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Int J Health Sci (Qassim). 2021 Nov-Dec; 15(6): 16–22. PMCID: PMC8589831
PMID: 34912184

Comparative diagnosis of premature rupture of membrane by nitrazine test, urea, and

creatinine estimation
Adebunmi O. Olarinoye,1 Noah O. Olaomo,2 Kike T Adesina,1 Grace G. Ezeoke,1 and Abiodun P. Aboyeji1

Abstract

Objective:

The study compared the reliability of nitrazine, urea, and creatinine in the diagnosis of premature
rupture of membrane (PROM). Nitrazine strip measures pH levels while urea and creatinine are
produced mainly in amniotic fluid and not in the maternal vagina.

Methods:

Sixty-four pregnant women with demonstrable passage or pooling of liquor par vaginum on
speculum examination and 64 with no liquor drainage at 28–42 weeks gestation were studied.
Vaginal fluid aspirates from both groups of patient were tested for urea, creatinine, and pH levels
using nitrazine strip. Receiver Operating Characteristic (ROC) curves were plotted to determine
cutoff values for urea and creatinine. Statistical analysis was done using SPSS version 23.0 and
setting statistical significance at P ≤ 0.05.

Results:

Nitrazine test showed high level of specificity (100%) but a sensitivity of 87.5%. Predictive ability of
urea at >12.7 mg/dl obtained as cutoff value from the ROC curve showed a sensitivity of 19.64%
and specificity of 94.44% while for creatinine the cutoff value was at >0.8 mg/dl with sensitivity of
48.21% and specificity of 65.28%.
Conclusion:

Nitrazine strip was the most reliable of the three in the diagnosis of PROM. This was followed by
creatinine and then urea. In view of the gaps in sensitivity and accuracy and the importance of
precise diagnosis to prevent maternal and fetal complications, there is still the need to find other
affordable, more sensitive and more accurate biochemical marker/s that will help in diagnosing
PROM especially in difficult cases.

Keywords: Creatinine, nitrazine specificity, sensitivity, urea

Introduction

Premature rupture of membrane (PROM) can be defined as rupture of membranes before onset of
labor, if this occurs before 37 weeks of gestation it is defined as preterm PROM (PPROM) and if at
37 weeks and beyond as PROM or term PROM.[1,2]

The incidence of PROM said to be between 5% and 10% of all deliveries while that of PPROM is
put at 3% and it is the cause of over a third of preterm deliveries.[3,4] It is a significant cause of
maternal and perinatal morbidity and mortality. Risk factors include polyhydramnios, low
socioeconomic status, and low body mass index. There was no increased risk of PROM among
obese patients in Ahmed et al.’s study.[5] Maternal effects include endometritis, salpingitis, and
sepsis.[6] Fetal complications may include fetal lung hypoplasia, umbilical cord compression,
chorioamnionitis, neonatal sepsis, and many others.[7,8]

Patients history may suggest PROM, although this has been shown to be reliable only in 10–50% of
patients.[9,10] This maybe because the significance or perception of the illness by the women may
be inadequate.[11] Observation of fluid leakage from cervix or accumulation in posterior fornix on
speculum examination has been the main method for definite diagnosis of PROM. Other diagnostic
measures used include pH test and microscopic examination of amniotic fluid. Amniotic fluid
typically has a pH of 7.1–7.3, while normal vaginal secretions have a pH of 4.5–6.0. pH test can be
done by use of nitrazine strips which turns dark blue from yellow in fluids with pH above 6.5.[12]
False-positive nitrazine may occur in presence of blood, semen, infections such as bacterial
vaginosis. Diagnostic challenge may arise in absence of demonstrable egress or accumulation of
fluid on speculum examination. Other confirmatory tests include ultrasound guided Instillation of
indigo carmine dye into the uterus and observation of a blue stain on perineal pad or tampon.[2]
This is however invasive and is associated with risks of intrauterine infection, bleeding, iatrogenic
PROM and miscarriage.

