A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy
by
, , and
Hideto Yamada
1,2,*
,
Shigeki Shimada
3,
Hajime Ota
4,
Yuta Kobayashi
2,
Yoshiyuki Fukushi
2,
Shinichiro Wada
2 and
Soromon Kataoka
5 1
Center for Recurrent Pregnancy Loss, Teine Keijinkai Hospital, Sapporo 006-8555, Hokkaido, Japan
2
Department of Obstetrics and Gynecology, Teine Keijinkai Hospital, Sapporo 006-8555, Hokkaido, Japan
3
Mommy’s Clinic Chitose, Chitose 066-0038, Hokkaido, Japan
4
Department of Obstetrics and Gynecology, Sapporo Toho Hospital, Sapporo 065-0017, Hokkaido, Japan
5
Department of Obstetrics and Gynecology, Hakodate Central General Hospital, Hakodate 040-8585, Hokkaido, Japan
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(10), 2110; https://doi.org/10.3390/microorganisms12102110
Submission received: 18 September 2024
/
Revised: 11 October 2024
/
Accepted: 16 October 2024
/
Published: 21 October 2024
(This article belongs to the Special Issue Detection of Pathogenic Microorganism)
Abstract
This prospective cohort study aimed to evaluate the performance of Flora select™ (FS), a newly developed real-time PCR test, for the assessment of the vaginal microbiome during early pregnancy. Five hundred and fifty-six pregnant women underwent examinations of FS, Nugent score—a Gram-staining scoring system for the diagnosis of bacterial vaginosis (BV)—and conventional bacterial culture between 8 weeks and 12 gestational weeks. Nugent scores of 0–3, 4–6, and ≥7 were found in 469 (84.2%), 41 (7.4%), and 47 (8.5%) of the women, respectively. Relative dominance rates of Lactobacillus species of high (≥80% medium (50%≤, <80%), and low (0.1≤, <50%), and no detection (<0.1%) were 63.0%, 8.8%, 17.1%, and 11.2%, respectively. Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species were detected in 23.9%, 17.6%, 17.1%, 7.0%, 23.0%, and 4.9% of the women, respectively. Gardnerella species were detected in all women with Nugent scores ≥7 and Ureaplasma were detected in 40.4% of them. BV-associated bacterial species were also detected in 70.7% of women with Nugent scores of 4–6. Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species were highly prevalent in women with Nugent scores ≥4 or Lactobacillus species <50%. FS detected Gardnerella, Prevotella, and Atopobium species more effectively than conventional bacterial culture. FS could determine relative dominance rates of Lactobacillus species in the vaginal microbiome, and simultaneously detect four kinds of BV-associated bacteria, Ureaplasma and Mycoplasma species. Therefore, FS may be clinically useful for the screening of the vaginal microbiome during pregnancy to prevent preterm birth and for the assessment of the vaginal microbiome after BV treatments.
1. Introduction
Preterm birth is a major cause of perinatal mortality and the long-term neurologic morbidity of newborns; the risk of preterm birth increases in pregnant women with bacterial vaginosis (BV) [1]. BV results from the stabilization or colonization of several vaginal bacteria such as Gardnerella, Bacteroides, Ureaplasma, and Mycoplasma species. To diagnose BV, classical clinical signs and symptoms of Amsel’s criteria [2] or the microscopically based Nugent score [3] have been used over the years. However, the morphologic assessment of bacteria species is time-consuming, somewhat subjective, and cannot accurately identify the pathogens [4].
The human vaginal microbiome plays a role in causing or defending against BV. Lactobacillus species have the beneficial function of protecting against ascending infections of microorganisms during pregnancy [5]. The vaginal microbiome profiles of healthy pregnant women shifted toward a Lactobacillus-dominant state during early pregnancy [6]. Decreases in the abundance of Lactobacillus species, and increases in BV-associated bacteria and Ureaplasma species in the vaginal microbiome, are associated with BV, vaginal infection, and ascending infections of microorganisms, which may cause intrauterine infections such as chorioamnionitis and preterm births [7].
