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Localization of genital mycoplasmas in women
Localization of genital mycoplasmas in women WILLIAM JOEL
M. S. RANKIN,
YHU-HSIUNG Boston,
McCORMACK,
M.D.*
M.D. LEE,
M.D.
Massachusetts
Cultures for T-mycoplasmas and Mycoplasma hominis were obtained from the urethra, vagina, cervix, and posterior fornix of 132 women. Vaginal cultures resulted in the most isolations of both species. As a measure of the reproducibility of this method, 4 separate vaginal cultures were obtained from 52 additional women. A single vaginal culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M. hominis.
T H E G E N I T A L mycoplasmas, T-mycoplasmas and Mycoplasma hominis, when present in women, colonize the genital mucosa, including the external cervical OS,vagina, vestibule, and distal urethra. In planning epidemiologic studies, we wanted to know how best to obtain cultures for these organisms. We were especially interested in From the Channing Laboratory, Thorndike Memorial Laboratory, Harvard Medical Unit, Department of Medical Microbiology, and Department of Obstetrics and Gynecology, Boston City Hospital, Departments of Medicine and Pediatrics, Harvard Medical School, and Department of Obstetrics and Gynecology, Boston University School of Medicine. Supported by Grants Nos. HD-03693 from the National Institute of Child Health and Human Development and TO1 AI-00068 from the National Institute of Allergy and Infectious Diseases. y;;ived
for publicafion
November
23,
f;;;pted
for
November
26,
publication
Reprint requests: Dr. Channing Laboratory, Hospital, 774 Albany Massachusetts 02118.
McCormack, Boston City St., Boston,
*Recipient of United States Health Service Post-Doctoral 1 FO.? 4144394-02.
Public Fellowship
exploring techniques for the collection of specimens that would lend themselves to population surveys. Urine cultures are quite acceptable for population studies, and urine has been used to culture mycoplasmas. As bladder urine is normalIy sterile, the organisms are presumably picked up in transit from the periurethral mucosa. Archer1 found that urine provided as many positive cultures as genital swabs from the same women. However, his technique required centrifugation of the urine before culturing, a laborious and time-consuming procedure. Braun and associate? subsequently showed that direct cultures of urine grew significantly fewer T-mycoplasmas than genital cultures from the same women. Thus, we decided that urine cultures would not be satisfactory for our studies. We then considered vaginal cultures. These can be taken by simply inserting a cotton-tipped swab into the vagina. As a vaginal speculum is not required, the procedure is reasonably acceptable to the subject. Several investigators have noted that the vagina was a satisfactory site for the isolation of genital mycoplasmas,s-s but only one of these studies” included T-mycoplas-
Localization
mas, and this study merely notes that vaginal cultures were better than cervical cultures without providing any data. To examine further the suitability of vaginal cultures for the isolation of genital mycoplasmas, we obtained cultures from the urethra, vagina, cervix, and posterior fornix of 132 women. We found that vaginal cultures resulted in the most isolations of both T-mycoplasmas and M. hominis. We then obtained 4 separate vaginal cultures from an additional 52 women as a measure of the reproducibility of this method. A single vaginal culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M.
