BVBlue as a diagnostic instrument for the diagnosis of bacterial vaginosis: a systematic review

Abstract

Background

Bacterial vaginosis (BV) refers to an imbalance in normal vaginal flora, affecting approximately 50–60% of individuals engaging in high-risk sexual behaviors. BV diagnosis traditionally relies on Amsel’s criteria or Nugent’s scoring, but these methods are sluggishly effective, leading to delayed treatment. BVBlue, a swift point-of-care test, holds promise as a potential diagnostic tool for BV. This systematic review aims to evaluate the accuracy of the BVBlue criteria compared with Nugent’s scoring in diagnosing BV.

Methods

We conducted a systematic search using designated terms in PubMed, ProQuest, and Scopus to identify all studies reporting BVBlue in diagnosing BV. We subsequently screened and critically evaluated the journals via the QUADAS-2 Quality Assessment Tool. This systematic review has been registered in PROSPERO (no. CRD42024545022).

Result

Out of 19 studies identified in our research, five were ultimately included. The quality assessment results revealed that all the included studies had good quality and low risk of bias. BVBlue has a sensitivity ranging from 38% to 91.7% and a specificity ranging from 91 to 100% in diagnosing BV. Four studies endorsed the BVBlue scale because of its high sensitivity, specificity, and rapidity in diagnosing BV. However, one study advised against its use in certain populations with mixed vaginal infections because of its inability to detect variation in composition and diversity of vaginal bacterial flora, that may be sialidase-negative.

Conclusion

Our systematic review revealed that BVBlue is a promising diagnostic tool for BV because of its accuracy and speed. Nevertheless, additional research is essential, particularly in elucidating the bacterial epidemiology of BV in certain countries, to validate its diagnostic credibility thoroughly.

Background

Bacterial vaginosis (BV) is a condition caused by the overgrowth of normal vaginal flora [1]. According to the four Amsel criteria, it is a common vaginal condition with the following characteristics: 1) thin, gray/white discharge; 2) malodorous “fishy” discharge upon the addition of 10% potassium hydroxide; 3) high vaginal pH (> 4.5); and 4) identification of vaginal epithelial cells heavily coated with bacteria (i.e., “clue cells”) [2]. Approximately 20–30% of women with vaginal discharge have BV, and 50–60% of high-risk sexual behavior populations are at high risk [1]. BV has detrimental effects on various aspects of women’s quality of life. A survey conducted with women experiencing bacterial vaginosis episodes for the past 4 years revealed a significant negative impact on their sexual health, physical health, mental health and social interactions with other people [3, 4]. BV is also an important cause of obstetric and gynecological adverse sequelae. In the field of obstetrics, bacterial vaginosis (BV) has been linked to a greater likelihood of preterm delivery, first-trimester miscarriage in women undergoing in vitro fertilization, preterm premature rupture of membranes, chorioamnionitis, amniotic fluid infections, postpartum and postabortal endometritis, and postabortal pelvic inflammatory disease (PID) [5]. In gynecology, BV increases the risk of posthysterectomy infections, including vaginal cuff cellulitis, pelvic cellulitis, pelvic abscess, and PID [5]. BV is also linked to human papillomavirus infection, as women with BV are 2.68 times more likely to have cervical HPV infection [6]. These detrimental effects prompt the use of an accurate and quick diagnostic tool.

Current diagnostic tools for BV include Amsel's diagnostic criteria and the Nugent scoring system. Both Amsel and Nugent require a microscopic examination of vaginal discharge samples. While most diagnosticians should be able to perform basic microscopic exams, those who have little/distant experience in using microscopes or who do not have access to a microscope would be at a disadvantage. Self-reports about the fishy odor of vaginal discharge are not considered diagnostic, as the Amsel criteria require a whiff test to be performed. The whiff test is considered positive if some “fishy” odor is present after adding a wet amount of potassium hydroxide solution to the vaginal sample. Thus, another disadvantage can be overcome by extending the diagnostic time. At least three criteria from the Amsel criteria are needed to confirm a diagnosis of BV. Some have issued a more simplified version that requires only two criteria. One study revealed that pairing two favorable Amsel criteria demonstrated specificity ranging from 99 to 100% [7]. Individually, the presence of clue cells had a specificity of 98% and a sensitivity of 89.9% for detecting bacterial vaginosis. The identification of at least three Amsel criteria has been linked with findings from Gram stain results. The sensitivity and specificity of the Amsel criteria range from 37 to 70% and 94% to 99%, respectively, compared with the Nugent score. The reason behind this inconsistency remains unknown and can be a subject of future studies [7].

