nearby poverty 45 125 170 Average 18 504 522 Well and above 16 482 498 Total 79 1,111 1,190 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 Strongyloides inf. Job Inf. (+) Non – inf. Total Farmer 45 249 294 Other 34 862 896 Total 79 1,111 1,190 p < 0.001; OR = 4.58; CI 95%: 2.87 – 7.31 Farmer was 5.58 times risk to be infected Strongyloides spp than other job. Table 3.7 Relation between Strongyloides spp infection and using toilet status Strongyloides inf. Using toilets Inf. (+) Non – inf. Total Unhygienic 41 96 137 Hygienic 38 1.015 1.053 Total 79 1,111 1,190 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 Yes 70 513 583 No 9 598 607 Total 79 1,111 1,190 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. 9 Table 3.9 Multivariate analysis of factors related to Strongyloides spp infection Variable Relation P value OR correction Sex (male) Yes < 0.01 3.26 Age group (> 60) Yes < 0.01 2.89 Educational level (below high school) No > 0.05 1.03 Economic status Yes < 0.01 2.08 Farmer Yes < 0.05 2.07 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 (%) Direct smear 46 58.2 Modified Harada Mori culture 74 93.7 Coordinate both techniques 79 100 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) Structure Mean ± SD Min – max Body length (µm) 279,9 ± 17,5 240.6 – 320.3 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 Pointed tail shape 79/79 (100%) 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) Structure Mean ± SD Min – max Body length (µm) 576.4 ± 24.9 510.0 – 632.0 Horizontal size (µm) 16.9 ± 1.1 15.3 – 19.6 Length of esophagus (µm) 244.7 ± 17.9 210.3 – 132.0 Bucal cavity length (µm) 4.5 ± 0.5 4.0 – 6.0 10 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) Structure Mean ± SD Min – max 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 Pointed tail shape (100%) 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) Structure Mean ± SD Min – max 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 body length) 49 ± 1.0 48.0 – 50.0 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) 11 Species No. Percentage (%) S. stercoralis 66 94,2 S. ratti 2 2,9 Co-infection S. stercoralis, S. ratti 2 2,9 Total 70 100 The ratio of S. stercoralis was 97.1% (68/70) dominantly, of which 2.9% was co-infected with S. ratti. 3.2.3 Results of Nested - PCR and genetic sequencing A B Figure 3.1 Electrophoresis products of PCR I (A) and PCR II on agarose gel 1,5%; M: scale of DNA 100 bp; 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 No Code Highest similarity (%) Gene code Species 1 1 99,5 AB923888.1 S. stercoralis 2 7 98,6 AB923888.1 S. stercoralis 3 11 99,4 AB923888.1 S. stercoralis 4 15 99,7 AB923888.1 S. stercoralis 5 20 95,6 MK369923.1 S. stercoralis 6 25 98,5/98 AB923888.1/ AB453329.1 S. stercoralis/ S. ratti 1002 bp 500 bp 500 bp 975 bp 12 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 7 26 91,3 LL999104.1 S. stercoralis 8 35 100,0 LL999088.1 S. stercoralis 9 42 100,0 LL999110.1 S. stercoralis 10 47 99,2 AB923888.1 S. stercoralis 11 50 100,0 MK369923.1 S. stercoralis 12 54 98,0 AB923889.1 S. ratti 13 65 99,3/98,0 AB923888.1/ AB453329.1 S. stercoralis/ S. ratti 14 66 98,0 LN609412.1 S. ratti 13 The number of patients infected with Strongyloides spp completely without clinical symptoms was 10.1%. Table 3.17 Clinical symptoms in human strongyloidiasis (n = 79) Symptoms No. Percentage (%) Detail No./Percentage (%) Abdominal pain 58 73.4 Epigastric 33/79 (41.7%) A round the navel 17/79 (21.5%) Hypogastrium 8/79 (10.1%) Diarrhea, 33 41.8 Urticaria 45 57.0 The arms 36/79 (45.6%) Body 9/79 (11.4%) Headache 49 62.0 Lose-weight 9 11.4 CLM 3 3.8 Gastrointestinal symptoms had a high rate include: abdominal pain accounted 73.4% and diarrhea symptom was 41.8% Table 3.18 Percentage of patients with hyper-eosinophilia (n = 79) Value Number Percentage (%) Eosinophil/µl blood (E) Normal (< 500) 32 40.5 Increase (≥ 500) 47 59.5 Total 79 100 Mean = 694.56 ± 461.92. t test= 3.744; p value < 0.01; Distance of mean = 194.5; CT 95% (91.1 –298.0) Level of hyper- eosinophilia Normal (<500) 32 40.5 Mild increase (500 - 1500) 41 51.9 High increase (>1500) 6 7.6 Total 79 100 There was 59.5% of patients had hyper- eosinophil in their blood. The mean of eosinophil was 694.56, significantly different from the normal threshold p <0.01. 