International Journal of STD & AIDS 1992; 3: 33-37
ORIGINAL ARTICLE
Prevalence of urogenital Chlamydia trachomatis infection in EI Salvador I. Infection during pregnancy and perinatal transmission Ana Berta Canas Posada BSCl, Jon Jonasson MD PhD2, Leonor de Linares MD MSC 1 and Solgun Bygdeman MD PhD2 IDepartment of Microbiology, Faculty of Medicine, University of El Salvador; 2Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, Stockholm, Sweden Summary: One-hundred and twenty-nine pregnant women in labour (age range 15-46 years; median age 23) and 42 infants born to chlamydia-positive mothers (age range 5-15 days; median age 10) were investigated to estimate the prevalence and incidence, respectively, of Chlamydia trachomatis infection in San Salvador, El Salvador. Urethral and cervical samples were obtained from all women and conjunctival specimens were taken from both eyes of each child. The chlamydial antigen was detected with the commercial Pharmacia Chlamydia EIA kit. Direct immunofluorescence (DFA) (Syva MicroTrak) was used for confirmation. In the newborns both EIA and OFA tests on direct preparations from ocular smears were performed on all the samples. The prevalence of chlamydia! infection in pregnant women was 44% (57/129). The incidence of chlamydial infection in neonates was 64% (27/42), and the majority of the infected children (56%) had conjunctivitis. Referring to individuals rather than specimens the sensitivity of EIA tests on conjunctival samples from the infants was low (37%) as compared with 91% on urethral and cervical specimens from the pregnant women. Keywords: Chlamydia trachomatis, El Salvador, perinatal transmission, chlamydia! ophthalmia neonatorum
INTRODUCTION The important role of Chlamydia trachomatis as an aetiological agent in infections among pregnant women and neonates was documented at the beginning of this century, long before the agent was isolated in culture. Today C. trachomatis seems to be one of the most prevalent sexually transmitted pathogens in the industrialized world-r'. In developing countries, an even higher incidence of chlamydial infection has been reportedv". Several studies have defined risk factors for chlamydial cervical infection in pregnant women-Z:". It has been reported that the prevalence of C. trachomatis is higher in pregnant women than in other population groups. The cause of this unusual finding is unknown, although several hypotheses have been proposed'P-!'. The potential role of transmission by direct spread from the infected cervix of the mother to the baby during labour and delivery has also been
studied1,3,11- 13. Several authors have demonstrated the role of C. trachomatis in pneumonitis, and conjunctivitis. It is well known that the so-called ophthalmia neonatorum or conjunctivitis is the most common ocular disease among newborns. Chlamydia! aetiology has also been suspected in cases of otitis media and gastroenteritis of neonates and infants3,7,12- 15. In El Salvador, a developing country located in Central America, the prevalence and incidence of chlamydial infection is not known accurately. The present work is the first in a series of papers that investigates the prevalence of urogenital C. trachomatis infection in El Salvador. The aims of the present study were: (1) to determine the prevalence of chlamydia! infection in a group of pregnant women attending the Maternity Hospital of San Salvador City, (2) to estimate the risk of infection in newborn babies born to chlamydia-positive mothers. MATERIALS AND METHODS
Correspondence to: Dr J [onasson, Department of Clinical Bacteriology, Huddinge Hospital F72, S-141 86 Huddinge, Sweden
Study population and performance of the study The study group consisted of 129 pregnant women in labour, aged 15-46 years, admitted to the
34
International Journal of STD & AIDS Volume 3 January/February 1992
Maternity Hospital of San Salvador City for delivery from April to July 1989. Only a minor proportion of the 6171 deliveries at the hospital during this period could be included in the study due to the fact that interviews and samplings could only be conducted after a doctor had examined a patient and pronounced her available for the study. History of genitourinary tract infection was not a prerequisite for enrolment in the study. Patients with a previous caesarean section, rupture of membranes or vaginal bleeding were excluded from the study. After explaining the aims of the study and receiving verbal consent, personal data from each patient was obtained using a standardized questionnaire including name, age, address, occupation, marital status, duration of pregnancy, parity, and genital infections. Forty-two infants born to chlamydia-positive mothers were investigated in their houses, 5-15 days after birth. Sex, birth weight and length were recorded and the eyes of the children were examined for the presence of secretions, or erythema.
used for DFA according to the instructions of the manufacturer. All DFA tests were examined by the same investigator using a Leitz Weitzlar Ortholux II fluorescence microscope. For confirmative DFA-C the EIA transport medium was centrifuged and the sediment deposited on a slide (Syva MicroTrak), which was allowed to air dry, and then was fixed in methanol. Criteria of interpretation
Pregnan t women A patient was classified as chlamydia-positive if: (a) one or both samples were positive by EIA and confirmed by the DFA-C test with at least one EB, or (b) negative by ErA (gray zone), in one or both samples, but positive by DFA-C with three or more EBs.
