As shown in table VII, at the cut-off level of 12.7mg/dl, the sensitivity and specificity of vaginal fluid level of urea were 19.6% and 94.4% respectively. It has a higher positive predictive value than negative predictive value (73.3% versus 60.2%). It has false positive rate of 5.6% and false negative rate of 80.4%. Its diagnostic accuracy was 61.7%. The high false negative rate of 80.4% and false positive rate of 5.6% with a low diagnostic accuracy of 61.7% showed that vaginal fluid level of urea above 12.7mg/dl is not of high diagnostic accuracy for PROM
clinically.
Also as seen in table VII, at the cut off of 0.8mg/dl, the sensitivity and specificity of vaginal fluid level of creatinine were 48.2% and 65.3% respectively. It has higher negative predictive value than positive predictive value (61.8% versus 51.9%). It has false positive rate of 34.7% and false negative rate of 51.8%. Its diagnostic accuracy was 57.8%. The high false negative rate of 51.8%
40
and false positive rate of 34.7% with a low diagnostic accuracy of 57.8% showed that vaginal fluid level of creatinine above 0.8mg/dl is not of high diagnostic accuracy for PROM clinically.
Table I: Socio demographic and obstetric variables of the participants
Variables Group I Group II Total χ2/t p value
n (%) n (%) n (%)
Age group(years)
< 25 9 (14.1) 8 (12.5) 17 (13.3) 1.586 0.811
26 – 30 27 (42.2) 25 (39.1) 52 (40.6)
31 – 35 17 (26.6) 16 (25.0) 33 (25.8)
36 – 40 9 (14.1) 14 (21.9) 23 (18.0)
> 40 2 (3.1) 1 (1.6) 3 (2.3)
Mean ± SD Educational status
30.55 ± 4.92 31.03 ± 5.378 -0.532t 0.596
None 4 (6.3) 1 (1.6) 5 (3.9) 2.993Y 0.393
Primary 1 (1.6) 5 (7.8) 6 (4.7)
Secondary 16 (25.0) 11 (17.2) 27 (21.1)
Tertiary Ethnicity
43 (67.2) 47 (73.4) 90 (70.3)
Yoruba 59 (92.2) 56 (87.5) 115 (89.5) 1.660Y 0.646
Hausa/ Fulani 2 (3.1) 0 (0.0) 2 (1.6)
Igbo 1 (1.6) 2 (3.1) 3 (2.3)
Others Occupation
2 (3.1) 6 (9.4) 8 (6.3)
Employed 22 (34.4) 33 (51.6) 55 (43.0) 4.210 0.122
Self employed 27 (42.2) 22 (34.4) 49 (38.3)
Unemployed
Gestational age (weeks)
15 (23.4) 9 (14.1) 24 (18.8)
28 – 32 11 (17.2) 17 (26.6) 28 (21.9) 2.484 0.289
33 – 36 20 (31.3) 22 (34.4) 42 (32.8)
37 – 42 33 (51.6) 25 (39.1) 58 (45.3)
Mean ± SD Gravidity
35.84 ± 3.58 34.75 ± 3.55 1.737t 0.085
1 21 (32.8) 12 (18.8) 33 (25.8) 3.664 0.160
2 – 4 29 (45.3) 38 (59.4) 67 (52.3)
≥ 5
Booking status
14 (21.9) 14 (21.9) 28 (21.9)
Booked 43 (67.2) 61 (95.3) 104 (81.3) 16.615 < 0.001*
Unbooked 21 (32.8) 3 (4.7) 24 (18.8)
χ2: Chi square; Y: Yates corrected Chi square; t: Independent Samples T test
42
Figure I: Duration of drainage of liquor in hours
Table II: Comparing vaginal fluid urea and creatinine based on the duration of PROM and nitrazine test results.
