Pregnant women worldwide are at a risk of developing complications, in particular, hypertensive disorders of pregnancy; the most common being pre-eclampsia (PE). Pre-eclampsia affects 5%–8% of pregnancies worldwide, leading to substantial complications such as foetal and maternal morbidity and mortality (1). In low-and-middle-income countries (LMICs), especially those in sub-Saharan Africa, hypertensive disorders of pregnancy (HDP) account for 16% of maternal deaths (2–4). An alarming 18% of maternal deaths in South Africa (SA) result from HDP (5) The high incidence in SA may be attributed to the high burden of HIV, especially in pregnant women (6). Studies have reported an association between HIV treatment and the pathogenesis of pre-eclampsia in SA (6–8).

The International Society for the Study of Hypertension in Pregnancy (ISSHP) classified HDP into different types: white coat hypertension, masked hypertension, Haemolysis, elevated liver enzymes, low-platelet count (HELLP) syndrome, chronic hypertension (CH), gestational hypertension (GH), pre-eclampsia, pre-eclampsia with severe features and eclampsia (9). Pre-eclampsia and eclampsia are the principal causes of maternal and foetal mortality among the other conditions (5).

The incidence of maternal and foetal deaths due to HDP are evidently high and it is believed that early diagnosis of these conditions may decrease maternal and perinatal mortality. However, the diagnosis and distinction among all HDP classes have been challenging, especially in LMICs. The maternal and foetal outcomes of the different classes of HPD and the level at which the classes affect pregnant women differ. Eclampsia and PE are more aggressive compared to the other categories of HDP.

Pre-eclampsia is commonly defined and diagnosed as elevated blood pressure (>140/90 mmHg) commencing at >20 weeks of pregnancy with subsequent protein (300 mg per 24 h or 1 + dipstick) in the urine (10). According to the latest guidelines from ISSHP, PE may occur in the absence of proteinuria with high blood pressure in combination with disorders such as: renal disease, liver disorders, pulmonary oedema, thrombocytopenia, or fetal growth restriction (11). Pre-eclampsia affects 5%–8% of all pregnancies worldwide (1). Approximately 50,000–60,000 maternal deaths occur yearly due to PE (1). Of all the different classifications of HDP, PE has the highest prevalence in SA in which it complicates 14.8% of all pregnancies (12). The risk features of PE include first pregnancy, obesity, advanced maternal age (>35), a family history of HDP, African ancestry, and paternal influence (if the father of the baby has a history of PE or born from a mother who had PE or fathered a pre-eclamptic pregnancy previously). Currently, PE has no cure, the only known way is iatrogenic termination of pregnancy since the aetiology is thought to arise during placentation (13). Symptoms of PE include, Severe headaches, oedema, especial in the facial area, blurry vision, abdominal pain, difficulty breathing, tension or confusion, and seizures (14).

Since an angiogenic imbalance also characterises PE, angiogenic markers have been suggested as early predictors of PE (15). However, this method of diagnosis is difficult to implement in LMICs due to limited resources (financial, technical) and possibly contradictory results in local studies (11, 16, 17).

Other categories of the HDP that commonly occur are GH (>20 weeks gestation) and CH (<20 weeks gestation) in the absence of proteinuria (18). Similar to CH, GH may progress to PE but the mechanism is yet to be elucidated (19). Gestational hypertension affects 10%–15% of all pregnancies worldwide (20). Also, similar to CH, GH results in adverse maternal and foetal outcomes. The similarities noted among the different HDP classes make it difficult for health workers and clinicians to make a definitive diagnosis. Pre-eclampsia for instance, may easily be misdiagnosed and only discovered when the disorder has rapidly progressed to eclampsia (seizures). It is therefore imperative that these conditions are detected in early pregnancy to eliminate adverse outcomes.

