「질 트리코모나스」감염(Trichomoniasis vaginalis)진단을 위한 제동반응 및 응괴반응의 응용

Abstract

[한글]

Applications of the Immobilization-Agglomeration Reaction to the diagnosis of

Trichomoniasis vaginalis

Chung, Pyung-Rim

Department of Public Health, The Graduate School, Yonsei University

Introduction

There has been considerable work on the diagnosis of Trichomonas vaginalis

infection since Donne first found the parasite in 1837. However, the available

serological method so far has not been reported in the references. since world War

Ⅱ, the immobilization-agglomeration reaction has been applied to the diagnosis of

some of the parasite infections (Grief, 1947; Cole and Kent, 1953; Brown et al.,

1955; Biagi F, 1961, 1966; Zaman, 1964; Hong et al., 1968; Prakash et al., 1969

etc.). However, no report has appeared on the diagnosis of vaginal trichomoniasis.

The diagnosis of vaginal trichmoniasis has been mainly dependent on the

laboratory demonstration of the parasite. However, there is often some difficulty

experience in the collection of specimens, especially in the case of a mass survey.

since the procedure of the immobilization-agglomeration test is comparatively

simple, the author examined it s applicability to the diagnosis of T. Vaginalis

infection.

The present study was designed to evaluate the sensitivity and specificity of the

immobilization-agglomeration test with the sera from human cases of T. vaginalis

infection. The author also carried on observations of the antigenic relationship

between T. vaginalis, T. hominis and Candida albicans with antiserum produced in

rabbits.

Materials and Methods

1. Cultivation of T. vaginalis and T. hominis:

In Trussel and Johnson's medium or Lash's casein hydrolysate-serum medium, T.

vaginalis was cultured at 37℃. and subcultured at a two day interval.

T. hominis was cultured in the Modified Diphasic Medium which is generally

utilized for Entamoeba histolytica cultivation.

2. Preparation of antigens and antisera used:

1) Antigen: The active trophozoites required for the immobilization tests were

obtained from each culture after 20-24 hours incubation at 37℃. The saline

suspensions, that were prepared containing 40,000 parasites per ml., were used as

antigens of T. vaginalis and T. hominis respectively.

A saline suspension of C. albicans, containing 10,000 organisms per ml., was used

for a cross-reaction test with trichomonas antiserum.

2) Antisera: All the t. vaginalis collected from the cultures were washed 3 times

with 0.85% NaCl solution and concentrated by centrifugation for 5 minutes at 2,500

r.p.m. They were then suspended in 0.85% NaCl solution, keeping the concentration

of organisms about 440,000 per ml. The suspension was injected intravenously into

healthy adult rabbits 3 times on alternate days. The animals were bled by cardiac

puncture one week after the last injection, the sera being separated aseptically

and stored at -20℃.

Human antisera were obtained from the professional women who periodically visited

the public health clinics in Yosu and Kunsan, Korea, and were proed T. vaginalis

positive by vaginal swab examinations. Sera from females under 13 years old and

from males without any age restriction, were used as healthy controls. As

additional controls, sera of amebiasis, ascariasis and trichiuriasis cases were

collected for this test.

3. Method of the immobilization-agglomeration test:

The immobiization-agglomeration reaction was carried out by combining a drop of

trichomonas-saline suspension with a drop of serum on a clean slide. A cover slip

was placed on top of the mixture and sealed with melted paraffin. The slide was

then placed in an incubator at 27℃. A mixture of trichomonas and isotonic saline

solution was used as a control for spontaneous immobilization.

4. Criterion of judgment;

One hundred parasites were counted on the slide, examining the number of ones

immobilized. The agglomeration phenomenon was observed simultaneously. The

criterion for a positive reaction was determined when more than 50% of the

trichmonas were immobilized accompanied by agglomeration, while the saline controls

maintained their motility. If there was no agglomeration phenomenon but more than

90% of the trichomonas were immobilized, then it was regarded as a positive

reaction.

Results

A. Immobilization-agglomeration reaction of trichomonas with antiserum from the

rabbit immunized with T. vaginalis.

When the organisms were mixed with fresh antiserum, they were soon immobilized.

