Coverage Policy Manual
Policy #: 2014021
Category: Laboratory
Initiated: November 2014
Last Review: March 2022
  Multitarget Polymerase Chain Reaction Testing for Diagnosis of Bacterial Vaginosis (e.g., SureSwab, NuSwab)

Description:
Bacterial vaginosis (BV) is a common medical condition resulting from an imbalance in the normal vaginal flora. Although the identification of Gardnerella vaginalis has traditionally been associated with BV, there is no single etiologic agent. Most cases are asymptomatic, and most symptomatic cases can be diagnosed using clinical and microscopic evaluation. Multitarget polymerase chain reaction (PCR) testing is proposed as an alternative to currently available laboratory tests to diagnose BV. This test may improve outcomes if it is a more accurate and reliable method to diagnose BV.
 
BV is a condition caused by an imbalance in the normal bacteria vaginal flora. It is a common disorder, especially in women of reproductive age. While there is no single known etiologic agent, there is a shift in vaginal flora that involves a depletion of Lacobacillus species with a rise in vaginal pH and overgrowth of other bacteria, including G vaginalis, Mycoplasma hominis, Peptostreptococcus, Mobiluncus species, and various other anaerobic Gram-negative rods.
 
Prevalence of the condition is high, and it is asymptomatic in most cases. According to data from a nationally representative sample of women surveyed in 2001 to 2004, the prevalence of BV among women ages 14 to 49 in the U.S. Is 29% (CDC, 2015). BV is often confused with nonbacterial causes of vaginitis, including candidiasis and trichomoniasis.
 
When symptomatic, BV is associated with characteristic signs and symptoms. The most common sign of BV is an abnormal grayish white vaginal discharge, generally with an unpleasant (often “fishy”) smell. Some women experience mild itching or irritation.
 
BV resolves spontaneously in a high percentage of women. Treatment for symptomatic BV is usually a course of oral antibiotics, either metronidazole or clindamycin. Antibiotic treatment results in a high rate of remission of symptoms, but recurrences are common within the first year after treatment.
 
In practice, the diagnosis of BV can be made based on the presence of at least 3 Amsel criteria (characteristic vaginal discharge, elevated pH, clue cells, fishy odor), which is simple and has a sensitivity of over 90% and specificity of 77% compared with Gram stain (Amsel, 1983, Landers, 2004).
 
More specifically, vaginal discharge is characterized as homogeneous, thin, and whitish-gray; clue cells are squamous epithelial cells that normally have a sharply defined cell border but in BV, have bacteria adherent to their surfaces and appear to be “peppered” with bacteria; pH of vaginal fluid greater than 4.5; and a “fishy” odor of vaginal discharge before or after addition of potassium hydroxide 10%.
 
Vaginal culture is not an appropriate diagnostic method to identify BV because it is not caused by the presence of a particular bacterial species.
 
Various commercial tests provide rapid and accurate pH evaluation and amine detection. For example, automated devices that measure the volatile gases produced from vaginal samples and a colorimetric pH test are commercially available.
 
Nucleic acid probes of DNA fragments are available to detect and quantify specific bacteria in vaginal fluid samples. Polymerase chain reaction (PCR) methods extract and amplify the DNA fragments using either universal or specific primers. The result can be qualitative (to assess whether a specific microorganism is present) or quantitative (to assess how many microorganisms are present). The technology can be used to measure multiple organisms (eg, those known to be associated with BV) at the same time and is commercially available as multitarget PCR testing.
 
Proposed multitarget PCR Test
The SureSwab Total (Quest Diagnostics) test involves obtaining vaginal swab specimens, extracting total DNA, and quantitating the 4 types of bacteria using PCR. Results are reported as log cells per mL for each organism and concentrations of all Lactobacilli species are reported together then classified into 1 of the following 3 categories: not supportive, equivocal, and supportive.
 
A classification of not supportive of BV diagnosis is based on:
 
    • The presence of Lactobacillus species, G. vaginalis levels < 6.0 log cells/mL and absence of A. vaginae and Megasphaera species; or
    • The absence of Lactobacillus species, G. vaginalis levels <6.0 log cells/mL and absence of A. vaginae and Megasphaera species; or
    • The absence of all targeted organisms.
 
A classification of equivocal is based on:
 
    • The presence of Lactobacillus species, plus G. vaginalis at least 6.0 log cells/mL and/or presence of A. vaginae and/or Megasphaera species.
 
A classification of supportive of BV diagnosis is based on the absence of Lactobacillus species, and presence of G. vaginalis levels of at least 6.0 log cells/mL, and presence of A. vaginae and/or Megasphaera species.
 