In addition to the Amsel criteria, multiple point-of-care (POC) tests are available for BV diagnosis. One of the POC tests is BVBlue [8]. It is a relatively quick diagnostic tool compared with the Amsel or Nugent criteria. BV blue is a chromogenic diagnostic test based on the presence of elevated sialidase enzymes in vaginal fluid samples. Identifying and quantifying microbial enzymes, especially sialidases, holds promise for swift BV diagnosis.

Sialidases, which are pivotal for bacterial nourishment, cellular interactions, and evasion of immune responses, also increase the capacity of bacteria to adhere to, invade, and deteriorate mucosal tissue. These enzymes are widespread across various microorganisms, including bacteria, viruses, Mycoplasma, fungi, and protozoa. Anaerobic gram-negative bacterial rods such as Bacteroides, Gardnerella, and Prevotella species are among those that secrete sialidases, and this sialidase activity in bacteria is specific to the BVBlue testing kit. BVBlue detects vaginal fluid sialidase activity at levels of ≥ 7.8 U [9, 10]. One unit of sialidase activity is defined as the amount of enzyme required to liberate 1 nmol of substrate/ml/min at 37 °C. The BVBlue test starts with the acquisition of vaginal secretions with cotton swabs, which are mixed in test tubes filled with sialidase-binding substances. The tube was then incubated at room temperature for 10 min. After 10 min, one or two drops of chromogenic reagents were added, and the mixture was left for 3 min. The blue or green color indicates positive results, whereas the yellow color indicates negative results. The key advantage of this test is that it provides a quick and easy diagnostic tool. A limitation of this test is its inability to detect bacteria that do not produce sialidase enzymes, as certain bacterial species associated with bacterial vaginosis (BV) are not sialidase producers. Additionally, this test does not exclude the possibility of coexisting vaginal yeast infections, Trichomonas vaginalis infections, or other sexually transmitted infections (STIs). Furthermore, akin to the Amsel criteria, it does not assess the severity of bacterial vaginosis (BV) symptoms.

Given the inconsistency of findings in previous studies and the potential for utilizing this diagnostic test to enable more rapid diagnosis and treatment of bacterial vaginosis (BV), a thorough evaluation of this test is warranted. Consequently, this systematic review aims to consolidate and synthesize the existing evidence regarding the efficacy of this diagnostic test in the diagnosis of BV.

Materials and methods

This systematic review has been registered in PROSPERO (no. CRD42024545022).

Search strategy

We performed thorough searches of titles and abstracts via keywords in three different databases, Pubmed, Proquest, and Scopus (Table 1) on 16th March 2024, which helped us to identify studies that investigated the accuracy of BVBlue in the diagnosis of BV compared with the gold standard, which is Nugent's scoring or Amsel’s criteria.

Table 1 Search terms per database

Eligibility criteria

The studies included are screened on the basis of the following criteria: women with bacterial vaginosis, which is defined by the presence of at least 3 criteria in the Amsel criteria or a score of 7–10 in Nugent's scoring, are older than 18 years old, where Nugent's scoring and/or Amsel criteria alongside the use of the BVBlue were measured. Studies investigating pediatric patients were excluded. Studies without accessible full texts or that utilized languages other than English were excluded.

Study selection

Study selection was performed simultaneously and independently by three people. First, the titles and abstracts of the studies were screened. The studies that passed the first phase were then screened again thoroughly in each of their full texts against the eligibility criteria that we prepared. In the case where there were conflicting opinions or decisions regarding the eligibility of an article, a discussion between the conflicting reviewers was performed to reach a consensus. In the case where a consensus was not reached, a third reviewer provided the final decision. Covidence©, a tool designed for systematic reviews, was used to perform the study selection to ensure the credibility and accuracy of the selected studies, by streamlining the process from initial search to reporting. It facilitates collaboration, minimizes bias through risk of bias assessment tools, and ensures data accuracy and transparency by enabling version control and generating comprehensive reports.