The rate of immobilization was proportional to the concentration of the serum used

(Table 1). In higher concentration, i.e. 1:2 and 1:4, the immobility was observed

within four minutes treatment. But in the sample of a 1:32 dilution remobilization

appeared 40 minutes after treatment. In a 1:16 dilution, the organisms

disintegrated within 6 hours, the timing being variable according to the titre of

serum used (Fig. 1). In this experiment, the agglomeration reaction was observed

even to a 256 times dilution of antiserum. The minimum required period to produce a

complete immobilization titre in the rabbit serum was 25 days after the last

inoculation of T. vaginalis (Table 2).

On cross reaction studies, there was no cross reaction between T. vaginalis, t.

hominis and c. albicans (Table 3 & 6).

B. Immobilization-agglomeration reaction with human antisera.

The rsults of a representative experiment with serial dilutions of antiserum are

given in Talbe 4 and Fig. 2. The number of immobilized trophozoites was inversely

proportional to the concentration of human antiserum, but varie considerably with

time.

The peak of the immobilizing reaction was observed after 25-30 minutes, then

reversed to normal. In the control, the organisms with normal serum remained

actively motile throughout the period of observation.

The inactivation of sera (56℃., 30 min.) showed no fundamental differences when

compared with the untreated sera (Table 5). All the positive cases showed more than

90% immobilization and strong agglomeration reactions throughout the study.

Positive reactions were detected in 57 out of 71 samples(80.3%) of sera collected

in T. vaginalis positive women. But only 2 out of 34 sera(5.9%) from females under

13 years of age, and 4 out of 43 sera (9.3%) from males who were T. vaginalis

negative by the routine method, were determined as positive.

In order to observe any relationship to other parasitic infections, sera of

amebiasis, ascariasis and trichiuriasis cases were examined. Two out of 12 sera

from amebiasis patients showed positive reaction. But no positive reaction appeared

either in the sera of ascariasis or of trichiuriasis (Table 7). The sensitivity and

specificity of immobilization and agglomeration were 80.3% and 92.2%, respectively

(Table 8).

Summary

The trichomonas immobilization-agglomeration reaction was studied using the sera

from women with vaginal trichomoniasis and from rabbits inoculated with cultures of

T. vaginalis.

It was found that the greatest amount of immobilization occurred at 25 to 30

minutes, and that inactivation of the sera did not affect the results. An evidence

of antigenic differences between T. vaginalis, T. hominis and C. albicans was

confiremd.

The immobilization reaction was positive in 80.3% of the 71 T. vaginalis positive

cases, but in only 7.7% of the 77 persons of T. vaginalis negative cases.

From the above results, it is suggested that the immobilization reaction is a

useful tool for the diagnosis of T. vaginalis infection.

[영문]

Introduction

There has been considerable work on the diagnosis of Trichomonas vaginalis infection since Donne first found the parasite in 1837. However, the available serological method so far has not been reported in the references. since world War

Ⅱ, the immobilization-agglomeration reaction has been applied to the diagnosis of some of the parasite infections (Grief, 1947; Cole and Kent, 1953; Brown et al., 1955; Biagi F, 1961, 1966; Zaman, 1964; Hong et al., 1968; Prakash et al., 1969 etc.). However, no report has appeared on the diagnosis of vaginal trichomoniasis.

The diagnosis of vaginal trichmoniasis has been mainly dependent on the laboratory demonstration of the parasite. However, there is often some difficulty experience in the collection of specimens, especially in the case of a mass survey.

since the procedure of the immobilization-agglomeration test is comparatively simple, the author examined it s applicability to the diagnosis of T. Vaginalis infection.

The present study was designed to evaluate the sensitivity and specificity of the immobilization-agglomeration test with the sera from human cases of T. vaginalis infection. The author also carried on observations of the antigenic relationship between T. vaginalis, T. hominis and Candida albicans with antiserum produced in rabbits.

Materials and Methods

1. Cultivation of T. vaginalis and T. hominis:

In Trussel and Johnson's medium or Lash's casein hydrolysate-serum medium, T. vaginalis was cultured at 37℃. and subcultured at a two day interval.

T. hominis was cultured in the Modified Diphasic Medium which is generally utilized for Entamoeba histolytica cultivation.

2. Preparation of antigens and antisera used:

1) Antigen: The active trophozoites required for the immobilization tests were obtained from each culture after 20-24 hours incubation at 37℃. The saline suspensions, that were prepared containing 40,000 parasites per ml., were used as

antigens of T. vaginalis and T. hominis respectively.

A saline suspension of C. albicans, containing 10,000 organisms per ml., was used for a cross-reaction test with trichomonas antiserum.