Newborns Only infants with mothers who were positive according to the criteria above, were included. An infant was considered chlamydia-positive if: (a) DFA-D (direct from the eye) was positive in one or both eyes, when 4 or more EBs and/or one or Sampling more IBs were found, (b) EIA was positive in one Specimens for the laboratory diagnosis of C. or both eyes and confirmed by DFA-C with at least trachomatis infection using the Pharmacia Chlamydia one EB, (c) negative by EIA (gray zone negative) in (EIA) test were taken from the urethra and cervix one or both eyes, but confirmed by DFA-C with one of the women using standard techniques. The or more EBs and at the same time positive by DFAMicroTrak direct immunofluorescence test (DFA-C) D with at least one EB. used for the confirmation of EIA positive and negative gray zone results was performed on the Statistical analysis transport media of these samples. Two independent samples were obtained from Descriptive statistics and correlation analysis with the each eye of the infants by carefully rubbing the inner chi-square test were performed using the computer surface of the lower and the upper eyelids with program Quest (University of Umea, Sweden). swabs. The first one was used for the EIA test and the second for direct immunofluorescence (DFA-D). RESULTS Enzyme immunoassay (EIA) test Following collection the samples were kept refrigerated at 4°C in transport medium and processed within the following 2-7 days. The Pharmacia Chlamydia EIA Test (Pharmacia Diagnostics AB, Uppsala, Sweden) was performed on all samples according to the manufacturer's instructions. Samples with an absorbance value within 0.02 units above or below the cut-off value were considered to be in the 'gray zone'. The absorbance ratio (absorbance value/cut-off value) was calculated. Specimens with absorbance values in the positive range (absorbance ratio > = 1.0) or within the negative gray zone were further investigated by direct immunofluorescence test (DFA-C). Direct immunofluorescence test (OFA) The MicroTrak C. trachoma tis Direct Specimen Test (Syva Micro'Irak, Palo Alto, California, USA) was
The median age of the 129 pregnant women studied was 23 years (range 15-46). Nearly all of them, 121 (94%) were 'housewives' or unemployed. Thirteen per cent were single and 75% were unmarried but living together with their 'fiance'. Only 12% were married. The mean number of earlier pregnancies was 1.5 (range 0-14). Fifty-six (43%) were pregnant for the first time and 59 (46%) had 1-3 earlier pregnancies. Pregnancy ended by vaginal delivery in 108 cases (84%) and by caesarean section in 21 (16%) cases. At follow-up, 45 out of a total of 57 chlamydiapositive mothers were found living at the address given by them in the hospital. However, only 42 infants were tested, because 3 children had already died at home for unknown reasons. Twenty-one infants were girls and 21 boys. The median age of the babies at the time of investigation was 10 days (range 5-15). The mean birth weight was 3154g (range 2240-4281) and the mean length at birth was 48 cm (range 45-51). Two children had jaundice at birth. The others were recorded as healthy.
Posada et al.
Thirty-two children (76%) of chlamydia-positive mothers were born by vaginal delivery. The remaining 10 babies were born by caesarean section due to fetal distress, cephalopelvic disproportion or abnormal presentation. All of these infants had received ointment with 1% chloramphenicol in the eyes at birth. Prevalence of chlamydial infection The prevalence of chlamydial infection in pregnant women was 44% (57/129) (Table 1). Thirty-two per cent (18/57) were positive in both the urethra and the cervix, 47% (27/57) in the cervix only and 21% (12/57) in the urethra only. There were no significant differences in age, marital status or parity between chlamydia-positive and chlamydia-negative women. Chlamydia-positive women Fifty-two out of the 57 (91%) chlamydia-positive women were positive in both the EIA and DFA-C tests in at least one site. However, one sample from each of 5 women was in the EIA negative gray zone, but positive for DFA-C according to our established criteria with 3-20 EBs (false EIA negative individuals) (Table 1).
Chlamydial infection during pregnancy
35
than specimens the sensitivity for EIA was 91% (52/57) and the specificity 89% (64/72). The positive predictive value was 87% (52/60) and the negative predictive value 93% (64/69). Incidence of chlamydial infection in the newborns The incidence of chlamydia infected children born to chlamydia-positive mothers was 64% (27/42); 14 females and 13 males. Forty-eight per cent (13/27) were infected in both eyes, 33% (9/27) in the right eye only and 19% (5/27) in the left eye only. Twenty-two (82%) chlamydia-positive children were born by vaginal delivery, and the remaining 5 by caesarean section. The reason for caesarean section was abnormal presentation in 2 cases, fetal distress in 2 cases and cephalopelvic disproportion in the last one. Before the caesarean section was performed, membranes had been deliberately ruptured as a procedure of help in childbirth in all 5 cases. Chlamydia positive newborns The results of EIA, DFA-C and DFA-D tests in the 27 chlamydia positive newborns are shown in Figure 1. Seventeen babies were EIA negative but positive by DFA-D and/or by DFA-C, and therefore were considered positive according to our criteria.
Chlamydia-negative women
Chlamydia negative newborns
Seventy-two women were considered chlamydianegative (see columns 2 and 3 in Table 1). For 51 of these women both EIA tests were negative below the gray zone and consequently the DFA-C test was not performed. In 13 women one of the EIA tests was in the negative gray zone, and no EBs were found in the DFA-C test. In the remaining 8 women one EIA test was positive, but the DFA-C test was negative (false EIA positive individuals) (Table 1).
A total of 15 infants were considered chlamydianegative. However, EBs were found in DFA-D (direct from the eye) in 6 of the children, but the numbers (1-3) were too low for inclusion into the chlamydia-positive group. Two of these children had conjunctivitis. The chlamydia-negative infants were not younger than the chlamydia-positives, nor was there any difference in the mean weight or length at birth.
