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Isolation of Mycoplasma hominis, T-strains, and cytomegalovirus from the cervix of pregnant women
volume
106
number 5
March 1, 1970
American
JOWMI
of Obstetrics and Gynecology
OBSTETRICS
Isolation of Mycoplasma T-strains/and
hominis,
cytomegalovirus
from the cervix
of pregnant women HJORDIS
M.
GEORGE
E.
BERTTINA L.
THOMAS
Seattle,
M.D.
KENNY, B.
WAYNE J.
FOY,
PH.D.
WENTWORTH,
JOHNSON,
PH.D. M.D.
GRAYSTON,
M.D.
Washington
Mycoplasma hominis was isolated from 37 of 199 cervical cultures from women attending the prenatal clinic at the University Hospital in Seattle. The isolation rate was higher among Negroes (32 per cent), than among Caucasians (16 per cent). T-strains were isolated from 56 per cent of the pregnant Caucasian women and 75 per cent of the pregnant Negro women. Nearly all (92 per cent) of those who carried M. hominis also carried T-strains. No adverse eflect of pregnancy or on the infant from the presence of either M. hominis or T-strains was observed. Both organisms were transmitted to infants of positive mothers (M, hominis to 2 of 11 such infants and T-strains to 4 of 15 such infants). Cytomegalovirus (CMV) was isolated from cervical cultures of 5 of 140 pregnant women. Congenital cytomegalovirus disease or transmission to the infant was not seen in any of these 5 patients. On the other hand, the presence of CMV in the cervix was associated with lower than average birth weight.
T H E R E s u L T s o F recent studies indicate that the female genital tract harbors a variety of microorganisms which might have pathogenic potentials. Some of these are mycoplasmata (previously organisms termed pleuropneumonia-like organismsPPLO) , cytomegalovirus, and trachoma inclusion conjunctivitis (TRIG) agent.
From the Department of Preventive Medicine and the Department of Obstetrics and Gynecology, University of Washington School of Medicine. This investigation was supported in part by United States Public Health Service Training Grant AZ-206 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by United States Army Contract DA-49-193-MD-2294. 635
636 Foy et al.
Amer.
of T-strains
Methods Media for cultivation
Soy peptonet (Gm.) NaCl (Gm.) Mes$ (Gm.) Tris$ (Cm.) l-LO (ml.) Final pH (adjusted with 1N NaOH) Agar (Difco Noble) Agarose (Bausch and Lomb)
of mycoplasmata.
The general composition of complete media for cultivation of T-strains and M. hominis was: basic medium 70 per cent, horse serum 20 per cent, fresh yeast dialysate 10 per cent,17 penicillin 200 units per milliliter, and other additions as specified. The formula-
and M. hominis Basic
Ingredient
1, 1970 Gynec.
tion, formerly termed cytomegalic inclusion disease, may have a serious, sometimes lethal effect on the newborn infant.ll Mothers of such infants rarely give a significant history of illness during pregnancy which can be related to CMV infection and circumstances of fetal transmission to the infant are poorly understood. CMV may cause a generalized infection, with virus present in saliva and urine. Although most adult infections are probably asymptomatic, atypical mononucleosis,12 hepatitis,13 and respiratory symptoms including pneumoniall have been reported. The present study was initiated in November, 1965, to search for TRIC agent and mycoplasmata in the cervices of pregnant women. Reports regarding TRIC agent and preliminary findings regarding M. hominis among pregnant women in Seattle have been given elsewhere. I41 I5 A search for CMV in this population was started in June, 1966, following the report of AlexandeP that cytomegaloviruses were isolated from the cervix of 18 per cent of pregnant women on Taiwan.
Three types of Mycoplasma have been recovered from the genital tract. Initially the isolation of classic “large colony” mycoplasmata from the genital tract was described.l This group of organisms has now been determined to be predominantly M. hominis (previously M. hominis type 1) with a rare isolate of M. fermentans.2 An additional organism resembling mycoplasmata, the T-strain (T for tiny colony) was recognized by Shepard3 in 1954. Adequate technology for isolation of T-strains has been available only recently. Therefore most studies where organisms were not serotyped can be considered to have been concerned with M. hominis, since the techniques ordinarily used would not have detected T-strains. M. hominis is usually considered a harmless saprophyte, although occasional case reports have indicated potential pathogenity for mycoplasmata. 2 Recently M. hominis was isolated from the blood of a patient suffering from pyrexia after parturition4 and in one case after abortion.5 Jones6 isolated M. hominis from aborted fetuses and associated pyrexia after delivery with antibody rise to the organism. These reports clearly indicate a need for prospective studies of the effect of M. hominis on pregnancy and fetus. Tstrains of mycoplasmata have been associated with nongonococcal urethritis.3, ?-lo The effect of carriage of T-strains in women is not yet known. Congenital cytomegalovirus (CMV) infec-
TabIe I. Basic media for isolation
March J, Obstet.
E”
1
20 5 1,000 7.4 10 -
*Sterilized by auto&wing at 121” C. for 15 minutes and store Vkeffield Chemical Company, New York. SMes, Z-(N-morpholino) ethane sulfonic acid (Calbiccbem) . $Tris, (hydroxy methyl) aminomethane (Calbiihem).
at room
media+
Mes
20 5 4.26 (20 mM.) 1,000 6.0 10 temperature.
Tris
20 5 4.8 (20 mM.) 1,000 7.0 10 -
Volume Number
106
5
tions for the basic media are presented in Table I. The complete media for cultivation of M. hominis were prepared from either “E” basic medium or “Tris” basic medium, and these complete media were referred to respectively as “E” agar and “Tris” agar. Medium for isolation of T-strains contained “Me? agar base; this complete medium was termed “Mes” agar. Although thallium acetate was usually employed at 0.33 mg. per milliliter in media to be used for isolation of M. hominis, it was never employed in T-strain media. The pH of Mes agar was 6.5 when in air at room temperature and 6.3 when incubated in a mixture of 95 per cent Nz and 5 per cent CO, at 37O C. The complete fluid medium for isolation of M. hominis was diphasic media”; for T-strains a complete fluid medium was prepared from soy peptonefresh yeast extract dialysate basic broth,ls containing additionally 0.04 per cent urea and 0.004 per cent phenol red, was employed. The formulation of Mes agar and use of a urea color medium was an adaptation of the basic soy peptone medium used in this laboratoryl’ to meet the unique requirements for isolation of T-strains as described by ShepardlO : low pH, agar free of sulfated polysaccharides, and omission of thallium acetate. Isolation of mycoplasmata. One-tenth milliliter of a fresh nonfrozen specimen was inoculated into Tris and/or E agar and 0.1 ml. was added to a diphasic culture. Agar cultures were incubated aerobically and anaerobically in 95 per cent N, + 5 per cent CO2 at 37’ C. The diphasic medium was incubated aerobically and subcultured to agar at 4 to 5 days. Organisms were identified as to species by inhibition of colony formation with paper disks impregnated with rabbit antisera18 as described by Clyde.19 This procedure appeared adequate for isolation of M. hominis and other human species of mycoplasmata encountered, but colonies resembling T-strains were not recovered. Since the description of sensitive methods” was available only recently, isolation of Tstrains could be attempted in this study only from frozen specimens which had been stored
M.