3.3.2 Effectiveness of treatment: Clinical – paraclinical Table 3.19 ELISA test results in gastrointestinal strongyloidiasis (n = 79) Value Number Percentage (%) ELISA test Positive 76 96.2 Negative 3 3.8 total 79 100 Mean of positive value = 32.37 ± 23.26 NTU.Test t = 15.2; p < 0.01; Distance of mean: 22.4; CI 95% (18.6 – 24.2) 14 There was only 96.2% of strongyloidiasis patient had positive results that found antibodies againts to Strongyloides spp. Table 3.20 Responds of clinical symptoms in patients after 6 weeks of treatment (n=57) Sign/symptom Before treat. Post – treatment Cured (%) Reductio n (%) No reduction (%) Three classical symptoms of strongyloidias is Abdomina l pain 48 24 /48 (50) 12/48 (25) 12/48 (25) Diarrhea 26 10/26 (38.4) 8/26 (30.8) 8/26 (30.8) Urticaria 39 4 /39 (10.3) 20/39 (51.2) 15/39 (38.5) Headache 42 10/42 (23.8) 2/42 (4.8) 30/42 (71.4) Lose-weight 8 2/8 (25.0) 0/8 (0) 6/8 (75.0) Cutaneous larva migrans 2 2/2 (100) 0 (0) 0 (0) The symptoms improved with more cured level than reduction level, conversely, urticaria was reducced more than cured level. Table 3.21 Ratio of larvae clearance post-treatment (n = 79) Progress of test results post- treatment Before treat. 2 weeks 4 weeks 6 weeks Number of sample 79 75 61 57 Number of infected cases (%) 79 2 (2.7%) 3 (4.9%) 3 (5.3%) Number of larvae cleared cases (%) 73/75 (97.3%) 58/61 (95.1%) 54/57 (94.7%) The prevalence of larvae clearance in faeces was 94.7% at 6 weeks post-treatment. Table 3.22 Ivermectine effectiveness in the treatment (n = 57) Detail Number Percentage (%) Cured Stool test (-) and clinical symptoms (cured) 18 31.6 Reduction Stool test (-) and clinical symptoms (Reduction) 32 56.1 No reduction Stool test (+) 3 5.3 Stool test (-) and clinical symptoms (No reduction) 4 7.0 Total 57 100 15 Effectiveness of Ivermectine reached 87.7% at level from reduced to cured, while the efficacy of larvae cleaning reached 94.7%. 3.3.3 Adverse effects of ivermectin Table 3.23 Ratio of side effects of ivermectin (n = 79) Side effects Number Percentage (%) Dizziness, increased headache 1 1.3 Nausea 1 1.3 Diarrhea, loose stools (increased) 4 5.1 Erythema rash on the skin 1 1.3 Increased itching 2 2.5 The adverse symptoms: diarrhea, loose stools (increased) accounted for 5.1%, nausea, dizziness accounted for a lower percentage (1.3%) and also recovered. CHAPTER 4 DICUSSIONS 4.1 Determine the actual situation and factors related to human Strongyloides spp infection in Duc Hoa district, Long An province in 2017-2018. 4.1.1 Actual situation of Strongyloides spp in Duc Hoa district 4.1.1.1 The prevalence of Strongyloides spp infection Summing up the data at 5 study sites, determine the general prevalence of Strongyloides spp infection in Duc Hoa district is 6.64%, which was classify as an endemic of the disease (table 3.1). In 2 study at Phu My Hung and Phu Hoa Dong communes of Cu Chi district, Ho Chi Minh city, located adjacent to the East with Duc Hoa district, by the same technique with this study, the authors determined the prevalence of infection turn were 12.6% (n = 294) and 9.2% (n = 766), higher than our study. Myo Pa Pa (2018) studied in Myanmar to determine the overall infection rate of 5.7%, nearly equal to prevalence of this study. This similarity was explained by the same culture techniques to applicable in diagnosis, although the author Myo Pa Pa applied the technique of agar culture while we used the technique of culture with filter paper. The infection rate found in Duc Hoa district in this study was still lower than P. Laoraksawong (2017) in Thailand, Virak Khieu (2014) in Cambodia, Senephansiri P. (2017) in Laos with the infection rate were 23%, 21% and 17.1% respectively. The high prevalence of this infection may lead to the conclusion that those in Southeast Asia were the endemic areas of Strongyloidiasis. Table 3.1 shows that Duc Lap Thuong commune has the highest prevalence of Strongyloides spp infection at 12.4%, the lowest is in Duc Hoa town (2.1%). An Ninh Tay and Hiep Hoa communes have approximately equal rate: 4.4% and 4.5%. This result shows that, even within a district, each other study site had 16 different results, possibly because the relevant factors have an impact and needed to analyze clearly in the subsequent results in the study. 4.1.1.2 Related factors with Strongyloides spp infection The study data was collected according to the design at each site, aggregated for the main target of Duc Hoa district. Therefore, to eliminate the general bias factors, after univariate analysis of each factor, the multivariate analysis model was included in the analysis of the relation between Strongyloides spp infection and related factors in Duc Hoa district. There was relation between Strongyloides spp infection and male (p <0.001) in table 3.2. Table 3.9 of additional multivariate analysis showed that sex was associated with Strongyloides spp infection (p <0.01) and had adjusted OR index. Thus, in the community of Duc Hoa district, sex was a related factor and male was 3.26 times at risk to be infected than women. This result was similar to study result