hominis. Methods Women who presented to the prenatal and gynecology clinics of the Boston City Hospital and who for any reason required a speculum examination of the vagina were studied. The examining physician obtained the following cultures from each subject: (1) Urethra-a sterile swab was placed into the distal urethra and rotated gently; (2) Vagina-a sterile swab was inserted 1 to 2 inches into the vagina and rubbed against the vaginal wall; (3) Cervixafter the speculum had exposed the cervix, a sterile swab was placed into the external OS and rotated; (4) Posterior fornixwith the speculum in place, a sterile swab was placed into the posterior fornix and rotated. The reproducibility of vaginal cultures was evaluated in patients on the Boston City Hospital obstetric service. The labia were spread with a gloved hand, and 4 separate vaginal cultures were taken by placing sterile swabs 1 to 2 inches into the vagina and rubbing them against the vaginal wall. The methods used in our laboratory for the cultivation and identification of the genital mycoplasmas have been described in detail by Braun and associates.2 Immediately after collection, swabs were placed into 4 ml. screw-rapped vials containing 2 ml. of
of
genital
mycoplasmas
921
basic mycoplasma broth (70 per cent PPLO broth, 20 per cent unheated horse serum, 10 per cent yeast extract, 0.002 per cent phenol red, and 500 U. per milliliter of benzyl penicillin) to which 50 pg per milliliter of pal)mixin B and 5 pg per milliliter of amphoteritin B had been added. The pH had been adjusted between 6.0 and 6.4 with IN Hcl. All specimens were frozen within 6 hr. of collection and stored at -80’ C. Specimens were thawed after no more than 7 days of storage, and 0.2 ml. of broth was inoculated into 1.2 ml. of urea broth (basic broth at pH 6.0 plus 0.5 mg. per milliliter of urea and 15 pg per milliliter of lincomycin) for T-mycoplasmas and into 1.2 ml. of arginine broth (basic broth at pH 7.2 plus 10 mg. per milliter of arginine and 100 /kg per milliliter of erythromycin) for M. hominis. The pH of the broth was measured by comparison with color standards at the time of inoculation and daily during incubation at 37’ C. Specimens in \vhich the pH had risen were subcultured to rrrycoplasma agar. A single streak was made with a Pasteur pipette. After the inoculum had dried, a blank sensitivity disc impregnated with undiluted antiserum to 111. hominlv was placed at one end of the streak and ;t disc containing erythromycin was placed at the other end. Plates were incubated in an atmosphere of 90 per cent N,-10 per cent CO? and examined after 2 to 4 days at 37” C. T-mycoplasmas were identified by their characteristic colonial morphology, inhibition of growth around the disc c-ontaining erythromycin, and persistence of growth around the disc impregnated with antiserum to M. hominis. M. hominis was identified by its colonial morphology and by inhibition of growth around the disc containing specific antiserum. Results Table I shows the percentage of isolations of T-mycoplasmas from each of the sites sampled. The greatest number of isolations (86 or 65.2 per cent) were obtained from \,agirrrrl cultures. Only 62 isolations (47.0 per cent) MYYC made from cervical cultures (p
922
McCormack,
Rankin,
and
Lee Am.
Table I. Isolation of T-mycoplasmas 4 sites in 132 women
Site
;$f
Urethra Vagina Cervix Posterior fomix
!CE;f
81 86 62 80
*One hundred women mycoplasmas by combining
4 vaginal
Urethra Vagina Cervix Posterior fornix ‘Seventy combining
Eif 64 68 59 66
Culture First Second Third Fourth
81 86 62 80 be positive for of alI 4 cultures.
Table II. Isolation of Mycoplasma from 4 sites in 132 women
Site
T-
48.5 51.5 44.7 50
“Forty-four women shown mas by combining the results
from
Per cent of total 75.0 71.2 65.4 69.2
Per cent of 44 women with positive cultures* 88.6 84.1 77.3 81.8
to be positive for T-mycoplasof all 4 cultures.
4 vaginal
of Mycoplasma hominis cultures in 52 women
Culture 1i?$%.&i, 17ti;i;;~~: 91.4 97.1 84.3 94.3
for M.
No. of isolations 39 37 34 36
from
Table IV. Isolation
hominis
~~~~1~~
women shown to be positive the results of all 4 cultures.
