Low risk of perinatal transmission of human papillomavirus: Results from a prospective cohort study

Low risk of perinatal transmission of human papillomavirus: Results from a prospective cohort study D. Heather Watts, M D , a Laura A. Koutsky, PhD, b King K. Holmes, MD, Phl), c Deborah Goldman, ARNp, a Jane Kuypers, PhD, d Nancy B. Kiviat, ~JID, d and Denise A. Galloway, PhD e, f Seattle, Washington OBJECTIVE: Our purpose was to evaluate the risk of perinatal transmission of human papillomavirus. STUDY DESIGN: Pregnant women were evaluated at <20 weeks' and between 34 and 36 weeks' gestation for genital human papillomavirus by clinical and colposcopic examination and by polymerase chain reaction. Their 151 infants were evaluated at birth, 6 weeks, and 6, 12, 18, 24, and 36 months of age for detection of human papillomavirus deoxyribonucleic acid by polymerase chain reaction on samples from the mouth, external genitalia, and anus. Polymerase chain reaction was performed with human papillomavirus L1 consensus primers and hybridization to human papillomavirus types 6, 11, 16, 18, 31, 33, 35, 39, and 45 and to a generic probe. RESULTS: During pregnancy 112 (74%) of 151 women had historic, clinical, or deoxyribonucleic acid evidence of genital human papillomavirus infection. At 479 infant visits, human papillomavirus deoxyribonucleic acid was detected from only five (1.5%) of the 335 genital, four (1.2%) of the 324 anal, and none of the 372 oral or nasopharyngeal specimens. A positively reacting specimen was obtained from three (4%) of 80 infants born to women with human papillomavirus deoxyribonucleicacid detected at 34 weeks' gestation and from five (8%) of 63 born to women without human papillomavirus deoxyribonucleic acid (p = 0.47). All positive results in the infants were positive only with the generic probe and were preceded or followed by negatively reacting specimens. No clinical manifestations of human papillomavirus infection were detected in any infant. CONCLUSIONS: The isolated detection of unclassified human papillomavirus types from infants at only single visits may represent low-level genital or nongenital human papillomavirus or may represent contamination. Although perinatal transmission of human papillomavirus is not ruled out by these data, the upper 95% confidence interval for detection of perinatal transmission from women with any evidence of genital human papillomavirus was only 2.8%. (Am J Obstet Gyneco11998;178:365-73)

Key words: H u m a n papillomavirus, perinatal transmission, pregnancy

Genital h u m a n papillomavirus (HPV), the most comm o n sexually transmitted infection, has been recognized as the cause of genital warts and as a major contributor to the development of genital tract cancers3 Recurrent laryngeal papillomatosis, a rare, potentially life-threatening condition, has been associated with the presence of HPV types 6 and 11, the HPV types most commonly detected in genital warts. 2 Recurrent laryngeal papillomatosis has a bimodal age distribution with the first peak occurring in infancy3 and is postulated to be related to HPV

From the Departments of Obstetrics and Gynecology,a Epidemiology,b Medicine, c Pathology,d and Microbiology/ University of Washington, and Fred Hutchinson CancerResearch Cent~ f Supported by Grant Pol-AI 29363 from the National Institutes of Allergy and Infectious Diseases. Received for publication March 11, 1997; revised June 9, 1997; accepted September 16, 1997. Reprint "requests: D. Heather Watts, MD, University of Washington, Department of Obstetrics and Gynecology, Box 356460, Seattle, WA 98195-6460. Copyright 9 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/86134

infection acquired from a mother with genital warts or subclinical HPV infection. O n the basis of the incidence of recurrent laryngeal papillomatosis in infancy and the frequency of HPV infection among pregnant women, recurrent laryngeal papillomatosis is estimated to occur in one in several h u n d r e d exposures. 4 The risk of recurrent laryngeal papillomatosis is postulated to be highest in a firstborn infant born vaginally to an adolescent mother. 5 The question of perinatal transmission of HPV versus sexual abuse or other exposure is also raised in the evaluation of older children with genital warts. 6 The risk of transmission of HPV resulting in asymptomatic infecfi .)n is even less well characterized. We hypothesize that perinatal transmission from women with detectable genital HPV deoxyribonucleic acid (DNA) may occur frequently at delivery, in most cases leading to subclinical infection of the infant. Several studies have tested infants for HPV DNA. 7-12 Rates of detection of HPV in the first 1 or 2 days of life have ranged between 4% and 72% among infants b o r n to women with genital HPV detected during pregnancy 365

366 Watts et al.

February 1998 AmJ Obstet Gynecol

Table I. Characteristics at e n r o l l m e n t of w o m e n with at

least one infant visit Patients

Age

16-20 yr 21-25 yr 26-30 yr >30 yr

No.