hominis
in pregnant
cervix
637
for at least one year at -75 C. One tenth milliliter aliquots of specimen were inoculated into Mes agar and into urea color test media. Fluid medium was incubated aerobically at 37’ C., whereas Mes agar was incubated at 37O C. in 95 per cent N, + 5 per cent CO, atmosphere. Urea broth cultures were subcultured to Mes agar whenever alkaline color change of the phenol red indicator occurred. Organisms were identified as T-strains if they formed tiny colonies on Mes agar and were propagatable entities which produced an alkaline color reaction from urea. Serology. Antibody to M. hominis was detected by complement fixation, with the use of techniques previously described.ls A wild strain of M. hominis (GU-43) was employed for CF antigen because it was the best strain of 4 tested for detecting antibody in the human sera tested. Although the prototype M. hominis strain (ATCC 14027) reacted well with homologous rabbit antisera, it did not appear to measure human serum antibody. Sera were tested for CMV antibody by a microtiter complement fixation technique.20 Cytomegalovirus isolation. The methods for isolation and characterization of CMV were essentially as described previously.2o The H-200 strain of human fetal fibroblasts was employed. The study population. This consisted of pregnant women who were examined by the staff in the obstetric clinic at the University of Washington Hospital. This clinic serves a heterogenous group composed of wives of staff and faculty members, students, and persons of low income. A few women are referred to the clinic as high-risk patients, and some come from a home for unwed mothers. The cultures for mycoplasmata were all obtained from 199 women who were in their first trimester between November, 1965, and August, 1966. An attempt was made to reculture and obtain blood specimens from 20 of the M. hominis positive women, and 20 patients matched by race, parity, and age in the last trimester and post par-turn. Cultures from multiple sites of the newborn infants
638
et al.
Foy
normal deviate test and differences means by Student’s t test.
of these women were obtained when feasible within 3 days of birth and at the age of 2 weeks. Specimens for CMV culture were obtained from 120 of the patients seen in their first trimester of pregnancy between June, 1966, and October, 1966. On the possibility that pregnancy might precipitate a latent CMV infection, 50 women were cultured during their last trimester in the fall of 1966. Three patients were cultured both in their first and third trimester. Specimen collection. The cervical swabs were obtained by rotating a cotton swab in the cervical OS after the cervix had been cleaned with cotton and a Papanicolaou smear obtained. In the last trimester the procedure was similar, although no cytologic smear was obtained at that time. The swabs were placed in vials containing 3 ml. of trypticase soy broth (Baltimore Biological Laboratories) supplemented with 0.5 bovine serum albumin, and 100 units penicillin per milliliter. The vials were stored in the refrigerator until transferred to the laboratory, where they were processed the same day. Statistical analysis. For differences in rates this analysis is made by the approximate
Table
II.
Characteristics
of pregnant
women
M.
according
Mean age Per cent married Per cent primigravida Per cent history of venereal disease Per cent history of abortions Infant death Abortion? Lost to follow-up Per cent delivery$ complications Mean gestation periods Mean Apgar scores Mean birth weight (Gm.)g
hominis
fFetsl
women death
ZIncludes $Two
before
ectampsia, twin
births
of “other”
racial
20 weeks’
0 33 0 1 1 13 37.1 8.0 2,924
37 21 80 46
138 22 84 51
3 19 0
3 14 8 5 9 9 39.5 7.8 3,276
: 10 39.3 7.8 3,241
Negro 19 23 63 32 12 21 1 1 1 17 38.4 8.3 2,850
in
febrile comparison
/
I -
origin.
,
gestation.
pm-eclampsia, excluded
Negative
Caucasian
Total”
9 18 56 56
4 12 0 1 5 10 40.1 7.7 3,377
of M.
to race and presence
I
Negro
26 22 81 42
Number
between
M. hominis. All large colony isolates of mycoplasmata from the cervix in this study have been typed as M. homink The outcome of culture according to race, age, marriage, previous pregnancies, and history of venereal disease and previous abortions is shown in Table II. M. hominis was isolated at a higher rate (32 per cent) from Negroes than Caucasians (16 per cent) (P < 0.05). None of the 5 Oriental women were positive. The isolation rate among nulliparous and multiparous women was not significantly different, Neither was there any difference in history of previous abortions when each race was considered by itself. A higher isolation rate (29 per cent) was observed among unmarried than married women ( 17 per cent), but this difference did not reach statistical significance. The outcome and characteristics of pregnancy, also shown in Table II, are similar in both groups. Several of the mycoplasmatapositive women moved away from Seattle or changed hospital before delivery. We made
and their infants
Caucasian
1, 1970 Gynec.
Results
Positive
*Includes
March J. Obstet.
Amer.
reactions of
post
gestation
partum, period,
hemorrhage, Apgar
score,
abrupti and
birth
placenta, weight.
and
*lacenta
previa.
Volume Number
106 5
M. hominis
special efforts to assure ourselves that these losses to follow-up were not due to fetal wastage. Complications of delivery such as sepsis, toxemia, excessive bleeding or placental detachment did not differ between M. hominis positive or negative groups. The gestation period, Apgar score, and birth weight of the infants were also similar in the two groups. The charts of the newborn infants of M. hominis positive mothers were reviewed for their first 2 weeks of life and no abnormalities were noticed. Outcome of repeated cultures. The original protocol called for cultures of throat and collection of blood specimens in the last trimester and post partum, from 20 M. hominis positive women and 20 negative women matched by race, age, and parity. This project was complicated by the loss to follow-up of several of the women originally selected. The results are as follows: In the third trimester, 10 (67 per cent) of 15 women with isolation of M. hominis in the first trimester were again positive. Of 5 originally positive women, 3 showed M. hominis 6 weeks post partum. Throat cultures were also obtained from the 15 with positive cervical culture. In no case was M. hominis isolated from the
and T-strains
hominis
in pregnant
cervix
throat, but M. salivarium was isolated from 7 and M. pharyngis from one of them. When 15 women, negative for M. hominis in the first trimester, were cultured again in the last trimester, 2 were positive. At 6 weeks post partum, 3 of 8 originally negative women were positive. The throat cultures of 15 originally cervix-negative women revealed M. salivarium in 4, M. pharyngis in one, and nontypable species in 2 patients. Serologic results. The sera of 15 women with, and 9 without M. hominis isolates in the first trimester were tested for antibodies for this agent. None of the 9 primigravidas, regardless of outcome of culture, had detectable antibodies. Six of 10 multigravidas with positive cultures and 2 of 5 multigravidas with negative cultures had CF antibodies. Colonization of infants with M. hominis. Eleven infants of M. hominis positive women were cultured at 6 different sites (eye, nose, throat, umbilicus, vagina or urethral opening, and rectum) within 2 days of birth, and in 5 instances again at the age of 2 weeks. M. hominis was recovered from the eye of one and from the vagina of another. The vaginal culture was also positive 2 weeks after birth.
