Nghiên cứu một số đặc điểm lâm sàng, cận lâm sàng, kết quả điều trị bệnh do Gnathostoma spp, định danh mầm bệnh trên người và vật chủ trung gian tại phía Nam Việt Nam (20162017) tt

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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:
1. To describe the clinical and paraclinical characteristics of human
Gnathostoma spp infection in Southern Vietnam (2016-2017).
2. To evaluate the treatment outcomes of ivermectin on
Gnathostoma spp at the study sites.

3. To provide species identification of Gnathostoma spp on humans
and ontermediated hosts using morphological and molecularbiological techniques.


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SCIENTIFIC SIGNICICANCE, PRACTICALITY,
AND INNOVATION OF THE THESIS
1. 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.
2. 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.
3. 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 7 annexes.
Chapter 1. GENERAL MEDICAL LITERATURE REVIEW
1.1. Introduction of the Gnathostoma spp.


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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).


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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 4
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
1..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.


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Chapter 2. SUBJECTS AND METHODS
2.1. Objective 1 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 về dạ dày tá tràng.
 A positive pregnancy test or breastfeeding women.

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 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


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+ 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 2 months and 6 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
2 months and 6 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 3. RESULTS


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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

3 (2.68)

Daklak

12 (10.71) Vinh Long

3 (2.68)

Gia Lai

12 (10.71) Bac Lieu

2 (1.79)

Quang Ngai

7 (6.25)

Ben Tre

2 (1.79)

Ho Chi Minh city

7 (6.25)

Binh Dương

2 (1.79)

An Giang

4 (3.57)

Đong Nai

2 (1.79)

Long An

4 (3/57)

Hau Giang

2 (1.79)

Tien Giang

4 (3.57)

Kon Tum

2 (1.79)

Ca Mau

3 (2.68)

Soc Trang

2 (2.79)

Can Tho

3 (2.68)

Đong Thap

1 (0.89)

Dak Nong

3 (2.68)

Kien Giang

1 (0.89)

Khanh Hoa

3 (2.68)

Lam Dong

1 (0.89)

Phu Yen

3 (2.68)

Ninh Thuan

1 (0.89)

Quang Nam

3 (2.68)

Tay Ninh

1 (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..2. Patient
distribution by age and gender (n = 112)
Age group

Male

Female

p-value

< 15

15 (75%)

5 (25%)

< 0.05

≥ 15 - < 30

5 (33.33%)

10 (66.67%)

> 0.05

≥ 30 - < 45

7 (25%)

21 (75%)

< 0.05

≥ 45

15 (30.61%)

34 (69.39%)

< 0.05

42 (37.5%)

70 (62.5%)

Total


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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..3. 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..4. 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

5

4.46

The Kinh ethnic represented the predominant incidence of Gnathosoma
spp., while other ethnic minority groups occupied the rest 4.46%.


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3.1.2. Risk factors for Gnathostoma spp. infestation
Table 3.6. Some possible risk factors (n = 112)
#

Risk factors

Pos.(+) Rate (%)

1

Raw fresh-water fish or salad

80

71.43

2

Uncooked frog meat

73

65.18

3

vegetables forms of single serves or salads

72

64.29

4

Steamed or sliced snails mixed with vegetables

69

61.61

5

Undercooked or rare fresh-water eels

49

43.75

6

Other aquatic foods salad

43

38.39

7

Snake salad or snake liquid blood

36

32.14

8

Sliced raw fish, prawn with wasabi

31

27.68

9

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 (%)

1

< 7 days

3

2.68

2

≥ 7 - < 15 days

13

11.61

3

≥ 15 - < 30 days

35

31.25

4

≥ 30 - < 45 days

35

31.25

5

≥ 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 (%)


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Mucocutaneous tissue

92

82.14

Neural system

50

44.64

Digestive system

37

33.04

Ocular organs

13

11.61

Respiratory tract

7

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

8

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


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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)

9

8.04

Anorexia plus nausea

5

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

4

3.57

Persistent cough (dry without sputum)

2

1.79

Short breath

2

1.79

Sweezing

2

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

7

6.25

Ocular disorder (blurred vision)

6

5.36

Blurred vision (diplopia)

5

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)

7

6.25


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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

3 (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 (<40 U/L)

99 (88.39)

Normal (<40 U/L)

Increased ≥40 U/L

13 (11.61)

Tăng ≥ 40 U/L

Tested samples
24.83 ± 14.43
102 (91.07)
10 (8.93)

The proportion of patients within the normal range of SGOT (<40 U/L)
was 88.39%, and higher the normal range (≥40 U/L) was 13.61%.
Similarly, the patients with normal range of SGPT (<40 U/L) represented
91.07%, and higher the normal range (≥ 40 U/L) was 8.93%.
Table 3.17. Immunodiagnosis result of ELISA test (n = 112)
S/Co index

n(%)