Recent studies have focused on biochemical agents with high concentration in amniotic fluid but
little or no quantity in cervicovaginal secretions.[13-16] Examples include insulin like growth factor
binding protein-1, fetal fibronectin Lactate and beta-subunit of human gonadotropin and placental
alpha macroglobulin. Studies on use of metabolomics to predict preterm births are also on going.
[17] However, cost and availability of required tests for these substances are important challenges in
our local settings and areas where tertiary care is not always affordable or available.[18] Possibility
of using urea and creatinine in the diagnosis of PROM was explored because of the relative ease and
affordability of testing of these electrolytes. Moreover, it is expected that accuracy of these test
should be higher. Amniotic fluid creatinine increases from 20 to 32 weeks and urea in third trimester
while they are absent in cervicovaginal fluid.[16] The aim of this study was to compare reliability of
nitrazine paper, urea, and creatinine in the confirmation of PROM.

Methods

Study background

This study was done over a period of 7 months in the Obstetrics and Gynaecology unit of University
of Ilorin Teaching Hospital, Ilorin, Kwara State, Nigeria, while the chemical analysis was carried
out in the chemical pathology department. Ethical approval was obtained from the University of
Ilorin Teaching hospital ethical review committee (Approval number: NHREC/02/05/2010). Written
informed consent was also obtained from all participants.

Study design

This was a comparative study.

Study population

The study population consisted of pregnant women between 28 and 42 weeks gestational ages.
Study group was those who presented with history and demonstrable passage of liquor per vaginum
and speculum examination revealed pooling of fluid in the posterior fornix of the vagina or trickling
of fluid from the cervix with or without valsalva maneuver but with no uterine contractions. The
control group was gestational age matched pregnant women without history or clinical evidence of
drainage of liquor.

Inclusion criteria

Pregnant women with gestational age 28–42 weeks with confirmed demonstrable PROM and
controls with no history nor suspicion of PROM.

Exclusion criteria

History suggestive of vaginitis, history or presentation with antepartum hemorrhage, Urinary

incontinence, polyhydramnios, chorioamnionitis, or coitus within 24 h before presentation were
excluded from the study. Others include history of intake of antibiotics or use of local pessaries as
well as recent vaginal examination or use of vaginal douches.

Sample size determination

The sample size was calculated using the Fischer formula.[19]

2 2
n = z2pq/d2

Where:

n=minimum sample size for the study

z= standard normal deviation usually set at 1.96 at 95% confidence interval.

p= the prevalence of PROM. For this study, a prevalence of 3.9%[20] was used.

q=1-p=1-0.039=0.961.

d= the degree of accuracy desired, usually set at 0.05.

n=(1.96)2 × 0.039 × 0.961/(0.05)2

n=57.59

n=58 was the minimum sample size.

To cater for attrition, 10% of the sample size was added to the initial sample size which was 6.
Hence, sample size was 64 in each arm of the study; 64 in the PROM group (Group I), and 64 in the
control group (Group II). This gave a total sample size of 128.

Sampling method

Purposive non-probability sampling was employed.

Study procedure

Patients were recruited from the antenatal, emergency, labor wards, and antenatal clinics of the
department. A history of the index pregnancy was obtained including booking status and antenatal
care received so far. The records of booked patients were reviewed. Participants and controls
underwent a general physical examination as well as sterile speculum examination and Nitrazine
testing. A positive Nitrazine test was a change in the color of nitrazine paper from yellow to blue.
All these were recorded in the pro forma which was the research instrument.

Sterile vaginal speculum examination, nitrazine testing, and sample collection

After detailed explanation of the study and procedure informed consent was obtained. Sterile
vaginal speculum examination was done for every patient.
In the study group, PROM was confirmed by visualizing pooling of fluid in the posterior fornix or
trickling of fluid from the cervical os on Valsalva maneuver. The liquor was checked for color and
smell, 3 ml was aspirated and put inside a sterile bottle for laboratory analysis. A cotton tip
applicator was then dipped inside the pool of liquor and applied to a nitrazine paper for testing.