Another product, the BD Max (Becton, Dickinson), tests for markers of BV and vaginitis. The test uses a similar process to that described for SureSwab. Vaginal swab specimens are collected, DNA is extracted, and real-time PCR is used to quantitate targeted organisms. Results of BV marker tests are not reported for individual organisms. Instead, qualitative BV results are reported as positive or negative for BV based on the relative quantity of the various organisms. The Aptima BV Assay was cleared by the U.S. Food and Drug Administration with the BD Max as the predicate device. The Aptima assay is a nucleic acid amplification test (NAAT) for detection and quantitation of ribosomal RNA.
 
Medical Diagnostics Laboratory offers a Bacterial Vaginosis Panel. Markers are assessed using real-time PCR and Lactobacillus is profiled using quantitative PCR. GenPath Diagnostics also offers a bacterial vaginosis test.
 
The NuSwab® Select BV test (Laboratory Corporation of America) uses semiquantitative PCR analysis of 3 predictive marker organisms of vaginal dysbiosis to generate a total score that is associated with the presence or absence of BV. In this test system, samples with a total score of 0 to 1 are considered negative for BV, samples with a score of 3 to 6 are positive for BV, and samples with a score of 2 are indeterminate for BV.
 
Several of the manufacturers of the BV tests also have extensions that include other causes of vaginitis such as Trichomonas vaginalis and Candidiasis species.
 
  
Regulatory Status
In October 2016, the U.S. Food and Drug Administration completed a review of a de novo request for classification of the BD Max™ Vaginal Panel (Becton, Dickinson). The test was granted class II designation, marketing authorization, and is indicated for the direct detection of DNA targets from bacteria associated with bacterial vaginosis (DEN160001). In 2019, the Aptima BV Assay (Hologic, Inc.) received 510(k) clearance (K190452) with the BD Max as the predicate device. Product code: PQA, NSU, PMN.
 
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests must meet the general regulatory standards of the Clinical Laboratory Improvement Act (CLIA). Laboratories that offer laboratory-developed tests must be licensed by the CLIA for high-complexity testing.
 
Coding
There is no one CPT code for this testing. It would be reported with CPT codes for the various infectious agents for which testing was performed. Below is an example of a possible list of codes:
 
87491: Infectious agent detection by nucleic acid (DNA or RNA); Chlamydia trachomatis, amplified probe technique
87591: Neisseria gonorrhoeae, amplified probe technique
87481: Candida species, amplified probe technique (3 units reported using the modifier -59 on 2 of them to indicate testing for different subspecies of Candida was performed)
87512: Gardnerella vaginalis, quantification
87661: Trichomonas vaginalis, amplified probe technique
87999: Unlisted microbiology procedure (4 units reported using modifier -59 on 3 of them to report different subspecies testing of Megasphaera was performed. This is incorrect coding as unlisted codes are only reported once since they do not have an assigned value.)
81513: Infectious disease, bacterial vaginosis, quantitative real time amplification of RNA markers for Atopobium vaginae, Gardnerella vaginalis, and Lactobacillus species, utilizing vaginal fluid specimens, algorithm reported as a positive or negative result for bacterial vaginosis (effective 1/1/2021)
81514: Infectious disease, bacterial vaginosis and vaginitis, quantitative real time amplification of DNA markers for Gardnerella vaginalis, Atopobium vaginae, Megasphaera type 1, Bacterial Vaginosis Associated Bacteria 2 (BVAB 2), and Lactobacillus species (L. crispatus and L. jensenii), utilizing vaginal fluid specimens, algorithm reported as a positive or negative for high likelihood of bacterial vaginosis, includes separate detection of Trichomonas vaginalis and/or Candida species (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis), Candida glabrata, Candida krusei, when reported (effective 1/1/2021)

Policy/
Coverage:
Effective July 2022
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis, or trichomonas vaginosis utilizing testing for multiple organisms (e.g., SureSwab, NuSwab, Aptima BV, BD Max) does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis, or trichinella vaginosis utilizing testing for multiple organisms (e.g., SureSwab, NuSwab, Aptima BV, BD Max) is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective January 2020 through June 2022
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis, or trichomonas vaginosis utilizing testing for multiple organisms (e.g., SureSwab, NuSwab) does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis, or trichinella vaginosis utilizing testing for multiple organisms (e.g., SureSwab, NuSwab) is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective March 2015 to January 2020
 
Multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis or trichomonas vaginosis does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis, fungal vaginosis or trichinella vaginosis is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective Prior to March 2015
 
Multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, multitarget polymerase chain reaction (PCR) testing for diagnosis of bacterial vaginosis is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.

