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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.

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

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



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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

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(%)

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)


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


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


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


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


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


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


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

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


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(%)



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(%)

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 (

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%)


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%)


(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%)


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%)

6 (5,9%)

> 0,05


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



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

44

44

42.26 ± 1.71

II

42 47

44

42

45

45

43

44.05 ± 1.65

III

44 48

49

47


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%.


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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


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

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%),


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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

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 (
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 (
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.
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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.