In controls, speculum examination was done as described above, the posterior fornix was irrigated
with 5 mls of sterile water and 3 ml was aspirated, put in a sterile bottle and sent for laboratory
analysis. A cotton tipped swab was also applied to the remaining sterile water in the posterior fornix
and put on a nitrazine paper for testing. Samples were taken to the chemical pathology laboratory
for analysis of urea and creatinine levels.

Urea and creatinine estimation

Urea estimation was based on the principle of enzymatic degradation by urease while creatinine was
based on reaction with picric acid. These reactions produced color changes, which were measured
by absorbance spectrophotometer against standard solutions of known concentration of urea and
creatinine.

Data management

Analysis was performed using SPPSS version 20.0 and P < 0.05 was termed significant. The data
were presented in frequency tables, histogram, and curve graphs. Chi-square analysis and odds
ratios with 95% confidence intervals were used to compare proportions and Student’s t-test for
continuous data.

Results

The socio-demographic and obstetric variables of the participants are shown in Table 1. The age
range of participants was 20–44 years. The mean age of the subjects was 30.55 ± 4.92 years while it
was 31.03 ± 5.38 years for the controls.
 Table 1

Socio demographic and obstetric variables of the participant

The majority of the subjects were self-employed while most of the controls were organized public or
private sector employees. The self-employed comprised of the artisans, traders and business women.
Of the subjects 27 (42.2%) were either public or private sector employees (employed) while only 15
(23.4%) were unemployed. On the other hand, 33 controls (51.6%) were either public or private
sector employees while only 9 controls (14.1%) were unemployed.

A total of 104 participants (81.3%) were booked, of these 43 (67.2%) were subjects while 61
(95.3%) controls were booked. The booking status of all the participants was statistically significant,
P < 0.001.

Duration of liquor drainage before presentation is shown in Figure 1. Majority 42 (65.6%) of the
subjects presented within 24 h of PROM, while 5 (7.8%) presented after 72 h of PROM, the least
number 4 (6.3%) presented between 48 and 72 h after onset of drainage of liquor.

Figure 1

Duration of drainage of liquor to presentation in hospital

Participants that presented between 48 and 72 h after onset of drainage of liquor had the highest
median level of vaginal fluid urea of 5.85 mg/dl (IQR 2.18–8.40 mg/dl) while those presenting
between 24 and 48 h had the least median level of 3.90 mg/dl (IQR 0.8–13.2 mg/dl). The median
vaginal fluid creatinine level was highest in those presenting between 24 and 48 h, 1.1 mg/dl (IQR:
0.3–2.25 mg/dl) while it was least in those who presented after more than 72 h, 0.3 mg/dl (IQR 0.1–
2.0 mg/dl). The duration of PROM had no significant effect on the vaginal fluid level of urea (P =
0.829) or creatinine (P = 0.634), this is shown in Table 2.

Table 2

Comparing vaginal fluid urea and creatinine levels with duration of PROM among study group participants

Table 3 reveals that the percentage of positive Nitrazine test was highest within 24 h of PROM, it
then reduced gradually with increasing duration of PROM, while the likelihood of getting a false
negative test in the presence of PROM increased with duration of liquor drainage from 9.5% at >24
h to 25% at 48–72 h PROM. Surprisingly all five cases with PROM duration >72 h were all truly
positive. These changes were however not significant. Neither urea or creatinine levels nor nitrazine
test were significantly affected by duration of PROM.

Table 3

Nitrazine positivity with duration of PROM among study group participants

Cutoff values for urea and creatinine in predicting PROM for this study was determined using the
Receiver Operating Characteristic Curve (ROC), this is shown in Figure 2. The area under the curve
(AUC) of the ROC curve for urea was 0.503 at P = 0.957. The AUC helps to determine the
discriminative power of a test and is a measure of diagnostic accuracy. The cutoff value of urea for
predicting PROM as determined from the ROC was at >12.7 mg/dl with sensitivity of 19.64% and
specificity of 94.44%.
 Figure 2

(a and b) Receiver operating characteristic curves for urea and creatinine. Area under the curve: 0.503; p value:
Area under the curve: 0.537; p value: 0.475; 0.957; 95%Confidence interval:0.413–0.592; 95%Confidence
interval: 0.447–0.626

The AUC of the ROC for creatinine was 0.537 at P = 0.475. The cutoff value for predicting PROM
as determined from the ROC curve was at >0.8 mg/dl with sensitivity of 48.21% and specificity of
65.28%.