Recently, molecular testing using real-time polymerase chain reaction (PCR) and 16S rRNA sequencing for BV diagnosis has been developed. A study found that log Lactobacillus crispatus/Gardnerella vaginalis was the best diagnostic indicator of BV [8]. A meta-analysis of cohort studies for the vaginal microbiome using 16S rRNA sequencing during pregnancy revealed that Lactobacillus crispatus reduced preterm birth, whereas Lactobacillus iners, Gardnerella and Prevotella species increased the risk of preterm birth [9]. A randomized clinical trial on pregnant women at low risk of preterm birth demonstrated that real-time PCR tests and treatment for BV, based on Gardnerella and Atopobium quantification, significantly reduced preterm birth rates in nulliparous women [10]. The development of culture-independent molecular diagnostics has enabled the detection of BV-associated non-cultivable bacterial species. Multiple nucleic acid amplification tests for BV diagnosis are commercially available in the United States [4].
This prospective cohort study evaluated the performance of Flora select™ (FS), a newly developed real-time PCR test, in the assessment of the vaginal microbiome during early pregnancy in comparison with the Nugent score and conventional bacterial culture.
2. Materials and Methods
2.1. Study Participants
This prospective cohort study was conducted between December 2023 and July 2024 according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Teine Keijinkai Hospital (No. 2-023272-00, Date of approval: 17 November 2023). Written informed consent was obtained from participants or the guardians of women under 18 years old in four hospitals/clinics.
Pregnant women underwent examinations of FS, together with Nugent scoring—a Gram-staining scoring system for BV diagnosis—and conventional bacterial culture of vaginal swabs between 8 weeks and 12 weeks of gestation at regular maternity checkups. Two swabs were used to obtain vaginal fluid. The first swab was used for the Nugent score and microbiological culture, and the second for FS.
2.2. Procedures
2.2.1. Nugent Score and Microbiological Culture
The Nugent scoring system evaluates bacterial morphotypes microscopically for BV diagnosis using Bartholomew and Mittwer methods (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and the Favor method (Shimadzu Diagnostics Co., Tokyo, Japan) for gram staining. Nugent scores range from 0 to 10, according to the quantitative presence of three bacterial morphotypes, which are assessed for Gram-stained vaginal fluid. The quantitative number of Lactbacillus morphotypes is scored 0–4, where 0 indicates the lowest amount. Small Gram-variable rods (Gardnerella vaginalis and Bacteroides morphotypes) are scored 0–4, where 4 indicates the highest amount, and curved Gram-variable rods (Mobiluncus morphotypes) are scored 0–2, where 2 indicates the highest amount. BV is diagnosed when Nugent scores are ≥7 [3,11]. In the present study, Nugent scores were classified into 0–3 (BV-negative), 4–6 (BV-intermediate), and ≥7 (BV-positive).
For microbiological culture, swabs of vaginal fluid were inoculated onto BD BBL™ chocolate II agar medium (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) for fastidious microorganisms—especially Neisseria and Haemophilus species; onto BD BBL™ TSAII 5% sheep blood agar medium/BTB lactose agar medium (Nippon Becton Dickinson Co., Ltd.) for Lactobacillus and Streptococcus species; onto AccuRate™ separated ABHK agar/BBE agar (Shimadzu Diagnostics Co.) for anaerobic culture and Prevotella species; and onto BD BBL™ CHROMagar™ Candida II agar medium (Nippon Becton Dickinson Co., Ltd.) for candida and fungus. The cultures of Ureaplasma and Mycoplasma species were not performed. Microbiological culture was performed at a temperature of 37 °C and humidity of 50–80% or 95% in CO2 incubation.
All bacteria and fungi isolated were identified using the VITEK™ MS PRIME (bioMérieux Japan Ltd., Tokyo, Japan), which can detect almost all bacteria.