in the cervix T-strains Positiue
Total*
I
Caucasian
Neeative
1
Negro
Total*
162 22 82 50
92 21 78 51
21 21 62 48
116 21 74 52
4 14 9 6 11 10 39.4 7.9 3,224
6 13 4 2 10 11 39.8 7.6 3,225
6 19 0 2 1 10 37.6 8.3 2,950
6 14 4 4 12 12 39.4 7.7 3,172
639
Caucasian 72 22 92 49 0 14 4 4 4 7 39.3 8.0 3,373
Negro 7 21 57 14 14 43 1 0 1 33 39.4 8.0 2,593
1
Total* 83 22 89 46 1 17 5 4 6 9 39.3 8.0 3,304
640
Foy et al.
Amer.
Description
of CMV-positive
1, 1970 Gynec.
from 3 infants, similarly cultured, but born to T-strain negative mothers. Cytomegalovirus. CMV was isolated from cultures of the cervix of 4 of 120 women in the first trimester, and from one of 50 women in the third trimester. The socioeconomic data regarding women tested in the first and third trimester have been compared to see to what extent they show different population characteristics. The distribution according to race, age, proportion married, and history of previous abortions was similar for these 2 groups. The main difference was that 59 per cent of those tested in their first trimester were nulliparous compared with 40 per cent in those tested in their last trimester. Comparison of the CMV positive women with the CMV negative women revealed the following differences: Three out of the 4 positive Caucasian women were unmarried compared with 22 per cent unmarried Caucasian women in the negative culture group. A history of abortions was found in only 26 per cent of Caucasian multigravida, but both multiparous CMV positive women (OB 685 and OB 737) had histories of 2 spontaneous abortions each (Table III). OB 685 in addition had been delivered of her 2 live children prematurely. At her last trimester examination she had a severe follicular cervicitis. The previous live birth of OB 737 was complicated by abruptio placentae. The mean age of CMV negative Caucasian women was 21.4 years, mean gestation period 39.7 weeks, mean Apgar score of in-
Neither infant showed any signs of illness. M. hominis was not isolated from 7 infants whose mothers had negative cervical cultures ante partum. T-strains. The outcome of culture for T-strains according to race, age, marital status, previous pregnancies, and history of venereal disease, and previous abortions is shown in Table II. T-strains were isolated at a higher rate from Negroes (75 per cent) than from Caucasians (56 per cent), and in higher rates from unmarried (77 per cent) than from married (54 per cent) women (P < 0.05). The outcome and characteristics of pregnancy, also shown in Table II, were similar for the T-strain positive and negative group. Of the 37 M. hominis positive women 34 (92 per cent) were also positive for Tstrains, compared with 51 per cent (82/ for M. hominis 162) of those negative (P < 0.01). Outcome of repeated cultures. Twenty-two women who were positive for T-strains in the first trimester were again cultured in their last trimester and 17 of them again had positive cultures. Eleven of them were tested 6 weeks post partum and 9 were positive. On the other hand, 4 of 8 women negative for T-strains in their first trimester were positive in their third trimester. Colonization of infants. Fifteen infants born to women with T-strains in the cervix were cultured at 6 different sites as described above shortly after birth. T-strains were recovered from the vagina of 3, and from the throat of one. T-strains were not recovered
Table III.
March J. Obstet.
women
Record MO.
Age
/
%$f
(
Race
OB 537
19
Single
Caucasian
OB 669
17
Separated
Caucasian
OB 685
26
Married
Oriental
/
Obstetric
Gravida
history
1 “z”’
/ $fz
ii::,
1
38
2
3,040
1
Gravida Gravida 5, para 2, abortions 2
36
7
2,090
36
9
2,935
42
8
3,190
39
5
2,750
OB 695
17
Single
Caucasian
Gravida
OB 737
20
Married
Caucasian
Gravida 4, para abortions 2
1 1,
Volume Number
M. hominis in pregnant
106 5
fant 7.7, and mean birth weight of infant 3,309 grams. For the four CMV positive Caucasian women the mean age was 18.2 years, mean gestation period 38.7 weeks, mean Apgar score of infant 5.5, and mean birth weight of infant 2,767 grams. The difference in birth weight and Apgar score between infants of CMV positive and CMV negative Caucasian mothers is marginally significant statistically (P = 0.025 and P = 0.08). The mean birth weight of infants of CMV negative unmarried Caucasian women was 3,302 grams, equal to the married group; thus the difference in birth weight for the CMV positive and negative women could not be explained by marital status alone. Results of repeated cultures. Three positive mothers (OB 537, OB 685, and OB 695) had cervical, urine, saliva, and blood specimens taken in the last trimester and 6 weeks post partum. OB 685 had positive cervical and urine cultures in the last trimester, and OB 695 had a positive culture 6 weeks post partum. No virus was isolated from the other specimens. Serologic testing for CF antibodies for CMV. Six of 16 women with negative cultures for CMV and 3 of 5 women with positive cultures had serum CF antibody titers of 1: 8 or greater. One (OB 695) with no detectable antibody 6 weeks prior to delivery had a titer of 1: 16 6 weeks post par-turn. Only a single antepartum serum specimen was available on OB 537, who lacked CF antibodies in the sixth month of gestation. Investigation of infants. Although one (OB 537) of the 5 infants born to mothers with isolates of CMV failed to thrive in the neonatal period, there was no sign of overt CMV disease in these infants. Infant OB 537 had urine, saliva, and spinal fluid cultures within one week of birth and repeated cultures of saliva and urine at 6 and 8 weeks of age. The cultures were all negative for CMV. Urine and saliva cultures were also obtained on infant OB 685 at age 2, 7, and 15 weeks, on infant OB 695, at 3 weeks, and on infant OB 737 at age 6 weeks. These cultures were all negative. The infants of OB 685 and OB 737 ap-
cervix
641
peared normally developed and healthy at the age of one year. FamiIy members of OB 685 and OB 737 were investigated for CMV excretion in urine and saliva specimens. Neither husband nor the siblings (aged 2v2, 2, and 1 years) in these families were found to be excreting CMV. Comment Mycoplasma hominis. The isolation rate of 19 per cent from women attending a prenatal clinic is significantly lower than the rate of 48 per cent observed among women of similar age distribution attending a venereal disease clinic in Seattle.14 Both investigations were made by the same laboratory using the same methods. This supports previous observations39 21 of a higher rate of occurrence of mycoplasmata among promiscuous persons. A higher rate among Negroes than Caucasians has also been previously reported. 3p I41 I5 Repeated cultures from the same women yielded somewhat irregular results, probably indicating that test results from a single cervical swab might be negative even when the organism is present. The rate of 19 per cent carriers is therefore a minimum estimate. Mycoplasmata were isolated from 2 of 11 infants born to women from whom M. hominis had previously been isolated. Since the isolations were made as early as one day after birth, it is impossible to tell whether infection occurred intrauterinely or by passage through the infected birth canal. The organism was isolated from the vagina of one infant on severa occasions during the first year of life, which indicates that genital M. hominis infection is not always obtained through sexual contact. Recent case reports51 6 have associated M. hominis infection with abortion. The organism was isolated from the bloodstream of an aborting woman and from the aborted fetus. The present study did not demonstrate any deleterious effect on pregnancy or infant from the presence of M. hominis in the cervix. If this Mycoplasma species is a cause of abortion, such complication does not appear to be a common event.