Table 4 shows the percentage of positivity and negativity obtained using nitrazine, urea, and
creatinine and the measure of agreement tests. Nitrazine was positive in 56 out of 64 cases in the
study group and negative in the entire control group. This test result was statistically significant with
P = 0.04.

Table 4

Nitrazine test versus vaginal fluid urea and creatinine levels of study participants in evaluating PROM

At the determined cut of value of 12.7 mg/dl, urea was positive in only 12 cases of study group and
negative in 52 cases. Among the controls 61 (95.3%) were truly negative for urea. Creatinine was
positive in 31 (48.4%) of the 64 study group participants and falsely negative in 33 (51.6%) while of
the control group, it was truly negative in 43 (67.2%) and falsely positive in 21 (32.8%). The k value
of 0.875 of nitrazine showed that it was the most reliable test for predicting PROM, followed by
creatinine 0.156 and finally urea at k of 0.141.

Table 5 shows the value of the various tests in detecting and eliminating PROM. Specificity is
defined as the ability of a test to correctly identify people without the disease, while the sensitivity
of the test is the ability of the test to correctly identify people with the disease. Nitrazine test showed
high level of specificity (100%) but a sensitivity of 87.5%. The next more sensitive test was
creatinine test while urea had the lowest sensitivity of the 3.
 Table 5

Evaluation of nitrazine and the cutoff points of urea and creatinine in the diagnosis of PROM

Discussion

Prompt and accurate diagnosis of PROM preterm and at term is important, although management
modalities vary. While conservative approach of management may be employed for preterm, for
term PROM (37–42 weeks) stimulation of labor with oxytocin or prostaglandin may be necessary if
spontaneous onset of labor fails to occurs, to avoid prolonged PROM, chorioamnionitis and other
complications. There may be associated antimicrobial use or misuse.[21,22]

A larger percentage (65%), of PROM cases presented before 24 h of drainage while the remaining
35% presented after 24 h of drainage. This is similar to what was obtained by Sharma et al. and
Khan et al. in which 91.6% and 61.67%, respectively, presented before 24 h of drainage.[6,23]

Vaginal fluid levels of urea and creatinine showed no significant difference with duration of PROM,
with P = 0.829 and 0.634 respectively, this may be due to the relative stability of urea and creatinine
in the vaginal fluid as compared to the proteins in ferning test and fetal cells which may be readily
degraded by microorganisms in the vagina flora which are known to produce proteases.[24] With
nitrazine, there was a definite, although also not statistically significant difference of increased
positivity with shorter duration of PROM. Percentage of false negative increased with longer
durations of PROM. However, in the five cases longer than 72 h, nitrazine test was surprisingly
positive in all, these cases could possibly be those with intermittent leakage of liquor.

The optimal cutoff value for urea for this study was 12.7 mg/dl. This cutoff value is comparable to
13.2 mg/dl obtained by Mohammed et al.[10] and 14.75 mg/dl reported by Hanfy.[25] However, it
is higher than the 6.0 mg/dl reported by Kariman et al.[26] The AUC obtained from the ROC curve
for urea was 50.3% (0.503). This finding is much lower when compared to the AUCs of 84% and
91% reported by Kariman et al.[26] and Hanfy,[25] respectively. The values for sensitivity,
specificity, PPV, NPV, and accuracy of vaginal fluid urea level from this study was 18.8%, 95.3%,
80.0%, 54.0%, and 57.0%, respectively, while other studies were 90%, 79%, 83%, 87.5%, and 85%,
respectively, by Kariman et al.,[26] 96% and 93%, respectively, for sensitivity and specificity of urea
and 97% and 98%, respectively, for creatinine by Malchi et al.,[27] 100% for all the parameters by
Hanfy[25] and Mohammed et al.[10]