2.2.2. Real-Time PCR Test (Flora SelectTM)
The samples for Nugent score, bacterial culture, and Flora selectTM (FS, Varinos Inc., Tokyo, Japan) were simultaneously obtained by swabbing the vaginal walls using two different swabs. Vaginal swab samples for FS were directly collected into a Copan eNAT collection tube (Copan Italia, Brescia, Italy). The samples were stored at room temperature until DNA extraction, which was performed within four weeks according to the manufacturer’s protocol. DNA extraction and the amplification of bacterial DNA by real-time PCR, using SYBR Green Method (TOYOBO, Osaka, Japan), was conducted by Varinos Inc. (Tokyo, Japan). The samples were treated with proteinase K of ≥600 U/mL (Kanto kagagu Co., Inc., Tokyo, Japan) and a lysozyme solution of 1.5 mg/mL (Merck KGaA, Darmstadt, Germany) for cell lysis as pretreatment. Genomic DNA was extracted using MagNA Pure 24 system (Pathogen 1000 hp 3.2 software/protocol, Roche Diagnostics GmbH, Mannheim, Germany).
The relative absolute abundance of Lactobacillus species was calculated by the ratio of the amount of Lactobacillus to the total amount of bacteria species. The cycle threshold (Ct) of each sample was compared with that of the standard curve made by diluting the genomic DNA of Lactobacillus crispatus. Lactobacillus abundance was classified into four categories, based on relative dominance rates of Lactobacillus species, comprising high (≥80%), medium (50%≤, <80%), low (0.1≤, <50%), and no detection (<0.1%). The detection of four BV-associated bacteria (Gardnerella, Atopobium, Streptococcus, and Prevotella) species and two miscarriage/preterm birth-associated bacteria (Ureaplasma and Mycoplasma) species was performed by multiplex PCR using specific primers for each bacterium. Primers for the four BV-associated bacteria and Ureaplasma were designed to amplify all known species in each genus and were targeted at the 16S or 23S rRNA gene region. Two sets of primers were designed to amplify Mycoplasma species including Mycoplasma genitalium and hominis, which are found in female reproductive organs.
The threshold for a positive result for each bacterium was adjusted to the threshold of a conventional bacterial culture test. The amplification reactions were performed on an CFX96 C1000 Touch Real-Time System (CFX Maestro software, Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a total volume of 20 μL containing THUNDERBIRD Next SYBR qPCR Mix (TOYOBO Co., Ltd., Osaka, Japan), forward and reverse primer sets (at 0.5 μM concentration), and the extracted bacterial DNA. A no-template control and a positive control were included on each plate.
The correlation coefficient between FS and next-generation sequencing for relative dominant rates of Lactobacillus species was 0.995. Both positive and negative predictive values of FS for known DNA samples of the six bacteria were found to be 100%.
2.3. Statistical Analysis
Categorical variables were compared using Fisher’s exact test. The prevalence of having Lactobacillus species <50%, BV-associated bacteria, Ureaplasma, and Mycoplasma species was compared among women with Nugent scores 0–3, 4–6, and ≥7. The prevalence of having Nugent scores ≥7/≥4, BV-associated bacteria, Ureaplasma, and Mycoplasma species was compared among women with high, medium, low, and no detection of relative dominance rates of Lactobacillus species. All p-values were two-sided, and a p-value < 0.05 was considered statistically significant. Statistical analyses were conducted using R software (version 4.3.2) (R Foundation for Statistical Computing, Vienna, Austria).
3. Results
A total of 556 pregnant women with a median age of 31 (range, 16–45) years were enrolled. Table 1 shows participant characteristics, Nugent scores, and results of FS. Of the 556 women, 19 (3.4%) had an obstetric history of only one preterm birth between 32 weeks and 36 weeks of gestation, and 119 (21,4%) had a history of miscarriage. Nugent scores of 0–3 (BV-negative), 4–6 (BV-intermediate), and ≥7 (BV-positive) were found in 468 (84.2%), 41 (7.4%), and 47 (8.5%) of the participants, respectively. Relative dominance rates of Lactobacillus species of high (≥80%), medium (50%≤, <80%), low (0.1≤, <50%), and no detection (<0.1%) were 63.0%, 8.8%, 17.1%, and 11.2%, respectively. BV-associated bacteria of Gardnerella, Prevotella, Atopobium, and Streptococcus species were detected in 23.9%, 17.6%, 17.1%, and 7.0% of the participants, respectively. Miscarriage/preterm birth-associated bacteria of Ureaplasma and Mycoplasma species were detected in 23.0% and 4.9% of the participants, respectively.