Sensitivity, specificity and predictive values of the EIA test on pregnant women
Sensitivity, specificity and predictive values of the EIA test in the newborn babies
According to our criteria 6% (8/129) of the women were false EIA positive individuals and 4% (5/129) false EIA negative by the combined results of EIA and DFA-C tests on cervical and urethral samples (Table 1). Thus referring to the individuals rather
Referring to the individuals rather than specimens, the sensitivity of the EIA was 37% (10/27) (Figure 1) DFA-D
Table 1. Enzyme immunoassay (EIA) vs direct immunofluorescence (DFA-C) tests on ceroical and uretJ:ral samples from 129 pregnant women (positive results were gIven higher priority than negative irrespective of site) DFA-C
EIA
EIA
+
+
52 5
8 13
Total
57
21
ND, not done
NO
Total
0 51
60 69
51
129
DFA-C
Figure 1. Outcome of direct immunofiuorescence (DFA·D), enzyme immunoassay (EIA) and confinnative DFA-C tests in 27 chlam~diapositive infants ('in accordance with the established criteria) .
36
International Journal of STD & AIDS Volume 3 January/February 1992
and the specificity was 100% (15/15). The positive predictive value was 100% (10/10), and the negative predictive value 47% (15/32). The sensitivity for DFA-D was 81% (22/27). Outcome of the EIA and DFA-D tests in symptomatic and asymptomatic newborns Out of a total of 27 chlamydia-positive infants, 15 (56%) (ie 36% of the 42 children born to chlamydiapositive mothers) had signs of conjunctivitis. Twelve of these 15 children had received ocular treatment in addition to prophylaxis; 6 were treated with chloramphenicol and 6 with tetracyclin ointment. DISCUSSION This is the first study of C. trachomatis infection in pregnant women in El Salvador. A relatively high prevalence (44%) was observed. Previous studies of pregnant women in other countries have revealed chlamydial infection in 8-50% of all cases 5,6,8,9. The highest prevalence observed (50%) was found among pregnant women in Suva on the Fiji islands>. Risk factors associated with chlamydial infection are promiscuity, low age, first pregnancy, low socioeconomic and poor hygienic conditions in crowded settings, etc. Several of these risk factors exist in El Salvador; the high prevalence of chlamydial infection that we have found should therefore not be totally unexpected. However, contrary to expectations we could not find any statistically significant differences in marital status, parity or age distributions between chlamydiapositive and chlamydia-negative women. We obtained two specimens from each woman, one from the urethra and one from the cervix. This procedure increased the fossibility of detection of C. trachoma tis antigen 1 , since the chlamydial infection would have been missed in 21% of the infected women if specimens had been taken from the cervix only. The transmission rate from mothers to infants reported in the literature has varied from 23% to 70%10 as compared with 64% in the present study. Of the chlamydia-positive infants 56% had signs of conjunctivitis. Holmes" proposed that there is a 50% risk that the normal child will develop a chlamydial conjunctivitis if it becomes infected with C. trachoma tis and a 10% risk that it will develop pneumonia. The risk reported for inclusion conjunctivitis in infants born to cervically infected mothers has ranged from 18% to 50% in various studies1,3,7 compared with 36% in the present study. Five of the chlamydia-positive infants were born by caesarean section. In all 5 cases the membranes had been deliberately ruptured which precludes any conclusions to be drawn as to whether intrauterine infection had occurred prior to the onset of labour. Frost et al,17 have detected chlamydial antigen in placentas which suggests possible intrauterine
infection. BelP8 has isolated C. trachomatis from twins delivered by caesarean section without prior rupture of the chorioamniotic membrane. The problem with laboratory diagnosis of chlamydial infection is how to judge the results. EIA false negative as well as false positive specimens have been reported'? and the results should therefore be confirmed by another test. The use of more than one test increases the possibility of obtaining a correct diagnosis. For DFA the main problem is to judge if the fluorescent dots seen are real EBs. This is the reason why in different studies different numbers of EBs (usually between 5 and 10) have been required for a specimen to be regarded as positive-''. The importance of a standardized sampling scheme and reading procedure as well as an experienced microscopist is evident. Chlamydial culture is generally regarded as the 'gold standard' although its sensitivity is not 100%. For the moment our laboratory in San Salvador is not adequately equipped in a way that permits us to perform such analysis. Neither EIA nor DFA have a sensitivity or a specificity of 100%. According to the manufacturer, Pharmacia Chlamydia EIA is intended for the detection of C. trachomatis in endocervical or urethral swab specimens where the sensitivity of the test in high risk female populations is claimed to be more than 90% as compared with culture. We thought it would also be of interest to investigate the sensitivity of the EIA test on conjunctival samples even if such use of the test kit has not been recommended by the manufacturer. The 37% sensitivity that we report here is discouraging. Unfortunately, there is a certain amount of subjectivity involved in the results of microscopy which makes DFA results from different laboratories and investigators difficult to