642
March
Foy et al.
JoneP reported on development of CF antibody titers for M. homink in pregnant women in relation to parturition. The findings in this study, where none of 15 primigravidas, but 8 of 15 multigravidas, had CF antibodies lends support to Jones’ theory that CF antibodies develop in association with parturition or abortion. T-strains. The over-all isolation rate, 58 per cent, for T-strains in this study is similar to the rate of 65 per cent reported among pregnant women by Czonka, Williams, and Corse.v The isolation system described for T-strains in this study appeared to be efficient because its facility in detecting T-strains certainly cannot be less than 58 per cent and is possibly close to the efficiency of isolation shown by “E” medium and diphasic broth for M. pneumoniae (65 per cent). The higher rate of T-strains among unmarried than married, and Negro than Caucasian, women suggests socioeconomic differences, an idea which is borne out by the close association of T-strain isolation with M. hominis positivity. While a recent report has associated a T-strain with abortion,23 the present data provide little basis for suspecting that T-strains would be a common cause for spontaneous abortions, prematurity, or
REFERENCES
Dienes, L., and Edsall, J.: Proc. Sot. Exp. Biol. Med. 36: 740, 1937. 2. Chanock, R. M.: New Eng. J. Med. 273: 1199, 1965. 3. Shepard, M. C.: Amer. J. Syph. Vener. Dis. 38: 113, 1954. 4. Tully, J., and Smith, L.: J. A. M. A. 204: 827, 1968. 5. Harwick, H. J., Tuppa, J. B., Purcell, R. H., and Fekety, R. R., Jr.: AMER. J. OBSTET. GYNEC. 99: 725, 1967. 6. Jones, D. M.: Brit. Med. J. 1: 388, 1967. 7. Ford, D., and DuVernet, M.: Brit. J. Vener. Dis. 39: 18, 1963. 8. Shepard, M., Alexander, C., Lunceford, C., and Campbell, P.: J. A. M. A. 188: 729, 1964. 9. Czonka, G., Williams, R., and Corse, J.: Ann. N. Y. Acad. Sci. 143: 794. 1967. 10. Shepard, M. C.: Ann. N. .Y. Acad. Sci. 143: 505, 1967.
Amer. J. Obstet.
1, 1970 Gym.
other
adverse effect on pregnancy or fetus. The 3 per cent isolation rate of CMV from the cervix of pregnant women is in agreement with the findings of Feldman”’ and Hildebrandt and associates,25 who isolated the virus from the urine of 3 and 3vz per cent of pregnant women, respectively. On the other hand, Alexander,lG who also used cervical cultures, obtained an isolation rate of 18 per cent among women on Taiwan. In 2 of the above-mentioned studies transmission to the infant was occasionally seen. In this study no transmission was observed. The fact that lower than average birth weight and low Apgar score were observed in the infants of positive mothers in this study suggests an adverse effect of maternal CMV infection on the fetus and indicates the need for a larger study to substantiate this association. Low birth weight is observed in congenital CMV disease of both severelI and mild*” type. While 3 previous investigations of CMV in pregnancy16T 24s25 have not reported findings similar to ours, Diosi and associates”’ recently reported on a case of abortion associated with CMV infection, discussing the possibility of the virus causing endometritis. Further studies are warranted.
CMV.
11.
1.
12.
13. 14.
15.
16. 17.
18.
Hanshaw, J. B.: Pediat. Clin. N. Amer. 13: 279, 1966. Klemola, E., Kaariaainen, L., von Essen, R., Haltia. K.. Koivuniemi. A.. and von Bonsdorff, ‘C. H.: Acta Med. &and. 182: 311, 1967. Lamb, S. G., and Stern, H.: Lancet 2: 1003, 1966. Holt, S., Pederson, A. H. B., Wang, S. P., Kenny, G. E., Fey; H. M., and Grayston, J: T.: Amer. J. Ophthal. 63: 1057, 1967. Foy, H. M., Wang, S. P., Kenny, G. E., Johnson, W. L., and Grayston, J. T.: Amer. T. Onhthal. 63: 1053. 1967. klex’ander, E. R.: P&at. Res. 1: 210, 1967. Grayston, J. T., Foy, H. M., and Kenny. . , G. E.: Mycoplasmas (PPLO) in Human Disease, in Dowling, H. F.. editor: Disease-a-Month. Chicago, 1367, Ye& Book Medical Publishers: Inc. Kenny, G. E.: Ann. N. Y. Acad. Sci. 143: 676, 1967.
Volume Number
19. 20.
21.
22. 23.
106 5
Clyde, W. A.: J. Immunol. 92: 958, 1964. Levinsohn, E. M., Foy, H. M., Kenny, G. E., Wentworth, B. B., and Grayston, J. T.: Proc. Sot. Exp. Biol. Med. In press. Klienberger-Nobel, E. : PPLO : Mycoplasmataceae, New York, 1962, Academic Press, Inc. Jones, D. M.: J. Clin. Path. 20: 633, 1967. Kundsin, R.: Science 157: 1573, 1967.
M. hominis
24. 25.
26. 27.
in pregnant
cervix
643
Feldman, R. A.: Proc. XI International Congress of Pediatrics, Tokyo, 1965, p. 215. Hildebrandt, R. J., Sever, J. L., Margileth, A. M., and Callagan, D. A.: AMER. J. OBSTET. GYNEC. 98: 1125, 1967. Emanuel, I., and Kenny, G. E.: Pediatrics 38: 957, 1966. Diosi, P., Babusceac, L., Nevinglovschi, O., and Kun-stoicu, G.: Lancet 2: 1063, 1967.
