Introduction

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Premature Rupture of Membranes (PROM) is usually associated with significant perinatal and maternal infectious morbidities.1,2 Failure to identify patients with PROM can result in failure to implement standard measures and conversely an incorrect diagnosis leads to inappropriate interventions.3,4 Accurate diagnosis of PROM remains a frequent clinical problem in obstetrics. Unfortunately, a non-invasive standard diagnostic test is not available.5,6 The absence of a non-invasive gold standard for diagnosis of PROM resulted in the appearance of several tests based on alternative biochemical markers. These biochemical markers include; vaginal prolactin, HCG (human chorionic gonadotropin), alpha feto protein, fetal fibronectin (fFN) and insulin like growth factor binding protein1(IGFBP 1).715 The quantitative evaluation of HCG as a marker for PROM is costly and time consuming.1618 fFN ia a complex adhesive glycoprotein expressed in the choriodecidual junction between maternal decidua and fetal membranes.19,20 fFN ia a complex adhesive glycoprotein (450 KD) and a member of extracellular matrix family at the maternal-fetal interface expressed in the choriodecidual junction between maternal decidua and fetal membranes. It is detectable during first 22 weeks of pregnancy and it should be absent between2234 weeks gestation, and is then again normally detectable after 34 gestational weeks, during the finalstage of pregnancy.1921 fFN is thought to be a trophoblast glue that promotes cellular adhesion at uterine-placental and decidual fetal membrane interface, it is released into cervicovaginal secretions when the chorionic/decidual interface is disrupted and in cases of preterm labor (PTL).1921 This study was designed to compare fFN using Quick Check fFN test versus IGFBP-1 using Actim PROM test for detection of PROM.

Patients and Methods

This study was carried out over two year, from March 2011 to March 2013. Two hundred and twenty pregnant women >34 weeks and <37 weeks gestation were included in this study after informed consent and approval of the study protocol by the institute ethical committee and divided into two groups; 110 patients with PROM were included in group I (study) and 110 patients without PROM were included from the antenatal ward, during control of blood pressure (hypertension) or blood sugar (diabetic) or assessment of fetal wellbeing (IUGR) in group II (controls). Patients with multiple pregnancies or more than 37 weeks or not sure of dates or fetal distress or vaginal bleeding or preterm labor (PTL) or chorioamnionitis were excluded from current study. The diagnosis of PROM in this study was based on; history of watery discharge, confirmed by positive ferning, positive nitrazine tests, visualization of fluid coming from the cervix during speculum examination and Trans-abdominal sonography (TAS) to measure the amniotic fluid index (AFI ≤ 5 cm). 6,9 The gestational age was calculated from the first day of last menstural period and confirmed by early ultrasound scan (before 20 weeks gestation). Patient of this study were examined by sterile speculum to detect amniotic fluid pooling through cervical canal and for collection of samples. Laboratory investigations were done to exclude chorioamnionitis (maternal fever, maternal tackycardia, fetal tackycardia, maternal leucocytosis, C-reactive protein).

Samples collection:

Patients were examined in dorsal Lithotomy position with good illumination using sterile speculum (without antiseptics). Four sterile swabs were used to collect the samples from posterior vaginal fornix (swabs should not touch the vaginal wall during insertion or removal).

Nitrazine test:

First sterile swab impregnated with nitrazine yellow dye was inserted in the posterior viginal fornix for 15 seconds, then the color of the swab was interpreted after removal of the swab from the vagina. Blue color was considered as positive (PROM).

Ferning test:

Sample collected from the posterior vaginal fornix using second swab was spreaded on a glass slide, creating a very thin smear and the smear was examined by low power microscope, crystallization of amniotic fluid to form a fern like pattern was considered as positive (PROM).

Quick Check fFN test:

Quick Check fFN test is a 10-minute, one-step, visual test. The patient should not had sexual intercourse or vaginal douching within the last 24 hours before the fFN test. According to manufacturer’s instructions, before collection the patient sample, the tube containing the extraction buffer was removed from the package and its cap was carefully removed. During speculum examination, prior to any vaginal examination, third polyster tipped applicator provided with the kit was rotated across posterior vaginal fornix for 10 seconds, then inserted into the tube containing the extraction buffer for 10-15 seconds. The test strip was removed from the foil pouch and its lower end (indicated by the arrows) was inserted into the tube containing the extraction buffer for 10 minutes.