%

53 58 29 11

35 38 19 7

60 60 31

40 40 20

102 25 18

67 17 12

6

4

128

85

93 45 11 2

62 30 7 1

45 57 31 18

30 38 20 12

72 34 12 33

48 22 8 22

40 5 10

26 3 7

53/151 34/145 31/146 20/145 5/144 2/26 12/146

35 23 21 14 3 1.6 8

15/145 89/151

10 59

Race White Black Other Marital status Single Married Divorced. separated, or widowed Living with partner >1 year Income <$1000 mo Parity 0 1-2 3-4 >4 Age at first intercourse <15 yr 15-16 vr 17-18 yr >18 yr No. of lifetime sexual partners 1-5 6-10 11-15 >15 Current drug use Marijuana Heroin Cocaine History of genital infectious C. trachomatis N. gvnorrhoeae T. vaginalis

Bacterial vaginosis Syphilis HIV Pelvic inflammatory disease Genital herpes Any of the above

by a p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) - b a s e d m e t h o d and between 0.6% and 20% a m o n g infants delivered by w o m e n w i t h o u t d e t e c t a b l e HPV d u r i n g pregnancy. 7-10 Rates of detection at 6 weeks have also varied and were n o t always significantly d i f f e r e n t for infants b o r n to mothers with positive or negative assays for genital HPV. 8, 10, 11 Two studies have r e p o r t e d detection rates of HPV

a m o n g small groups of children older than 6 weeks who had been b o r n to w o m e n with known HPV status at delivery, again with conflicting results. I~ 12 Thus data evaluating transmission o f HPV f r o m m o t h e r to infant are conflicting, and follow-up has been of limited duration and numbers. O u r objective was to evaluate prospectively the risk of p e r i n a t a l transmission o f genital HPV i n f e c t i o n f r o m m o t h e r to infant in a p o p u l a t i o n with a high prevalence of HPV d u r i n g pregnancy. Infants were evaluated until 3 years of age for the detection of HPV DNA by sampling from multiple sites. Extensive data collected systematically from the mothers and infants also allowed assessm e n t of risk factors for perinatal transmission of HPV.

Material and methods English-speaking w o m e n seeking t r e a t m e n t at the H a r b o r v i e w Medical C e n t e r W o m e n ' s Clinic b e f o r e 20 weeks' gestation were eligible. They were assigned in rotation to the study's w o m e n ' s health care specialist according to h e r s c h e d u l e d availability. After routine obstetric e v a l u a t i o n patients were given i n f o r m a t i o n regarding the study and asked to participate. Those who agreed gave written i n f o r m e d consent, as a p p r o v e d by the University of Washington Institutional Review Board. O f 372 new obstetric patients assigned to the study clinician, 49 were n o t eligible because they did n o t speak English (14), they were beyond 20 weeks' gestation (11), they were younger than 16 years (six), they had a miscarriage or ectopic p r e g n a n c y (nine), or for o t h e r reasons (nine). Ninety-nine eligible patients d e c l i n e d a n d 235 enrolled. F u r t h e r standardized interview data were t h e n o b t a i n e d a n d physical a n d c o l p o s c o p i c e x a m i n a t i o n s were p e r f o r m e d . Any abnormalities visible on genital examination were recorded, and vulvar and vaginal specim e n s were o b t a i n e d with p o l y e t h y l e n e t e r e p h t h a l a t e fiber (Dacron) swabs and c o m b i n e d for HPV D N A detection. Cervical swabs for HPV d e t e c t i o n were o b t a i n e d separately with polyethylene t e r e p h t h a l a t e fiber swabs. Swabs were placed into 1 ml specimen transport m e d i u m (STM; Digerie Diagnostics, Silver Springs, Md.) for vulvovaginal and cervical specimens and transported to the Harborview Medical C e n t e r HPV laboratory. C o l p o s c o p i c e x a m i n a t i o n o f the vulva, vagina, a n d cervix was t h e n p e r f o r m e d before and after application of 5% acetic acid solution. W o m e n were asked to r e t u r n for the same evaluation and s p e c i m e n collection at 34 weeks' gestation, 6 weeks post partum, and 1 year post partum. I n f o r m a t i o n was collected at each visit concerning frequency of sexual contact, new sexual partners, and i n t e r v e n i n g genital infections. T r e a t m e n t for visible genital warts was offered to patients. Cervical biopsy samples were obtained only if a high-grade squamous intraepithelial lesion was suspected on the basis of colposcopy or detected by Papanicolau smear, la

Watts et al. 367

Volume 178, Number 2 AmJ Obstet Gynecol

Table II. History a n d c u r r e n t clinical a n d l a b o r a t o r y e v i d e n c e o f genital H P V i n f e c t i o n d u r i n g p r e g n a n c y Enrollment (<20 weeks' gestation) No. History Genital warts 43/145 Abnormal Papanicolaou smear 45/145 Current genital warts 16/146 Papanicolaou smear ASCUS 17/141 LGSIL 14/141 HGSIL 3/141 Cervical HPV DNA positive n = 144 Any 59 Type 6/11 12 Type 16 17 Type 18 5 Type 45 2 Type 31, 33, 35 15 Nontyped 22 Vulvovaginal HPV positive n = 143 Any 73 Type 6/11 20 Type 16 18 Type 18 6 Type 45 6 Type 31, 33, 35 18 Nontyped 33 Any cervical or vulvovaginal 80/144 HPV DNA positive Any history or current evidence 109/151 of genital warts or abnormal Papanicolaou smear, or detection of HPV DNA

34 weeks' gestation

%

No.