compare-", In the present study all DFAs were read by the same investigator and great efforts were made in comparing suspected EBs in the specimens with those in the positive and negative controls. Our interpretation criteria were arbitrarily chosen in order to optimize the detection of chlamydial infection. Differences in the interpretation criteria could drastically affect the results. For example, raising the cut-off to 5 EBs when reading the DFAC and DFA-D results in the present series (but otherwise using the same criteria) would have changed the proportion of chlamydia-positive pregnant women from 44% to 30%. However, the data obtained with these more stringent criteria would appear to represent under-estimates because the majority of the 18 women who would thus be re-classified as chlamydia-negative were clearly EIA positive and 4 of them also ga~e birth to chlamydiapositive children (data not given). In conclusion, the observed high prevalence of Chlamydia trachomatis infection in Salvadorean pregnant women, and the high risk of transmission of the infection to their newborns, suggest that
Posada et al. Chlamydial infection during pregnancy
routine C. trachomaiis testing of patients at maternity clinics is clearly warranted. It seems necessary to create possibilities for the laboratory identification of urogenital chlamydial infection in women attending clinics run by the Ministry of Public Health. Specific treatment should be provided in order to prevent subsequent complications and postpartum endometritis in mothers and neonatal infections of the infants. Other alternatives include the possibility of treating all pregnant women between the 34th and 38th week of pregnancy with erythromycin. Although this might be the most cost-effective approach in the short term, focused case detection including treatment of sexual partners would be more appropriate. Furthermore, we would recommend erythromycin (0.5%) ophthalmic ointment or tetracyclin (1%) ointment in the eyes of the newborns as prophylactic treatment. The present use of chloramphenicol (1%) ophthalmic ointment does not protect against chlamydial infection of the eyes",
6
7
8
9
10
11 12
13
Acknowledgements: The present study was supported by grants from the Swedish Agency of Research Cooperation with Developing Countries (SAREC), under the Karolinska International Research Training (KIRT) Programme. The Pharmacia Chlamydia EIA kit was kindly supplied by Pharmacia Diagnostics AB. We thank the medical staff and the administrative personnel of the Maternity Hospital of San Salvador, for their valuable assistance in the collection of the specimens from pregnant women.
14
15
16 17
References 1 Alexander ER, Harrison HR. Role of Chlamydia trachomatis in perinatal infection. Rev Infect Dis 1983;5:713-19 2 Harrison HR, Boyce TW, Haffner WHJ, et al. The prevalence of genital Chlamydia trachomatis and mycoplasmal infection during pregnancy in an American Indian population. Sex Transm Dis 1983;10:184-6 3 Holmes KK. The chlamydia epidemic. JAMA 1981;17:1718-23 4 Chungue E, Cartel JL, Tourneaux A, et al. Chlamydia trachomatis genital infections in Tahiti. Eur J Clin Microbiol Infect Dis 1988;7:635-8 5 Gyaneshwar R, Nsanze H, Singh KP, Pillay S, Seruvatu 1. The prevalence of sexually transmitted diseases agents in
18 19
20
37
pregnant women in Suva. Aust NZ J Obstet Gynaecol 1987;27:213-15 [ama Ahmed H. Infections associated with sexual transmission in Somalia. Studies with special attention to women of reproductive age. MSc Thesis, 1986, Karolinska Institute, Stockholm, Sweden Sandstrom 1. Ophthalmia neonatorum with special reference to Chlamydia trachomatis. Diagnostic and treatment. Acta Paediatr Scand 1986;suppl 300:1-24 Ismail MA, Chandler AE, Beem MO, Moawad AH. Chlamydial colonization of the cervix in pregnant adolescents. J Reprod Med 1985;30:549-53 Khurana CM, Deddish PA. DelMundo F. Prevalence of Chlamydia trachomatis in the pregnant cervix. Obstet Gynecol 1985;&&:241-3 Heggie AD, Lumicao GG, Stuart LA, Gyves TM. Chlamydia trachomatis infections in mothers and infants. Am J Dis Child 1981;135:507-11 Rettig PJ. Perinatal infection with Chlamydia trachomatis. Clin Perinatal 1988;15:321-50 Schachter J, Grossman M, Sweet RL, et al, Prospective study of perinatal transmission of Chlamydia trachomatis. JAMA 1986;255:3374-7 Schaefer C, Harrison HR, Boyce WT, Lewis M. Illnesses in infants born to women with Chlamydia trachomatis infection. Am J Dis Child 1985;139:127-33 Datta P, Laga M, Plummer FA, et al. Infection and disease after perinatal exposure to Chlamydia trachomatis in Nairobi Kenya. J Infect Dis 1988;158:524-8 Harrison HR, Alexander ER. Chlamydia trachomatis infections of the infant. In: Holmes KK, Mardh PA, Wiesner PI, eds. Sexually transmitted diseases. New York: McGraw-Hill Book Co, 1984:270-80 Paavonen J, Vesterinen E. Chlamydia trachomatis in cervicitis and urethritis in women. Scand J Infect Dis 1982;32(Suppl):45-54 Frost E, Leclerc A, Gioanni G, et al. Chlamydiae infected placenta less often than gonococci. Genitourin Med 1988; 64:349-50 Bell TA. Chlamydia trachomatis infection in dizygotic twins delivered by caesarean section. Genitourin Med 1988;64:347-8 Bygdeman SM, Lidbrink P, Nahkiaisoja M, Parkhede U. Comparison of Pharmacia Chlamydia EIA, IDEIA and cell culture in the detection of urogenital chlamydia! infection. Int J STD AIDS 1990;1:199-204 Williams T, Maniar AC, Brunham RC, Hammond GW. Identification of Chlamydia irachomatis by direct immunofluorescence applied in specimens originating in remote areas. J Clin MicrobioI1985;22:1053-4