Table 2 shows the results of FS compared with Nugent scores. BV-associated bacteria and Gardnerella species were detected in all 47 women with Nugent scores ≥7, and Ureaplasma species was detected in 19 (40.4%) of the 47 women. BV-associated bacteria species were detected in 29 (70.7%) of 41 women with Nugent scores of 4–6. The prevalence of having Lactobacillus species <50%, BV-associated bacteria, Ureaplasma, and Mycoplasma species was significantly different among women with Nugent scores of 0–3, 4–6, and ≥7.
Table 3 shows the results of FS compared with the relative dominance rates of Lactobacillus species. BV-associated bacteria species were detected in 80 (84.2%) of 95 women with low (0.1≤, <50%) and in 41 (66.1%) of 62 women with no detection (<0.1%). The prevalence of having Nugent scores ≥7/≥4, BV-associated bacteria, Ureaplasma, and Mycoplasma species was significantly different among women with high, medium, low, and no detection of relative dominance rates of Lactobacillus species.
Table 4 shows the comparison between conventional bacterial culture and FS to detect BV-associated bacteria species. FS detected Gardnerella, Prevotella, and Atopobium species more than conventional bacterial culture. Conventional bacterial cultures detected 46 of 133 women to be FS-positive for Gardnerella species, only 4 of 98 women as FS-positive for Prevotella species, and none of 95 women as FS-positive for Atopobium species. However, the conventional bacterial culture detected Streptococcus species more than FS, although PCR primers were set for all Streptococcus species.
Figure 1 shows the prevalence of Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species, and relative dominance rates of Lactobacillus species—≥80% (high) and <50% (low and no detection)—according to the Nugent score. Of 556 women, 468 with Nugent scores of 0–3 (84.2%) had a prevalence of Gardnerella species of <31.3%, while 51 (9.2%) women with Nugent scores of 5–10 had a prevalence of Gardnerella species of 100% and a prevalence of Atopobium and Prevotella species of ≥50%, except for 2 women with Nugent scores 9–10. The prevalence of relative dominance rates of Lactobacillus species ≥80% (high) declined from 82.5% at Nugent score 0 to 18.8% at the score 3, and further to 0% at the scores 5–10. The prevalence of relative dominance rates of Lactobacillus species <50% (low and no detection) increased from 9.4% at a Nugent score of 0, to 68.8% at a score of 3, to 75.7% at a score of 4, and further to 100% at scores of 5–10.
Of 37 women with a Nugent score of 4, Lactobacillus ≥80% was detected in 18.9%, Lactobacillus <50% in 75.7%, Gardnerella in 48.7%, Atopobium in 48.7%, and Prevotella in 51.4%.
4. Discussion
This prospective study, for the first time, evaluated the performance of Flora select™ (FS), a real-time PCR test, for the assessment of the vaginal microbiome between 8 weeks and 12 weeks of gestation in women at relatively low risk of preterm birth. This study revealed that the relative dominance rates of Lactobacillus species—classified as high, medium, low, and no detection—were 63.0%, 8.8%, 17.1%, and 11.2%, respectively. Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species were detected in 23.9%, 17.6%, 17.1%, 7.0%, 23.0%, and 4.9% of the pregnant women, respectively (Table 1). Gardnerella species were detected in all women with a Nugent score ≥7, and 40.4% of them had Ureaplasma species. BV-associated bacteria species were also detected in 70.7% of women with Nugent scores of 4–6. The prevalence of Gardnerella, Prevotella, Atopobium, Streptococcus, Ureaplasma, and Mycoplasma species was very high in women with BV scores ≥4 (Table 2) or Lactobacillus species <50% (Table 3). FS also detected Gardnerella, Prevotella, and Atopobium species more effectively than conventional bacterial culture (Table 4). FS could determine relative dominance rates of Lactobacillus species in the vaginal microbiome, and simultaneously detect four kinds of BV-associated bacteria, particularly Ureaplasma and Mycoplasma species, which cannot be detected by Gram staining or conventional bacterial culture. Therefore, FS may be clinically useful for the screening of the vaginal microbiome during pregnancy to prevent preterm labor and for the assessment of the vaginal microbiome after BV treatments.
