China: A novel nematode discovered through investigation of an unknown disease

[Guest guest]

http://www.aiabeijing.org/netprints/01/01/010116.htm

 

 

 

A novel nematode discovered through investigation of an unknown disease

 

 

 

ZHU Naishuo (Ph.D & MD)

 

SHA Zhe

 

YU Shanqian

 

Lab. Of Virology & Immunology

Dept. of Microbiology

School of Life Sciences

Fudan University

Shanghai, 200433

P.R.China

 

Tel: 86-21-65641215(Lab)

 

E-mail: nszhu

 

Author responsible for correspondence about the manuscript: ZHU Naishu

Source of grants: supported by Fudan University and Mrs LI Hongliu

 

 

 

CMJ 2001 114 & #65288;1 & #65289; 89-95

 

Abstract

 

Objective: To find out the pathogenic parasite of an unknown disease

whose symptoms are creeping eruptions on the skin, migratory piercing

pain and hard itch in muscle and tissue, and look into the

classification of the parasite.

 

Methods: Using laboratory methods such as blood diagnosis, urine

analysis, mucous excretion analysis to obtain and observe the

pathogenic parasite and in the mean time referring to the patients & #8242;

medical record and physical examinations for further information's.

 

Results: Using gene detection of numerous kinds of viruses gave out

negative results. From the blood of the patient one kind of infant

nematode with a single hook on its head was found under microscope,

but the species is not yet determined.

 

Conclusions: The disease resembles LM in many aspects but the

pathogenic parasite and some certain symptoms differ from these two.

The nematode cannot be G.spinigerum but most probably is a close

relative to it. Judging from its outlook, especially the single hook

on its head, we denominate the worm as " Strongylus monospinigerum "

temporaly.

 

Key Words: Strongylus monospinigerum, Creeping eruption, Gnathostoma

hispidum (G.hispidum), Gnathostoma spinigerum(G.spinigerum), larva

migrans (LM)

 

Introduction

 

A strange disease was met in Shanghai, P.R.China. After careful and

repeated examination of the bloods of three patients from one family,

some kind of nematode was discovered.

 

The main symptoms of the patients are creeping eruption on skin,

migratory piercing and hard itch in muscle and tissue. Trying therapy

before the discovery of the parasite including Diethylcarbamazine,

Albendazole, Levamisole and Praziquantel had no effect.

 

The symptoms of the disease and the parasite found resemble LM and

G.spinigerum in some aspects, but also have marked differences in

others. The nematode is most probably of an undiscovered species.

 

Methods

 

Patients & Symptoms:

 

The first found case of patient is a female, 35 years old. The disease

was caught through contact with dirty toilet water during the period

of menstruation in Oct.1994 and acute symptoms started after several

days. The most striking symptoms are intermittent twinges as if neuro

filament was scratched and itchy sensations as if ants were crawling

on skin and flesh. Itching started 7-12 days after infection and

twinge started later, about 19 days after infection. Then numerous

other symptoms began, which included dyspepsia and constant feelings

of languor. However, in the mean time her body swelled remarkably.

 

The disease lasted more than two years and invaded the whole body. The

symptoms became more and more serious as time went on. The disease

seems pretty contagious. Infected people consequently include the

following:

 

1. Family members of the patient, including her parents, her husband

and her 6-year-old daughter;

 

2. Two close friends of the girl in her infantry;

 

3. A most intimate roommate of the patient during her

3-week-hospitalization;

 

As the patient claimed, people who came into contact with objects she

used to wipe eyes such as handkerchief, towel, etc always caught the

disease more seriously. It seems that the disease is highly contagious

through contact with mucous membrane such as that of eyes and vagina.

 

Moreover, people whose sexual ability is fully developed are more

vulnerable to the disease than both infants and old persons.

 

Physical Examinations & Signs

 

The most outstanding sign discovered through ocular observation was

numerous creeping eruptions on skin in all parts of body (usually 3-4

cm long, the longest 14-cm, usually straight.)(Fig 1).

 

Fig 1: The creeping eruption on the patient's leg

 

The marks were found on every body's skin in her family and infected

people as introduced above. The marks are usually accompanied by

epidermis bleeding which form intermittent blood dots along these

marks. There are also marks that show no signs of epidermis bleeding.

These marks are white and look like scratches made by outer force but

careful examination of these marks showed that the cuticle is mostly

intact which points out that the marks are caused by epidermis force.

Those marks usually display themselves for a period and then

disappear. The amount of marks occurring at the same time varies

according to the patient's emotion. More marks occurred when the

patient was in high spirit.