(Accepted 2 July 1991)
ORIGINAL ARTICLE
Prevalence of urogenital Chlamydia trachomatis infection in EI Salvador I. Infection during pregnancy and perinatal transmission Ana Berta Canas Posada BSCl, Jon Jonasson MD PhD2, Leonor de Linares MD MSC 1 and Solgun Bygdeman MD PhD2 IDepartment of Microbiology, Faculty of Medicine, University of El Salvador; 2Karolinska Institute, Department of Clinical Bacteriology, Huddinge Hospital, Stockholm, Sweden Summary: One-hundred and twenty-nine pregnant women in labour (age range 15-46 years; median age 23) and 42 infants born to chlamydia-positive mothers (age range 5-15 days; median age 10) were investigated to estimate the prevalence and incidence, respectively, of Chlamydia trachomatis infection in San Salvador, El Salvador. Urethral and cervical samples were obtained from all women and conjunctival specimens were taken from both eyes of each child. The chlamydial antigen was detected with the commercial Pharmacia Chlamydia EIA kit. Direct immunofluorescence (DFA) (Syva MicroTrak) was used for confirmation. In the newborns both EIA and OFA tests on direct preparations from ocular smears were performed on all the samples. The prevalence of chlamydia! infection in pregnant women was 44% (57/129). The incidence of chlamydial infection in neonates was 64% (27/42), and the majority of the infected children (56%) had conjunctivitis. Referring to individuals rather than specimens the sensitivity of EIA tests on conjunctival samples from the infants was low (37%) as compared with 91% on urethral and cervical specimens from the pregnant women. Keywords: Chlamydia trachomatis, El Salvador, perinatal transmission, chlamydia! ophthalmia neonatorum
INTRODUCTION The important role of Chlamydia trachomatis as an aetiological agent in infections among pregnant women and neonates was documented at the beginning of this century, long before the agent was isolated in culture. Today C. trachomatis seems to be one of the most prevalent sexually transmitted pathogens in the industrialized world-r'. In developing countries, an even higher incidence of chlamydial infection has been reportedv". Several studies have defined risk factors for chlamydial cervical infection in pregnant women-Z:". It has been reported that the prevalence of C. trachomatis is higher in pregnant women than in other population groups. The cause of this unusual finding is unknown, although several hypotheses have been proposed'P-!'. The potential role of transmission by direct spread from the infected cervix of the mother to the baby during labour and delivery has also been
studied1,3,11- 13. Several authors have demonstrated the role of C. trachomatis in pneumonitis, and conjunctivitis. It is well known that the so-called ophthalmia neonatorum or conjunctivitis is the most common ocular disease among newborns. Chlamydia! aetiology has also been suspected in cases of otitis media and gastroenteritis of neonates and infants3,7,12- 15. In El Salvador, a developing country located in Central America, the prevalence and incidence of chlamydial infection is not known accurately. The present work is the first in a series of papers that investigates the prevalence of urogenital C. trachomatis infection in El Salvador. The aims of the present study were: (1) to determine the prevalence of chlamydia! infection in a group of pregnant women attending the Maternity Hospital of San Salvador City, (2) to estimate the risk of infection in newborn babies born to chlamydia-positive mothers. MATERIALS AND METHODS
Correspondence to: Dr J [onasson, Department of Clinical Bacteriology, Huddinge Hospital F72, S-141 86 Huddinge, Sweden
Study population and performance of the study The study group consisted of 129 pregnant women in labour, aged 15-46 years, admitted to the
34
International Journal of STD & AIDS Volume 3 January/February 1992
Maternity Hospital of San Salvador City for delivery from April to July 1989. Only a minor proportion of the 6171 deliveries at the hospital during this period could be included in the study due to the fact that interviews and samplings could only be conducted after a doctor had examined a patient and pronounced her available for the study. History of genitourinary tract infection was not a prerequisite for enrolment in the study. Patients with a previous caesarean section, rupture of membranes or vaginal bleeding were excluded from the study. After explaining the aims of the study and receiving verbal consent, personal data from each patient was obtained using a standardized questionnaire including name, age, address, occupation, marital status, duration of pregnancy, parity, and genital infections. Forty-two infants born to chlamydia-positive mothers were investigated in their houses, 5-15 days after birth. Sex, birth weight and length were recorded and the eyes of the children were examined for the presence of secretions, or erythema.
used for DFA according to the instructions of the manufacturer. All DFA tests were examined by the same investigator using a Leitz Weitzlar Ortholux II fluorescence microscope. For confirmative DFA-C the EIA transport medium was centrifuged and the sediment deposited on a slide (Syva MicroTrak), which was allowed to air dry, and then was fixed in methanol. Criteria of interpretation
Pregnan t women A patient was classified as chlamydia-positive if: (a) one or both samples were positive by EIA and confirmed by the DFA-C test with at least one EB, or (b) negative by ErA (gray zone), in one or both samples, but positive by DFA-C with three or more EBs.