Rationale:
Tests for Lactobacillus species and G. vaginalis are currently used in laboratory diagnosis of BV. Therefore, this review will focus on tests for additional organisms tested included in multitarget tests, as well as the value of a summary categorization of test results. In the case of the SureSwab (Quest Diagnostics) test, which is commercially available, additional organisms are A. vaginae and Megasphaera species. The policy will briefly review the evidence on the accuracy of each of these markers for diagnosing BV and will then review evidence on the accuracy of multitarget polymerase chain reaction (PCR) tests. In addition, the policy will evaluate the evidence on the clinical utility of multitarget PCR tests, including summary categorization based on multiple markers, compared with currently accepted techniques for diagnosing BV. As with other diagnostic tests, diagnostic accuracy refers to the sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of the tests in relevant populations of patients. Clinical utility requires demonstration that the diagnostic information can be used to improve patient management decisions and/or patient health outcomes, compared with standard laboratory and examination-based methods of diagnosis.
 
Moreover, the review will focus on the patient population for whom a multitarget PCR testing for bacterial vaginosis might be clinically indicated. Most cases of BV can be identified using clinical examination and microscopic examination, and screening asymptomatic patients is not recommended by national organizations (see Practice Guidelines and Position Statements section). Therefore, the most relevant potential target population for multitarget PCR testing is women with an indeterminate diagnosis after standard workup ie women in whom a Gram stain is currently indicated.
 
Diagnostic Accuracy of A. vaginae and/or Megasphaera species
There are several studies on the newer markers, A. vaginae and Megasphaera species, and these have generally found that the sensitivity and specificity for diagnosing BV that are equal to or greater than G vaginalis. (Menard, 2010; Shipitsyna, 2013; Fredricks). This highlights the potential use of these markers, alone or in combination, to improve the accuracy of laboratory-based diagnosis. For example, a 2010 prospective study by Menard et al evaluated a quantitative PCR (qPCR) assay for A. vaginae and G. vaginalis and compared results with the Amsel criteria and Nugent Score (Menard, 2010). The study included 163 pregnant women with singletons who reported vaginal symptoms before 20 weeks’ gestation. For the novel marker A. vaginae, the sensitivity and specificity (95% confidence interval [CI]) of qPCR analysis for predicting a clinical diagnosis of BV were 96% (88% to 100%) and 96% (92% to 99%). This compares with a sensitivity of 72% (54% to 90%) and a specificity of 97% (94% to 99%) for G. vaginalis. When the 2 markers were considered together, elevated levels of 1 or both had high diagnostic accuracy for predicting BV. The sensitivity was 100%, specificity was 93% (89% to 97%), PPV was 73% (59% to 88%) and NPV was 100%.
 
A 2013 study by Shipitsyna et al (Shipitsyna, 2013). prospectively evaluated the accuracy of 16 bacterial species/genera in the diagnosis of BV, using real-time PCR assays. The Amsel criteria were used as the reference standard for BV. The study included 163 non-pregnant women undergoing routine check-ups (n=90) or evaluation of vaginal symptoms (n=73). A total of 73 women were diagnosed with BV, 11 were categorized by the Amsel criteria as intermediate cases, and 79 were healthy controls. Both G. vaginalis and A. vaginae were detected in 100% of the women with BV. However, these bacteria were also detected in a large proportion of women without BV, yielding low specificities (22% and 37%, respectively). Diagnostic accuracy was higher when bacterial genera/species were quantified and diagnostic thresholds were determined. The sensitivity and specificity of qPCR assays for detecting A. vaginae was 96% and 97%, respectively. For Megasphaera type 1, the sensitivity was 88% and the specificity was 100%. The qPCR assays were also highly accurate at detecting other several other organisms, including G. vaginalis and Bacterial Vaginosis-Associated Bacterium 2 (BVAB2). In combination, the investigators found the highest sensitivities and specificities for diagnosing BV when the depletion of Lacobacillus species was combined with the presence of either G. vaginalis or A. vaginae.
 
Other studies evaluating vaginal microbiota but not reporting diagnostic accuracy information have also been published (Zozay-Hinchliffe, 2010; De Backer, 2007). For example, a 2007 study by De Backer et al evaluated the levels of several well-known bacterial species in vaginal samples using real-time PRC and also developed primers for evaluating novel bacteria including A. vaginae (De Backer, 2007). The study included 71 women, 32 of whom were pregnant. (The authors did not report whether these women had vaginal symptoms, but this does not appear to be the case.) The reference test was analysis of Gram stains using Hays criteria. Samples from 12 of 71 women (17%) were categorized by Gram staining as Grade III, BV. All Grade III samples were positive for G. vaginalis and 10 were also positive for A. vaginae.
 