30 31 11

--16/146

12 10 2

51 14 13 4 4 13 23 56

9/130 13/130 1/130 n = 142 54 10 15 6 3 12 20 n = 146 73 15 15 4 5 15 35 79/146

72

106/146

41 8 12 3 1.4 10 15

%

Either enrollment or 34 weeks' gestation No.

%

16/146

11 16 18 3

50 10 10 3 3 10 24 54

23/141 25/141 4/141 n = 147 73 17 22 9 4 15 35 n = 149 88 24 21 7 7 21 49 95/149

59 16 14 5 5 14 33 64

73

112/151

74

11 7 10 0.8 38 7 11 4 2 8 14

50 12 15 6 3 10 24

ASCUS, Atypical squamous ceils of undetermined significance; LGSIL, low-grade squamous intraepithelial lesions; HGSIL, high-grade squamous intraepithelial lesions.

Infant visits. A s u b s e t o f 34 i n f a n t s u n d e r w e n t nas o p h a r y n g e a l a s p i r a t i o n with a b u l b syringe in t h e delivery r o o m ; aspirates were p l a c e d i n t o STM for H P V D N A d e t e c t i o n . M o t h e r s o f e n r o l l e d i n f a n t s were a s k e d to b r i n g t h e i n f a n t s to t h e H a r b o r v i e w M e d i c a l C e n t e r W o m e n ' s C l i n i c for follow-up e x a m i n a t i o n s a n d specim e n collection at 6 weeks, 6 m o n t h s , 1 year, 2 years, a n d 3 years o f age. A n i n t e r i m visit at 18 m o n t h s was a d d e d halfway t h r o u g h t h e study. A t e a c h visit, m o t h e r s were int e r v i e w e d r e g a r d i n g any visible wartlike lesions o n t h e m selves, family m e m b e r s , o r t h e infant, a b o u t day care situations for t h e infant, a n d a b o u t any o t h e r illnesses o r n e w s y m p t o m s i n t h e infant. I n f a n t s were e x a m i n e d for any e v i d e n c e of warts o n t h e body. A soft t o o t h b r u s h was t h e n u s e d to b r u s h t h e inside o f t h e m o u t h o n t h e b u c c a l a n d l i n g u a l surfaces. T h e t o o t h b r u s h was t h e n a g i t a t e d in STM a n d r e m o v e d . M o i s t e n e d swabs were u s e d to collect s p e c i m e n s f r o m t h e i n f a n t ' s e x t e r n a l genitalia a n d separate m o i s t e n e d swabs were u s e d for p e r i a n a l s p e c i m e n collection. Swabs were p l a c e d s e p a r a t e l y i n t o STM for t r a n s p o r t to t h e H P V laboratory. G e n i t a l a n d anal specim e n s were o b t a i n e d u n t i l t h e child was 2 years old o r was

toilet t r a i n e d , w h i c h e v e r c a m e first. As a p p r o p r i a t e for age, a heel-stick o r finger-stick b l o o d s p e c i m e n was obt a i n e d f r o m t h e c h i l d at e a c h visit for later serologic determination. P a r t i c i p a t i o n in t h e study was also o f f e r e d for siblings y o u n g e r t h a n 6 years living in t h e h o u s e with t h e i n d e x i n f a n t . T w e n t y - t h r e e s i b l i n g s were e x a m i n e d w i t h t h e same sampling protocol used for the index infants. E x a m i n a t i o n o f t h e siblings b e g a n at t h e 34 weeks' gestat i o n visit for t h e m o t h e r a n d t h e n at t h e a n n u a l visit o f t h e infant.

Laboratory methods PCR analysis. I n t h e l a b o r a t o r y 200 gl of e a c h s a m p l e was digested with p r o t e i n a s e K, e t h a n o l p r e c i p i t a t e d , a n d r e s u s p e n d e d in 50 gl Tris e t h y l e n e d i a m i n e t e t r a a c e t a t e . PCR was p e r f o r m e d in d u p l i c a t e o n 5 gl o f e a c h s a m p l e with H P V L1 c o n s e n s u s p r i m e r s MY09 a n d MY11 (PerkinE l m e r Corp., Norwalk, C o n n . ) , as previously described. 14 E a c h s a m p l e was s i m u l t a n e o u s l y a m p l i f i e d w i t h t h e h u m a n [3-globulin p r i m e r s P C O 4 a n d G H 2 0 ( P e r k i n E l m e r ) to assess s p e c i m e n adequacy. PCR p r o d u c t s were analyzed by p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s a n d sam-

368 Watts et al.

February 1998 A m J Obstet Gynecol

Table Ill. A g e a n d a n a t o m i c s i t e - s p e c i f i c H P V D N A d e t e c t i o n r a t e s a m o n g i n f a n t s a n d siblings

Site Visit

Anal

Birth 6 wk 6mo 12mo 18mo 24mo 36mo Siblings Tot~

Genital

1/102 2/93 1/73 0/30 0/24 0/2 0/2 4/326 (1.2%)

0/109 1/90 2/73 1/32 1/29 0/2 0/5 5/340 (1.5%)

Oral

Total infants

0/34* 0/79 0/85 0/66 0/34 0/49 0/25 0/27 0/399

145 122 93 37 51 26 26 151infants, 26siblings

Total infants with any positive result: 8/151 (5.3%). Specimens negatively reacting to ~-globulin on amplification were excluded from these results, including 129 anal, 73 genital, and 46 oral specimens. *Birth specimens consisted only of nasopharyngeal aspirates obtained in the delivery room.