Isolation of T-mycoplasmas cultures in 52 women
zgkk$
61.4 65.2 47.0 60.6
shown to the results
Table III.
from
April 1, 1’372 J. Obstet. Gynecol.
komink
First Second Third Fourth by
= < 0.01). Cultures of the urethra and posterior fornix showed intermediate results. T-mycoplasmas were isolated from a total of 100 different women if the results of the cultures from all 4 sites are combined. An estimate of the sensitivity of a single culture taken from a given area can be made by comparison with the 100 patients shown to be positive for T-mycoplasmas by combining the results of all 4 cultures. This is also shown in Table I. Vaginal cultures detected 86 per cent and cervical cultures detected 62 per cent of the women shown to be positive for T-mycoplasmas. Vaginal and urethral cultures together detected 94 per cent of these women. Table II presents the rates of isolation of M. hominis. Again, vaginal cultures (68 or 51.5 per cent) were more productive than cervical cultures (59 or 44.7 per cent) although this difference is not statistically significant. A single vaginal culture detected 68 (97.1 per cent) of the 70 women who were shown to be positive by combining the results of all 4 cultures. Four separate vaginal cultures were then
21 22 22 22
*Twenty-three women hominis by combining the
40.4 42.3 42.3 42.3 shown results
to be positive of all 4 cultures.
91.3 95.7 95.7 95.7 for
M.
obtained at the same time from each of 52 women. Tables III and IV show the results of the isolations from these cultures of Tmycoplasmas and M. hominis, respectively. A single culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M. hominis by combining the results of all 4 cultures. Comment These data show that for the isolation of the genital mycoplasmas vaginal cultures are at least as productive as cultures taken from the posterior fornix, urethra, or cervix. This has allowed us to use vaginal cultures in our epidemiologic studies. Several women on our staff have been trained to take these cultures. The technique involves simply spreading the labia with a gloved hand to expose the vaginal opening, inserting a sterile swab 1 to 2 inches into the vagina with the other hand, and rubbing the swab against the vaginal wall. The procedure is rapid and painless and has been well accepted by our
Volume Number
112 7
subjects. It has been especially useful for postpartum women where a specuhrm examination would have been quite uncomfortable. In one survey, a group of student and graduate nurses took their own vaginal cultures with the use of this technique. A disquieting observation is that although vaginal cultures resulted in the most isolations of T-mycoplasmas the vaginal culture was positive in only 86 per cent of the women who were shown to be carrying Tmycoplasmas by combining the results of cultures from all 4 sites. This relative insensitivity of a single vaginal culture for the isolation of T-mycoplasmas is also apparent from the results obtained with 4 vaginal cultures taken at the same time. Here, a single culture detected between 77.3 and 88.6 per cent of the women who were shown to be colonized with T-mycoplasmas by combining the results of all 4 cultures. Apparently, the number of T-mycoplasmas present is so small or the organisms are so fastidious that with the currently available laboratory methods a single vaginal culture will fail to identify T-mycoplasmas in as many as 22 per cent of colonized women.
Localization
of genital
mycoplasmas
923
A higher yield can be obtained by culturing more than one site. Combining urethral and vaginal cultures detected 94 per cent of women shown to be positive for T-mycoplasmas. Results can also be improved by obtaining 2 cultures from the same site. Any combination of 2 vaginal cultures revealed over 90 per cent of women who were shown to be positive for T-mycoplasmas by combining the results of all 4 vaginal cultures. Cultures for Mycoplasma hominis seem to be more reproducible. When vaginal cultures were compared, a single culture detected between 91.3 and 95.7 per cent of women shown to be positive by combining the results of all 4 cultures. The valuable technical assistance of Miss Vicki Crockett and Mrs. Penrhyn Bailey is gratefully acknowledged. This study would not have been possible without the cooperation of the house officers on the obstetric and gynecology service at the Boston City Hospital. The horse serum was supplied by the Biological Laboratories of the Massachusetts Department of Health.
REFERENCES
1. Archer, J. F.: Br. J. Vener. Dis. 44: 232, 1968. 2. Braun, P., Klein, J. O., Lee, Y-H., and Kass, E. H.: J. Infect. Dis. 121: 391, 1970. 3. Dunlop, E. M. C., Hare, M. J., Jones, B. R., and Taylor-Robinson, D.: Br. J. Vener. Dis. 45: 274, 1969.
4. Freundt, E. A.: Acta PathoI. Microbic& Sand. 32: 468, 1953. 5. Horoszewicz, J.: Br. J. Vener. Dis. 37: 183, 1961.
M. S. RANKIN,
YHU-HSIUNG Boston,
McCORMACK,
M.D.*
M.D. LEE,
M.D.