Diagnostic accuracy of multitarget PCR test
There are no published studies on the diagnostic accuracy of any commercially available multitarget qPCR test. The SureSwab (Quest) test quantifies levels of Lactobacillus species, G. vaginalis, A. vaginae and Megasphaera species. Moreover, none of the studies evaluating the diagnostic accuracy of multiple qPCR markers included the most relevant population, women who had already undergone standard workup and whose diagnosis was indeterminate.
 
Cartwright et al, in 2012, developed and validated a multitarget semiquantiative PCR test including 3 organisms: A. vaginae, Megasphaera type 1 and BVAB2 (Cartwright, 2012). The investigators appropriately used separate samples for the development and validation phases and compared the diagnostic accuracy of the multitarget panel with an accepted reference standard. The patient population consisted of 402 women presenting at an STD clinic (n=299) or a personal health clinic (n=103). The reference standard was a combination of the Nugent and Amsel criteria. First, Nugent scores were generated and samples were categorized as BV positive, BV negative or intermediate. Then, samples with intermediate Nugent scores were reanalyzed using Amsel criteria. The intermediate samples that met Amsel criteria were considered positive for BV and those that did not meet Amsel criteria were considered negative for BV.
 
Samples from 169 women were included in the development phase, of which 108 (64%) were positive for BV and 61 (36%) were negative for BV. The investigators initially targeted 5 organisms, A. vaginae BVAB2, G. vaginalis, L. crispatus and Megasphaera 1 using PCR assays. They conducted logistic regression analysis to identify the optimal combination of 2, 3 or 4 markers and selected the combination of A. vaginae, BVAB2 and Megasphaera 1. Together, these 3 markers yielded a sensitivity of 93.3% and specificity of 92.9% for diagnosing BV, compared with the reference standard. The investigators also calculated a composite score based on the levels of these 3 organisms in the sample. Cutoff values were determined by analysis of frequency distributions obtained during qPCR testing.
 
In the validation phase, the multitarget PCR test was validated using an additional 227 samples. Compared with the samples used in the development phase, more were collected from the lower-prevalence general health clinic. According to the reference test, 100 of 227 cases (48.5%) were positive for BV and 107 (51.5%) were negative. Using the composite score generated in the developmental phase, 14 of the 227 samples (6.2%) yielded a composite score of 2 and were categorized as indeterminate (this included 5 BV positive and 9 BV negative samples). Of the 213 evaluable samples, 104 of 105 (99.05) BV-positive samples had a positive composite score and 98 of 108 (90.7%) BV-negative samples had a negative composite score. When the entire study population was analyzed, the multitarget PCR assay had a sensitivity of 96.7%, specificity of 92.2%, PPV of 94.0% and NPV of 95.6%, and an indeterminate rate of 5.3%.
 
In addition, the 2013 study by Shipitsyna et al (Shipitsyna, 2013), previously described, included relevant analyses. This study, which included women having routine checkups and those seeking care for vaginal symptoms, evaluated the diagnostic accuracy 16 individual bacterial species/genera using qPCR technology and investigated the optimal combination of markers.. The investigators found that the combination with the highest sensitivities and specificities for diagnosing BV was Lacobacillus species, G. vaginalis, and A. vaginae. BV was diagnosed when the level of Lactobacillus species was low and there were increased levels (above a set threshold) of either G. vaginalis or A. vaginae.
 
Clinical utility of multitarget PCR test
No published studies were identified that evaluated changes in patient management and/or health outcomes when a multitarget PCR test was used to diagnose BV, compared with standard methods of diagnosis.
 
Summary of Evidence
No published evidence was available on the diagnostic accuracy of any commercially available multitarget PCR test compared with alternatives. There is some evidence that PCR tests for individual bacterial genera/species and combinations of these organisms may have high sensitivity and specificity for diagnosing BV, but it is not possible to determine the true diagnostic accuracy with certainty due to limited research. Moreover, approaches to using PCR in the various studies differ, and there no generally accepted cutoffs for the levels of novel species such as Atopobium vaginae. Furthermore, studies were not conducted with the most clinically relevant target population, symptomatic women with indeterminate diagnoses after standard workup.
 