 

Another striking symptom is blister with diameter up to 0.5cm found in

mouth. When it is pricked, transparent liquid like lymphatic liquid

flow out. & #914;-ultrasonography also showed two cysts in matrix with

radius approximately 3-4 cm.

 

Physical Examination also revealed gum bleeding, conjunctivitis in

eyes, increased leucorrhoea similar to cheese, a little hepatomegaly

and splenomegaly, inflammation in mucous membrane of all body, etc.

The patients also claimed that migratory shade could be observed in

her eyes occasionally.

 

Other results obtained from physical examination are supplied below:

 

96.6 Assay: serum calcium: 2.6mmol.L-1, (normal: (1.12-1.23)mmol.L-1).

 

96.9 Assay: adrenocorticotropic hormone: 6.3ng.L-1, (normal:

(10-80)ng.L-1);

 

thyrotropic hormone: 1.38mIU.L-1, (normal: (2-10)mIU.L-1);

 

luteinizing hormone: 0.48mIU.ml,(normal: follicular period:(5-30)mIU.ml-1

 

ovulate period: (75-150)mIU.ml-1

 

luteal period: (3-30)mIU.ml-1).

 

(menstruate: sept.19th)

 

12. Urine radial immunological assay: b 2-microglobulin:

625ng.ml-1, (normal: (23-159)ng.ml-1);

 

albumin: 13.42m g.ml-1, (normal: (0.62-6.7)m g.ml-1).

 

96.12 Serum radial immunological assay: collagen: 237.1ng.ml-1,

(normal: (49.77± 5)ng.ml-1);

 

fibronectin: 200.1ng.ml-1, (normal: (115.7± 17.3)ng.ml-1).

 

96.12 X-ray assay: Degradation of vertebra cervicalis and lumbar.

 

The patients sought treatment in almost every major hospitals and

medical institutes in Shanghai for more than three years.

Possibilities of the cause as fungus, trichomonad, filariasis have

been denied due to the failure of corresponding treatments. Trying

therapy including Diethylcarbamazine, Albendazole, Levamisole and

Praziquantel also had no effect.

 

Laboratory Experiments

 

& #8544;.Microscopic Observation

 

1. Blood Diagnosis

 

1. Mix 250ul venous blood with 1ml di-diluted water (ddH2O)

together, centrifugalize (8000-10000rpm, 2-3min).

 

2. .Wash the sediments with 1ml ddH2O, centrifugalize

6000-8000rpm for 1-2 min (this procedure can be repeated until red

blood cells are completely removed).

 

3. Mix the sediments with 50-100ul normal saline, observe the

mixture under ordinary microscope

 

2. Urine analysis

 

1. Precipitate newly excreted urine for 20minutes.

 

2. Draw 1.5ml upper-layer solution, centrifugalize 6000rpm

for 2-3 min). Wash the sediments with 1ml normal saline,

centrifugalize 6000rpm for 2min .

 

2.3 Observe the sediments under ordinary microscope.

 

3. Mucous excretion analysis

 

Directly observe mucous excretion under ordinary microscope.

 

(All instruments, vessels used must be strictly sterilized and water

used must be completely pure lest extraneous microorganisms will

interfere.)

 

& #8545;. Using PCR method to carry out gene detection of the patient's

blood, urine and mucous excretion for Hepatitis A Virus & #65292;Hepatitis B

Virus & #65292;Hepatitis C Virus & #65292;Hepatitis E Virus & #65292;Rubeila

Virus & #65292;

Respiratory Syhcytial Virus & #65292;Tubercle Bacillus & #65292;Human

Cytomegalovirus & #65292;Herpes Simplex

Virus & #65292;Toxoplasma & #65292;N.Gonorrhoeae & #65292;

Human Papillomavirus & #65292;Treponema pallidum & #65292;Chlamydia

Trochomatis & #65292;

Ureaplasma & #65292;Epstein-Barr Virus and Human parvovirus B19 to obtain

important information concerning the involvement of these pathogens in

the disease we study.

 

Result

 

Gene detection of all the suspected pathogens invariably gave out

negative results, obviating the involvement of these pathogens in the

disease we study. Under microscope, a kind of worm was found in the

blood of the patient, her husband and her 6-year-old daughter. The

worm was white or orange, 140-300um long and 4-10um broad

(approximately 30:1), resembling nematode(Fig 2-1); with single thorn

in tail (Fig 2-2), single hook in head(Fig2-4)and skewed lines on

skin(Fig 2-3, Fig 2-6, Fig 2-7). It may get off sheath, which indicate

its being a larvae. The cells and their cores, intestine, coupling

thorn, hidden organ in the male worm can be seen under microscope(Fig

2-5).