2) Antisera: All the t. vaginalis collected from the cultures were washed 3 times with 0.85% NaCl solution and concentrated by centrifugation for 5 minutes at 2,500 r.p.m. They were then suspended in 0.85% NaCl solution, keeping the concentration

of organisms about 440,000 per ml. The suspension was injected intravenously into healthy adult rabbits 3 times on alternate days. The animals were bled by cardiac puncture one week after the last injection, the sera being separated aseptically and stored at -20℃.

Human antisera were obtained from the professional women who periodically visited the public health clinics in Yosu and Kunsan, Korea, and were proed T. vaginalis positive by vaginal swab examinations. Sera from females under 13 years old and

from males without any age restriction, were used as healthy controls. As additional controls, sera of amebiasis, ascariasis and trichiuriasis cases were collected for this test.

3. Method of the immobilization-agglomeration test:

The immobiization-agglomeration reaction was carried out by combining a drop of trichomonas-saline suspension with a drop of serum on a clean slide. A cover slip was placed on top of the mixture and sealed with melted paraffin. The slide was

then placed in an incubator at 27℃. A mixture of trichomonas and isotonic saline solution was used as a control for spontaneous immobilization.

4. Criterion of judgment;

One hundred parasites were counted on the slide, examining the number of ones immobilized. The agglomeration phenomenon was observed simultaneously. The criterion for a positive reaction was determined when more than 50% of the trichmonas were immobilized accompanied by agglomeration, while the saline controls maintained their motility. If there was no agglomeration phenomenon but more than 90% of the trichomonas were immobilized, then it was regarded as a positive reaction.

Results

A. Immobilization-agglomeration reaction of trichomonas with antiserum from the rabbit immunized with T. vaginalis.

When the organisms were mixed with fresh antiserum, they were soon immobilized. The rate of immobilization was proportional to the concentration of the serum used (Table 1). In higher concentration, i.e. 1:2 and 1:4, the immobility was observed

within four minutes treatment. But in the sample of a 1:32 dilution remobilization appeared 40 minutes after treatment. In a 1:16 dilution, the organisms disintegrated within 6 hours, the timing being variable according to the titre of serum used (Fig. 1). In this experiment, the agglomeration reaction was observed

even to a 256 times dilution of antiserum. The minimum required period to produce a complete immobilization titre in the rabbit serum was 25 days after the last inoculation of T. vaginalis (Table 2).

On cross reaction studies, there was no cross reaction between T. vaginalis, t. hominis and c. albicans (Table 3 & 6).

B. Immobilization-agglomeration reaction with human antisera.

The rsults of a representative experiment with serial dilutions of antiserum are given in Talbe 4 and Fig. 2. The number of immobilized trophozoites was inversely

proportional to the concentration of human antiserum, but varie considerably with time.

The peak of the immobilizing reaction was observed after 25-30 minutes, then reversed to normal. In the control, the organisms with normal serum remained actively motile throughout the period of observation.

The inactivation of sera (56℃., 30 min.) showed no fundamental differences when compared with the untreated sera (Table 5). All the positive cases showed more than 90% immobilization and strong agglomeration reactions throughout the study.

Positive reactions were detected in 57 out of 71 samples(80.3%) of sera collected in T. vaginalis positive women. But only 2 out of 34 sera(5.9%) from females under 13 years of age, and 4 out of 43 sera (9.3%) from males who were T. vaginalis negative by the routine method, were determined as positive.

In order to observe any relationship to other parasitic infections, sera of amebiasis, ascariasis and trichiuriasis cases were examined. Two out of 12 sera from amebiasis patients showed positive reaction. But no positive reaction appeared

either in the sera of ascariasis or of trichiuriasis (Table 7). The sensitivity and specificity of immobilization and agglomeration were 80.3% and 92.2%, respectively (Table 8).

Summary

The trichomonas immobilization-agglomeration reaction was studied using the sera from women with vaginal trichomoniasis and from rabbits inoculated with cultures of T. vaginalis.

It was found that the greatest amount of immobilization occurred at 25 to 30 minutes, and that inactivation of the sera did not affect the results. An evidence of antigenic differences between T. vaginalis, T. hominis and C. albicans was confiremd.

The immobilization reaction was positive in 80.3% of the 71 T. vaginalis positive cases, but in only 7.7% of the 77 persons of T. vaginalis negative cases.

From the above results, it is suggested that the immobilization reaction is a useful tool for the diagnosis of T. vaginalis infection.