Massachusetts
Cultures for T-mycoplasmas and Mycoplasma hominis were obtained from the urethra, vagina, cervix, and posterior fornix of 132 women. Vaginal cultures resulted in the most isolations of both species. As a measure of the reproducibility of this method, 4 separate vaginal cultures were obtained from 52 additional women. A single vaginal culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M. hominis.
T H E G E N I T A L mycoplasmas, T-mycoplasmas and Mycoplasma hominis, when present in women, colonize the genital mucosa, including the external cervical OS,vagina, vestibule, and distal urethra. In planning epidemiologic studies, we wanted to know how best to obtain cultures for these organisms. We were especially interested in From the Channing Laboratory, Thorndike Memorial Laboratory, Harvard Medical Unit, Department of Medical Microbiology, and Department of Obstetrics and Gynecology, Boston City Hospital, Departments of Medicine and Pediatrics, Harvard Medical School, and Department of Obstetrics and Gynecology, Boston University School of Medicine. Supported by Grants Nos. HD-03693 from the National Institute of Child Health and Human Development and TO1 AI-00068 from the National Institute of Allergy and Infectious Diseases. y;;ived
for publicafion
November
23,
f;;;pted
for
November
26,
publication
Reprint requests: Dr. Channing Laboratory, Hospital, 774 Albany Massachusetts 02118.
McCormack, Boston City St., Boston,
*Recipient of United States Health Service Post-Doctoral 1 FO.? 4144394-02.
Public Fellowship
exploring techniques for the collection of specimens that would lend themselves to population surveys. Urine cultures are quite acceptable for population studies, and urine has been used to culture mycoplasmas. As bladder urine is normalIy sterile, the organisms are presumably picked up in transit from the periurethral mucosa. Archer1 found that urine provided as many positive cultures as genital swabs from the same women. However, his technique required centrifugation of the urine before culturing, a laborious and time-consuming procedure. Braun and associate? subsequently showed that direct cultures of urine grew significantly fewer T-mycoplasmas than genital cultures from the same women. Thus, we decided that urine cultures would not be satisfactory for our studies. We then considered vaginal cultures. These can be taken by simply inserting a cotton-tipped swab into the vagina. As a vaginal speculum is not required, the procedure is reasonably acceptable to the subject. Several investigators have noted that the vagina was a satisfactory site for the isolation of genital mycoplasmas,s-s but only one of these studies” included T-mycoplas-
Localization
mas, and this study merely notes that vaginal cultures were better than cervical cultures without providing any data. To examine further the suitability of vaginal cultures for the isolation of genital mycoplasmas, we obtained cultures from the urethra, vagina, cervix, and posterior fornix of 132 women. We found that vaginal cultures resulted in the most isolations of both T-mycoplasmas and M. hominis. We then obtained 4 separate vaginal cultures from an additional 52 women as a measure of the reproducibility of this method. A single vaginal culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M.