Urea Creatinine
Variable Median (IQR) Median (IQR) Duration of
(hours)
PROM
0 – 24 5.40 (2.25-10.38) 0.80 (0.2-2.00) >24 – 48 3.90 (0.80-13.20) 1.10 (0.30-2.25)
0 5 10 15 20 25 30 35 40 45
24 -
0 >24 -48 >48 -72 > 72
) 42 (65.6%
) 13 (20.3%
(6.3%
4 ) 5(7.8%)
- 0 24
>24 - 48 72
>48 - 72
>
Duration of drainage of liquor (hours)
> 48 – 72 5.85 (2.18-8.40) 0.60 (0.53-0.68) > 72 5.50 (0.90-6.90) 0.30 (0.10-2.00) K/ F (p
value)
Nitrazine test
0.885K (0.829)
1.712K (0.634)
Positive 5.40 (1.80 – 8.83) 0.80 (0.23–1.45) Negative 4.50 (1.60 – 12.68) 1.30 (0.60–4.38) U/t (p value) 209.500U (0.768) 183.000U (0.402)
IQR: Inter-quartile range; K: Kruskal Wallis test; F; U: Mann Whitney U test; t:
Independent Samples
Table III: Comparing vaginal fluid urea and creatinine results based on gestational age and gravidity of study participants.
Group I Group II U p value
Variable Median (IQR) Median (IQR)
Urea Gestational
(completed weeks) age
28 – 32 3.90 (3.60 – 7.30) 4.80 (1.85 – 6.30) 89.000 0.832 33 – 36 2.75 (1.00 – 6.30) 6.30 (2.93 – 9.50) 152.500 0.089
37 – 42 Gravidity
6.40 (2.70 – 14.65) 5.30 (2.10 – 10.50) 353.000 0.350
44
1 6.30 (1.30-13.20) 5.05 (2.00 – 10.33) 125.000 0.985
2 – 4 5.50 (3.35-9.10) 5.09 (2.33 – 7.95) 507.000 0.577
≥ 5 3.00 (1.48-6.85)
Creatinine
5.05 (2.10 – 9.50) 76.500 0.323
Gestational
(completed weeks) age
28 – 32 0.70 (0.00 – 3.00) 0.30 (0.05 – 2.10) 85.000 0.686 33 – 36 1.00 (0.53 – 4.25) 0.75 (0.00 – 1.48) 174.000 0.242
37 – 42 Gravidity
0.60 (0.25 – 1.85) 0.60 (0.25 – 1.50) 394.000 0.771
1 0.70 (0.30-1.60) 0.50 (0.02 – 2.98) 119.000 0.792
2 – 4 1.00 (0.25-2.70) 0.65 (0.15 – 1.00) 445.000 0.177
≥ 5 0.75 (0.45-1.15) 0.60 (0.30 – 1.73) 90.000 0.712
IQR: Inter-quartile range; U: Mann Whitney U test
Table IV: Comparing vaginal fluid urea and creatinine in PROM and control groups
Variable PROM Control U p value
n = 64 n = 64
Urea(mg/dl)
Median
(Interquartile range) Creatinine(mg/dl)
5.35
(1.80 – 9.93)
5.30
(2.10 – 9.50)
2035.00
0.951
Median
(Interquartile range)
0.80
(0.30 – 1.93)
0.70
(0.20 – 1.83)
1898.500 0.474
U: Mann-Whitney U test
46
Area under the curve: 0.503; p value: 0.957; 95% Confidence interval: 0.413 – 0.592 Figure II: ROC Curve of Vaginal fluid Urea in predicting PROM
Area under the curve: 0.537; p value: 0.475; 95% Confidence interval: 0.447 – 0.626
Figure III: ROC Curve of Vaginal fluid Creatinine in predicting PROM Table V: Sensitivity and Specificity of Vaginal fluid Urea and creatinine in predicting PROM (Criterion values and coordinates of the ROC curve)
Criterion(mg/dl) Sensitivity(%) 95% CI Specificity(%) 95% CI