Principles of the Quick Check fFN test:

Immunoassays specific for fFN can detect fFN in the cervicovaginal secretions of the pregnant women during the first 22 weeks of pregnancy until fetal membranes completely fuse to decidua. By 37 weeks’ gestation, fFN loses its adhesive properties, and can be execreted in cervical secretions (this why women more than 37 weeks gestation were excluded from this study). The patient should not have vaginal examination or manipulation such as sexual intercourse or vaginal douching within the last 24 hours before fFN test. Fetal fibronectin in the cervicovaginal secretions was determined using a qualitative enzyme-linked immunosorbent assay test. fFN swab was collected as third swab because the fFN test detect fFN in amniotic fluid at higher concentration than 50 ng/ml, and fFN test results can be affected by semen, blood, and vaginal infections. Actim PROM test gives positive result for PROM when the sample contains IGFBP-1 above 25 mg/l and not affected by affected by semen, urine, blood or vaginal infection. fFN test is a solid-phase immunogold assay. Specimens obtained from the posterior fornix are placed into an extractor buffer. Then fetal fibronectin test strip lower end (indicated by the arrows) was inserted into the tube containing the extraction buffer for 10 minutes.

A positive sample contain fFN will result in two lines in test strip and a negative sample will result in one control line in test strip.2224

Actim PROM test:

According to the manufacturer’s instructions, fourth polyester swab provided with the kit was inserted into the vagina for 10-15 seconds, then rinsed in the provided specimen extraction solution or buffer for 10 seconds, then removed from the solution and disposed. The yellow area of the dipstick was dipped into the specimen extraction solution, removed when the liquid reaches result window and result was interpreted as soon as it was visible. If only one blue line was visible, the test result was negative (no PROM). If two blue lines were visible, the test result was positive (PROM). Actim PROM test for detection of IGFBP-1 gives positive result for PROM when the sample contains IGFBP-1 above 25 mg/l. Unlike fFN and Nitrazine tests, vaginal discharge, urine or seminal fluids were found to have no effect on the performance of IGFBP-1 test.4 On the other hand, presence of vaginal bleeding may give a positive result due to presence of IGFBP-1 in the blood.4

Sample Size and Statistics

The required sample size was calculated using G* Power software version 3.17 for sample size calculation (*Heinrich Heine Universität; Düsseldorf; Germany), setting the α-error probability at 0.05, power (1-β error probability) at 0.95 %, and effective sample size (w) at 0.3. The effective size (w) was calculated as follows: w = , where χ2 is the chi-square test and N is the total sample size. Number of participants needed to produce a statistically acceptable figure was 220 women (110 women in each group).

After delivery the collected data on admission were statistically analyzed using Statistical Package for Social Sciences; version 15. Data were presented as mean, standard deviation (±SD), number and percentage. Chi-square test (χ2) was used for comparison between groups as regard qualitative variables; Un-paired student (t) test was used for comparison between groups as regard numerical variables and a significant level of p value was set at 0.05.

Results

In this study; there was no significant difference between two studied groups regarding; mean age and mean gestational. (Table 1)

Table. 01

Maternal age and gestational age for the studied population.

Variables Group I (PROM Group = 110) Group II (Control Group = 110) p Value test used (Significance)
Maternal age (years) Mean + SD 30.7 + 4.15 31.4 + 6.27 p >0.05 (0.16)t test (Non-Significant)
Gestational age (weeks)Mean + SD 35.0 + 2.56 35.4 + 4.23 p >0.05 (0.86)t test (Non-Significant)

In group I (PROM); fFN test was true positive (based on history, confirmed by positive ferning, positive nitrazine tests, visualization of fluid coming from the cervix and AFI≤ 5 cm measued by TAS) in 104 patients (94.5% = sensitivity) and it was false negative in 6 patients (5.5%), while in group II (No PROM); fFN test was true negative in 98 patients (89.1% – specificity) and it was false positive in 12 patients (10.9%). (Table 2)

In group I; Actim PROM test was true positive in 101 patients (91.8% = sensitivity) and it was false negative in 9 patients (8.2%), while in group II; Actim PROM test was true negative in 94 patients (85.5% = specificity) and it was false positive in 16 patients (14.5%).