 

Fig 2-1: The whole body Fig 2-3: the middle part

 

Fig 2-2 & #65306;the tail Fig 2-4: the head

 

Fig 2-6: the head Fig 2-7: the head

 

& #12288;

 

Fig 2-5 & #65306;the tail

 

Fig 2-1~2-4, Fig 2-6, Fig 2-7: under interference microscope & #65292;Fig 2-5:

under phase-contrast microscope.

 

& #12288;

 

Discussion

 

It is now quite certain that the disease and following symptoms are

caused by this parasite. The migration of the parasite may lead to

system invasion. The parasite can creep through cuticle and muscle. In

the mean time it will tear blood vessels and engender creeping

eruptions; it can also creep through the wall of stomach, abdominal

membrane, etc, which will cause stomachache and dyspepsia; still it

can creep into matrix and vagina, which will result in abnormal

leucorrhea. As the larva migrates through some certain location, it

will tear and scratch corresponding neuro filaments. The worm

(140-300um long, 4-10 um broad) is big enough to scratch even the

broadest neuro filament( appox.20um), which can bring about the

feelings of twinge and itching. When the bodies of parasite or wastes

excreted by them accumulate, swelling and hard knobs come into being.

The accompanying symptoms is a feeling of languor.

 

Those creeping eruptions can be attributed to the hook in the its head

by which the parasite can tear tissues as it creeps through body. The

parasite should have the ability of excreting proteinase of high

activity as it ploughs its way ahead. As it migrates, corresponding

symptoms and signs starts throughout human body.

 

Blisters have sometimes occurred in mouth and matrix where mucous

membrane abounds.This pathogenic phenomenon may be caused by lymphotic

vessel being blocked up. Migratory shade in eyes is also claimed by

the patient which is probably also caused by the creeping parasite. It

seems that the parasite has a predisposition for the environment of

mucous membrane. The infection of the disease is also caused mainly

through mucous membrane: ocular mucous membrane, vaginal mucous

membrane, etc.

 

Specialists of Chinese hospitals have not yet met with such cases,

which suggest that the nematode might be of a completely new species.

Although similar symptoms appear in an considerable portion of human

population in Shanghai, the disease has long been neglected because

the symptoms are not specific.

 

Judging from the characteristics of the infection, symptoms and signs

of the disease, we put forth the hypothesis concerning the life cycle

of this parasite.

 

Most parasites cannot complete their life cycles inside human body and

have to experience part of their life cycle outside, due to unfit

environments such as unfit temperature and lack of sufficient oxygen

necessary for certain periods of their development. But this parasite

maybe a completely a different one, and can probably reproduce inside

human body. Evidence can be gained through investigating the patient's

symptoms and signs during different stages of the disease. For one

thing, the disease was caught through contact with contaminated water

only once, which indicated that the original amount of the parasite

cannot be very large. For another, the parasite is so small that only

a few parasites are not enough to generate such a serious, systemic

and all-time deteriorating disease. Therefore, we can conclude that

the amount of the parasites is increasing as time goes by. Only two

possibilities can explain this increase:

 

1. The parasite can reproduce and proliferate inside human body.

 

2. The patient is infected more than once during disease.

 

If the second explanation is true, the parasites that infected the

patient later came most presumably from the patient herself. The eggs

laid and fertilized inside the body of the patient contaminated outer

objects. Then they hatched and formed contagious pathogen like

contagious zygote or larva in the outer environment such as water,

towel, handkerchief and even on moist surface parts of the body.

 

But in this occasion, parasites must experience two periods in

different environments in order to reproduce, which will limit their

proliferation greatly and slow down the pace of their accumulation. On

the contrary, the disease was spreading and deteriorating at a rather

quick speed. From another aspect, the symptoms didn't appear

simultaneously. Lighter symptoms such as itching appeared first

whereas heavier ones such as twinge appeared later. It seems that the

parasite experienced a period of development inside the patient.

Smaller larva formed first. When they migrate, lighter itching will

take place because the comparatively small size of them. Later, these

larva mature and their size grow bigger. When they migrate, heavier

sensation such as twinge will start.

 

Judging from those aspects, the second explanation seems far from the

truth. By comparison, the first explanation is more presumable. We

postulate the life cycle of this parasite as follows:

 

Zygotes are laid inside human body. As symptoms and signs shown, the

eggs most likely are laid in mucous membrane especially that of

vagina, intestine and eyes. Those zygotes can contaminate outer

objects through contact with these mucous membrane and can also give

birth to larva. Larva can then mature inside human body and can again

lay eggs and start another life cycle. During that period, both larva

and mature parasites can migrate and cause the disease. Zygotes in

outer environment cannot hatch but can infect other people thus spread

the disease.