Although the routine screening or treatment of BV for asymptomatic women at low risk for preterm birth is generally not recommended [12], there is evidence from one trial that BV screening and treatment programs for pregnant women before 20 weeks of gestation reduces preterm birth and preterm low birthweight [13,14]. In the present study, none of the women with Nugent scores 5–10 had Lactobacillus ≥80% (high), but all had Lactobacillus <50% and Gardnerella species. (Figure 1). Therefore, all women with Nugent scores 5–10 should receive treatment for BV during pregnancy, while women with a Nugent score of 4 and with positive tests for BV-associated bacteria and Lactobacillus <50% can be treated for BV.
In the 556 women, 73 (13.1%)/80 (14.4%) had Gardnerella/BV-associated bacteria species with low levels of Lactobacillus (0.1≤, <50%) and 34 (6.1%)/41 (7.4%) had Gardnerella/BV-associated bacteria species with no detection (<0.1%) of Lactobacillus species (Table 3). When FS is used for the screening of the vaginal microbiome during the first trimester, these women are prime candidates for antibiotic therapy to prevent preterm labor.
Ureaplasma species are the most frequently isolated microorganisms from the amniotic fluid and placentae of women who deliver preterm, and are also associated with miscarriages, neonatal respiratory diseases, and chorioamnionitis [15]. A retrospective cohort study found that the presence of Ureaplasma species in the vaginal microbiome at <11 weeks of gestation was causally associated with late miscarriages and early preterm birth [16]. Recently, a prospective cohort study of pregnant women with threatened miscarriage/preterm labor demonstrated that the presence of Ureaplasma species and decreased relative dominance rate of Lactobacillus species in the vaginal microbiome predicted a high possibility of subsequent preterm birth [17]. In the uterine endometrium microbiome of nonpregnant women with recurrent pregnancy loss, the presence of Ureaplasma species increased the risk of the miscarriage of fetuses with normal chromosomes in their subsequent pregnancies, and the presence of Ureaplasma and Gardnerella species and a decreased relative dominance rate of Lactobacillus species increased the risk of preterm birth in the subsequent pregnancies [18]. These prospective cohort studies have revealed that the presence of Ureaplasma species in the vagina during pregnancy and in the uterine endometrium before pregnancy increased the risk of adverse pregnancy outcomes. Although Ureaplasma species cannot be detected by Gram staining or conventional bacterial culture, the present study using FS identified Ureaplasma species in the vaginal microbiome in 21.2%, 24.4%, and 40.4% of pregnant women with Nugent scores 0–3, 4–6, and ≥7, respectively (Table 2). Because metronidazole, as a standard treatment for BV, is not effective against Ureaplasma species, these women could be treated further with macrolides. FS may be clinically useful because it can detect Ureaplasma species and BV-associated bacteria simultaneously.
This prospective study has several limitations. All participants were Japanese; the human vaginal microbiome changes according to race, diet, living environment, and life cycle stage. Differences in Lactobacillus species (L. crispatus, gasseri, jensenii, and iners) were not assessed. Repeated FS tests were needed to confirm the results. Pregnancy outcomes (miscarriage, preterm birth, and term delivery) of participants in relation to results of FS and Nugent score were not evaluated. The efficacy of prophylactic antibiotic therapy or the cost-effectiveness of FS as a screening compared to Nugent score were not determined. These should be clarified in further investigations, and the cohort study is now in progress.
5. Conclusions
This study demonstrated that FS could determine the relative dominance rates of Lactobacillus species in the vaginal microbiome, and simultaneously detect four kinds of BV-associated bacteria, together with Ureaplasma and Mycoplasma species. FS may be clinically useful for the screening of the vaginal microbiome during pregnancy to prevent preterm birth and for the assessment of the vaginal microbiome after BV treatments. When FS is used for the screening of the vaginal microbiome during the first trimester, women having Gardnerella/BV-associated bacteria species with low/no detection of Lactobacillus species are prime candidates for treatment with antibiotic therapy to prevent preterm birth.