There is also a lack of evidence on the clinical utility of PCR testing for BV ie, studies showing that testing leads to better patient management decisions and/or better health outcomes. Studies of diagnostic accuracy alone are inadequate, especially because most symptomatic women can be diagnosed with a standard workup and/or a trial of empiric therapy. Screening asymptomatic women or screening of all pregnant women is not recommended by national organizations.
 
Practice Guidelines and Position Statements
In 2010, the Centers for Disease Control and Prevention issued guidelines in 2010 on sexually transmitted diseases characterized by vaginal discharges (Centers for Disease Control, 2010). Regarding diagnosis of bacterial vaginosis, the guidelines state:
 
“BV can be diagnosed by the use of clinical criteria (i.e., Amsel’s Diagnostic Criteria) or Gram stain. Other tests, including a DNA probe-based test for high concentrations of G. vaginalis (Affirm VP III, Becton Dickinson, Sparks, Maryland), a prolineaminopeptidase test card (Pip Activity TestCard, Quidel, San Diego, California), and the OSOM BVBlue test have acceptable performance characteristics compared with Gram stain. Although a card test is available for the detection of elevated pH and trimethylamine, it has low sensitivity and specificity and therefore is not recommended. PCR also has been used in research settings for the detection of a variety of organisms associated with BV, but evaluation of its clinical utility is uncertain.”
 
In 2012, the American College of Obstetricians and Gynecologists published a Practice Bulletin, “Prediction of Preterm Birth”. The bulletin stated that bacterial vaginosis testing is not recommended as a screening strategy in asymptomatic pregnant women at increased risk of preterm birth (American College of Obstetricians and Gynecologists, 2012).
 
U.S. Preventive Services Task Force Recommendations
2008 recommendations on screening for BV in pregnancy (U.S. Preventive Services Task Force, 2008) are:
“The USPSTF recommends against screening for bacterial vaginosis in asymptomatic pregnant women at low risk for preterm delivery”. (Grade D recommendation).
 
“The USPSTF concludes that the current evidence is insufficient to assess the balance of benefits and harms of screening for bacterial vaginosis in asymptomatic pregnant women at high risk for preterm delivery.” (I statement)
 
2016 Update
A literature search conducted through January 2016 did not reveal any new information that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
Kusters and colleagues reported on a semiquantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (Kusters, 2015). The research was conducted in the Netherlands and the investigators did not report any affiliation with any company marketing a multitarget PCR test in the U.S. The study included 159 women at least 18 years old who were presenting with complaints of abnormal vaginal discharge. Women underwent examination and had a vaginal swab for PCR and a vaginal smear for microscopy. Eight women (5%) had missing data and were not included in the analysis. The investigators tested for 5 bacterial species, A. vaginae G. vaginalis, Megasphaera 1 and 2 Lactobacillus species (L. crispatus and L. iners). They also calculated a lactobacillus index (L-index) based on the relative presence of the 2 species. An index <1, when there was a higher load of L crispatus, indicated a shift to disturbed vaginal microflora.
 
In the analysis, 83 of 151 women (55%) had normal vaginal microflora, 13 (9%) had intermediate vaginal microflora and 55 (36%) had BV according to the Nugent Score. In women with a Nugent Score indicating BV (score of 7 to 10), G. vaginalis, A. vaginae and Megasphaera phylotype 1 were present in 96%, 87% and 60%, respectively of the vaginal specimens. In women with normal vaginal microflora (Nugent score of 0 to 3), these proportions were 27%, 6% and 2%, respectively. The investigators also evaluated the diagnostic accuracy of the multiplex qPCR assay compared with the Nugent Score as the reference standard. The multiplex qPCR test was considered positive when at least 2 BV-associated bacteria were detected and indeterminate when 1 BV-associated bacterium was detected and the L-index was less than 1. In the remaining situations, the test was scored negative. Fifty-five samples were scored BV-PCR positive, 10 were BV-PCR indeterminate and 86 were BV-PCR negative. The sensitivity and specificity of the multiplex BV-PCR test was 92% and 96%, respectively.
 