Table IV. Results o f m a t e r n a l a n d i n f a n t t e s t i n g f o r H P V by P C R by site a n d visit, a m o n g i n f a n t s w i t h a n y positive H P V P C R test

Patient (mother) No. 77 Dates Visit No. Cx g/v

10/31/91 1 . .

3/24/92 2

. .

. .

6/2/92 3

10/13/92 4

4/6/93

4/11/94

4/10/95

6 wk -I I

6 mo WNT -I

1 yr ND -ND

2 yr I I I

3 yr

-I I

3/22/93 4 NT NT 1 yr NT ----

10/18/93

11/8/93

4/20/94

10/26/4

4/14/5

1 yr --

18 mo --

2 yr --

30 mo I

. .

Infant age Anal Oral Genital

Patient (mother) No. 92 Dates Visit No. Cx V/v Infant age Anal Back Oral Genital

1/2/92 1 ---

5/1/92 2 NT --

7/17/92 3 I -6 wk -I I

18 mo I I I

Patient (mother) No. 99 Dates Visit No. Cx V/v Infant age Anal Oral Genital

3/4/92 1 --7 --

9/9/92 2 -WNT

11/24/92 3 ND WNT 6 wk -. I

4/7/93 4 --6 mo I .

. --

.

.

--

WNT

--

I

12/28/93 1 .

5/20/94 2

8/5/94 3

3/22/95

1 yr (baby 1) I . --

18 mo (baby 1) I . I

Patient (mother) No. 114 Dates Visit No. Cx V/v Infant age Anal Oral Genital

5/27/92 1 I .

10/23/92 2 . .

2/3/93 3

6/9/93

. .

.

.

.

.

6 wk (baby 1) -. --

6 mo (baby 1) -. --

.

I 6 wk (baby 2) I

9 mo (baby 2) WNT

I

WNT

.

continued

Watts et al. 3 6 9

Volume 178, Number 2 AmJ Obstet Gynecol

Table IV---cont'd Patient (mother) No. Dates Visit No. Cx V/v Infant age Anal Oral Genital Patient (mother) No. Dates Visit No. Cx V/v Infant age Anal Oral Genital Patient (mother) No. Dates Visit No. Cx V/v Infant age Anal Oral Genital Patient (mother) No. Dates Visit No. Cx V/v Infant age Anal Oral Genital

128 8/26/92 1 . . . .

1/19/93 2 . . . .

4/19/93 3 6 wk . . .

143 12/2/92 1 6/11 16, 18, 30s

5/7/93 2 NT WNT

9/14/93 4

. . .

6/25/93

192 10/8/93 1 ---

2/9/94 2 --

--

2/11/94 2 --

9/30/94

1 yr

18 mo

. . .

2 yr NT

6/6/94 4 NT WNT 1 yr --WNT

5/13/94 3 W30s 30s 6 wk i -I

9/29/94 4 --6 mo ----

3/10/95

6/8/94 3 I --

10/10/94 4 --6 wk ----

WNT ---

3/13/95

I

8/12/93 3 18 18 6 wk I ND I

-176 7/28/93 1 NT WNT

6 mo . . .

3/9/94

12/14/94

18 mo ----

1 yr --WNT 4/5/95

6 mo ----

1 yr

Cx, Cervical; V/v, vulvovaginal; ~ weakly hybridized, HPV nontyped; ND, not done;/, insufficient specimen; N~, HPV nontyped. *PCR testing with amplification for HPV types 6, 11, 16, 18, 31, 33, 35, 39, and 45 by L1 consensus primer and hybridization.

ples with positive results for t h e ~-globulin D N A b a n d were d o t b l o t t e d a n d h y b r i d i z e d w i t h a b i o t i n - l a b e l e d g e n e r i c H P V p r o b e c o n t a i n i n g specific H P V D N A seq u e n c e s i n t e r n a l to t h e L1 c o n s e n s u s p r i m e r s , w h i c h hybridize w i t h P C R p r o d u c t s f r o m at least 27 genital H P V types. 15 P C R p r o d u c t s t h a t were H P V reactive with t h e g e n e r i c p r o b e were h y b r i d i z e d with b i o t i n - l a b e l e d H P V type-specific o l i g o n u c l e o t i d e s i n five h y b r i d i z a t i o n p r o b e mixes: H P V types 6 a n d 11; H P V type 16; H P V type 18; H P V types 31, 33, 35, a n d 3 9 ; a n d H P V type 45. S p e c i m e n s t h a t h y b r i d i z e d only with t h e g e n e r i c p r o b e were c o n s i d e r e d to have a positive r e a c t i o n o f unclassified H P V type. Hybrid capture analysis. Samples t h a t were H P V reactive with t h e g e n e r i c p r o b e after PCR amplification were analyzed w i t h o u t PCR amplification with t h e h y b r i d c a p t u r e assay (Digene Diagnostics, Inc., Silver Spring, Md.) to detect h i g h e r levels of H P V DNA. H y b r i d c a p t u r e is a p p r o x imately 1000 times less sensitive t h a n PCR for d e t e c t i o n o f H P V DNA. A 100/zl a l i q u o t o f t h e original sample in STM was d e n a t u r e d a n d hybridized with m i x t u r e s of 14 whole g e n o m i c H P V r i b o n u c l e i c acid p r o b e s . 14 D N A - r i b o n u -