Author Contributions
Conceptualization, H.Y.; methodology, S.S., H.O., S.K. and H.Y.; data acquisition, S.S., H.O., Y.K., Y.F., S.W. and H.Y.; formal analysis, H.Y.; investigation, S.S., H.O., Y.K. and H.Y.; writing—original draft preparation, H.Y.; writing—review and editing, S.S., H.O., Y.K., S.K. and H.Y. All authors have read and agreed to the published version of the manuscript.
Funding
This work was funded by the Japan Agency for Medical Research and Development (grant JP21gk0110047, JP23fk0108682, and JP22gn0110061 to Yamada) and the Japan Society for the Promotion of Science (grants 20K09642, 23K08888, and 24K02691 to Yamada).
Data Availability Statement
The data underlying this study cannot be shared publicly for privacy reasons. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Acknowledgments
We acknowledge and thank all members of Teine Keijinkai Hospital, Mommy’s Clinic Chitose, Sapporo Toho Hospital, Hakodate Central General Hospital, and Varinos Inc. for their contributions to this study.
Conflicts of Interest
The authors declare no conflicts of interest.
References
- Hillier, S.L.; Nugent, R.P.; Eschenbach, D.A.; Krohn, M.A.; Gibbs, R.S.; Martin, D.H.; Cotch, M.F.; Edelman, R.; Pastorek, J.G., 2nd; Rao, A.V.; et al. The Vaginal Infections and Prematurity Study Group. N. Engl. J. Med. 1995, 333, 1737–1742. [Google Scholar] [CrossRef] [PubMed]
- Amsel, R.; Totten, P.A.; Spiegel, C.A.; Chen, K.C.S.; Eschenbach, D.; Holmes, K.K. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am. J. Med. 1983, 74, 14–22. [Google Scholar] [CrossRef] [PubMed]
- Nugent, R.P.; Krohn, M.A.; Hillier, S.L. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J. Clin. Microbiol. 1991, 29, 297–301. [Google Scholar] [CrossRef] [PubMed]
- Muzny, C.A.; Cerca, N.; Elnaggar, J.H.; Taylor, C.M.; Sobel, J.D.; van der Pol, B. State of the art for diagnosis of bacterial vaginosis. J. Clin. Microbiol. 2023, 61, e0083722. [Google Scholar] [CrossRef] [PubMed]
- Witkin, S.S. The vaginal microbiome, vaginal anti-microbial defence mechanisms and the clinical challenge of reducing infection-related preterm birth. BJOG Int. J. Obstet. Gynaecol. 2015, 122, 213–218. [Google Scholar] [CrossRef] [PubMed]
- Serrano, M.G.; Parikh, H.I.; Brooks, J.P.; Edwards, D.J.; Tom, J.; Edupuganti, L.; Huang, B.; Girerd, P.H.; Bokhari, Y.; Bradley, S.P.; et al. Racioethnic diversity in the dynamics of the vaginal microbiome during pregnancy. Nat. Med. 2019, 25, 1001–1011. [Google Scholar] [CrossRef] [PubMed]
- Goldenberg, R.L.; Culhane, J.F.; Iams, J.D.; Romero, R. Epidemiology and causes of preterm birth. Lancet 2008, 371, 75–84. [Google Scholar] [CrossRef] [PubMed]
- Deng, T.; Shang, A.; Zheng, Y.; Zhang, L.; Sun, H.; Wang, W. Log (Lactobacillus crispatus/Gardnerella vaginalis): A new indicator of diagnosing bacterial vaginosis. Bioengineered 2022, 13, 2981–2991. [Google Scholar] [CrossRef] [PubMed]
- Huang, C.; Gin, C.; Fettweis, J.; Foxman, B.; Gelaye, B.; MacIntyre, D.A.; Subramaniam, A.; Fraser, W.; Tabatabaei, N.; Callahan, B. Meta-analysis reveals the vaginal microbiome is a better predictor of earlier than later preterm birth. BMC Biol. 2023, 21, 199. [Google Scholar] [CrossRef] [PubMed]