106
number 5
March 1, 1970
American
JOWMI
of Obstetrics and Gynecology
OBSTETRICS
Isolation of Mycoplasma T-strains/and
hominis,
cytomegalovirus
from the cervix
of pregnant women HJORDIS
M.
GEORGE
E.
BERTTINA L.
THOMAS
Seattle,
M.D.
KENNY, B.
WAYNE J.
FOY,
PH.D.
WENTWORTH,
JOHNSON,
PH.D. M.D.
GRAYSTON,
M.D.
Washington
Mycoplasma hominis was isolated from 37 of 199 cervical cultures from women attending the prenatal clinic at the University Hospital in Seattle. The isolation rate was higher among Negroes (32 per cent), than among Caucasians (16 per cent). T-strains were isolated from 56 per cent of the pregnant Caucasian women and 75 per cent of the pregnant Negro women. Nearly all (92 per cent) of those who carried M. hominis also carried T-strains. No adverse eflect of pregnancy or on the infant from the presence of either M. hominis or T-strains was observed. Both organisms were transmitted to infants of positive mothers (M, hominis to 2 of 11 such infants and T-strains to 4 of 15 such infants). Cytomegalovirus (CMV) was isolated from cervical cultures of 5 of 140 pregnant women. Congenital cytomegalovirus disease or transmission to the infant was not seen in any of these 5 patients. On the other hand, the presence of CMV in the cervix was associated with lower than average birth weight.
T H E R E s u L T s o F recent studies indicate that the female genital tract harbors a variety of microorganisms which might have pathogenic potentials. Some of these are mycoplasmata (previously organisms termed pleuropneumonia-like organismsPPLO) , cytomegalovirus, and trachoma inclusion conjunctivitis (TRIG) agent.
From the Department of Preventive Medicine and the Department of Obstetrics and Gynecology, University of Washington School of Medicine. This investigation was supported in part by United States Public Health Service Training Grant AZ-206 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by United States Army Contract DA-49-193-MD-2294. 635
636 Foy et al.
Amer.
of T-strains
Methods Media for cultivation
Soy peptonet (Gm.) NaCl (Gm.) Mes$ (Gm.) Tris$ (Cm.) l-LO (ml.) Final pH (adjusted with 1N NaOH) Agar (Difco Noble) Agarose (Bausch and Lomb)
of mycoplasmata.
The general composition of complete media for cultivation of T-strains and M. hominis was: basic medium 70 per cent, horse serum 20 per cent, fresh yeast dialysate 10 per cent,17 penicillin 200 units per milliliter, and other additions as specified. The formula-
and M. hominis Basic
Ingredient
1, 1970 Gynec.
tion, formerly termed cytomegalic inclusion disease, may have a serious, sometimes lethal effect on the newborn infant.ll Mothers of such infants rarely give a significant history of illness during pregnancy which can be related to CMV infection and circumstances of fetal transmission to the infant are poorly understood. CMV may cause a generalized infection, with virus present in saliva and urine. Although most adult infections are probably asymptomatic, atypical mononucleosis,12 hepatitis,13 and respiratory symptoms including pneumoniall have been reported. The present study was initiated in November, 1965, to search for TRIC agent and mycoplasmata in the cervices of pregnant women. Reports regarding TRIC agent and preliminary findings regarding M. hominis among pregnant women in Seattle have been given elsewhere. I41 I5 A search for CMV in this population was started in June, 1966, following the report of AlexandeP that cytomegaloviruses were isolated from the cervix of 18 per cent of pregnant women on Taiwan.
Three types of Mycoplasma have been recovered from the genital tract. Initially the isolation of classic “large colony” mycoplasmata from the genital tract was described.l This group of organisms has now been determined to be predominantly M. hominis (previously M. hominis type 1) with a rare isolate of M. fermentans.2 An additional organism resembling mycoplasmata, the T-strain (T for tiny colony) was recognized by Shepard3 in 1954. Adequate technology for isolation of T-strains has been available only recently. Therefore most studies where organisms were not serotyped can be considered to have been concerned with M. hominis, since the techniques ordinarily used would not have detected T-strains. M. hominis is usually considered a harmless saprophyte, although occasional case reports have indicated potential pathogenity for mycoplasmata. 2 Recently M. hominis was isolated from the blood of a patient suffering from pyrexia after parturition4 and in one case after abortion.5 Jones6 isolated M. hominis from aborted fetuses and associated pyrexia after delivery with antibody rise to the organism. These reports clearly indicate a need for prospective studies of the effect of M. hominis on pregnancy and fetus. Tstrains of mycoplasmata have been associated with nongonococcal urethritis.3, ?-lo The effect of carriage of T-strains in women is not yet known. Congenital cytomegalovirus (CMV) infec-
TabIe I. Basic media for isolation
March J, Obstet.
E”
1
20 5 1,000 7.4 10 -
*Sterilized by auto&wing at 121” C. for 15 minutes and store Vkeffield Chemical Company, New York. SMes, Z-(N-morpholino) ethane sulfonic acid (Calbiccbem) . $Tris, (hydroxy methyl) aminomethane (Calbiihem).
at room
media+
Mes
20 5 4.26 (20 mM.) 1,000 6.0 10 temperature.
Tris
20 5 4.8 (20 mM.) 1,000 7.0 10 -
Volume Number
106
5
tions for the basic media are presented in Table I. The complete media for cultivation of M. hominis were prepared from either “E” basic medium or “Tris” basic medium, and these complete media were referred to respectively as “E” agar and “Tris” agar. Medium for isolation of T-strains contained “Me? agar base; this complete medium was termed “Mes” agar. Although thallium acetate was usually employed at 0.33 mg. per milliliter in media to be used for isolation of M. hominis, it was never employed in T-strain media. The pH of Mes agar was 6.5 when in air at room temperature and 6.3 when incubated in a mixture of 95 per cent Nz and 5 per cent CO, at 37O C. The complete fluid medium for isolation of M. hominis was diphasic media”; for T-strains a complete fluid medium was prepared from soy peptonefresh yeast extract dialysate basic broth,ls containing additionally 0.04 per cent urea and 0.004 per cent phenol red, was employed. The formulation of Mes agar and use of a urea color medium was an adaptation of the basic soy peptone medium used in this laboratoryl’ to meet the unique requirements for isolation of T-strains as described by ShepardlO : low pH, agar free of sulfated polysaccharides, and omission of thallium acetate. Isolation of mycoplasmata. One-tenth milliliter of a fresh nonfrozen specimen was inoculated into Tris and/or E agar and 0.1 ml. was added to a diphasic culture. Agar cultures were incubated aerobically and anaerobically in 95 per cent N, + 5 per cent CO2 at 37’ C. The diphasic medium was incubated aerobically and subcultured to agar at 4 to 5 days. Organisms were identified as to species by inhibition of colony formation with paper disks impregnated with rabbit antisera18 as described by Clyde.19 This procedure appeared adequate for isolation of M. hominis and other human species of mycoplasmata encountered, but colonies resembling T-strains were not recovered. Since the description of sensitive methods” was available only recently, isolation of Tstrains could be attempted in this study only from frozen specimens which had been stored
M.