Also in 2015, Rumyantseva evaluated a multitarget quantitative real-time PCR test (Rumyantseva, 2015).  The study was conducted in Russia and included 100 women undergoing routine annual clinical examination. Thirteen women had complained of vaginal discharge and the rest were asymptomatic. Using PCR assays, the investigators estimated the total amount of bacteria in vaginal samples, and the relative amount of lactobacillus, A. vaginae and G. vaginalis. Findings were compared with Nugent score evaluation and with wet mount microscopy. A positive PCR test was defined as a concentration of A. vaginae and/or G. vaginalis that was equal to or exceeded the Lactobacillus species concentration. For 72 of the 98 evaluable samples, the 3 methods gave the same results (agreement=73.5%). The investigators calculated the sensitivity and specificity of each of the diagnostic tests. They defined a true positive sample as having at least 2 of 3 positive findings and a true negative test as having at least 2 out of 3 negative findings. In this analysis, the sensitivity of the PCR assay was 92.8% and the specificity was 85.7%. This compared to a sensitivity and specificity of 75% and 97.1%, respectively for the Nugent score and 96.4% and 94.3%, respectively for wet mount microscopy.
 
Ongoing and Unpublished Clinical Trials
A search of ClinicalTrials.gov in November 2015 did not identify any ongoing or unpublished trials that would likely influence this review.
 
The evidence for multitarget polymerase chain reaction testing in patients who have signs or symptoms of bacterial vaginosis includes several prospective studies on the diagnostic accuracy of PCR assays of individual markers or combinations of markers, and several prospective studies validating the diagnostic accuracy of multitarget tests. Relevant outcomes are test accuracy, test validity, symptoms and change in disease status. None of the studies evaluated a multitarget PCR test that is commercially available in the U.S. The available studies suggest that the multitarget PCR tests that were evaluated may have high sensitivity and specificity for diagnosing BV, but it is not possible to determine the true diagnostic accuracy with certainty due to limited research and heterogeneity in methodology eg, differences in the included markers, scoring systems and/or reference tests. However, studies of diagnostic accuracy alone in unselected populations of women with BV are inadequate, because most symptomatic women can be diagnosed with a standard workup and/or a trial of empiric therapy. Studies have not been conducted in the most clinically relevant target population, symptomatic women with indeterminate diagnoses after standard workup. Furthermore, there is a lack of evidence on the clinical utility of PCR testing for BV, ie, studies showing that testing leads to better patient management decisions and/or better health outcomes than current approaches. The evidence is insufficient to determine the effects of the technology on health outcomes.
 
2017 Update
A literature search conducted using the MEDLINE database through February 2017 did not reveal any new information that would prompt a change in the coverage statement.
 
2018 Update
 
A literature search conducted using the MEDLINE database did not reveal any new information that would prompt a change in the coverage statement.
 
In 2016, Hilbert et al reported on a quantitative real-time PCR test (Hilbert, 2016). The study was funded through MDL and evaluated markers in that laboratory’s BV Panel. The study included 400 samples from 149 premenopausal women ages 18 and older who presented with vaginitis. Fifteen samples were excluded, including 8 samples with discordant findings using the Amsel criteria and the Nugent score, leaving 385 samples from 146 women in the analysis. Samples were obtained at the initial and at follow-up clinic visits. Participants were evaluated for BV using the Amsel criteria, and vaginal smears were evaluated by Nugent score. Additionally, vaginal samples were sent to MDL and tested for multiple organisms. In multivariate analysis, the model that best predicted BV included A. vaginae, G. vaginalis, Megasphaera type 1, and Megasphaera type 2. The selected model incorporating these 4 markers had a sensitivity of 92% and a specificity of 95% for predicting BV. When only samples from the first visit were analyzed, for which 84% were from BV-positive women, the model had a sensitivity of 91% and a specificity of 96%. At the second visit, when 94% of samples were from BV-negative women, the sensitivity was 100% and specificity was 96%.
 
Centers for Disease Control and Prevention
In 2015, the Centers for Disease Control and Prevention updated its guidelines on sexually transmitted diseases (cdc, 2015). Regarding diagnosis of bacterial vaginosis (BV), the guidelines stated: “BV can be diagnosed by … clinical criteria (i.e., Amsel’s Diagnostic Criteria) or Gram stain. A Gram stain (considered the gold standard laboratory method for diagnosing BV) is used to determine the relative concentration of lactobacilli … PCR [polymerase chain reaction] has been used in research settings for the detection of … organisms associated with BV, but evaluation of its clinical utility is still underway. Detection of specific organisms might be predictive of BV by PCR. Additional validation is
needed….”
 
2019 Update
Annual policy review completed with a literature search using the MEDLINE database through February 2019. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
In practice, the diagnosis of BV can be made based on the presence of at least 3 Amsel criteria (characteristic vaginal discharge, elevated pH, clue cells, fishy odor), which is simple and has a sensitivity of over 90% and specificity of 77% compared with Gram stain (Amsel, 1983; Landers, 2004).
 