cleic acid hybrids were c a p t u r e d in tubes a n d d e t e c t e d with a n enzyme-labeled a n t i b o d y a n d c h e m i l u m i n e s c e n t substrate. T h r e e p r o b e mixes were u s e d for typing in t h e h y b r i d c a p t u r e assay: types 6, 11, 42, 43, a n d 44 (design a t e d as low-risk types); types 31, 33, 35, 51, a n d 52 (desi g n a t e d as i n t e r m e d i a t e - r i s k types); a n d types 16, 18, 45, a n d 56 ( d e s i g n a t e d as high-risk types). A n a l i q u o t o f o r i g i n a l s p e c i m e n in STM f r o m 20 infants was s e n t to a s e c o n d l a b o r a t o r y for H P V testing by P C R with t h e same p r i m e r s a n d p r o b e s . S p e c i m e n s inc l u d e d five n a s o p h a r y n g e a l s p e c i m e n s f r o m delivery, five s p e c i m e n s with positive r e s u l t s in o u r l a b o r a t o r y , five s p e c i m e n s f r o m infants b o r n to w o m e n with H P V 6 o r 11, 16, 18, o r 30 d e t e c t e d d u r i n g p r e g n a n c y , a n d five specim e n s f r o m i n f a n t s b o r n to w o m e n with n o d e t e c t e d H P V at e i t h e r p r e g n a n c y visit. Statistical m e t h o d s . Rates o f positively r e a c t i n g specim e n s by site a n d age o f the i n f a n t are d e s c r i b e d f o r e a c h interval. Clinical factors in t h e m o t h e r s potentially assoc i a t e d w i t h H P V t r a n s m i s s i o n w e r e c o m p a r e d w i t h ~2 tests b e t w e e n infants with any positive H P V D N A detection a n d infants with all negatively r e a c t i n g s p e c i m e n s .

370 Watts et al.

February 1998 AmJ Obstet Gynecol

Table V. Clinical characteristics of m o t h e r s and m e t h o d o f delivery by HPV detection in the infant

Infant HPV Any positive (n = 8)

Maternal age 16-20 yr 21-25 yr 26-30 yr ->31 Parity 0 1-2 >3 Ethnicity White Black Other Current warts in mother Current Papanicolaou smear Normal ASCUS SIL Genital HPV positive by PCR Cesarean delivery

All negative (n = 143)

No.

%

No.

%

Significance

1 4 3 0

13 50 38 0

52 54 26 11

38 38 18 7

p= 0.31

6 2 0

75 25 0

84 42 11

61 31 8

p = 0.62

4 3 1 0/8

50 38 12 0

56 57 30 16/143

39 39 22 11

p = 0.78

8 0 0 2/7 2/8

100 0 0 29 25

102 19 16 77/141 23/136

77 13 10 55 17

p = 0.26

p = 0.68

p = 0.25 p = 0.63

ASCUS, Atypical squamous ceils of undetermined significance; SIL, squamous intraepithelial lesion.

Results O f the 235 w o m e n enrolled, 151 had live births and were available for follow-up, 25 (11%) had a spontaneous o r i n d u c e d a b o r t i o n or stillbirth, f o u r (2%) d e l i v e r e d after study closure, and 55 (23%) m o v e d f r o m the area or transferred their obstetx-ic care to a n o t h e r facility and were therefore unavailable for study follow-up. W o m e n whose infants were seen in follow-up did n o t differ f r o m those with p r e g n a n c y loss or n o follow-up with respect to age, ethnicity, age at first intercourse, n u m b e r o f lifetime partners, parity, history o f g e n i t a l warts or a b n o r m a l Papanicolaou smear, or rate of genital HPV DNA detection d u r i n g pregnancy. Characteristics at the e n r o l l m e n t visit for w o m e n with infants with study follow-up are summarized in Table 1. In general most had low income, and there was g o o d representation o f white, black, and o t h e r ethnic groups. It is noteworthy that 35% of the w o m e n were y o u n g e r than 21 years and 61% were nulliparous. A l t o g e t h e r 89 (59%) of 151 gave a history of o n e or m o r e p r e v i o u s sexually t r a n s m i t t e d diseases, a n d 55 (36%) had c u r r e n t evidence of an active sexually transmitted disease. Genital infections d e t e c t e d at enrollment i n c l u d e d Chlamydia trachomatis in e i g h t (6%) o f 136, Neisseria gonorrhoeae in t h r e e (2%) of 136, Trichomonas vaginalis in 10 (7%) o f 144, bacterial vaginosis in 47 (31%) o f 151, a n d h u m a n i m m u n o d e f i c i e n c y virus (HIV) infection in two (16%) of 126. T h e history and c u r r e n t clinical or laboratory evidence of genital HPV infection a m o n g the 151 p r e g n a n t w o m e n are s u m m a r i z e d in Table II. Results of HPV D N A testing were rather con-