Recently, a way to solve this predicament was found by the introduction of a new way to test for PE and most importantly, to distinguish it from other classes of HPD. The CRDPT was initially introduced by Buhimschi et al. (21). This test was used to detect misfolded proteins in the urine of women suspected to have HDP. Misfolded proteins have a high binding affinity for CRD. Thus the CRDPT was manufactured and its effectiveness tested in women with HDP (21). In the study by Buhimschi et al., prominent levels of congophillia were discovered in the urine of women with PE, indicating a high level of misfolded proteins compared to controls and triggered an interest in the use of the CRDPT to detect PE or superimposed PE in CH.

Several ways to diagnose PE have been proposed previously (21). However, most tests were time-consuming, expensive, and some reports showed contradictory results (22). Besides elevated BP, the commonly practiced way of diagnosing PE is through detecting proteinuria. Buhimschi et al. reported that proteinuria indicated that PE was a protein configuration disorder (21). Rood et al. reported using the CRDPT to distinguish PE from the other HDP classes (22). The advantages of CRDPT are that they are quick and easy to perform, accurate, non-invasive and cost-effective (22). This theory was tested in a group of pregnant women with HDP. Congophilia was found in 12% of pregnant women with a tentative diagnosis, 58% of patients admitted for PE confirmed positive on the CRDPT and only 10% did not have congophilia resulting in a change in diagnosis (10). These results demonstrate that the CRDPT is effective and has the potential to be used as an accurate and early predictor of PE. Furthermore, it was reported that this test can be used to rule out PE in women with other categories of HPD (10).

In India, a group of researchers conducted a study using the Rood et al. protocol (22). The study population consisted of women with early-and late-onset PE. These sub-divisions of PE are based on gestational age. Early-onset PE occurs at ≤34 weeks of gestation while late-onset occurs at ≥34 weeks. Studies have reported that early-onset PE results in severe features of PE compared to late-onset PE (23, 24). Nonetheless, the CRDPT indicated that misfolded proteins were higher in late-onset PE compared to early-onset PE and the normotensive group (25). These findings designate the variance in pathology of the two classes of PE (26). In contradiction to results reported thus far, Dobert et al. reported that the CRDPT results were “poor” in women between 35 and 37 weeks of gestation (27).

Most studies report positive results on the effectiveness of CRDPT. However, very little has been done in this area of research since no studies have been published in Africa where HDP increases maternal and foetal morbidity and mortality. Table 1 summarizes all prospective studies in different countries that used the CRDPT to diagnose HDP. This table includes the different authors, the year of publication, country, the study population, ethnicity, study design, results, and conclusions. The purpose of this table is to indicate the number of studies that used the test globally and the results reported. In addition, the systematic review aims to conduct a meta-analysis on the studies listed in Table 1 to investigate whether the test is effective. This summary will also reveal areas that require further research with respect to the CRDPT.

To evaluate the accuracy of the Congo Red Dot Paper Test in diagnosing hypertensive disorders of pregnancy.

recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-analysis for Protocols (PRISMA-DTA) guidelines 2020 (27). The results were reported based on the PRISMA 2015 (28), statement and article screening and the selection process was demonstrated through a PRISMA-P flow diagram. Furthermore, the current protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42021283679.

A table was used to collect background information and process the data items from each selected article. To confirm that all the relevant information regarding significant aspects of the study were gathered, a data charting form was created, piloted, and updated.

A study classified as high risk of bias indicates low confidence that the results reflect the true treatment effect. The quality and risk of bias of selected articles was performed by both reviewers (WNP and OPK) using Cochrane risk of bias tool (28). This tool takes into consideration the following, random sequence generation, allocation concealment blinding of participants and personnel, blinding of results assessment, incomplete outcome data, and selective reporting.