More specifically, vaginal discharge is characterized as homogeneous, thin, and whitish-gray; clue cells are squamous epithelial cells that normally have a sharply defined cell border but in BV, have bacteria adherent to their surfaces and appear to be “peppered” with bacteria; pH of vaginal fluid greater than 4.5; and a “fishy” odor of vaginal discharge before or after addition of potassium hydroxide 10%.
 
Both comparator diagnostic methods (ie, clinical diagnosis using the Amsel criteria and laboratory diagnosis using Nugent or Ison and Hay criteria) have subjective components and, therefore, may be imprecise (Nugent, 1991; Ison, 2002). Moreover, Gram stain examination is time-consuming, requires substantial training, and it is difficult to determine an appropriate clinical response for intermediate scores. The two methods of diagnosis can also be used in combination to increase diagnostic accuracy.
 
BD Max Test
The FDA decision summary and Gaydos et al (2017) for the BD Max test includes a description of a prospective clinical diagnostic accuracy study (FDA, 2016;, Gaydos, 2017).The study included 1763 women with symptoms of BV or vaginitis. Both clinician-collected and self-collected vaginal swabs were obtained, and were analyzed independently. A total of 1559 (88%) clinician-detected and 1582 (90%) self-detected samples were available for analysis.
 
NuSwab
Cartwright et al published data on a multitarget semiquantitative PCR test including 3 organisms: Atopobium vaginae, Megasphaera type 1, and BVAB2 (Cartwright, 2012). The investigators used separate samples for the development and validation phases and compared the diagnostic accuracy of the multitarget panel with an accepted reference standard. The patient population consisted of 402 women presenting at a clinic for sexually transmitted infections (n=299) or a personal health clinic (n=103). Samples from 169 women were included in the development phase, of which 108 (64%) were positive for BV and 61 (36%) were negative for BV. In the validation phase, the multitarget PCR test was assessed using an additional 227 samples. Results were similar in Cartwright et al, which reported on a multicenter study of 1579 women of whom 538 were positive and 1041 were negative for BV (Cartwright, 2018). In this publication, the authors proposed an α-diversity score generated from next-generation sequencing that could be used to resolve discordant PCR and Nugent/Amsel results.
 
2020 Update
A literature search was conducted through January 2020.  There was no new information identified that would prompt a change in the coverage statement.  
 
2021 Update
Annual policy review completed with a literature search using the MEDLINE database through February 2021. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
Schwebke et al compared the Aptima BV assay (Hologic, Inc.) to Nugent score as reference standard in 1,417 symptomatic women (Schwebke, 2020). Both clinician- and patient-collected swabs were assessed. Clinicians utilized modified Amsel criteria for the resolution of indeterminate Nugent scores.
 
Richter et al compared the accuracy of testing with Aptima BV, Hologic Analyte Specific Reagent, and the direct-probe BD Affirm test to Nugent score as the reference standard in 111 symptomatic women (Richter, 2019). Modified Amsel criteria were used for the resolution of indeterminate Nugent scores.
 
In individuals who have signs or symptoms of BV who receive multitarget PCR testing, the evidence includes several prospective studies on technical performance and diagnostic accuracy. Relevant outcomes are test validity, symptoms, and change in disease status. Several studies have evaluated the diagnostic accuracy of multitarget PCR tests for BV, including 5 studies evaluating commercially available tests. The studies found sensitivities between 84% and 95% and specificities between 85% and 97% compared with standard methods of diagnosis. Most studies used a combination of the Amsel criteria and Nugent scoring as the reference standard. There is a lack of direct evidence on the clinical utility of PCR testing for BV (ie, studies showing that testing leads to better patient management decisions and/or better health outcomes than current approaches). Moreover, a chain of evidence does not currently support multitarget testing because most symptomatic women can be diagnosed with a standard workup. The evidence is insufficient to determine that the technology results in an improvement in the net health outcomes.
 
2022 Update
Annual policy review completed with a literature search using the MEDLINE database through February 2022. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
In 2021, the Centers for Disease Control and Prevention updated its guidelines on sexually transmitted infections (Workowski, 2021). Regarding the diagnosis of bacterial vaginosis (BV), the guidelines stated:
 
“BV can be diagnosed by....clinical criteria (i.e., Amsel’s Diagnostic Criteria) or by determining the Nugent score from a vaginal Gram stain. Vaginal Gram stain, considered the reference standard laboratory method for diagnosing BV, is used to determine the relative concentration of lactobacilli …"
 
The guidelines state that multiplex PCR assays are available, but noted that traditional methods of BV diagnosis, including the Amsel criteria, Nugent score, and the Affirm VP III assay, remain useful for diagnosing symptomatic BV because of their lower cost and ability to provide a rapid diagnosis. The guidelines also stated that BV nucleic acid amplification tests should be used among symptomatic women only (eg, women with vaginal discharge, odor, or itch) because their accuracy is not well defined for asymptomatic women.
 