Quality assessment

QUADAS-2 was utilized to assess the methodological quality in terms of the risk of bias to determine the quality of the studies included. The QUADAS-2 tool consists of 4 main domains that address specific parameters or domains to identify the potential bias of the studies, namely, patient selection, index test, reference standard, flow and timing. The assessment was performed by three independent investigators, with conflicts being resolved through the consensus of all the authors.

Results

Search results

We performed searches on three different databases via the advanced search method with the search strategy shown in Table 1. We managed to retrieve 30 studies from these databases that consisted of 11 duplicates, as shown in Fig. 1. The remaining 19 studies underwent title/abstract screening against our eligibility criteria. A total of 8 studies passed through title/abstract screening and underwent full-text screening. A total of 3 studies were excluded from the full-text screening for various reasons: 2 studies did not have a full-text version (only abstracts), and 1 study did not compare BVBlue with Amsel’s criteria or Nugent’s scoring. The remaining 5 studies that were excluded from the full-text screening were selected and critically appraised.

Fig. 1

PRISMA Flowchart

Quality assessment

The quality and risk of bias assessment of the studies included in this systematic review are depicted in detail in Appendix Table 1 and Table 2. Overall, all the studies assessed with QUADAS-2 tools were of good quality and had a low risk of bias. However, 2 of the studies showed some concerns in the Index Test domain due to unclear methods of whether blindings were performed in the study or not.

Table 2 QUADAS-2 Quality assessment results

BVBlue in the diagnosis of bacterial vaginosis

Overall, 5 studies compared the accuracy of the BVBlue score in diagnosing BV with that of the Nugent score, as shown in Tables 3 and 4. Of the five articles identified, two compared the BV Blue test with Amsel's criteria and Nugent's scoring system. Five studies except that of Madhivanan et al. reported promising results for the use of the BVBlue in the diagnosis of BV, with a sensitivity ranging from 79–91.7%, specificity ranging from 91–100%, PPV (Positive Predictive Value) ranging from 85–100%, and NPV (Negative Predictive Value) ranging from 90.6–98.1% [11]. Madhivanan et al. performed a study in India, and the results are different from those of other studies, with a BVBlue sensitivity of 38%, specificity of 95%, PPV of 88.4%, and NPV of 60.5% compared with Nugent’s scoring and a sensitivity of 51%, specificity of 94%, PPV of 89.5%, and NPV of 65.7% compared with Amsel’s criteria. The likelihood ratio was also calculated manually by the reviewers and showed significant favorable results, with values ranging from 10.7—∞, with the highest likelihood ratio shown in the study performed by Foessleitner et al. and the lowest likelihood ratio shown in the study performed by Lokken et al.

Table 3 Patient characteristics in the selected studies

Table 4 Summary of findings in the selected studies

Discussion

BV poses a significant challenge to women's health and demands accurate and prompt diagnosis for effective management. The chromogenic BVBlue test, a point-of-care (POC) diagnostic tool for detecting sialidase activity in vaginal fluid, has emerged as a potential solution [17]. This systematic review aims to comprehensively evaluate the efficacy, advantages, and limitations of the BVBlue in diagnosing BV, drawing insights from recent studies and considering the implications of demographic variations in test performance.