hominis
in pregnant
cervix
637
for at least one year at -75 C. One tenth milliliter aliquots of specimen were inoculated into Mes agar and into urea color test media. Fluid medium was incubated aerobically at 37’ C., whereas Mes agar was incubated at 37O C. in 95 per cent N, + 5 per cent CO, atmosphere. Urea broth cultures were subcultured to Mes agar whenever alkaline color change of the phenol red indicator occurred. Organisms were identified as T-strains if they formed tiny colonies on Mes agar and were propagatable entities which produced an alkaline color reaction from urea. Serology. Antibody to M. hominis was detected by complement fixation, with the use of techniques previously described.ls A wild strain of M. hominis (GU-43) was employed for CF antigen because it was the best strain of 4 tested for detecting antibody in the human sera tested. Although the prototype M. hominis strain (ATCC 14027) reacted well with homologous rabbit antisera, it did not appear to measure human serum antibody. Sera were tested for CMV antibody by a microtiter complement fixation technique.20 Cytomegalovirus isolation. The methods for isolation and characterization of CMV were essentially as described previously.2o The H-200 strain of human fetal fibroblasts was employed. The study population. This consisted of pregnant women who were examined by the staff in the obstetric clinic at the University of Washington Hospital. This clinic serves a heterogenous group composed of wives of staff and faculty members, students, and persons of low income. A few women are referred to the clinic as high-risk patients, and some come from a home for unwed mothers. The cultures for mycoplasmata were all obtained from 199 women who were in their first trimester between November, 1965, and August, 1966. An attempt was made to reculture and obtain blood specimens from 20 of the M. hominis positive women, and 20 patients matched by race, parity, and age in the last trimester and post par-turn. Cultures from multiple sites of the newborn infants
638
et al.
Foy
normal deviate test and differences means by Student’s t test.
of these women were obtained when feasible within 3 days of birth and at the age of 2 weeks. Specimens for CMV culture were obtained from 120 of the patients seen in their first trimester of pregnancy between June, 1966, and October, 1966. On the possibility that pregnancy might precipitate a latent CMV infection, 50 women were cultured during their last trimester in the fall of 1966. Three patients were cultured both in their first and third trimester. Specimen collection. The cervical swabs were obtained by rotating a cotton swab in the cervical OS after the cervix had been cleaned with cotton and a Papanicolaou smear obtained. In the last trimester the procedure was similar, although no cytologic smear was obtained at that time. The swabs were placed in vials containing 3 ml. of trypticase soy broth (Baltimore Biological Laboratories) supplemented with 0.5 bovine serum albumin, and 100 units penicillin per milliliter. The vials were stored in the refrigerator until transferred to the laboratory, where they were processed the same day. Statistical analysis. For differences in rates this analysis is made by the approximate
Table
II.
Characteristics
of pregnant
women
M.
according
Mean age Per cent married Per cent primigravida Per cent history of venereal disease Per cent history of abortions Infant death Abortion? Lost to follow-up Per cent delivery$ complications Mean gestation periods Mean Apgar scores Mean birth weight (Gm.)g
hominis
fFetsl
women death
ZIncludes $Two
before
ectampsia, twin
births
of “other”
racial
20 weeks’
0 33 0 1 1 13 37.1 8.0 2,924
37 21 80 46
138 22 84 51
3 19 0
3 14 8 5 9 9 39.5 7.8 3,276
: 10 39.3 7.8 3,241
Negro 19 23 63 32 12 21 1 1 1 17 38.4 8.3 2,850
in
febrile comparison
/
I -
origin.
,
gestation.
pm-eclampsia, excluded
Negative
Caucasian
Total”
9 18 56 56
4 12 0 1 5 10 40.1 7.7 3,377
of M.
to race and presence
I
Negro
26 22 81 42
Number
between
M. hominis. All large colony isolates of mycoplasmata from the cervix in this study have been typed as M. homink The outcome of culture according to race, age, marriage, previous pregnancies, and history of venereal disease and previous abortions is shown in Table II. M. hominis was isolated at a higher rate (32 per cent) from Negroes than Caucasians (16 per cent) (P < 0.05). None of the 5 Oriental women were positive. The isolation rate among nulliparous and multiparous women was not significantly different, Neither was there any difference in history of previous abortions when each race was considered by itself. A higher isolation rate (29 per cent) was observed among unmarried than married women ( 17 per cent), but this difference did not reach statistical significance. The outcome and characteristics of pregnancy, also shown in Table II, are similar in both groups. Several of the mycoplasmatapositive women moved away from Seattle or changed hospital before delivery. We made
and their infants
Caucasian
1, 1970 Gynec.
Results
Positive
*Includes
March J. Obstet.
Amer.
reactions of
post
gestation
partum, period,
hemorrhage, Apgar
score,
abrupti and
birth
placenta, weight.
and
*lacenta
previa.
Volume Number
106 5
M. hominis
special efforts to assure ourselves that these losses to follow-up were not due to fetal wastage. Complications of delivery such as sepsis, toxemia, excessive bleeding or placental detachment did not differ between M. hominis positive or negative groups. The gestation period, Apgar score, and birth weight of the infants were also similar in the two groups. The charts of the newborn infants of M. hominis positive mothers were reviewed for their first 2 weeks of life and no abnormalities were noticed. Outcome of repeated cultures. The original protocol called for cultures of throat and collection of blood specimens in the last trimester and post partum, from 20 M. hominis positive women and 20 negative women matched by race, age, and parity. This project was complicated by the loss to follow-up of several of the women originally selected. The results are as follows: In the third trimester, 10 (67 per cent) of 15 women with isolation of M. hominis in the first trimester were again positive. Of 5 originally positive women, 3 showed M. hominis 6 weeks post partum. Throat cultures were also obtained from the 15 with positive cervical culture. In no case was M. hominis isolated from the
and T-strains
hominis
in pregnant
cervix
throat, but M. salivarium was isolated from 7 and M. pharyngis from one of them. When 15 women, negative for M. hominis in the first trimester, were cultured again in the last trimester, 2 were positive. At 6 weeks post partum, 3 of 8 originally negative women were positive. The throat cultures of 15 originally cervix-negative women revealed M. salivarium in 4, M. pharyngis in one, and nontypable species in 2 patients. Serologic results. The sera of 15 women with, and 9 without M. hominis isolates in the first trimester were tested for antibodies for this agent. None of the 9 primigravidas, regardless of outcome of culture, had detectable antibodies. Six of 10 multigravidas with positive cultures and 2 of 5 multigravidas with negative cultures had CF antibodies. Colonization of infants with M. hominis. Eleven infants of M. hominis positive women were cultured at 6 different sites (eye, nose, throat, umbilicus, vagina or urethral opening, and rectum) within 2 days of birth, and in 5 instances again at the age of 2 weeks. M. hominis was recovered from the eye of one and from the vagina of another. The vaginal culture was also positive 2 weeks after birth.