UREA
>10.9 19.64 10.2 - 32.4 83.33 72.7 - 91.1
>11.2 19.64 10.2 - 32.4 88.89 79.3 - 95.1
>11.6 19.64 10.2 - 32.4 91.67 82.7 - 96.9
>12.6 19.64 10.2 - 32.4 93.06 84.5 - 97.7
>12.7 19.64 10.2 - 32.4 94.44 86.4 - 98.5
>13.7 17.86 8.9 - 30.4 95.83 88.3 - 99.1
>14.4 16.07 7.6 - 28.3 95.83 88.3 - 99.1
>15.6 14.29 6.4 - 26.2 95.83 88.3 - 99.1
>17.6
12.50
CREATININE
5.2 - 24.1
97.22
90.3 - 99.7
>0.4 67.86 54.0 - 79.7 41.67 30.2 - 53.9
>0.5 66.07 52.2 - 78.2 44.44 32.7 - 56.6
>0.6 57.14 43.2 - 70.3 50.00 38.0 - 62.0
48
>0.7 51.79 38.0 - 65.3 58.33 46.1 - 69.8
>0.8 48.21 34.7 - 62.0 65.28 53.1 - 76.1
>0.9 46.43 33.0 - 60.3 65.28 53.1 - 76.1
>1 39.29 26.5 - 53.2 72.22 60.4 - 82.1
>1.1 28.57 17.3 - 42.2 72.22 60.4 - 82.1
>1.2 26.79 15.8 - 40.3 72.22 60.4 - 82.1
Youden index (J): 0.1409; Associated criterion: >12.7mg/dl (Urea) ; Youden index (J):
0.1349; Associated criterion: > 0.80mg/dl (Creatinine)
Table VI: Nitrazine test versus vaginal fluid urea and creatinine levels of study participants in evaluating PROM
Nitrazine test Κ p
value
OR (95% CI)
Variable
Positive n (%)
Negative n (%)
Total n (%)
Urea test (>12.7mg/dl)
Positive 11 (19.6) 4 (5.6) 15 (11.7) 0.153 0.014 4.156 (1.246 -13.863) Negative
Creatinine (>0.8mg/dl)
45 (80.4)
68 (94.4)
113(88.3)
Positive 27 (48.2) 25 (34.7) 52 (40.6) 0.136 0.123 1.750 (0.857 – 3.575) Negative 29 (51.8) 47 (65.3) 76 (59.4)
κ:Kappa; OR: Odds Ratio; 95% CI: 95% Confidence Interval
Table VII: Evaluation of the cut-off points of Urea and Creatinine in the diagnosis of PROM
Evaluation Urea test (>12.7mg/dl) Creatinine (> 0.8mg/dl)
Sensitivity 19.6% 48.2%
Specificity 94.4% 65.3%
Positive Predictive Value 73.3% 51.9%
Negative Predictive Value 60.2% 61.8%
False Positive rate 5.6% 34.7%
False Negative rate 80.4% 51.8%
Accuracy 61.7% 57.8%
50
CHAPTER SIX DISCUSSION
The diagnosis of PROM could be challenging; posing a serious dilemma especially in equivocal cases.2 When the diagnosis of PROM is uncertain, it could result in unnecessary clinical interventions with undesirable consequences.18 PROM predisposes mothers, fetuses and neonates to increased incidence of polybacterial infections,78 problems of prematurity, sepsis and even mortalities.3, 6, 9, 79, 80 These problems are even more pronounced before term. Presently, it is difficult to effectively prevent PROM.80 Therefore, establishing a diagnostic method of greater accuracy than preexisting methods is of utmost obstetric importance. Vaginal fluid urea and creatinine measurement is one of the advocated promising biochemical marker method for diagnosing PROM.