BVBlue is a chromogenic test, operating on the principle of detecting increased sialidase activity (≥ 7.8 U), an enzyme produced by certain bacteria associated with BV, primarily Gardnerella vaginalis. Other forms of sialidase testing, such as immunoassays, PCR testing, fluorescence spectrometry, and biochemiluminescence, have been expressed in various studies. However, this review focuses specifically on BVBlue to critically evaluate its sensitivity, specificity, and clinical utility. The decision to include only BVBlue was based on several considerations. Firstly, BVBlue is widely recognized as a point-of-care diagnostic tool, designed for rapid and simple detection of sialidase activity, making it highly relevant in clinical settings where immediate results are required. Secondly, while other sialidase testing methods demonstrate high sensitivity and specificity in controlled laboratory conditions—such as biochemiluminescence with a reported sensitivity of 95.40% and specificity of 94.94% [18], or immunoassays with a sensitivity and clinical specificity of 96.29% [9] (both compared to the Amsel method)—these approaches often require sophisticated instrumentation, trained personnel, or extended processing times, which limit their practical applicability in routine clinical practice. Lastly, the inclusion of multiple diagnostic tools would have diluted the review's focus and introduced significant variability due to differences in methodologies and validation standards. Future research could address this gap by systematically comparing multiple sialidase-based diagnostic tools.

Sialidase activity contributes to the breakdown of mucins in the vaginal epithelium, leading to an elevated vaginal pH, a hallmark of BV. By detecting sialidase activity, BVBlue provides a rapid and reliable indication of the presence of BV, facilitating prompt diagnosis and treatment initiation. However, there may be various etiologies of BV, in addition to the common anaerobic Gardnerella vaginalis, such as Atopobium vaginae, Ureaplasma urealyticum, Mycoplasma hominis, and Escherichia coli, hence the limitations of this testing method [10].

When evaluating patients who exhibit symptoms suggestive of BV, four out of five studies reported that the BVBlue test performed better than Amsel’s criteria and the Nugent scoring system did. Other traditional diagnostic techniques listed in the Amsel’s criteria relying only on increased vaginal pH and a positive amine test result, may also cause several drawbacks. If pH was the only factor considered, many women would be overtreated, especially in populations where BV is not as common. A positive amine test result is a highly reliable indicator of the disease; however, owing to its low sensitivity, a significant number of women with BV are undiagnosed [14].

Additionally, BVBlue offers several advantages over traditional diagnostic methods. Its POC nature enables rapid diagnosis, reducing the turnaround time and facilitating the immediate initiation of treatment. Compared with microscopy, BVBlue provides diagnostic information much faster (10 min bedside vs a few hours to two days delay of Gram staining, depending on the laboratory) [10]. Moreover, its simplicity and ease of use make it suitable for deployment in various healthcare settings, including resource-limited environments [15]. Importantly, BVBlue specifically targets bacteria that produce sialidases, primarily Gardnerella vaginalis. However, other bacteria associated with BV, such as Prevotella spp. and Atopobium vaginae, may also contribute to sialidase activity, enhancing the test's sensitivity in detecting BV. Understanding the spectrum of bacteria detected by BVBlue is crucial for accurately interpreting test results and optimizing their clinical utility [11].

The results of the study by Foessleitner et al. (2021) were consistent with the high sensitivity (81%) and specificity (100%) of BVBlue compared with Nugent's scoring, indicating its accuracy in diagnosing BV, particularly in asymptomatic pregnant women [12]. Similarly, Bradshaw et al. [14] reported high sensitivity (88%) and specificity (91%) of the BVBlue criteria compared with Amsel's criteria, supporting its effectiveness in diagnosing BV in clinical settings [14]. These findings align with those of Myziuk et al. [15], who reported the high sensitivity (91.7%) and specificity (97.8%) of the BVBlue score compared with Nugent's scoring, underscoring its reliability in diagnosing BV in nonmenstruating women attending sexually transmitted disease clinics [14, 15]. The promising results were achieved under controlled conditions by on-site experts, which may result in varying outcomes when the test is conducted under different conditions or by non-expert personnel.

However, the study by Madhivanan et al. [13] in Mysore, India, presented a different perspective, with a lower sensitivity (38%) of the BVBlue score than the Nugent score. This discrepancy may be attributed to variations in the study population, including differences in vaginal flora composition and the prevalence of BV-associated bacteria, which could affect the test performance. The vaginal biota of women with BV in this study population may have included sialidase-negative G. vaginalis strains or other bacteria that do not produce sialidases (e.g., Atopobium vaginae, Prevotella spp., Mobiluncusspp., and other anaerobic bacteria) [13]. According to previous studies, only 75% to 84% of women with BV have sialidase activity, indicating that sialidase expression is not consistent [13, 19]. Additionally, research has shown that racial/ethnic differences exist in the composition of BV flora, suggesting that the bacteria linked to BV in this population may differ from those in other racial/ethnic groups [20].