In group I; Ferning test was true positive in 93 patients (84.5% = sensitivity) and it was false negative in 17 patients (15.5%), while in group II; Ferning test was true negative in 86 patients (78.2% = specificity) and it was false positive in 24 patients (21.8%).

In group I; Nitrazine test was true positive in 96 patients (87.3% = sensitivity) and it was false negative in 14 patients (12.7%), while in group II; Nitrazine test was true negative in 89 patients (80.9% = specificity) and it was false positive in 21 patients (19.1%).

Table. 02

Fetal Fibronectin (fFN), IGFBP-1 (Actim PROM), Ferning and Nitrazine positive & negative cases in the two studied groups.

Variables Group I (PROM = 110 patients) Group II (Controls = 110 patients)
Positive casesTrue PositiveNumber (%) Negative casesFalse NegativeNumber (%) Positive casesFalse PositiveNumber (%) Negative casesTrue NegativeNumber (%)
Fetal Fibronectin (fFN) test 104 (94.5%) 6 (5.5%) 12 (10.9%) 98 (89.1%)
IGFBP-1 (Actim PROM test) 101 (91.8%) 9 (8.2%) 16 (14.5%) 94 (85.5%)
Ferning test 93 (84.5%) 17 (15.5%) 24 (21.8%) 86 (78.2%)
Nitrazine test 96 (87.3%) 14 (12.7%) 21 (19.1%) 89 (80.9%)

IGFBP-1 = Insulin growth factor binding protein-1

The sensitivity & specificity of fFN test to diagnose PROM was 94.5% & 89.1%; respectively compared with 91.8% & 85.5%; respectively for Actim PROM test, 84.5% & 78.2%; respectively for Ferning test and 87.3% & 80.9%; respectively for Nitrazine test. The PPV, NPV & accuracy of fFN test to diagnose PROM were 89.7%, 94.2% & 91.8%; respectively compared with 86.3%, 91.3% & 88.6%; respectively for Actim PROM test, 79.5%, 83.5% & 81.4%; respectively for Ferning test and 82.1%, 86.4% & 84.1%; respectively for Nitrazine test. (Table 3)

Table. 03

Sensitivity, specificity, predictive values & accuracy of IGFBP-1 (Actim PROM), fetal Fibronectin (fFN), Ferning & Nitrazine tests in diagnosis PROM.

Variables Fetal Fibronectin (fFN) test IGFBP-1 (Actim PROM) test Ferning test Nitrazine test
Sensitivity = True positive /(True positive + false negative) X 100 104/(104 + 6) X 100 = 94.5% 101/(101 + 9) X 100 = 91.8% 93/(93 + 17) X 100= 84.5% 96/(96 + 14) X 100= 87.3%
Specificity =True negative/(True negative + false positive) X 100 98/(98 + 12) X 100= 89.1% 94/(94 + 16) X 100 = = 85.5% 86/(86 + 24) X 100 = 78.2% 89/(89 + 21) X 100 = 80.9%
Positive Predicative Value (PPV) =True positive /(True positive + false positive) X 100 104/(104 + 12) X100 = 89.7% 101/(101 + 16) X100 = 86.3 % 93/(93 + 24) X 100= 79.5% 96/(96 + 21) X 100= 82.1%
Negative Predictive Value (NPV) =True negative /(True negative + false negative) X 100 98/(98 + 6) X 100= 94.2% 94/(94 + 9) X 100= 91.3 % 86/(86 + 17) X 100= 83.5% 89/(89 + 14) X 100= 86.4%
Accuracy = True positive + true negative/(True positive + true negative + false positive + false negative) X 100 104 + 98/(104 + 98 + 12 + 6) X 100= 91.8% 101 + 94/(101 + 94 + 16 + 9) X 100= 88.6% 93 + 86/(93 + 86 + 24 + 17) X 100= 81.4% 96 + 89/ (96 + 89 + 21 + 14) X 100= 84.1 %

Although, sensitivity, specificity, predictive values and accuracy of fFN test to diagnose PROM were higher than sensitivity, specificity, predictive values & accuracy of Actim PROM, Ferning and Nitrazine tests, this difference was statistically insignificant (P >0.05). Tables 4 and 5.