hominis. Methods Women who presented to the prenatal and gynecology clinics of the Boston City Hospital and who for any reason required a speculum examination of the vagina were studied. The examining physician obtained the following cultures from each subject: (1) Urethra-a sterile swab was placed into the distal urethra and rotated gently; (2) Vagina-a sterile swab was inserted 1 to 2 inches into the vagina and rubbed against the vaginal wall; (3) Cervixafter the speculum had exposed the cervix, a sterile swab was placed into the external OS and rotated; (4) Posterior fornixwith the speculum in place, a sterile swab was placed into the posterior fornix and rotated. The reproducibility of vaginal cultures was evaluated in patients on the Boston City Hospital obstetric service. The labia were spread with a gloved hand, and 4 separate vaginal cultures were taken by placing sterile swabs 1 to 2 inches into the vagina and rubbing them against the vaginal wall. The methods used in our laboratory for the cultivation and identification of the genital mycoplasmas have been described in detail by Braun and associates.2 Immediately after collection, swabs were placed into 4 ml. screw-rapped vials containing 2 ml. of
of
genital
mycoplasmas
921
basic mycoplasma broth (70 per cent PPLO broth, 20 per cent unheated horse serum, 10 per cent yeast extract, 0.002 per cent phenol red, and 500 U. per milliliter of benzyl penicillin) to which 50 pg per milliliter of pal)mixin B and 5 pg per milliliter of amphoteritin B had been added. The pH had been adjusted between 6.0 and 6.4 with IN Hcl. All specimens were frozen within 6 hr. of collection and stored at -80’ C. Specimens were thawed after no more than 7 days of storage, and 0.2 ml. of broth was inoculated into 1.2 ml. of urea broth (basic broth at pH 6.0 plus 0.5 mg. per milliliter of urea and 15 pg per milliliter of lincomycin) for T-mycoplasmas and into 1.2 ml. of arginine broth (basic broth at pH 7.2 plus 10 mg. per milliter of arginine and 100 /kg per milliliter of erythromycin) for M. hominis. The pH of the broth was measured by comparison with color standards at the time of inoculation and daily during incubation at 37’ C. Specimens in \vhich the pH had risen were subcultured to rrrycoplasma agar. A single streak was made with a Pasteur pipette. After the inoculum had dried, a blank sensitivity disc impregnated with undiluted antiserum to 111. hominlv was placed at one end of the streak and ;t disc containing erythromycin was placed at the other end. Plates were incubated in an atmosphere of 90 per cent N,-10 per cent CO? and examined after 2 to 4 days at 37” C. T-mycoplasmas were identified by their characteristic colonial morphology, inhibition of growth around the disc c-ontaining erythromycin, and persistence of growth around the disc impregnated with antiserum to M. hominis. M. hominis was identified by its colonial morphology and by inhibition of growth around the disc containing specific antiserum. Results Table I shows the percentage of isolations of T-mycoplasmas from each of the sites sampled. The greatest number of isolations (86 or 65.2 per cent) were obtained from \,agirrrrl cultures. Only 62 isolations (47.0 per cent) MYYC made from cervical cultures (p
922
McCormack,
Rankin,
and
Lee Am.
Table I. Isolation of T-mycoplasmas 4 sites in 132 women
Site
;$f
Urethra Vagina Cervix Posterior fomix
!CE;f
81 86 62 80
*One hundred women mycoplasmas by combining
4 vaginal
Urethra Vagina Cervix Posterior fornix ‘Seventy combining
Eif 64 68 59 66
Culture First Second Third Fourth
81 86 62 80 be positive for of alI 4 cultures.
Table II. Isolation of Mycoplasma from 4 sites in 132 women
Site
T-
48.5 51.5 44.7 50
“Forty-four women shown mas by combining the results
from
Per cent of total 75.0 71.2 65.4 69.2
Per cent of 44 women with positive cultures* 88.6 84.1 77.3 81.8
to be positive for T-mycoplasof all 4 cultures.
4 vaginal
of Mycoplasma hominis cultures in 52 women
Culture 1i?$%.&i, 17ti;i;;~~: 91.4 97.1 84.3 94.3
for M.
No. of isolations 39 37 34 36
from
Table IV. Isolation
hominis
~~~~1~~
women shown to be positive the results of all 4 cultures.
Isolation of T-mycoplasmas cultures in 52 women
zgkk$
61.4 65.2 47.0 60.6
shown to the results
Table III.
from
April 1, 1’372 J. Obstet. Gynecol.