sistent at e n r o l l m e n t and at 34 weeks' gestation. Thus 59 (41%) had HPV DNA d e t e c t e d by PCR-based assay of the cervical specimen, and 73 (51%) had HPV D N A d e t e c t e d in the vulvovaginal s p e c i m e n at e n r o l l m e n t , and overall 95 (64%) were PCR positive in either s p e c i m e n at either visit. Overall 112 (74 %) had historical, clinical, cytologic, or m o l e c u l a r e v i d e n c e o f H P V i n f e c t i o n d u r i n g pregnancy. T h e rates o f HPV DNA detection by site and by age of infant are shown in Table III. Anal and genital specimens c o l l e c t e d f r o m the i n f a n t early in the study w e r e frequently negatively reacting for [5-globulin and were considered insufficient for HPV DNA detection. T h e n u m bers of insufficient specimens for each site are shown at the b o t t o m of that table and are n o t i n c l u d e d in the denominators. Yield increased w h e n s p e c i m e n collection was c h a n g e d f r o m use of dry swabs to use of swabs moiste n e d with saline solution. Overall n i n e specimens f r o m eight infants reacted positively for PCR HPV DNA. In all n i n e cases specimens only reacted positively for unclassified HPV types, n o t for HPV 6, 11, 16, 18, 31, 33, 35, 39, or 45. A s u m m a r y o f the m a t e r n a l and infant results by visit for e a c h of the e i g h t infants with a positive HPV DNA result is shown in Table IV. Note that subject 114 was studied d u r i n g two pregnancies. She did n o t have HPV D N A d e t e c t e d d u r i n g e i t h e r pregnancy, a n d h e r first infant c o n t i n u e d to have a negative HPV D N A result. H e r second infant h a d a weak positive reactivity for an unclassified HPV type at 9 months. All three of the e n r o l l e d w o m e n who were seropositive

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for HIV had genital HPV detected. O n e woman was seronegative for HIV antibody at enrollment but had antibodies detected at 34 weeks' gestation. She had HPV 6 or 11 in the vulvovaginal specimen and a nontyped HPV in the cervix at enrollment and had HPV types 16 and 45 at both sites at 34 weeks' gestation, when she was also noted to have primary genital herpes simplex virus type 1 according to culture and serologic studies. H e r Papanicolaou smears showed atypia at enrollment and a low-grade squamous intraepithelial lesion at 34 weeks' gestation. The two patients whose HIV infection antedated pregnancy had no symptoms; both had atypical Papanicolaou smears at 34 weeks' gestation and had nontyped HPV detected from both the cervical and vulvovaginal sites at both visits during pregnancy. M1 women maintained CD4 lymphocyte counts >500 cells/ml during pregnancy. Each of the three babies born to the women who were HIM seropositive had negative results of HPV DNA detection at all sites at four visits each and are not infected with HIV. O n e of the HIV seropositive women had a second baby who had negative results for HPV DNA at 6 weeks of age. In one case where unclassified HPV DNA was detected from both mother and infant, the amplified DNA PCR products from each were tested in the hybrid capture assay, which has an extended spectrum of types. By hybrid capture assay the infant's PCR product was identified as type 56 and the mother's as type 51 or 52. Two of the 20 specimens tested in the second laboratory had positive reactions for HPV DNA by the generic probe, but neither was typable. O n e was an anal specime n from an infant born to a mother with no HPV DNA detected during pregnancy; this infant had negative resuits in the primary laboratory but positive results in the second laboratory. The second specimen, a genital specime n from a l-year-old infant born to a woman with HPV 6 or 11 in the vulvovaginal specimen and HPV 16, 18, and 31, 33, 35, or 39 in the cervix at enrollment and nontyped HPV DNA in vulvovaginal and cervical specimens at 34 weeks' gestation, had a weakly positive reaction for a nontyped HPV in both laboratories. The infant had a nonreactive nasopharyngeal specimen at birth, inadequate specimens at 6 weeks, nonreactive anal and oral specimens at 1 year, and nonreactive anal, oral, and genital specimens at 18 months. Twenty-six siblings younger than 6 years of 24 index infants were evaluated at a total of 46 visits for HPV DNA detection. Twenty-seven specimens from the mouth, five from the genitals, and two from the anus were nonreactive for HPV DNA by PCR. To further evaluate the likelihood that HPV DNA detection in the infants was related to vertical transmission of HPV, clinical characteristics that could influence transmission were evaluated (Table V). HPV DNA detection in the infant was not associated with maternal age, parity,

m o d e of delivery, genital warts or a b n o r m a l Papanicolaou smear during pregnancy, or maternal genital HPV DNA detection during pregnancy.