Similarly, the lower sensitivity (25.5%) of the BVBlue criteria than that of Amsel's criteria in HIV-negative Kenyan women, as reported by Lokken et al. [16], underscores the influence of demographic factors and regional differences on BVBlue performance. This observation aligns with the variation in sensitivity across different populations. For example, the lower performance of the BVBlue test among women in sub-Saharan Africa may be attributed to variations in the vaginal microbiota, including differences in bacterial strains and women's intravaginal practices [16, 20].

BVBlue is not without limitations, as are most diagnostic tests. The existence of sialidase activity does not rule out the presence of yeast, T. vaginalis, or other organisms because mixed vaginal infections can occur [10]. Sialidase production is also not uniform among women with BV, and the vaginal biota of BV may contain sialidase-negative strains or a low number of sialidase-producing bacteria [13]. The presence of intermediate flora consistently reduces the performance of other diagnostic methods compared with that of the Nugent method. The high percentage of sensitivity and sensitivity reported in the study by Bradshaw et al. can also be correlated with the test being performed by experienced lab operators [14]. The color spectrum between yellow and green could confuse the examiner between positive and negative results [11]. Moreover, as stated by Myziuk et al. [15], women who have recently douched, have vaginal sex, or used spermicides, vaginal lubricants, or feminine deodorant sprays within 72 h before testing should not use BVBlue to avoid unfavorable results [15]. Other factors, such as methodological differences among studies, such as sample size, diagnostic criteria, and geographical locations, may have contributed to discrepancies in sensitivity findings [21]. Variability in BV prevalence and microbial diversity among study populations could also impact test performance [21, 22]. Factors such as age, hormonal status, and sexual behavior may further influence the sensitivity of BVBlue, emphasizing the need for tailored diagnostic approaches on the basis of demographic characteristics [23].

In summary, while all five studies support the high specificity of the BVBlue for diagnosing BV, variations in sensitivity across different populations highlight the influence of demographic factors and methodological differences in test performance. Further research is needed to explain these factors and optimize the clinical utility of the BVBlue in diverse demographic populations. BVBlue holds significant promise as a rapid and accessible diagnostic tool for BV. Research to map the vaginal flora is also essential for ensuring efficient diagnosis and treatment, especially in Indonesia. Its simplicity and ease of use make it suitable for integration into routine clinical practice, particularly in settings where laboratory infrastructure is limited. However, clinicians should exercise caution when interpreting BVBlue results, considering the potential impact of demographic variations on test performance, highlighting the need for contextualized interpretation of results and further research to optimize its clinical utility. Future research should focus on refining the diagnostic accuracy of the BVBlue scale and exploring its utility in diverse demographic settings to ensure equitable access to accurate BV diagnosis and management.

Recommendation

Given the findings above, it is recommended that the BVBlue test be utilized primarily as a confirmatory diagnostic tool for BV in symptomatic individuals or those already suspected of having BV. Its high specificity makes it particularly suitable for this purpose; however, owing to the variability in sensitivity observed across different populations, healthcare providers should be cautious and consider demographic and regional factors when interpreting BVBlue results. To increase its clinical utility, further research is necessary to map the epidemiology of BV-associated flora in diverse populations. Additionally, clinicians should be aware of factors that may affect test performance, such as recent intravaginal practices, and should integrate BVBlue results with other clinical findings and diagnostic criteria to make well-informed decisions about patient care.

Conclusion

The BVBlue test has been reported as a diagnostic tool that is both accurate and rapid in diagnosing BV. Its diagnostic value is favorable; however, this result is strongly influenced by the epidemiology of the flora causing BV in certain populations. Hence, studies to determine the epidemiology of the flora causing BV are strongly recommended before this diagnostic tool can be used to assure its credibility in diagnosing BV.