Newborns Only infants with mothers who were positive according to the criteria above, were included. An infant was considered chlamydia-positive if: (a) DFA-D (direct from the eye) was positive in one or both eyes, when 4 or more EBs and/or one or Sampling more IBs were found, (b) EIA was positive in one Specimens for the laboratory diagnosis of C. or both eyes and confirmed by DFA-C with at least trachomatis infection using the Pharmacia Chlamydia one EB, (c) negative by EIA (gray zone negative) in (EIA) test were taken from the urethra and cervix one or both eyes, but confirmed by DFA-C with one of the women using standard techniques. The or more EBs and at the same time positive by DFAMicroTrak direct immunofluorescence test (DFA-C) D with at least one EB. used for the confirmation of EIA positive and negative gray zone results was performed on the Statistical analysis transport media of these samples. Two independent samples were obtained from Descriptive statistics and correlation analysis with the each eye of the infants by carefully rubbing the inner chi-square test were performed using the computer surface of the lower and the upper eyelids with program Quest (University of Umea, Sweden). swabs. The first one was used for the EIA test and the second for direct immunofluorescence (DFA-D). RESULTS Enzyme immunoassay (EIA) test Following collection the samples were kept refrigerated at 4°C in transport medium and processed within the following 2-7 days. The Pharmacia Chlamydia EIA Test (Pharmacia Diagnostics AB, Uppsala, Sweden) was performed on all samples according to the manufacturer's instructions. Samples with an absorbance value within 0.02 units above or below the cut-off value were considered to be in the 'gray zone'. The absorbance ratio (absorbance value/cut-off value) was calculated. Specimens with absorbance values in the positive range (absorbance ratio > = 1.0) or within the negative gray zone were further investigated by direct immunofluorescence test (DFA-C). Direct immunofluorescence test (OFA) The MicroTrak C. trachoma tis Direct Specimen Test (Syva Micro'Irak, Palo Alto, California, USA) was
The median age of the 129 pregnant women studied was 23 years (range 15-46). Nearly all of them, 121 (94%) were 'housewives' or unemployed. Thirteen per cent were single and 75% were unmarried but living together with their 'fiance'. Only 12% were married. The mean number of earlier pregnancies was 1.5 (range 0-14). Fifty-six (43%) were pregnant for the first time and 59 (46%) had 1-3 earlier pregnancies. Pregnancy ended by vaginal delivery in 108 cases (84%) and by caesarean section in 21 (16%) cases. At follow-up, 45 out of a total of 57 chlamydiapositive mothers were found living at the address given by them in the hospital. However, only 42 infants were tested, because 3 children had already died at home for unknown reasons. Twenty-one infants were girls and 21 boys. The median age of the babies at the time of investigation was 10 days (range 5-15). The mean birth weight was 3154g (range 2240-4281) and the mean length at birth was 48 cm (range 45-51). Two children had jaundice at birth. The others were recorded as healthy.
Posada et al.
Thirty-two children (76%) of chlamydia-positive mothers were born by vaginal delivery. The remaining 10 babies were born by caesarean section due to fetal distress, cephalopelvic disproportion or abnormal presentation. All of these infants had received ointment with 1% chloramphenicol in the eyes at birth. Prevalence of chlamydial infection The prevalence of chlamydial infection in pregnant women was 44% (57/129) (Table 1). Thirty-two per cent (18/57) were positive in both the urethra and the cervix, 47% (27/57) in the cervix only and 21% (12/57) in the urethra only. There were no significant differences in age, marital status or parity between chlamydia-positive and chlamydia-negative women. Chlamydia-positive women Fifty-two out of the 57 (91%) chlamydia-positive women were positive in both the EIA and DFA-C tests in at least one site. However, one sample from each of 5 women was in the EIA negative gray zone, but positive for DFA-C according to our established criteria with 3-20 EBs (false EIA negative individuals) (Table 1).
Chlamydial infection during pregnancy
35
than specimens the sensitivity for EIA was 91% (52/57) and the specificity 89% (64/72). The positive predictive value was 87% (52/60) and the negative predictive value 93% (64/69). Incidence of chlamydial infection in the newborns The incidence of chlamydia infected children born to chlamydia-positive mothers was 64% (27/42); 14 females and 13 males. Forty-eight per cent (13/27) were infected in both eyes, 33% (9/27) in the right eye only and 19% (5/27) in the left eye only. Twenty-two (82%) chlamydia-positive children were born by vaginal delivery, and the remaining 5 by caesarean section. The reason for caesarean section was abnormal presentation in 2 cases, fetal distress in 2 cases and cephalopelvic disproportion in the last one. Before the caesarean section was performed, membranes had been deliberately ruptured as a procedure of help in childbirth in all 5 cases. Chlamydia positive newborns The results of EIA, DFA-C and DFA-D tests in the 27 chlamydia positive newborns are shown in Figure 1. Seventeen babies were EIA negative but positive by DFA-D and/or by DFA-C, and therefore were considered positive according to our criteria.
Chlamydia-negative women
Chlamydia negative newborns
Seventy-two women were considered chlamydianegative (see columns 2 and 3 in Table 1). For 51 of these women both EIA tests were negative below the gray zone and consequently the DFA-C test was not performed. In 13 women one of the EIA tests was in the negative gray zone, and no EBs were found in the DFA-C test. In the remaining 8 women one EIA test was positive, but the DFA-C test was negative (false EIA positive individuals) (Table 1).
A total of 15 infants were considered chlamydianegative. However, EBs were found in DFA-D (direct from the eye) in 6 of the children, but the numbers (1-3) were too low for inclusion into the chlamydia-positive group. Two of these children had conjunctivitis. The chlamydia-negative infants were not younger than the chlamydia-positives, nor was there any difference in the mean weight or length at birth.