This study has shown the range of values of vaginal fluid levels of urea and creatinine in women with and without clinical features of PROM. The median value for vaginal fluid urea level for pregnant women with clinical features of PROM (GROUP I) in this study was 5.35mg/dl (IQR:
1.80-9.93). This is lower than the value of 13.77mg/dl reported by Kariman et al65 in Iran. In Egypt, Mohammed et al14 reported a value of 37.8mg/dl which is comparable to a value of 33.79mg/dl reported by Hanfy66 also in Egypt. This difference in value might be partly due to the difference in selection criteria for the participants (GROUP I). In their studies, all the participants in group I must have a positive nitrazine test apart from the other clinical features of PROM. This narrowed the selection of their participants to those who satisfied the clinical and nitrazine test criteria for the diagnosis of PROM. However, analyzing the results of participants who are nitrazine positive showed that this might not be the main reason for this observed difference. The difference in the values of vaginal fluid urea obtained in this study might also be due to racial differences between the participants in this study and the participants used by the earlier researchers. A literature search did not yield any similar local study using the same set of participants as in this study. In Brazil, Oliveira et al20 found that race has no effect on the amniotic fluid urea and creatinine level.
However, in Brazil, the intense biological miscegenation81 (racial interbreeding) over decades
might have weakened the effect of race on biological parameters among white, mixed race and black people.81 Therefore, racial differences might still account for this observed difference.
The median value of urea for women in group II (controls) in this study was 5.30mg/dl (IQR 2.10-9.50mg/dl). This is comparable to the value of 5.13mg/dl reported by Kariman65 et al in Iran but higher than the values of 1.1mg/dl14 and 1.86mg/dl66 obtained in Egypt. The higher value of urea obtained in this study may be due to the presence of substances that cause positive interference with urea assay in higher concentration or quantity in the normal cervicovaginal discharge of this patient population. Normal cervicovaginal discharge can be contaminated by urea produced by vulva sweat glands72 and endogenous ammonia production73 and these if present will lead to positive interference. Also, there are no documented values of urea level in cervicovaginal secretions of pregnant women in Ilorin. This is perhaps the first study to evaluate this in our setting.
In this study, the median value of creatinine in the vaginal fluid in PROM group (group I) was 0.8mg/dl (IQR 0.3-1.93mg/dl). This is comparable to the median value of 0.95mg/dl obtained by Li et al58 but is higher than the finding of 0.4mg/dl reported by Sekhavat et al.64 However, other researchers reported higher levels of 1.23-1.86mg/dl.14, 18, 65, 66 The lower value of creatinine in the participants with PROM may also be partly due to the difference in selection criteria between this study and theirs as earlier outlined. It is also probably due to the effects of interfering substances which may reduce (negative interference) or increase (positive interference) the values of creatinine measured. Amniotic fluid contains glucose, bilirubin, albumin and other proteins.82 These components of amniotic fluid interfere with creatinine assay. Glucose, albumin and other proteins showed positive interference with creatinine measurement while bilirubin showed negative interference.73, 83 However, in some instances the influence of these interfering substances could be complexinterfering either negatively or positively.83 Also, the normal cervicovaginal secretion is rich in different types of proteins.73 Therefore, the lower values of creatinine obtained from this study probably could have been from the lowering effect of the negatively interfering bilirubin or a complex interplay between the negatively and positively interfering substances or both. Also, as earlier explained ethnicity could also play a role in explaining above finding.
Therefore, further research is indicated to determine the amniotic fluid and normal cervicovaginal fluid constituents in our patient populations. These facts may also explain the findings in the controls. The median value of creatinine in the vaginal fluid in the control group (group II) was 0.7mg/dl (IQR 0.2-1.83mg/dl). The reported range of values of creatinine in the vaginal fluid of controls was 0.05-0.27mg/dl.14, 18, 58, 64- 66
The median level of urea among group I participants was 5.35mg/dl (IQR:1.8-9.93mg/dl) while it was 5.3mg/dl (IQR:2.10-9.50mg/dl) among the controls. The median level for creatinine was 0.8mg/dl (IQR: 0.3-1.93) among group I participants while it was 0.7mg/dl (IQR: 0.2-1.83 mg/dl) among the controls. There was no statistically significant difference in the vaginal fluid levels of urea and creatinine in both groups; p values for urea and creatinine were 0.951 and
0.474 respectively.