 

Careful examination of past medical records found out that the

symptoms and signs of the disease resemble those of LM in the

following aspects

 

1. Systematic invasion to the body

 

2. Creeping eruptions observed in various parts of the body.

 

3. Myalgia and Stomachache

 

4. Abnormal leucorrhea

 

5. Itching and pain felt by the patient.

 

6. Swelling

 

7. Hard knobs felt by touching the patient's skin.

 

Judging from those symptoms and signs. We postulate that the illness

was caused in similar means comparing with LM, that is, by migrating

parasites.

 

However, these diseases and their pathogenic parasite differs in

various aspects:

 

1. The ways through which the disease is caught:

 

LM caused by G.spinigerum and G.hispidum is infected through

mouth, especially through eating uncooked seafish while this disease

is far more contagious. It infect people through numerous ways by the

contact of mucous membrane.

 

2. The outlook and size of the parasite involved (table 1)

 

Table 1 Comparison of the parasite in this study with G.hispidum and

G.spinigerum(3rd-stage-larva)

& #12288;

 

The parasite in This study

 

 

G.hispidum

 

(3rd–stage-larva)

 

 

G.spinigerum

 

(3rd–stage-larva)

 

Color

 

 

White or orange

 

 

Red or pink

 

 

Red or pink

 

Length

 

 

140-300 um

 

 

Approx. 4mm

 

 

2.5-3.2mm

 

Width

 

 

4-10um

 

 

0.28mm

 

 

0.37-0.47mm

 

Length/width

 

Structures on the surface of body

 

 

Approx. 30

 

Only one hook on the head, has a tail thorn

 

 

Approx. 15

 

With many spines on head ball

 

 

Approx. 8

 

Many spines on the suface of body

 

Through the comparison between these parasites, we discovered that

obvious differences exist among them. It might be that this parasite

is in former stages of the development of G.spinigerum or G.hispidum,

but the ratio of length to breadth is not in accord. Such ratio of the

2nd-stage-larva of G.spinigerum whose lengths(223-275um) mostly close

to this parasite is 15-20(223-275 & 15.8-17.4) while this parasite has

the ratio approximately 30.

 

3. 3.The stage of growth of the pathogenic parasite involved

 

4. Larva migrans is caused by the larva of its pathogenic parasite,

for instance, the 3rd-stage-larva of G.spinigerum or G.hispidum. These

parasites must experience the period inside secondary intermediate

host in order to reproduce. In comparison, the larva and mature

parasite of this parasite can both migrate so as to cause disease. In

fact, the parasite can reproduce inside human body so as to be

exempted of the period outside.

 

4. Symptoms and signs of the disease. (table 2)

 

Table 2: Comparison of symptoms and signs of this disease and the LM

of other nemotodes

& #12288;

 

The Strange Disease(This Study)

 

 

LM

 

Migrant Marks

 

 

 

Mostly Straight ,Intermittent bleeding along the marks

 

 

Continuous,Always Crooked

 

Location of the marks

 

 

Near surface(cuticle)

 

 

Deep

 

Pain

 

 

Twinge

 

 

Pressing and swelling pain in addition to twinge

 

Suppuration

 

 

No

 

 

Yes

 

Tumor

 

 

No

 

 

Yes

 

Weight

 

 

Increase

 

 

Decrease

 

Through comparison of the main symptoms and signs of these diseases,

we find out the main differences between those parasites regarding

their ways of causing disease.

 

a. We found two most outstanding characteristics of the marks.

Intermittent tearing open of the cuticle and intermittent bleeding

along marks. First, we observed that in a very small portion of the

marks, the cuticle is torn open and this implies that the parasite can

migrate so near to the surface as to tear open the outmost layer of

human skin. However, the parasite we obtained is merely 4-10 um wide,

much smaller than the average thickness of the cuticle of human

body(about 0.1mm). We deduced that when the marks are formed, the

parasite is creeping in or alongside cuticle. In another aspect, we

observe intermittent blood stains along the marks which indicates that

capillary is occasionally torn open. Because epidermis capillary is

located in realskin, the parasite should be able to approach realskin

in order to tear open the capillary.

 

Therefore we deduce that the parasite creep in cuticle when making the

mark. It can move in different layers of the cuticle. Sometimes it can

approach the surface and sometimes it can approach the realskin where

capillaries lay. G.spinigerum and G.hispidum, however, usually migrate

in deeper layers where blood vessels are more abundant so that

creeping eruptions cause by them is deeper located and continuous.