Sensitivity, specificity and predictive values of the EIA test on pregnant women
Sensitivity, specificity and predictive values of the EIA test in the newborn babies
According to our criteria 6% (8/129) of the women were false EIA positive individuals and 4% (5/129) false EIA negative by the combined results of EIA and DFA-C tests on cervical and urethral samples (Table 1). Thus referring to the individuals rather
Referring to the individuals rather than specimens, the sensitivity of the EIA was 37% (10/27) (Figure 1) DFA-D
Table 1. Enzyme immunoassay (EIA) vs direct immunofluorescence (DFA-C) tests on ceroical and uretJ:ral samples from 129 pregnant women (positive results were gIven higher priority than negative irrespective of site) DFA-C
EIA
EIA
+
+
52 5
8 13
Total
57
21
ND, not done
NO
Total
0 51
60 69
51
129
DFA-C
Figure 1. Outcome of direct immunofiuorescence (DFA·D), enzyme immunoassay (EIA) and confinnative DFA-C tests in 27 chlam~diapositive infants ('in accordance with the established criteria) .
36
International Journal of STD & AIDS Volume 3 January/February 1992
and the specificity was 100% (15/15). The positive predictive value was 100% (10/10), and the negative predictive value 47% (15/32). The sensitivity for DFA-D was 81% (22/27). Outcome of the EIA and DFA-D tests in symptomatic and asymptomatic newborns Out of a total of 27 chlamydia-positive infants, 15 (56%) (ie 36% of the 42 children born to chlamydiapositive mothers) had signs of conjunctivitis. Twelve of these 15 children had received ocular treatment in addition to prophylaxis; 6 were treated with chloramphenicol and 6 with tetracyclin ointment. DISCUSSION This is the first study of C. trachomatis infection in pregnant women in El Salvador. A relatively high prevalence (44%) was observed. Previous studies of pregnant women in other countries have revealed chlamydial infection in 8-50% of all cases 5,6,8,9. The highest prevalence observed (50%) was found among pregnant women in Suva on the Fiji islands>. Risk factors associated with chlamydial infection are promiscuity, low age, first pregnancy, low socioeconomic and poor hygienic conditions in crowded settings, etc. Several of these risk factors exist in El Salvador; the high prevalence of chlamydial infection that we have found should therefore not be totally unexpected. However, contrary to expectations we could not find any statistically significant differences in marital status, parity or age distributions between chlamydiapositive and chlamydia-negative women. We obtained two specimens from each woman, one from the urethra and one from the cervix. This procedure increased the fossibility of detection of C. trachoma tis antigen 1 , since the chlamydial infection would have been missed in 21% of the infected women if specimens had been taken from the cervix only. The transmission rate from mothers to infants reported in the literature has varied from 23% to 70%10 as compared with 64% in the present study. Of the chlamydia-positive infants 56% had signs of conjunctivitis. Holmes" proposed that there is a 50% risk that the normal child will develop a chlamydial conjunctivitis if it becomes infected with C. trachoma tis and a 10% risk that it will develop pneumonia. The risk reported for inclusion conjunctivitis in infants born to cervically infected mothers has ranged from 18% to 50% in various studies1,3,7 compared with 36% in the present study. Five of the chlamydia-positive infants were born by caesarean section. In all 5 cases the membranes had been deliberately ruptured which precludes any conclusions to be drawn as to whether intrauterine infection had occurred prior to the onset of labour. Frost et al,17 have detected chlamydial antigen in placentas which suggests possible intrauterine
infection. BelP8 has isolated C. trachomatis from twins delivered by caesarean section without prior rupture of the chorioamniotic membrane. The problem with laboratory diagnosis of chlamydial infection is how to judge the results. EIA false negative as well as false positive specimens have been reported'? and the results should therefore be confirmed by another test. The use of more than one test increases the possibility of obtaining a correct diagnosis. For DFA the main problem is to judge if the fluorescent dots seen are real EBs. This is the reason why in different studies different numbers of EBs (usually between 5 and 10) have been required for a specimen to be regarded as positive-''. The importance of a standardized sampling scheme and reading procedure as well as an experienced microscopist is evident. Chlamydial culture is generally regarded as the 'gold standard' although its sensitivity is not 100%. For the moment our laboratory in San Salvador is not adequately equipped in a way that permits us to perform such analysis. Neither EIA nor DFA have a sensitivity or a specificity of 100%. According to the manufacturer, Pharmacia Chlamydia EIA is intended for the detection of C. trachomatis in endocervical or urethral swab specimens where the sensitivity of the test in high risk female populations is claimed to be more than 90% as compared with culture. We thought it would also be of interest to investigate the sensitivity of the EIA test on conjunctival samples even if such use of the test kit has not been recommended by the manufacturer. The 37% sensitivity that we report here is discouraging. Unfortunately, there is a certain amount of subjectivity involved in the results of microscopy which makes DFA results from different laboratories and investigators difficult to compare-", In the present study all DFAs were