The optimal cutoff for creatinine level in vaginal fluid was 0.8 mg/dl. This figure is lower than 1.05
mg/dl obtained by Hanfy[25] but it is higher than 0.05–0.5 mg/dl reported by others.[26,28,29] The
AUC from the ROC curve for creatinine from this study was 53.7% (0.537). This is in contrast to the
AUCs of 99.99% by Kariman et al.[26] and 82% by Hanfy.[25] For creatinine, sensitivity,
specificity, PPV, NPV, and accuracy obtained from the study are 48.2%, 65.3%, 51.9%, 61.8%, and
57.8%, respectively. The values reported for sensitivity, specificity, PPV, NPV, and accuracy for
creatinine in other studies ranged between 90 and 100%[10,19-22] Several studies obtained 90% and
above for sensitivity, specificity PPV and NPV of urea and creatinine.

Sensitivity, specificity, PPV, NPV, and accuracy of nitrazine test for PROM in this study were
87.5%, 100%, 100%, 88.9%, and 93.7%, respectively. This is similar but slightly higher than in the
study by Agbara et al. in which nitrazine had an accuracy of 89.3%.[30] It was also comparable to
the study by Haseli et al. with the values 94.1%, 90.5%, 98.2%, and 73.1%.[31]

Urea and creatinine have not been shown to be reliable in predicting PROM this is because the AUC
obtained from the ROC curves are low 50.3% (0.503) for urea and 53.7% (0.537) for creatinine
suggest unreliability. The sensitivity levels (the ability of the test to correctly identify the disease)
are also low, urea’s sensitivity was 18.8%, and creatinine sensitivity was 48.3%.

These results are quite different from several other studies as mentioned above. Possible reasons for
such low values could be a result of interference from components of amniotic fluid[32,33] the
influence of these interfering substances could be complex interfering either negatively or positively.
Furthermore, ethnicity, differences in selection criteria and laboratory techniques could also
possibly be factors responsible for the differences. Of the two, creatinine showed to be more reliable
than urea in predicting PROM (k values of 0.156 and 0.141 and diagnostic accuracy of 57.8% and
57.0% respectively). This is similar to the finding of Kariman et al.[26] However Hanfy reported
that[25] urea was more reliable than creatinine while Mohammed et al.[2] discovered that they were
both of equal reliability.

Nitrazine, on the other hand, remains a reliable instrument for detection of PROM and especially
with proper selection of patient. In the analysis of the relationship between the three methods,
Nitrazine had a k value of 0.875 which is fairly close to 1 and much higher than was obtained for
urea and creatinine.

Conclusion

According to our findings, nitrazine was more reliable than vaginal fluid levels of urea and
creatinine in the diagnosis of PROM, but, creatinine is comparatively more reliable than urea in the
diagnosis of PROM. Further research efforts should be geared towards perfecting urea and
creatinine estimation with minimal interference and finding other new novel but affordable
biochemical markers with higher sensitivity in the vaginal fluid that will help in diagnosing PROM
in difficult cases.

Authors’ Declaration Statements

Ethics approval and consent to participate

Ethical approval was obtained from the University of Ilorin Teaching hospital ethical review
committee (Approval number: NHREC/02/05/2010). Written informed consent was also obtained
from all participants.

Consent for publication

None.

Availability of data and material

The data used in this study are available and will be provided by the corresponding author on a
reasonable request.

Competing interests

The authors declare no conflict of interest.

Funding Statement

The research was funded by the researchers, there was no external financial support.

Authors’ Contributions

Study conception and design and data collection: Dr. Olaomo, Prof Adesina; Analysis and
interpretation of results: Dr. Olaomo and Olarinoye A.O; manuscript preparation and revision: Dr.
Olarinoye, Ezeoke, Prof Aboyeji; all authors reviewed the results and approved the final version of
the manuscript.

ORCID link of the submitting author: https://orcid.org/0000-0002-9784-0596

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