The quality and scientific evidence of the selected articles was determined using the Grading of recommendations assessment, development and evaluation (GRADE) tool to assess risk of bias (29), inaccuracy, variability and indirectness, and placed into one of four categories, ranging from very low (where the true effect is probably markedly different from the estimated effect) to very high (that which was observed is most likely to be true). Randomised controlled trials without limitations therefore, provided the most relevant evidence, with observational studies falling into the lowest category, given the potential for confounding. The strength of the recommendations identified from these articles were evaluated according to the quality of evidence presented. The following authors (WNP and OPK) independently evaluated the quality of the articles using the data extraction tool, with consensus on disagreement being achieved with the assistance of another author (JM).

The meta-analysis on the effectiveness of CRDPT is displayed in Figure 2. Only five studies were included in our meta-analysis.

Our meta-analysis revealed a significant difference between the HDP and normotensive group. This is noted by a significantly decreased in the effectiveness of CRDPT in detecting HDP as compared to normotensive group [(Risk Ratio (RR) = 6.32 (2.17, 18.43) p < 0.00001]. However, the included studies had a high nature of heterogeneity [I² = 98%, p < 0.00001] partially due to different study designs included in the analysis and different regions where studies were conducted given that none of these studies were conducted in African countries where HDP is prominent. More importantly, sensitivity analysis was conducted using one study exclusion approach, and the result showed a minimal change in the level of heterogeneity [[RR = 4.18 (1.67, 10.46) p < 0.00001] [I² = 96%, p < 0.00001] (Figure 3).

In this systematic review and meta-analysis, we examined the effectiveness of CRDPT in the diagnosis of HDP. Our findings showed that CRDPT is not effective in the diagnosis of HDP (Figure 2). Our findings are in support of Dorbert et al., who reported that the performance of CRDPT in diagnosing PE is poor (27). Furthermore, a study by McCarthy et al. that evaluated urinary congophilia levels in women with PE, chronic kidney disease without PE, and lupus nephritis in non-pregnant women, confirmed that the CRDPT cannot differentiate between these conditions, suggesting that CRDPT is not a reliable tool in the prediction of PE or HDP (26).

Even though there are studies which found CRDPT to be effective in the detection of PE (21, 22, 30–32), most of these studies had sampling bias (21, 22, 31, 32), whereby higher sampling probability in the PE group was noted compared to the normotensive group (21) or higher sampling probability in the normotensive group compared to PE group (22, 31, 32) (Figures 5, 6). In addition, most of these studies included a mixture of ancestry, mostly Caucasian, and Asian, and a minority of the groups were women of African ancestry (Table 2). In addition, the included studies had a high nature of heterogeneity (I² = 97%, p < 0.00001) because of the different countries that explored the CRDPT. However, none of these were African countries where HDP is prominent. Furthermore, most of the included studies were cohort studies.

Disorders of pregnancy due to hypertension are one of the causes of maternal morbidity and mortality in LMICs (33). The prevalence of these disorders is higher in women of African ancestry than their counterparts (34). Women of African ancestry in LMICs, mainly in the African continent experience HDP more often than women of a different race and geographic location (high-income-countries). The screening and the detection of hypertension in pregnancy has been difficult in these countries for various reasons like poor infrastructure and equipment. Pre-eclampsia for instance, affects the placenta and the mother systemically leads to more than just elevated blood pressure and protein in the urine, sometimes presenting with hypertension in fusion with by other disorders involving the liver the brain, and thrombocytopenia, resulting in pernicious complications for the mother and her unborn baby (11) such as placenta hypoxia, placental ischemia, and foetal growth restriction.