of Laoraksawong P. et al (2018) in Thailand (n= 526 ) with 4 times higher risk in male. Compared to the study in Cu Chi in 2004, men were 2.96 times at risk than women. In Cambodia, in two studies at different districts, Virak Khieu et al (2014) also identified that men was 1.7 times at risk more than women. Thus, from the data of this study, in collaboration with many of other studies found an association between male sex and the prevalence of Strongyloides spp infection, may lead to sex is a related factor with Strongyloides spp infection in community. This study surveyed the relation between Strongyloides spp infection and groups of age (below 15, 15 - 60 and > 60). The results of multivariate analysis also noted a significant relation (p <0.01), the risk in people over 60 years was 2.89 times higher than others. This result was different from the three studies in Cu Chi in 2001, 2004 and 2017. In that studies, the authors did not identify the related to age although the studies only interested in two age groups in and out of labor. There was relation between Strongyloides spp infection and p <0.05 in table 3.4, those with education level below high school was 1.98 times at risk compared to the group had higher educational level. However, when included in the multivariate analysis model, Table 3.9 showed that education levels above and below high school are not related to the Strongyloides spp infection situation in Duc Hoa district, the OR index was adjusted equal to 1.03. Thus, the relation found in univariate analysis is not strong enough, or due to other factors affecting and causing interference. In 2018, Myo Pa Pa studied in Myanmar, Suntaviritun P. et al studied in Thailand, determined that there was no relation in education level and Strongyloides spp infection. This study gave similar results, although the mentioned authors used secondary school level to divide group in that studies. In tables 3.5 and 3.9, there was relation between economic status and Strongyloides spp infection (p <0.01). Infection was more present in the poor and nearby-povety group with 2.08 times at risk than other. Although Duc Hoa district 17 has been developing economic strongly in recent years, but the index identifying poor, neaby-povety and average households applied in the study, was generally prescribed for the rural level nationwide. This index may not really suitable for fast changing of economic conditions. But the study results found consistent with the result from many studies in the world showed that the Strongyloides spp infection was associated with poverty. There was a relation between Strongyloides spp infection and farmer. The results in tables 3.6 and 3.9 showed that farmer was really related to this infection (p <0.05) and the adjusted OR index was 2.08 times. Thus, when working in farm, the probability of larvae from contaminated soil to invasive the body and causing disease would be higher. This result was similar to the study of Senephansiri P. in Laos (2017), two studies of Virak Khieu et al (2014) at 2 different locations in Cambodia, identified that farmer was at higher risk for Strongyloides spp infection than other jobs. Table 3.7 and Table 3.9 showed that there was a significant different between the using unhygienic toilet with other group in Strongyloides spp infection (p <0.01). People who used unhygienic toilets was 3.3 times at risk higher than using hygienic toilets. So, in Duc Hoa district, using toilets was the related factor. Investigation of the relation between the direct contact with soil in daily activities and the status of Strongyloides infection, identified the relevance and OR index as 2.69. This result is similar to the authors V.T.L Binh studied in 2 communes of Cao Dien, Phu Tho and Duong Thanh in Thai Nguyen province in 2014, Senephansiri P in Laos and Myo Pa Pa in Myanmar. Therefore, the direct contact with soil in daily activities is an important risk factor for the infection of Strongyloides spp in Duc Hoa district. 4.2 Determine component species of Strongyloides in human strongyloidiasis disease 4.2.1 Survey pathogens by morphology Table 3.10 shows that in 79 patients with a gastrointestinal strongyloidiasis, the first times direct smear test is only able to detect 58.2%. This prove that the detection ability of direct smear technique in strongyloidiasis diagnosis is quite low. Therefore, this is not recommended as the main technique to be used to study to screen Strongyloides spp infection for community. Modified culture technique (Sasa 1986) in the study detected 93.7% cases at the first test. This result was higher than 78.4% in a study at Cu Chi district (2004), 47.8% of Rayzan H. Z et al (2012) in Egypt. There were still 5 cases (6.3%) in the first culture had negative result test while the direct smear technique had positive result. For that reason, the combination of the two techniques has resulted in better detection and proves that no technique was absolute perfect. 