komink
First Second Third Fourth by
= < 0.01). Cultures of the urethra and posterior fornix showed intermediate results. T-mycoplasmas were isolated from a total of 100 different women if the results of the cultures from all 4 sites are combined. An estimate of the sensitivity of a single culture taken from a given area can be made by comparison with the 100 patients shown to be positive for T-mycoplasmas by combining the results of all 4 cultures. This is also shown in Table I. Vaginal cultures detected 86 per cent and cervical cultures detected 62 per cent of the women shown to be positive for T-mycoplasmas. Vaginal and urethral cultures together detected 94 per cent of these women. Table II presents the rates of isolation of M. hominis. Again, vaginal cultures (68 or 51.5 per cent) were more productive than cervical cultures (59 or 44.7 per cent) although this difference is not statistically significant. A single vaginal culture detected 68 (97.1 per cent) of the 70 women who were shown to be positive by combining the results of all 4 cultures. Four separate vaginal cultures were then
21 22 22 22
*Twenty-three women hominis by combining the
40.4 42.3 42.3 42.3 shown results
to be positive of all 4 cultures.
91.3 95.7 95.7 95.7 for
M.
obtained at the same time from each of 52 women. Tables III and IV show the results of the isolations from these cultures of Tmycoplasmas and M. hominis, respectively. A single culture detected from 77.3 to 88.6 per cent of women shown to be positive for T-mycoplasmas and from 91.3 to 95.7 per cent of women shown to be positive for M. hominis by combining the results of all 4 cultures. Comment These data show that for the isolation of the genital mycoplasmas vaginal cultures are at least as productive as cultures taken from the posterior fornix, urethra, or cervix. This has allowed us to use vaginal cultures in our epidemiologic studies. Several women on our staff have been trained to take these cultures. The technique involves simply spreading the labia with a gloved hand to expose the vaginal opening, inserting a sterile swab 1 to 2 inches into the vagina with the other hand, and rubbing the swab against the vaginal wall. The procedure is rapid and painless and has been well accepted by our
Volume Number
112 7
subjects. It has been especially useful for postpartum women where a specuhrm examination would have been quite uncomfortable. In one survey, a group of student and graduate nurses took their own vaginal cultures with the use of this technique. A disquieting observation is that although vaginal cultures resulted in the most isolations of T-mycoplasmas the vaginal culture was positive in only 86 per cent of the women who were shown to be carrying Tmycoplasmas by combining the results of cultures from all 4 sites. This relative insensitivity of a single vaginal culture for the isolation of T-mycoplasmas is also apparent from the results obtained with 4 vaginal cultures taken at the same time. Here, a single culture detected between 77.3 and 88.6 per cent of the women who were shown to be colonized with T-mycoplasmas by combining the results of all 4 cultures. Apparently, the number of T-mycoplasmas present is so small or the organisms are so fastidious that with the currently available laboratory methods a single vaginal culture will fail to identify T-mycoplasmas in as many as 22 per cent of colonized women.
Localization
of genital
mycoplasmas
923
A higher yield can be obtained by culturing more than one site. Combining urethral and vaginal cultures detected 94 per cent of women shown to be positive for T-mycoplasmas. Results can also be improved by obtaining 2 cultures from the same site. Any combination of 2 vaginal cultures revealed over 90 per cent of women who were shown to be positive for T-mycoplasmas by combining the results of all 4 vaginal cultures. Cultures for Mycoplasma hominis seem to be more reproducible. When vaginal cultures were compared, a single culture detected between 91.3 and 95.7 per cent of women shown to be positive by combining the results of all 4 cultures. The valuable technical assistance of Miss Vicki Crockett and Mrs. Penrhyn Bailey is gratefully acknowledged. This study would not have been possible without the cooperation of the house officers on the obstetric and gynecology service at the Boston City Hospital. The horse serum was supplied by the Biological Laboratories of the Massachusetts Department of Health.
REFERENCES
1. Archer, J. F.: Br. J. Vener. Dis. 44: 232, 1968. 2. Braun, P., Klein, J. O., Lee, Y-H., and Kass, E. H.: J. Infect. Dis. 121: 391, 1970. 3. Dunlop, E. M. C., Hare, M. J., Jones, B. R., and Taylor-Robinson, D.: Br. J. Vener. Dis. 45: 274, 1969.
4. Freundt, E. A.: Acta PathoI. Microbic& Sand. 32: 468, 1953. 5. Horoszewicz, J.: Br. J. Vener. Dis. 37: 183, 1961.