52
It has been documented that there is usually a significant rise in error rate with increasing duration between membrane rupture and sampling for other methods of PROM diagnosis like amniotic fluid crystallization (ferning test) and fetal cell staining.36, 40 However, findings from this study showed that the duration from rupture of membrane to sampling has no statistically significant effect on the vaginal fluid levels of urea and creatinine; p values of 0.829 and 0.634 respectively. This may be probably due to the relative stability of urea and creatinine over time in the vaginal fluid as compared to the proteins (ferning test) and fetal cells (fetal cell staining) which may be readily degraded by microorganisms (microflora) in the vagina microenvironment. Vagina microflora are known to produce proteases84 which can be involved in cellular and protein degradation.
This study also showed that the gestational age of both groups I and group II has no statistically significant effect on the vaginal fluid levels of urea and creatinine in both groups. The p values for urea and creatinine in the PROM group and controls in relation to various gestational ages were greater than 0.05. This probably means that at 28weeks and beyond the vaginal fluid level of urea and creatinine is not dependent on the gestational age. This might indicate a reasonable degree of fetal renal maturity.
The relationship between gravidity and the vaginal fluid levels of urea and creatinine has not been specifically documented in the literature. In this study, gravidity has no clinically significant effect on the vaginal fluid levels of urea and creatinine in both group I and group II (controls) participants. The p values for urea and creatinine in the PROM group and controls in relation to gravidity were greater than 0.05. This is not surprising as in every pregnancy, amniotic fluid production is dependent primarily on the renal status of each fetus and not on the total number of pregnancies. Therefore, gravidity is not expected to have any effect on the amniotic fluid contents.
In this study, the urea level of vaginal fluid in nitrazine positive participants is higher than that of nitrazine negative participants. This may be because the nitrazine positive participants fulfil more criteria for diagnosing PROM than the nitrazine negative participants and are more likely to be true positive cases of PROM than the nitrazine negative participants. Also, urea estimation is less prone to the effects of interfering substances compared to creatinine.73 However, this finding is not statistically significant; p value: 0.768. On the other hand, the creatinine level of vaginal fluid in nitrazine positive participants is lower than those of nitrazine negative participants. This finding is also not statistically significant; p value:0.402. The possible complex nature of interferences with creatininine estimation may account for this. Possibly, urea is a comparatively better agent than creatinine in predicting PROM. This finding is not yet conclusive and needs further evaluation.
The optimal cut-off value for urea in the diagnosis of PROM from this study using ROC curve is at 12.7mg/dl. This is comparable to 13.2mg/dl obtained by Mohammed et al14 and 14.75mg/dl reported by Hanfy.66 However, it is higher than the 6.0mg/dl reported by Kariman et al.65 The Area under the Curve (AUC) obtained from the ROC curve for urea was 50.3% (0.503). This is low and indicates that urea may not be reliable in predicting PROM. This finding is in contrast to the AUCs of 84% and 91% reported by Kariman et al65and Hanfy65 respectively. In this study, at the cut-off of 12.7mg/dl of urea, the sensitivity and specificity were 19.6% and 94.4% respectively while
positive predictive value (PPV), negative predictive value (NPV) and accuracy obtained were 73.3%, 60.2%, and 61.7% respectively. The values of sensitivity, specificity, PPV, NPV and accuracy of vaginal fluid urea level from other studies were 90%, 79%, 83%, 87.5% and 85%
respectively by Kariman et al,65 100% for all the parameters by Hanfy65 and Mohammed et al.14 The probable reasons for these findings are as stated earlier.