in the cervix T-strains Positiue
Total*
I
Caucasian
Neeative
1
Negro
Total*
162 22 82 50
92 21 78 51
21 21 62 48
116 21 74 52
4 14 9 6 11 10 39.4 7.9 3,224
6 13 4 2 10 11 39.8 7.6 3,225
6 19 0 2 1 10 37.6 8.3 2,950
6 14 4 4 12 12 39.4 7.7 3,172
639
Caucasian 72 22 92 49 0 14 4 4 4 7 39.3 8.0 3,373
Negro 7 21 57 14 14 43 1 0 1 33 39.4 8.0 2,593
1
Total* 83 22 89 46 1 17 5 4 6 9 39.3 8.0 3,304
640
Foy et al.
Amer.
Description
of CMV-positive
1, 1970 Gynec.
from 3 infants, similarly cultured, but born to T-strain negative mothers. Cytomegalovirus. CMV was isolated from cultures of the cervix of 4 of 120 women in the first trimester, and from one of 50 women in the third trimester. The socioeconomic data regarding women tested in the first and third trimester have been compared to see to what extent they show different population characteristics. The distribution according to race, age, proportion married, and history of previous abortions was similar for these 2 groups. The main difference was that 59 per cent of those tested in their first trimester were nulliparous compared with 40 per cent in those tested in their last trimester. Comparison of the CMV positive women with the CMV negative women revealed the following differences: Three out of the 4 positive Caucasian women were unmarried compared with 22 per cent unmarried Caucasian women in the negative culture group. A history of abortions was found in only 26 per cent of Caucasian multigravida, but both multiparous CMV positive women (OB 685 and OB 737) had histories of 2 spontaneous abortions each (Table III). OB 685 in addition had been delivered of her 2 live children prematurely. At her last trimester examination she had a severe follicular cervicitis. The previous live birth of OB 737 was complicated by abruptio placentae. The mean age of CMV negative Caucasian women was 21.4 years, mean gestation period 39.7 weeks, mean Apgar score of in-
Neither infant showed any signs of illness. M. hominis was not isolated from 7 infants whose mothers had negative cervical cultures ante partum. T-strains. The outcome of culture for T-strains according to race, age, marital status, previous pregnancies, and history of venereal disease, and previous abortions is shown in Table II. T-strains were isolated at a higher rate from Negroes (75 per cent) than from Caucasians (56 per cent), and in higher rates from unmarried (77 per cent) than from married (54 per cent) women (P < 0.05). The outcome and characteristics of pregnancy, also shown in Table II, were similar for the T-strain positive and negative group. Of the 37 M. hominis positive women 34 (92 per cent) were also positive for Tstrains, compared with 51 per cent (82/ for M. hominis 162) of those negative (P < 0.01). Outcome of repeated cultures. Twenty-two women who were positive for T-strains in the first trimester were again cultured in their last trimester and 17 of them again had positive cultures. Eleven of them were tested 6 weeks post partum and 9 were positive. On the other hand, 4 of 8 women negative for T-strains in their first trimester were positive in their third trimester. Colonization of infants. Fifteen infants born to women with T-strains in the cervix were cultured at 6 different sites as described above shortly after birth. T-strains were recovered from the vagina of 3, and from the throat of one. T-strains were not recovered
Table III.
March J. Obstet.
women
Record MO.
Age
/
%$f
(
Race
OB 537
19
Single
Caucasian
OB 669
17
Separated
Caucasian
OB 685
26
Married
Oriental
/
Obstetric
Gravida
history
1 “z”’
/ $fz
ii::,
1
38
2
3,040
1
Gravida Gravida 5, para 2, abortions 2
36
7
2,090
36
9
2,935
42
8
3,190
39
5
2,750
OB 695
17
Single
Caucasian
Gravida
OB 737
20
Married
Caucasian
Gravida 4, para abortions 2
1 1,
Volume Number
M. hominis in pregnant
106 5
fant 7.7, and mean birth weight of infant 3,309 grams. For the four CMV positive Caucasian women the mean age was 18.2 years, mean gestation period 38.7 weeks, mean Apgar score of infant 5.5, and mean birth weight of infant 2,767 grams. The difference in birth weight and Apgar score between infants of CMV positive and CMV negative Caucasian mothers is marginally significant statistically (P = 0.025 and P = 0.08). The mean birth weight of infants of CMV negative unmarried Caucasian women was 3,302 grams, equal to the married group; thus the difference in birth weight for the CMV positive and negative women could not be explained by marital status alone. Results of repeated cultures. Three positive mothers (OB 537, OB 685, and OB 695) had cervical, urine, saliva, and blood specimens taken in the last trimester and 6 weeks post partum. OB 685 had positive cervical and urine cultures in the last trimester, and OB 695 had a positive culture 6 weeks post partum. No virus was isolated from the other specimens. Serologic testing for CF antibodies for CMV. Six of 16 women with negative cultures for CMV and 3 of 5 women with positive cultures had serum CF antibody titers of 1: 8 or greater. One (OB 695) with no detectable antibody 6 weeks prior to delivery had a titer of 1: 16 6 weeks post par-turn. Only a single antepartum serum specimen was available on OB 537, who lacked CF antibodies in the sixth month of gestation. Investigation of infants. Although one (OB 537) of the 5 infants born to mothers with isolates of CMV failed to thrive in the neonatal period, there was no sign of overt CMV disease in these infants. Infant OB 537 had urine, saliva, and spinal fluid cultures within one week of birth and repeated cultures of saliva and urine at 6 and 8 weeks of age. The cultures were all negative for CMV. Urine and saliva cultures were also obtained on infant OB 685 at age 2, 7, and 15 weeks, on infant OB 695, at 3 weeks, and on infant OB 737 at age 6 weeks. These cultures were all negative. The infants of OB 685 and OB 737 ap-
cervix
641
peared normally developed and healthy at the age of one year. FamiIy members of OB 685 and OB 737 were investigated for CMV excretion in urine and saliva specimens. Neither husband nor the siblings (aged 2v2, 2, and 1 years) in these families were found to be excreting CMV. Comment Mycoplasma hominis. The isolation rate of 19 per cent from women attending a prenatal clinic is significantly lower than the rate of 48 per cent observed among women of similar age distribution attending a venereal disease clinic in Seattle.14 Both investigations were made by the same laboratory using the same methods. This supports previous observations39 21 of a higher rate of occurrence of mycoplasmata among promiscuous persons. A higher rate among Negroes than Caucasians has also been previously reported. 3p I41 I5 Repeated cultures from the same women yielded somewhat irregular results, probably indicating that test results from a single cervical swab might be negative even when the organism is present. The rate of 19 per cent carriers is therefore a minimum estimate. Mycoplasmata were isolated from 2 of 11 infants born to women from whom M. hominis had previously been isolated. Since the isolations were made as early as one day after birth, it is impossible to tell whether infection occurred intrauterinely or by passage through the infected birth canal. The organism was isolated from the vagina of one infant on severa occasions during the first year of life, which indicates that genital M. hominis infection is not always obtained through sexual contact. Recent case reports51 6 have associated M. hominis infection with abortion. The organism was isolated from the bloodstream of an aborting woman and from the aborted fetus. The present study did not demonstrate any deleterious effect on pregnancy or infant from the presence of M. hominis in the cervix. If this Mycoplasma species is a cause of abortion, such complication does not appear to be a common event.