4.2.1.1 1st stage larvae (rhabditiform) Table 3.11 shows that the larvae has average length of 279.9 µm, an average width of 18.47 µm. Thus, compared to Grove DI (1989), Prayong R. et al. (2013), 18 the body length of larvae in this study tend to be longer because that authors recorded that the length is from 200 - 250 µm, while the horizontal size of larvae is similar with this study. The reason of difference is explained by the larvae that cause disease in the community are often chronic, the density of larvae is low, the symptoms cause not massively equivalent to the longer time of larvae in human colon, will grow longer leading to body length is longer. The average length of the esophagus is 75.7 µm, with the bulge forms, and the average ratio compare to the body length is 27.1%, completely consistent with the structure of stage 1 larvae. 100% larvae have pointed tail, indicating that all measured larvae ear stage 1 larvae. The average length of 1st stage larvae bucal cavity is 4.4 µm, the min - max is 3.9 µm - 5.3 µm. This is an important structure to distinguish with 1st stage hookworm larvae that have long length bucal cavity, suitable for the authors Grove D. I (1989), T. T. Hong (2017) and Prayong R. (2013). From the above results, they are confirmed that all surveyed larvae were 1st stage larvae of the Strongyloides spp are confirmed that all surveyed larvae are 1st larvae of the Strongyloides spp. 4.2.1.2 2nd stage larvae 2nd stage larve of Strongyloides spp has 576.4 µm body length in average, the average horizontal size is 16.9 µm. The results is consistent with Grove D.I (1989), Prayong R. (2013), which reported from 450 - 600 µm, and horizontally slender than 1st stage larvae. Average length of esophagus is 244.7 µm, also tubular, and has an average ratio with body length as 42.5%. This is entirely consistent with the structure of 2nd stage larvae with tubular esophagus and over one-third of the body length from literature. 100% of the larvae has not pointed tail, of which 86.1% has split tail in 2, indicating that all larvae molted in past. Thus, these 2nd stage larvae had blunt or split 2 in shape(100%) , an average endpoint width is 2.6 µm, there is a necessary indicator to show that their tail was not as sharp as the tail of hookworm larvae. This indicator accurately identify all surveyed larvae collected from the culture sample are belonged to Strongyloides spieces. 4.2.1.3 Free-living adult worms males and females Table 3.13 shows that the average length of the male is 778.8 µm, the average horizontal size is 45.1 µm. Although this result was higher than reporting of Prayong R. (about 0.7mm), completely consistent with Grove D.I. (1989) is from 700 to 900 µm. Different from the 2nd stage larvae, the esophagus of adult worms grow to be horizontal and shorter in length. The average length of the esophagus is 131.3 µm, accounted for the average ratio 17% compared with the body length, the intercourse spines are 33.4 µm average of length, determining the sex of the worm as an adult male. Table 3.14 shows that the average length of female worms is 916.7 µm, the average horizontal size is 46.2 µm. This result is within the threshold but at a low 19 level compared to the reported of Grove D.I. (1989). The esophagus of female worms has 130.6 µm of an average length, similar to those of male worm, but the average ratio compared to the average body length is only as 14.3% because the female's body length is longer. Two uterine branches contain eggs lying symmetrically through the vulva. According to Grove D.I. (1989), the distinction of Strongyloides spieces including S. stercoralis, S. ratti, S. fuellebornii, etc. and others, can only be based on the shape of the oral structure, which is difficult to observe. For the above reasons, in terms of morphology corresponding to the design in this study, accurate samples were identified as 1st, 2nd stage larvae, male and female adult worm of Strongyloides spp in limited. 4.2.2 Results of real-time PCR in identification of Strongyloides spp Table 3.15 statics 70 samples, records that S. stercoralis account for 97.1%, of which 2.9% is co-infected with S. ratti. Results were also found in components species with 2.9% of S. ratti infection alone. The dominant S. stercoralis (97.1%) were consistent with the N.V.DE (2017) and D.T. Hong (2018) identified 100% as S. stercoralis, although the authors did not use t