Comment Data from this study do not suggest a high risk of perinatal transmission of HPV. Despite a high prevalence of HPV infection at 34 weeks' gestation am o n g pregnant' women, few positive results were obtained from their infants. M1 eight infants with a positively reactive specimen had positive results only at a single visit, and seven of eight had positive reactions only in a single specimen. Positive test results appeared late, rather than earlier in the neonatal period, and all eight were with HPV DNA not typable as c o m m o n genital types. Th e absence of any detectable positive reaction in neonatal nasopharyngeal specimen suggests that transient prenatal contamination of infant specimens by maternal HPV, without actual infant infection, was not occurring. In addition, when unclassified HPV-positive specimens from a m o t h e r and infant pair were typable by hybrid capture, the types were not concordant. The low rate of HPV DNA positivity may reflect either transient genital infection or a low rate of specimen contamination with nongenital HPV types. In addition, women with negative results for genital HPV at all visits were as likely to have an infant with HPV detected as were those with HPV detected during pregnancy. If the observed low rate of positivity represents transient infection or contamination of oral or genital specimens with nongenital HPV types, testing of serum specimens obtained serially from infants in this study may demonstrate d ev el o p m en t of new immunoglobulin G antibody with time. In addition to the largely negative results in the study infants, none of the older siblings tested had positive results for HPV. This offers evidence against intrafamilial, as well as perinatal, transmission of c o m m o n genital HPV types to young children. It is unlikely that the results of this study are related to inadequate specimen collection or insensitive testing. As noted, specimens collected from the genital and anal area of infants early in the study were frequently negatively reacting for [~-globulin, and these were n o t included in the analysis. After the specimen-collection technique was changed, the [5-globulin positivity rate increased markedly. Only specimens positively reacting for ~-globulin were tested for HPV DNA, so it seems unlikely that the low rate of HPV detection in this study was related to inadequate specimen collection. Furthermore, with the same collection techniques and assays HPV DNA was detected in >50% of the women during pregnancy, and in the mothers HPV DNA detection correlated well with cytologic evidence of HPV infection and with current genital warts. M1 women with genital warts during pregnancy had HPV DNA detected from the vulvovaginal

372 ~/att$ ~t a].

specimen, and all women with squamous intraepithelial lesions on Papanicolaou smear had HPV DNA detected from the cervical specimen. Laboratory personnel performing HPV DNA testing were unaware of the clinical findings. Previous studies of infection with genital HPV types during the neonatal period have yielded highly variable results. In an early study Sedlacek et al. 7 detected HPV DNA in nasopharyngeal specimens by Southern blot in 11 (44%) of 25 infants b o r n to mothers with detectable HPV d u r i n g pregnancy and in four (20%) of infants b o r n to women without detectable HPV (p > 0.05). Fredericks et al. 11 evaluated oral HPV DNA detection by PCR at 6 weeks of age among infants born to mothers with and without genital HPV at delivery. They detected HPV DNA in eight (72%) of 11 oral specimens from infants born to mothers with HPV and one (5%) of 19 infants born to mothers without HPV (p < 0.05). Pakarian et al. 8 evaluated oral specimens at 1 day of age and genital specimens at 6 weeks of age by PCR in infants born to mothers with known HPV status. At 1 day of age, oral specimens had positive results in 10 (50%) of 20 infants b o r n to mothers with detectable HPV DNA and one (9%) of 11 infants born to mothers without detectable HPV DNA (p < 0.05). At 6 weeks of age HPV DNA was detected in six (30%) of the 20 infants born to mothers with HPV compared with two (18%) of the 11 infants born to mothers without HPV (p = 0.77). O n the other hand, Smith et al. 9 evaluated oral and genital specimens by PCR for HPV between 1 and 3 days of age and found a much lower rate of positivity. One (4%) of 25 infants born to mothers with detectable HPV DNA and one (0.6%) of 178 infants b o r n to mothers without detectable HPV DNA had positive results (p = 0.58), a finding more consistent with this study. The frequent detection by Pakarian et al. 8 of HPV DNA from infants within a few days of birth may represent transient mucosal colonization, although high rates of positivity in infants born to women without HPV suggest contamination. The widely varying prevalence estimates for infants shortly after delivery and several weeks later suggest large differences in populations, differences in sampling methods, or a wide range of specificity of laboratory assays. Specimens from the backs of infants in this study all yielded negative results, confirming specificity of the laboratory assays used in this study. Two other studies evaluating children for HPV DNA detection beyond the neonatal period also yielded varied results. In a study of oral HPV DNA detection by PCR in children aged between 4 months and 111/2 years born to mothers who had been studied for HPV DNA detection during pregnancy, Puranen et al. 12 showed that 14 (32%) of 44 children b o r n to mothers with detectable HPV DNA had oral HPV DNA detected, versus seven (50%) of 14 b o r n to mothers without HPV DNA. Cason et al. 1~