642
March
Foy et al.
JoneP reported on development of CF antibody titers for M. homink in pregnant women in relation to parturition. The findings in this study, where none of 15 primigravidas, but 8 of 15 multigravidas, had CF antibodies lends support to Jones’ theory that CF antibodies develop in association with parturition or abortion. T-strains. The over-all isolation rate, 58 per cent, for T-strains in this study is similar to the rate of 65 per cent reported among pregnant women by Czonka, Williams, and Corse.v The isolation system described for T-strains in this study appeared to be efficient because its facility in detecting T-strains certainly cannot be less than 58 per cent and is possibly close to the efficiency of isolation shown by “E” medium and diphasic broth for M. pneumoniae (65 per cent). The higher rate of T-strains among unmarried than married, and Negro than Caucasian, women suggests socioeconomic differences, an idea which is borne out by the close association of T-strain isolation with M. hominis positivity. While a recent report has associated a T-strain with abortion,23 the present data provide little basis for suspecting that T-strains would be a common cause for spontaneous abortions, prematurity, or
REFERENCES
Dienes, L., and Edsall, J.: Proc. Sot. Exp. Biol. Med. 36: 740, 1937. 2. Chanock, R. M.: New Eng. J. Med. 273: 1199, 1965. 3. Shepard, M. C.: Amer. J. Syph. Vener. Dis. 38: 113, 1954. 4. Tully, J., and Smith, L.: J. A. M. A. 204: 827, 1968. 5. Harwick, H. J., Tuppa, J. B., Purcell, R. H., and Fekety, R. R., Jr.: AMER. J. OBSTET. GYNEC. 99: 725, 1967. 6. Jones, D. M.: Brit. Med. J. 1: 388, 1967. 7. Ford, D., and DuVernet, M.: Brit. J. Vener. Dis. 39: 18, 1963. 8. Shepard, M., Alexander, C., Lunceford, C., and Campbell, P.: J. A. M. A. 188: 729, 1964. 9. Czonka, G., Williams, R., and Corse, J.: Ann. N. Y. Acad. Sci. 143: 794. 1967. 10. Shepard, M. C.: Ann. N. .Y. Acad. Sci. 143: 505, 1967.
Amer. J. Obstet.
1, 1970 Gym.
other
adverse effect on pregnancy or fetus. The 3 per cent isolation rate of CMV from the cervix of pregnant women is in agreement with the findings of Feldman”’ and Hildebrandt and associates,25 who isolated the virus from the urine of 3 and 3vz per cent of pregnant women, respectively. On the other hand, Alexander,lG who also used cervical cultures, obtained an isolation rate of 18 per cent among women on Taiwan. In 2 of the above-mentioned studies transmission to the infant was occasionally seen. In this study no transmission was observed. The fact that lower than average birth weight and low Apgar score were observed in the infants of positive mothers in this study suggests an adverse effect of maternal CMV infection on the fetus and indicates the need for a larger study to substantiate this association. Low birth weight is observed in congenital CMV disease of both severelI and mild*” type. While 3 previous investigations of CMV in pregnancy16T 24s25 have not reported findings similar to ours, Diosi and associates”’ recently reported on a case of abortion associated with CMV infection, discussing the possibility of the virus causing endometritis. Further studies are warranted.
CMV.
11.
1.
12.
13. 14.
15.
16. 17.
18.
Hanshaw, J. B.: Pediat. Clin. N. Amer. 13: 279, 1966. Klemola, E., Kaariaainen, L., von Essen, R., Haltia. K.. Koivuniemi. A.. and von Bonsdorff, ‘C. H.: Acta Med. &and. 182: 311, 1967. Lamb, S. G., and Stern, H.: Lancet 2: 1003, 1966. Holt, S., Pederson, A. H. B., Wang, S. P., Kenny, G. E., Fey; H. M., and Grayston, J: T.: Amer. J. Ophthal. 63: 1057, 1967. Foy, H. M., Wang, S. P., Kenny, G. E., Johnson, W. L., and Grayston, J. T.: Amer. T. Onhthal. 63: 1053. 1967. klex’ander, E. R.: P&at. Res. 1: 210, 1967. Grayston, J. T., Foy, H. M., and Kenny. . , G. E.: Mycoplasmas (PPLO) in Human Disease, in Dowling, H. F.. editor: Disease-a-Month. Chicago, 1367, Ye& Book Medical Publishers: Inc. Kenny, G. E.: Ann. N. Y. Acad. Sci. 143: 676, 1967.
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19. 20.
21.
22. 23.
106 5
Clyde, W. A.: J. Immunol. 92: 958, 1964. Levinsohn, E. M., Foy, H. M., Kenny, G. E., Wentworth, B. B., and Grayston, J. T.: Proc. Sot. Exp. Biol. Med. In press. Klienberger-Nobel, E. : PPLO : Mycoplasmataceae, New York, 1962, Academic Press, Inc. Jones, D. M.: J. Clin. Path. 20: 633, 1967. Kundsin, R.: Science 157: 1573, 1967.
M. hominis
24. 25.
26. 27.
in pregnant
cervix
643
Feldman, R. A.: Proc. XI International Congress of Pediatrics, Tokyo, 1965, p. 215. Hildebrandt, R. J., Sever, J. L., Margileth, A. M., and Callagan, D. A.: AMER. J. OBSTET. GYNEC. 98: 1125, 1967. Emanuel, I., and Kenny, G. E.: Pediatrics 38: 957, 1966. Diosi, P., Babusceac, L., Nevinglovschi, O., and Kun-stoicu, G.: Lancet 2: 1063, 1967.