≥ 1,0 - < 1,2

95 (84.82%)

≥ 1,2 - <1,5

11 (9.82%)

≥ 1,5

6 (5.35%)

And all patients with Gnathostoma spp. infection were screened and
selected if their sample/cut-off values ≥ 1.0.
3.2. Evaluation of ivermectine in the treatment for human
gnathostomiasis


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Table Error! No text of specified style in document..18. Clinical and
laboratory manifestations before and after treatment 2 months posttreatment
Clinical and lab symptom

Befor Rx

After 2 mos

(n = 112)

(n = 107)

Mucocutaneous tissue

92 (82%)

71 (66.40%)

< 0,05

Digestive tract

37 (33%)

19 (17.80%)

< 0,05

Neural system

50 (44.60%)

41 (38.30%)

< 0,05

7 (6.30%)

4 (3.80%)

> 0,05

Ocular vision

13 (11.60%)

8 (7.50%)

> 0,05

ELISA titer ≥ 1.0

112 (100%)

49 (45.80%)

< 0,05

9 (8.0%)

5 (4.70%)

> 0,05

Eosinophil >500 cells/ mm3

31 (27.7%)

19 (17.80%)

< 0,05

SGOT ≥ 40 U/L

13 (11.6%)

31 (29%)

< 0,05

SGPT ≥ 40 U/L

10 (8.9%)

15 (14%)

> 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 6 months post-treatment
Before Tx

After Tx 6 mos.

(n = 112)

(n = 102)

Mucocutaneous

92 (82%)

8 (7.8%)

< 0,05

Digestive tract

37 (33%)

1 (1%)

< 0,05

Respiratory tract

7 (6,3%)

1 (1%)

> 0,05

Ocular vision

13 (11.6%)

1 (1%)

< 0,05

Neural system

50 (44.6%)

10 (9.8%)

< 0,05

ELISA ≥ 1.0

112 (100%)

12 (11.8%)

< 0,05

9 (8.0%)

5 (4.9%)

> 0,05

Organs/Tissues

WBC >10.000/ mm3

p-value


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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%)

6 (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 6 months (n=102)
Treatment outcome

n

Rate (%)

Cure

94

92.16%

Partial recovery, reduction

4

3.92%

Not recovery

4

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

7

6,25 Early: 1h; Late: 48hs

Abdomen pain, nausea

8

7,14 Early: 2h; Late: 24hs

Loose stool or diarrhea

1

0,89 1 ca: 24hs

Muscle pain

1

0,89 1 ca: 48hs

Fever

0

Itching, skin rash

6

0
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.


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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)

6

10

12

18

20

2,4

2,8

8

7

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
2

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

6

12

18

20

8

10

7

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..3. 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..4. 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..5. 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
1
5
6
4
Larva
sample

1

Homogenous
tương
5 đồng
6 (%)
4

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

1

5

6

4

effect
Mã số gen của Ngân hàng
gen thế giới
AB181156 G.doloresi
AB180100 G.doloresi
JN408299 G.doloresi
AB181158 G. hispidum


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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 4. 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 (<7 days) in our study
(2.68%) was much lower than that of Huynh Hong Quang et al. (28.57%).
This might be due to the differences in terms of study participants and
time. Gnathostoma patients with healthy status represented up to 98.21%,
whereas malnutritious patients stood for only 1.79%. This result was in
agreement with the study conducted by Nguyen Van Chuong et al. (2010)
reporting the similar result (97.4% of healthy patients of gnathostomiasis).
Our data revealed various reasons for hospitalization: 82.14% of patients
reportedly needed to be hospitalised upon having symptoms pertaining to
the cutaneous and subcutaneous form; 44.64% of neurological form, and
33.04% of the gastrointestinal manifestations. Other reasons included
ocular form (11.61%) and symptoms of the respiratory system (6,25%).
- Clinical manifestations
Patients with cutaneous and subcutaneous symptoms represented the
highest proportion (81.25%), followed by those of the neurological system
(45.53%), digestive system (34.82%), occular symptoms (11.6%), and
respiratory system as lowest incidence (7.14%). The involvement of
multiple organs of the disease is as a result of the migration of
Gnathostoma spp that caused cutaneous or visceral symptoms depending
on the location of migration and the reaction of the body as well. Twentyfour hours after being digested by the patients, the larvae will live coiled
up in the patient’s intestinal wall. 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 manifestation (11.61%). The frequencies of clinical
features may vary from regular occurrence (41.96%) to intermittence