The optimal cut-off for creatinine level in vaginal fluid was 0.8mg/dl. This was obtained from the ROC curve. This figure is lower than 1.05mg/dl obtained by Hanfy65 but it is higher than 0.05-0.5mg/dl reported by others.14, 18, 58, 64, 65, 68 The AUC from the ROC curve for creatinine from this study was 53.7% (0.537). This finding is in contrast to the AUCs of 99.99% by Kariman et al65 and 82% by Hanfy.65 The optimal creatinine cut-off of 0.8mg/dl obtained from this study gives sensitivity, specificity, PPV, NPV and accuracy of 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 90-100% 14, 18, 58, 64, 65 The lower figures obtained in this study may be due to the earlier factors mentioned.
The analysis of the relationship between nitrazine test and urea test using Kappa test at the optimum cut off point of 12.7mg/dl showed that the nitrazine test is more reliable than urea test; κ value of 0.153 which is far less than 1. Also, Kappa analysis also showed that nitrazine is more reliable than creatinine level of vagina fluid in diagnosing PROM; κ value of 0.136 which is also far less than 1. These two κ values for urea and creatinine; 0.153 and 0.136 respectively are far less than 1 and showed that they are not reliable in diagnosing PROM. Therefore, the null hypothesis is accepted.
This study showed that of the two, urea may be more reliable than creatinine in predicting PROM (κ values of 0.153 and 0.136, specificity of 94.44% and 65.28% and diagnostic accuracy of 61.7%
and 57.8% respectively). This is similar to the finding of Hanfy.65 However, Kariman et al65 reported that creatinine is more reliable than urea while Mohammed et al14 discovered that they were both of equal reliability.
54
CONCLUSION.
The median value for the vaginal fluid level of urea in pregnant women with PROM from this study was 5.35mg/dl while it was 5.30mg/dl in the normal pregnant women (controls). Also, the median value for the vaginal fluid level of creatinine in pregnant women with PROM from this study was 0.80mg/dl while it was 0.70mg/dl in the controls.
When vaginal fluid levels of urea and creatinine were compared in pregnant women with PROM with their controls, there was no statistical difference. Similarly, the duration of PROM, gravidity and gestational age have no significant effect on the values of vaginal fluid levels of urea and creatinine.
Urea is comparatively more reliable than creatinine in the diagnosis of PROM. However, nitrazine is more reliable than vaginal fluid levels of urea and creatinine in the diagnosis of PROM.
RECOMMENDATIONS.
1. Nitrazine strips should be made readily available in UITH and other centres as an immediate bedside test for the diagnosis of PROM.
2. Efforts should be made to determine the constituents of normal cervicovaginal fluid in our pregnant patient populations. This is particularly important in order to determine the preponderance of the constituents that can cause positive and negative interference thus leading to falsely high and low urea and creatinine levels.
3. Also, the constituents of amniotic fluid should be determined in our setting. This will also help in determining components that interfere with the measurement of urea and creatinine in vaginal fluids in PROM.
4. This study should be carried out in multiple centres and with larger and diversified patient populations in order to further assess the reliability of nitrazine versus urea and creatinine in confirming PROM.
56
5. Further research efforts should be geared towards finding other biochemical markers in the vaginal fluid that will help in diagnosing PROM and which will be more reliable and cheaper than nitrazine.
LIMITATIONS.
1.The group of pregnant women who may present with history of drainage of fluid per vaginam but without pooling of fluid in the posterior fornix or trickling of fluid from the cervical os on Valsalva manouvre (suspected/unconfirmed PROM) were excluded from this study. This group usually presents the obstetricians with the greatest dilemma in management. The pattern of vaginal fluid level of urea and creatinine in them would also have been informative.
2. This study was done in only one centre. A multicentre study with a larger number of participants may be more representative.
3. The nitrazine negative participants were small compared to the nitrazine positive participants and all the controls were nitrazine negative.
4. The participants were predominantly of one ethnicity and most resided in the same geographical area. This deprived the study of diversity of patient selection.
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