Discussion

PROM is usually associated with significant perinatal & maternal infectious morbidities.25 Ferning has been associated with false-positive results in 5-30% and false-negative results in 5-12.9%. 4,6 Nitrazine evaluation has been associated with false-positive results in 17.4% and false negative results in 12.9%.4,6

Recent studies suggested that fFN in vaginal fluid is a good marker for prediction of PTL, 25,26 and few old studies suggested its role in PROM. 2731 This study was designed to compare fFN test versus IGFBP-1 test for detection of PROM. fFN can detect in the cervical secretions of the pregnant female during first 22 weeks of pregnancy. By 37 weeks’ gestation, fFN loses its adhesive properties and can be execreted in cervical secretions, 1921 this why pregnant women > 37 weeks gestation were excluded from this study.

IGFBP-1 regulates cellular growth and metabolism and is secreted from human liver, decidua and placenta. Its concentrations in amniotic fluid are 100-1000 fold higher than in serum. 4,32 The Actim PROM test is immunochromatography test based on the use of two monoclonal antibodies to human IGFBP-1 gives positive result for PROM when the sample contains IGFBP-1 above 25 mg/l. Unlike fFN and Nitrazine tests, vaginal discharge, urine or seminal fluids were found to have no effect on the performance of IGFBP-1 test.4 On the other hand, presence of vaginal bleeding may give a positive result due to presence of IGFBP-1 in the blood.4

In this study, although, sensitivity, specificity, predictive values and accuracy of fFN test to diagnose PROM were higher than sensitivity, specificity, predictive values and accuracy of Actim PROM, Ferning, Nitrazine, this difference was statistically insignificant.

One hundred and sixteen (116) women with history of PROM and without visible amniotic fluid leakage were included in Nisell et al.27 study to evaluate the role of fFN to detect equivocal PROM. Although, Nisell et al.27 concluded that the fFN had limited value compared by currently available methods in detection of equivocal PROM, Salfelder et al.28 found that 97.1% patients with clearly visible amniotic fluid in the vagina had a positive fFN test results, whereas 96.5% of controls without any signs of ruptured membranes had negative fFN test results and they concluded that a positive fFN test confirms the diagnosis of PROM especially in equivocal cases.

Table. 04

Sensitivity, specificity, predictive values & accuracy of fetal Fibronectin (fFN) test compared to Ferning & Nitrazine tests in diagnosis PROM.

Variables Fetal Fibronectin (fFN) test Ferning test Nitrazine test p Value (test used) Significance
Sensitivity 94.5% 84.5% 87.3% *p >0.05 (0.57)**p >0.05 (0.69)(X2) Non-Significant
Specificity 89.1% 78.2% 80.9% *p >0.05 (0.52)**p >0.05 (0.63)(X2) Non-Significant
Positive Predicative Value (PPV) 89.7% 79.5% 82.1% *p >0.05 (0.55)**p > 0.05 (0.66)(X2) Non-Significant
Negative Predictive Value (NPV) 94.2% 83.5% 86.4% *p >0.05 (0.55)**p >0.05 (0.66)(X2) Non-Significant
Accuracy 91.8% 81.4% 84.1 % *p > 0.05 (0.55)**p >0.05 (0.66)(X2) Non-Significant

*P-value when fetal fibronectin (fFN) test compared to Ferning test.**P-value when fetal fibronectin (fFN) test compared to Nitrazine test.

Also, Eriksen et al.29 concluded that the sensitivity of fFN in women with ROM was 98.2% in a multicenter clinical trial comparing, pooling, ferning, and nitrazine tests with fFN in 339 women (study group) with ROM and in 67 control women at term. Gu et al.30 found that fFN was detected in 95% of the patients with PROM and they concluded that fFN test is a sensitive test that can confirm the diagnosis of PROM, also, Pascal Gaucherand et al.31 concluded that fFN represents a diagnostic test of PROM with good sensitivity (94%) and specificity (97%) and Aaron et al.9 stated that the detection of fFN in amniotic fluid had 97-98% sensitivity, 70-97% specificity, 74-93% PPV and 98-100% NPV for detection of PROM.