Misfolded proteins (Amyloid) arise from hypoxia, placental ischemia and pro-inflammatory cytokines leading to endoplasmic reticulum stress in the placenta during pregnancy. These proteins have a high affinity for congophilia and can be detected in the placenta and in circulation in women with PE (35, 36), hence the establishment of the CRDPT to detect these proteins in PE (17). Different tests were used to confirm that the presence of misfolded proteins may be used as a predictor of PE. A11 and polyclonal aAPF antibodies were used to detect amyloid-protofibrils and prefibrillar oligomers in urine specimens of women with PE. Interestingly, these fibrils were not found in women with CH and women without PE (17). In addition, Buhimschi et al. used the CRDPT in women > 34 weeks of gestation to detect misfolded proteins to diagnose PE and as an indication for delivery. The use of the CRDPT proved significant in the study (17). However, another study conducted in China reported a statistically higher detection rate in early-onset (28 weeks to <34 weeks) compared to late-onset (ref 38). This included a cohort of PE women (n = 140) and had no control group. Of these PE women, the CRDPT detected misfolded proteins in 83% of severe PE cases, 86% of preterm births, 49% of mild PE cases and 50% at term (37). The findings of this study correspond to that of Dorbet et al. who reported that the CRDPT was a poor predictor of PE in women >35 weeks (27).

Rodriguez et al. used the CRDPT to determine proteinuria in 50 Brazilian women with PE (38). The findings indicated that the dye had high beta protein selectivity and was directly related to proteinuria. The authors concluded that the CRDPT is effective in diagnosing PE, simple, fast and affordable to use in LMICs (38) Furthermore, Sammar et al. used the CRDPT to prediction PE in 81 women from Israel and 642 women from the UK with a gestational age of 26–41 weeks (31). The test proved effective with a higher odd ratio (OR) in obese women and women with a high mean arterial pressure (MAP) and lower in women of African ancestry and a previous history of PE. An adjusted OR of all the parameters was calculated as 13.92 (p < 0.001) to predict PE. The study concluded that the CRDPT can be used to predict PE and has been reported as accurate in obese, women, women with high MAP, African ancestry and a previous history of PE (31). These findings contradict our findings because Sammar et al. had a larger population group of women with African ancestry and had no control group (healthy pregnant women) to compare with (31).

Sergeeva et al. conducted a study in Russian women with severe PE (28–36 weeks of gestation). The CRDPT was able to detect misfolded proteins in these women and concluded that the test may be used as a predictor of PE (39). However, this study did not investigate the effectiveness of the CRDPT in pregnant healthy women compared to women with other HDP.

In the current study, Table 2 includes all the countries that used the test and none of the studies were conducted in Africa. Notably, research conducted on the efficacy of the CRDPT globally are limited as the articles found were only twenty and were further reduced to suit the criteria of the research question. Interestingly, as far as we are aware, there are no studies conducted in Africa on the effectiveness of CRDPT in Africa. These findings require confirmation in LMICs where most of the population is of African Ancestry like South Africa.

South Africa is a LMIC and yet faces a high incidence of HDP due to several factors including poor infrastructure, poor health care services, a high prevalence of HIV and delayed hospital care for pregnant women in rural settlements (40). The CRDPT would be of critical importance in SA due to the urgent need to screen, diagnose, and treat HDP early in pregnancy.

The current study has few limitations, there is few studies published that evaluated the accuracy of CRD test in HDP, the included studies had different study designs, sample size, risk of bias and were conducted from different countries. Noteworthy, form of bias that may arise due to poor study methodology. We also noted a very significant rate of high heterogeneity among the included studies, which may be due to the patient characteristics, and samples. However, we evaluated and explored this by performing subgroup and sensitivity analyses, which showed minor significant changes from the initial analysis. Sensitivities and specificities were performed, but these statistics depend on the populations studied, the reference tests used. Quality of studies was analysed using the Cochrane risk of bias.

This study concludes that the CRDPT proved to be ineffective in the studies presented in the forest plot (Figure 2) due to bias noted in five of the six studies. Furthermore, the studies had larger normotensive groups compared to HDP. Hypertensive disorders of pregnancy are more common in women of African Ancestry. However, the ethnic groups included in this study had a minority of women of African ancestry. Therefore, more research in African countries with a higher prevalence of HDP is required to confirm the effectiveness of the CRDPT.