read by the same investigator and great efforts were made in comparing suspected EBs in the specimens with those in the positive and negative controls. Our interpretation criteria were arbitrarily chosen in order to optimize the detection of chlamydial infection. Differences in the interpretation criteria could drastically affect the results. For example, raising the cut-off to 5 EBs when reading the DFAC and DFA-D results in the present series (but otherwise using the same criteria) would have changed the proportion of chlamydia-positive pregnant women from 44% to 30%. However, the data obtained with these more stringent criteria would appear to represent under-estimates because the majority of the 18 women who would thus be re-classified as chlamydia-negative were clearly EIA positive and 4 of them also ga~e birth to chlamydiapositive children (data not given). In conclusion, the observed high prevalence of Chlamydia trachomatis infection in Salvadorean pregnant women, and the high risk of transmission of the infection to their newborns, suggest that
Posada et al. Chlamydial infection during pregnancy
routine C. trachomaiis testing of patients at maternity clinics is clearly warranted. It seems necessary to create possibilities for the laboratory identification of urogenital chlamydial infection in women attending clinics run by the Ministry of Public Health. Specific treatment should be provided in order to prevent subsequent complications and postpartum endometritis in mothers and neonatal infections of the infants. Other alternatives include the possibility of treating all pregnant women between the 34th and 38th week of pregnancy with erythromycin. Although this might be the most cost-effective approach in the short term, focused case detection including treatment of sexual partners would be more appropriate. Furthermore, we would recommend erythromycin (0.5%) ophthalmic ointment or tetracyclin (1%) ointment in the eyes of the newborns as prophylactic treatment. The present use of chloramphenicol (1%) ophthalmic ointment does not protect against chlamydial infection of the eyes",
6
7
8
9
10
11 12
13
Acknowledgements: The present study was supported by grants from the Swedish Agency of Research Cooperation with Developing Countries (SAREC), under the Karolinska International Research Training (KIRT) Programme. The Pharmacia Chlamydia EIA kit was kindly supplied by Pharmacia Diagnostics AB. We thank the medical staff and the administrative personnel of the Maternity Hospital of San Salvador, for their valuable assistance in the collection of the specimens from pregnant women.
14
15
16 17
References 1 Alexander ER, Harrison HR. Role of Chlamydia trachomatis in perinatal infection. Rev Infect Dis 1983;5:713-19 2 Harrison HR, Boyce TW, Haffner WHJ, et al. The prevalence of genital Chlamydia trachomatis and mycoplasmal infection during pregnancy in an American Indian population. Sex Transm Dis 1983;10:184-6 3 Holmes KK. The chlamydia epidemic. JAMA 1981;17:1718-23 4 Chungue E, Cartel JL, Tourneaux A, et al. Chlamydia trachomatis genital infections in Tahiti. Eur J Clin Microbiol Infect Dis 1988;7:635-8 5 Gyaneshwar R, Nsanze H, Singh KP, Pillay S, Seruvatu 1. The prevalence of sexually transmitted diseases agents in
18 19
20
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pregnant women in Suva. Aust NZ J Obstet Gynaecol 1987;27:213-15 [ama Ahmed H. Infections associated with sexual transmission in Somalia. Studies with special attention to women of reproductive age. MSc Thesis, 1986, Karolinska Institute, Stockholm, Sweden Sandstrom 1. Ophthalmia neonatorum with special reference to Chlamydia trachomatis. Diagnostic and treatment. Acta Paediatr Scand 1986;suppl 300:1-24 Ismail MA, Chandler AE, Beem MO, Moawad AH. Chlamydial colonization of the cervix in pregnant adolescents. J Reprod Med 1985;30:549-53 Khurana CM, Deddish PA. DelMundo F. Prevalence of Chlamydia trachomatis in the pregnant cervix. Obstet Gynecol 1985;&&:241-3 Heggie AD, Lumicao GG, Stuart LA, Gyves TM. Chlamydia trachomatis infections in mothers and infants. Am J Dis Child 1981;135:507-11 Rettig PJ. Perinatal infection with Chlamydia trachomatis. Clin Perinatal 1988;15:321-50 Schachter J, Grossman M, Sweet RL, et al, Prospective study of perinatal transmission of Chlamydia trachomatis. JAMA 1986;255:3374-7 Schaefer C, Harrison HR, Boyce WT, Lewis M. Illnesses in infants born to women with Chlamydia trachomatis infection. Am J Dis Child 1985;139:127-33 Datta P, Laga M, Plummer FA, et al. Infection and disease after perinatal exposure to Chlamydia trachomatis in Nairobi Kenya. J Infect Dis 1988;158:524-8 Harrison HR, Alexander ER. Chlamydia trachomatis infections of the infant. In: Holmes KK, Mardh PA, Wiesner PI, eds. Sexually transmitted diseases. New York: McGraw-Hill Book Co, 1984:270-80 Paavonen J, Vesterinen E. Chlamydia trachomatis in cervicitis and urethritis in women. Scand J Infect Dis 1982;32(Suppl):45-54 Frost E, Leclerc A, Gioanni G, et al. Chlamydiae infected placenta less often than gonococci. Genitourin Med 1988; 64:349-50 Bell TA. Chlamydia trachomatis infection in dizygotic twins delivered by caesarean section. Genitourin Med 1988;64:347-8 Bygdeman SM, Lidbrink P, Nahkiaisoja M, Parkhede U. Comparison of Pharmacia Chlamydia EIA, IDEIA and cell culture in the detection of urogenital chlamydia! infection. Int J STD AIDS 1990;1:199-204 Williams T, Maniar AC, Brunham RC, Hammond GW. Identification of Chlamydia irachomatis by direct immunofluorescence applied in specimens originating in remote areas. J Clin MicrobioI1985;22:1053-4
(Accepted 2 July 1991)