February1998 AmJ ObstetGynecol

evaluated infants at 1 day, 6 weeks, and 6 months of age by collecting specimens from the oral and genital areas for testing for HPV types 16 and 18 DNA by PCR. Although the rate of HPV DNAdetection among infants b o r n to mothers with positive results was significantly higher at 1 day and 6 weeks than for infants b o r n to mothers without HPV DNA, the rate of HPV DNA detection in both groups was high. The HPV DNA detection a m o n g the infants b o r n to mothers with HPV DNA ranged between 67% a n d 79% at the different ages, whereas among the infants born to mothers without HPV DNA the rate was between 22% and 25%. The rate of HPV 16 positivity among the pregnant women was 69% and the rate of HPV 18 positivity was 21%, both higher than in our p o p u l a t i o n (15% and 5%, respectively). Because of the high rate of HPV DNA detection among the infants born to mothers without genital HPV detection in that study, one must question the specificity of their PCR or consider the possibility of horizontal transmission between infants and Caregivers. This study has several strengths compared with previous published studies of HPV detection in children. The subjects enrolled were an unselected cohort with a high prevalence of genital HPV and a high frequency of previously postulated risk factors for transmission, including nulliparity, young age, and low cesarean section rate. 5 The primary laboratory method for HPV detection, PCR, has a high sensitivity, and a subset of specimens was tested in a second laboratory to confirm sensitivity and specificity. Mothers were tested serially, with a high concordance of results between visits and with clinical findings. Infants were tested serially from multiple sites. All these factors increase the likelihood of detection of perinatal transmission of HPV if it occurs with a frequency of 2% or more. Weaknesses of the study include the relatively small n u m b e r of infants studied, especially at older ages, although this is the largest study yet reported. Serum for antibody testing has been collected but not yet tested. Serologic profiles may be helpful in clarifying the risk of infant infection, which may be transient and n o t detected by the sampling schedule used in this protocol. A high proportion of inadequate specimens was found, especially early in the study, but these samples were not included in the results. Thus a larger study with more frequent sampling of children through a longer period may help to narrow the range of risk of transmission but is unlikely to detect a risk high enough to justify a change in c u r r e n t obstetric practice for women with subclinical HPV infection. Results of this study do not support a high risk of vertical transmission of genital HPV at delivery. The few positive results obtained from children studied across time are consistent with background positivity seen in a cohort of virgins. No compelling evidence of transmission from

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m o t h e r to i n f a n t was s e e n in the study. A l t h o u g h transmission o f H P V f r o m t h e m a t e r n a l genital tract to the inf a n t has b e e n a s s u m e d in cases o f laryngeal p a p i l l o m a t o sis, the risk for any given w o m a n - i n f a n t pair a p p e a r s to b e small a n d d o e s n o t justify c h a n g e in c u r r e n t o b s t e t r i c practice. We t h a n k Dr. Cosette W h e e l l e r for retesting 20 specim e n s f r o m infants a n d Shu-Kuang Lee, MS, for h e l p in data analysis. REFERENCES

1. Koutsky LA, Wolner-Hanssen P. Genital papillomavirus infections: current knowledge and future prospects. Obstet Gynecol Clin North Am 1989;16:541-64. 2. LeviJE, Delcelo R, Alberti VN, Torloni H, Villa LL. Human papillomavirus DNA in respiratory papillomatosis detected by in situ hybridization and the polymerase chain reaction. Am J Patho11989;135:1179-84. 3. Kashima HK, Shah K. Recurrent respiratory papillomatosis: clinical overview and management principles. Obstet Gynecol Clin North Am 1987;14:581-8. 4. Shah K, Kashima H, Polk BF, Shah F, Abbey H, Abramson A. Rarity of cesarean delivery in cases ofjuvenile-onset respiratory papillomatosis. Obstet Gyneco11986;68:795-90. 5. Kashima HK, Shah F, Lyles A, Glackin R, Muhammad N, Turner L, et al. A comparison of risk factors in juvenile-onset and adultonset recurrent respiratory papillomatosis. Laryngoscope 1992;102:9-13. 6. Gutman LT, Herman-Giddens MD, Phelps WC. Transmission of

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human genital papillomavirus disease: comparison of data forms adults and children. Pediatrics 1993;91:31-8. 7. Sedlacek TV, Lindheim S, Eder C, Hasty L, Woodland M, Ludomirsky A, et al. Mechanism for human papillomavirus transmission at birth. AmJ Obstet Gyneco11989;161:55-9. 8. Pakarian F, KayeJ, Cason J, Kell B, Jewers R, Derias NW, et al. Cancer associated human papillomaviruses: perinatal transmission and persistence. BrJ Obstet Gynaeco11994;101:514-7. 9. Smith EM, Johnson SR, Cripe T, Perlman S, McGuinness G, Jiang D, et al. Perinatal transmission and maternal risks of human papillomavirus infection. Cancer Detect Prev 1995;19:196-205. 10. Cason J, KayeJN, Jewers RJ, Kambo PK, Bible JM, Kell B, et al. Perinatal infection and persistence of human papillomavirus types 16 and 18 in infants.J Med Viro11995;47:209-18. 11. Fredericks BD, Balkin A, Daniel HW, Schonrock J, Ward B, Frazer IH. Transmission of human papiIlomavirus from mother to child. Aust N ZJ Obstet Gynaeco11993;33:30-2. 12. Puranen M, Yliskoski M, Saarikoski S, Syrjanen K, Syljanen S. Vertical transmission of human papillomavirus from infected mothers to their newborn babies and persistence of the virus in childhood. AmJ Obstet Gyneco11996;174:694-9. 13. The 1988 Bethesda System for reporting cervical/vaginal cytological diagnoses. National Cancer Institute Workshop. JAMA 1989;262:931-4. 14. Kuypers JM, Critchlow CW, Gravitt PE, Vernon DA, Sayer JB, Manos MM, et al. Comparison of dot filter hybridization, Southern transfer hybridization, and polymerase chain reaction amplification for diagnosis of anal human papillomavirus infection. J Clin Microbiol 1993;31:1003-6. 15. Bauer HA, Green CE, Manus MM. Detection of genital HPV infection using PCR. In: Harrington C, McGee J, editors. Diagnostic molecular pathology: a practical approach. Oxford, England: Oxford University Press; 1992. p. 131-52.

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