 

b. The difference of the kind of pains suffered by the patient between

these diseases should due to the different sizes of these parasites

and routes of their migration. The one in study has a length only

one-tenth to the 3rd-stage-larva of G.spinigerum and G.hispidum and

the width of the former is only one twentieth of the latter. On

another aspect, these parasites migrate in different parts of human

tissue so as to stimulate different neuro filaments. The parasite

mostly stimulates the neuro filaments associated with pains of skin

and flesh. These neuro filaments are especially sensitive to

scratching and tearing and are responsible for the sensation of

twinge. The parasite can also migrate through mucous membrane of

entrails. But because of its relatively small size, it can't stretch

visceral wall. Since stretching is the most effective stimulation to

visceral pain neuro filaments, the parasite cannot cause such pressing

and swelling pain------visceral pain. G.spinigerum and G.hispidum,

however, creep in deeper levels and always through visceral wall so

that they have more chances to contact with visceral pain neuro

filaments. Additionally, because of their bigger size, they can

stretch visceral wall and will stimulate corresponding neuro filaments

and cause the feelings of pressing and swelling pain.

 

c. The fact that the parasites can occur both in cuticle, blood, urine

and in mucous membrane indicates that it can migrate through the wall

of blood vessels esp. capillary. The parasite is small enough to

travel to various parts of human body swiftly through the circulation

of blood and can tear open the vessel wall as it move into tissue

where it do damage. There is even sign that the parasite can migrate

into lymphatic vessels and will block lymphatic circulation. When this

occurs, lymphatic liquid will accumulate and form blisters as those in

mouth. But G.spinigerum and G.hispidum are too big to do such damages.

 

Based on those analysis. We conclude that this parasite is certainly

not G.spinigerum and G.hispidum but may be of an unknown species

rather close to them. The disease is certainly not LM but has a

similar cause as the latter. The differences come from the different

size and type of migration of corresponding parasites.

 

We must point out that despite the much smaller size of this novel

parasite, it is perhaps more tremendous. First, it is far more

contagious. One can avoid being infected by G.spinigerum and G.

hispidum by avoid eating uncooked seafood only while this disease can

invade human body through various channels. One may be infected

through contact with contagious water, contaminated towel, through

blood contact, etc. That is to say, one may catch the disease

innocently. Second, the parasite is in fact rather hazardous. Its hook

on head may tear human tissue. It can migrate by itself or move to all

parts of human body through blood circulation so as to damage numerous

parts of body. If, unfortunately, it injures some most important

organs such as sinoatrial node and some vital neuro filaments,

momentous aftermath will follow. Third, the parasite is rather

difficult to eliminate. Scince it can proliferate inside human body,

it impossible to slay them by taking outside measures. We can spread

drug into earth to eliminate hookworm, and we can kill schistosoma by

eliminating water-snails, but we can't make similar approach to

eliminate this parasite. In order to control the disease, we must call

forth social effort from a wider scope.

 

We'd like to stress that the disease and accompanying symptoms and

signs are quite prevalent in Shanghai, and perhaps,China. But because

the symptoms are mostly less serious and signs are not specific, the

disease is always misdiagnosed and neglected. If no attention is paid

to the disease, the whole population will be threatened in near

future. Therefore, we sincerely hope the parasitologists,

microbiologists all over the world can put forward opinions on the

identification of the parasite, therapy and further research. We also

hope that attention of the whole human society to be paid to the

disease and the parasite so that further protection and diagnose will

become available.

 

& #12288;

 

References

 

1.Fang Xue, Wang Xiasheng, et al. Report of 10 cases of cutaneous

gnathostomiasis.Chin J

 

Dermatol1998;31(6):390

 

2.Sohn WM, Lee HA, et al Surface ultrastrustures of the third-stage

larvae of Gnathostoma

 

hispidum. Korean J Parasitol 1996 ;34(3):169-176

 

3.Daengsvang S. Gnathostomiasis in Southeast Asia. Southeast Asian J

Trop Med public

 

Health. 1981 ;12(3):319-332.

 

4.Ando K,Tanaka H,Taniguchi Y, et al.Two human cases of gnathomiasis

and discovery of a

 

second intermediate host of gnathostoma nipponicum in Japan.J

Parasitol 1988;74(4):623-627.

 

5.Hira PR, NeafieR, Prakash B,et al.Human gnathostomiasis:infection

with an immature male

 

gnathostoma spinigerum.Am J Trop Med Hyg 1989;41(1):91-94.

 

6.Nitidandhaprabhas P. Hanchansin S,Vongsloesvidhya Y.A case of

expectoration of gnathostoma

 

spinigerum in Tailand. Am J Trop Med Hyg 1975;24(3):547-548.