June 2022 Update
 
In individuals who have signs or symptoms of BV who receive multitarget PCR testing, the evidence includes several prospective studies on technical performance and diagnostic accuracy. The relevant outcomes are test validity, symptoms, and change in disease status. Several studies have evaluated the diagnostic accuracy of multitarget PCR tests for BV, including 5 studies evaluating commercially available tests. The studies found sensitivities between 84% and 95% and specificities between 85% and 97% compared with standard methods of diagnosis. Most studies used a combination of the Amsel criteria and Nugent scoring as the reference standard. There is a lack of direct evidence on the clinical utility of PCR testing for BV (ie, studies showing that testing leads to better patient management decisions and/or better health outcomes than current approaches). Moreover, a chain of evidence does not currently support multitarget testing because most symptomatic women can be diagnosed with a standard workup. The evidence is insufficient to determine that the technology results in an improvement in the net health outcome
 
Published in 2012 and reaffirmed in 2018, the American College of Obstetricians and Gynecologists (ACOG) has produced a Practice Bulletin on the prediction of preterm birth. The Bulletin stated that BV testing is not recommended as a screening strategy in asymptomatic pregnant women at increased risk of preterm birth (ACOG, 2012).
 
Published in 2020, the ACOG has issued a Practice Bulletin on vaginitis in nonpregnant patients (ACOG, 2020). The Bulletin made the following recommendations on the initial evaluation of patients with symptoms of vaginitis, citing CDC guidelines:
 
"A complete medical history, physical examination of the vulva and vagina, and clinical testing of vaginal discharge (ie, pH testing, a potassium hydroxide "whiff test," and microscopy) are recommended for the initial evaluation of patients with vaginitis symptoms."
 
The Bulletin noted that single-swab multiplex PCR testing "may be a promising alternative to microscopy," but that its clinical utility is still under evaluation.

CPT/HCPCS:
0330UInfectious agent detection by nucleic acid (DNA or RNA), vaginal pathogen panel, identification of 27 organisms, amplified probe technique, vaginal swab
0352UInfectious disease (bacterial vaginosis and vaginitis), multiplex amplified probe technique, for detection of bacterial vaginosis–associated bacteria (BVAB-2, Atopobium vaginae, and Megasphera type 1), algorithm reported as detected or not detected and separate detection of Candida species (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis), Candida glabrata/Candida krusei, and trichomonas vaginalis, , vaginal-fluid specimen, each result reported as detected or not detected
0353UInfectious agent detection by nucleic acid (DNA), Chlamydia trachomatis and Neisseria gonorrhoeae, multiplex amplified probe technique, urine, vaginal, pharyngeal, or rectal, each pathogen reported as detected or not detected
81513Infectious disease, bacterial vaginosis, quantitative real time amplification of RNA markers for Atopobium vaginae, Gardnerella vaginalis, and Lactobacillus species, utilizing vaginal fluid specimens, algorithm reported as a positive or negative result for bacterial vaginosis
81514Infectious disease, bacterial vaginosis and vaginitis, quantitative real time amplification of DNA markers for Gardnerella vaginalis, Atopobium vaginae, Megasphaera type 1, Bacterial Vaginosis Associated Bacteria 2 (BVAB 2), and Lactobacillus species (L. crispatus and L. jensenii), utilizing vaginal fluid specimens, algorithm reported as a positive or negative for high likelihood of bacterial vaginosis, includes separate detection of Trichomonas vaginalis and/or Candida species (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis), Candida glabrata, Candida krusei, when reported
87481Infectious agent detection by nucleic acid (DNA or RNA); Candida species, amplified probe technique
87512Infectious agent detection by nucleic acid (DNA or RNA); Gardnerella vaginalis, quantification
87661Infectious agent detection by nucleic acid (DNA or RNA); Trichomonas vaginalis, amplified probe technique
87999Unlisted microbiology procedure

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American College of Obstetricians and Gynocologists (ACOG).(2012) Prediction and Prevention of Preterm Birth: ACOG Practice Bulletin No. 130. Obstet Gynecol. 2012;120(4):964-973. PMID

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Centers for Disease Control and Prevention (CDC). Bacterial Vaginosis (BV) Statistics. http://www.cdc.gov/std/bv/stats.htm. Accessed September 5, 2014.

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