22

(39.28%). Cutaneous and subcutaneous symptoms are the most
predominant clinical manifestations of gnathostomiasis, which typically
presents with intermittent migratory swellings, vary in size and may be
pruritic, painful, or erythematous. These proportions were in agreement
with the study conducted by Huynh Hong Quang, reporting proportions of
nodular migratory swellings in 56.0-78.2% of the patients.
On the digestive tract, epigastric pains represented the highest
proportion (31.25%), followed by digestive disorders such as loose and
thick stools (8.04%), and poor appetite and nausea (4.46%). Less common
respiratory symptoms were found in the patients infected with
Gnathostoma spp (10/112), including a small proportion of patients with
angina (3.57%). In the studied patients, the ocular symptoms represented
relatively small proportions, including pains of the eyelids (6.25%), vision
impairment or blindness (5.36%), and diplopia (4.46%). Our study results
were in line with those studies conducted in the country and abroad.
Major neurological manifestations of the studied patients with
gnathostomiasis included headache (possible with diziness) as the highest
proportion (35.71%), diziness (27.68%), and sleep disorders (6.25%) .
4.1.3. Laboratory parameters
Most patients (92.0%) 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%, 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 (<40 U/L)
was 88.39%, and higher the normal range (≥40 U/L) was 13.61%.
Similarly, the patients with normal range of SGPT (<40 U/L) represented
91.07%, and higher the normal range (≥ 40 U/L) was 8.93%. No patients
with twice values of normal SGOT and SGPT levels were reported so far.
And all patients with Gnathostoma spp. infection were screened and
selected if their sample/cut-off values ≥ 1.0.


23

4.2. Evaluation of ivermectin following two months and six months of
treatment for gnathostomiasis patients
Post-treatment follow-ups were conducted on patients treated with
ivermectin (IVM) for Gnathostoma spp at single doses to evaluate any
changes in clinical and paraclinical manifestations prior to treatment and
two and six months following IVM administration. 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%. 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 and erythema, 1/112
(0.89%) had loose stools, 1/112 (0.89%) had mild myalgia. Particularly, no
cases of increased transaminase, reduced WBCs, or fever.

4.3. Species identification of Gnathostoma spp on intermediate hosts
On the morphological method, 81 larvae of Gnathostoma spp were
collected from livers of eels and fish using the renovated myolysis
technique; and 1 Gnathostoma larvae was collected from human sample.
And by molecular identification, 6/10 samples were identified as G.
spinigerum, 3/10 samples as G. doloresi, and 1/10 sample as G. hipidum.
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSION
1. Description of the clinical and sub-clinical characteristics of human
Gnathostoma spp. in the Southern Vietnam (2016-2017)
1.1. Clincal manifestations
- Dermal and mucocutaneous lesions acounted for 82.14% with pruritus,
urticaria 75%, skin welts 33.39%. Manifestations in the nervous system
accounted for 44.64%, with headache 35.71%, vertigo 27.68%.
Manifestations in the digestive organs accounted for 33.04%.
- Manifestations in the visual organs accounted for 11.61%, and
manifestations in the respiratory organs accounted for 6.25%.
1.2. Sub-clinical characteristics
- Patients with eosinophilia accounted for 27.68%, and raised SGOT,
SGPT enzymes accounted for 11.61% and 8.93%, respectively.


24

2. The utcome treatment for human Gnathostomiasis by ivermectin
Single dose 0.2mg/kg IVM treatment regime is highly effective in
treatment of human gnathostomiasis. Proportion of cured patients at 6
months post-treatment accounted for 92.16%. The positive ELISA reduced
from 100% to 11.8%, eosinophilia reduced from 27.68% to 9.9%.
Unexpected events of IVM single dose 0.2mg/kg were low: vergito,

headache (6.25%), stomachache, nausea (7.14%), itch/erythema (5.36%).
3. Identification of Gnathostoma species in human and intermediate
host by morphology and molecular biology
Proportion of freshwater ells collected from the Ho Chi Minh city’s
wholesale markets infected with Gnathostoma accounted for 2.57%. On the
Gnathostoma spp. morphology: Gnathostoma larvae have average length of
2.5 ± 0.64 mm, average width of 0.24 ± 0.05 mm; have 4 rows of spines on
the head bulb, the first row has average 42.26 ± 1.71 spines, the second row
has average 44.05 ± 1.65 spines, the third row has 48.65 ± 1.41 spines, the
fourth row has 51.28 ± 2.49 spines. Bio-molecular species identification of
Gnathostoma was conducted by genetic sequencing, data showed that three
species of Gnathostoma in the intermediate host were identified including
G. spinigerum, G. doloresi and G. hipidum. G. Spinigerum was also
identified in the human host. The 5.8S rRNA-ITS2 sequence region was
100% homologous with the 3 species G. spinigerum, G. doloresi and G.
hipidum that were published on the world GenBank.
RECOMMENDATIONS
The study results showed that G. spinigerum, G. doloresi, and G.
hispidum were detected on an additional intermediate freshwater eels host.
Unfortunately, only one gene-confirmed human G. spinigerum larva was
collected during the study, however, according to literature, G. doloresi
infected human as well. Therefore, it is necessary to carry out further
studies to determine if Vietnamese people are infected with this species or
not.