Recently, Two hundred and twenty (110 with PROM & 110 control) pregnant women >34 and <37 weeks gestation were included in Abdelazim study to detect the accuracy of fFN to diagnose PROM. Abdelazim found that fFN was more sensitive & specific to diagnose PROM compared with Ferning & Nitrazine tests.21 Dilbaz et al.4 found that the Actim PROM test had 88.2% sensitivity, 81% specificity, 79% PPV and 90% NPV in diagnosis of PROM and Erdemoglu et al.12 concluded that the Actim PROM test was more specific and more accurate than Nitrazine test in diagnosis of PROM.4,21

Table. 05

Sensitivity, specificity, predictive values & accuracy of fetal Fibronectin (fFN) test compared to IGFBP-1 Actim PROM) test in diagnosis PROM.

Variables Fetal Fibronectin (fFN) test IGFBP-1 (Actim PROM) test p Value (test used) Significance
Sensitivity 94.5% 91.8% P >0.05 (0.88)(X2) Non-Significant
Specificity 89.1% 85.5% P >0.05 (0.83)(X2) Non-Significant
Positive Predicative Value (PPV) 89.7% 86.3 % P >0.05 (0.84)(X2) Non-Significant
Negative Predictive Value (NPV) 94.2% 91.3 % P >0.05 (0.87)(X2) Non-Significant
Accuracy 91.8% 88.6% P >0.05 (0.85)(X2) Non-Significant

Also, sensitivity & specificity, predictive values and accuracy of Actim PROM t

est to diagnose PROM were higher than sensitivity & specificity predictive values & accuracy of Ferning and Nitrazine tests, but, this difference was statistically insignificant (P >0.05). Table 6

Cessation of amniotic fluid leakage for more than 12 hours before specimen collection may give a false negative result as IGFBP-1 is degraded by vaginal proteases. IGFBP-1 level increases in cervical and vaginal secretions of women with threatened PTL in absence of ROM due to amnion-decidual disruption. 13,33 This explains why in this study; Actim PROM test gave false negative result in nine (8.2%) cases of definite PROM and false positive result in 16 (14.5%) cases without PROM. Darj et al.34 found that the Actim PROM test had 95.7% sensitivity, 93.1% specificity for cases with definite PROM and had less sensitivity (70.8%) & less specificity (88.2%) for cases with suspected PROM and they concluded that Actim PROM test is a complimentary test to confirm PROM, but patients with a negative test should be followed up by C-reactive protein and white blood cells, to avoid intra-amniotic infections. 4,34 The performance of two rapid tests for diagnosis of PROM based on IGFBP-1 and placental alpha microglobulin in cervicovaginal secretions were compared by many authors and they concluded that both tests have similar performance to diagnose PROM.10,35,36

Only one recent study was available to detect fFN role in detection of PROM21 and the old avaliable studies 2731 did not compare or statistically analyzed the fFN accuracy or fFN predictive values with accuarcy or predictive values of Ferning or Nitrazine or IGFBP-1 (Actim PROM) tests.

In this study, both Quick Check fFN and Actim PROM tests were simple bedside tests to diagnose PROM and although fFN test was more accurate with high sensitivity, specificity and high predictive values than Actim PROM, Ferning and Nitrazine tests, this difference was statistically insignificant. Both Quick Check fFN & Actim PROM tests can be used as complimentary tests to confirm the clinical diagnosis of PROM.

Table. 06

Sensitivity, specificity, predictive values & accuracy of IGFBP-1 (Actim PROM) test compared to Ferning & Nitrazine tests in diagnosis PROM.

Variables IGFBP-1 (ActimPROM) test Ferning test Nitrazine test p Value (test used) Significance
Sensitivity 91.8% 84.5% 87.3% *p >0.05 (0.68)**p >0.05 (0.80) (X2) Non-Significant
Specificity 85.5% 78.2% 80.9% *p >0.05 (0.66)**p >0.05 (0.78) (X2) Non-Significant
Positive Predicative Value (PPV) 86.3 % 79.5% 82.1% *p >0.05 (0.69)**p >0.05 (0.80)(X2) Non-Significant
Negative Predictive Value (NPV) 91.3 % 83.5% 86.4% *p >0.05 (0.65)**p >0.05 (0.78) (X2) Non-Significant
Accuracy 88.6% 81.4% 84.1 % *p >0.05 (0.67)**p >0.05 (0.79)(X2) Non-Significant

Note. *P-value when IGFBP-1 compared to Ferning test **P-value when IGFBP-1 compared to Nitrazine test