- Bretelle, F.; Loubière, S.; Desbriere, R.; Loundou, A.; Blanc, J.; Heckenroth, H.; Schmitz, T.; Benachi, A.; Haddad, B.; Mauviel, F.; et al. Groupe de Recherche en Obstetrique et Gynécologie (GROG) Investigators. Effectiveness and costs of molecular screening and treatment for bacterial vaginosis to prevent preterm birth: The AuTop Randomized Clinical Trial. JAMA Pediatr. 2023, 177, 894–902. [Google Scholar] [CrossRef] [PubMed]
- Brookheart, R.T.; Lewis, W.G.; Peipert, J.F.; Lewis, A.L.; Allsworth, J.E. Association between obesity and bacterial vaginosis as assessed by Nugent score. Am. J. Obstet. Gynecol. 2019, 220, 476.e1–476.e11. [Google Scholar] [CrossRef] [PubMed]
- American College of Obstetricians and Gynecologists. Practice bulletin No. 130: Prediction and prevention of preterm birth. Obstet. Gynecol. 2012, 120, 964–973. [Google Scholar] [CrossRef] [PubMed]
- Sangkomkamhang, U.S.; Lumbiganon, P.; Prasertcharoensuk, W.; Laopaiboon, M. Antenatal lower genital tract infection screening and treatment programs for preventing preterm delivery. Cochrane Database Syst. Rev. 2015, 2015, CD006178. [Google Scholar] [CrossRef] [PubMed]
- Kiss, H.; Petricevic, L.; Husslein, P. Prospective randomized controlled trial of an infection screening programme to reduce the rate of preterm delivery. BMJ 2004, 329, 371. [Google Scholar] [CrossRef] [PubMed]
- Sweeney, E.L.; Dando, S.J.; Kallapur, S.G.; Knox, C.L. The human Ureaplasma species as causative agents of chorioamnionitis. Clinl. Microbiol. Rev. 2017, 30, 349–379. [Google Scholar] [CrossRef] [PubMed]
- Kataoka, S.; Yamada, T.; Chou, K.; Nishida, R.; Morikawa, M.; Minami, M.; Yamada, H.; Sakuragi, N.; Minakami, H. Association between preterm birth and vaginal colonization by mycoplasmas in early pregnancy. J. Clin. Microbiol. 2006, 44, 51–55. [Google Scholar] [CrossRef] [PubMed]
- Shi, Y.; Tanimura, K.; Sasagawa, Y.; Yamada, H. Vaginal microbiota associated with preterm delivery. J. Infect. Chemother. 2020, 26, 1134–1138. [Google Scholar] [CrossRef] [PubMed]
- Shi, Y.; Yamada, H.; Sasagawa, Y.; Tanimura, K.; Deguchi, M. Uterine endometrium microbiota and pregnancy outcome in women with recurrent pregnancy loss. J. Reprod. Immunol. 2022, 152, 103653. [Google Scholar] [CrossRef] [PubMed]
Figure 1.
The prevalence of each bacterium according to Nugent score.
Table 1.
Participant characteristics, Nugent score, and results of Flora select™.
| Characteristics of Participants n = 556 | Median | Range |
|---|---|---|
| Age, years | 31 | 16–45 |
| Body mass index, kg/m2 | 21.3 | 15.4–37.8 |
| Gravidity | 2 | 1–8 |
| Parity | 1 | 0–6 |
| Number of previous miscarriages | 0 | 0–4 |
| Number of previous preterm births | 0 | 0–1 |
| Gestational week (w) and day (d) of examinations | 10w4d | 8w0d–12w6d |
| Number | % | |
| Number of women with a history of miscarriage | 119 | 21.4 |
| Number of women with a history of preterm birth | 19 | 3.4 |
| Nugent score | Number | % |
| 0–3 | 468 | 84.2 |
| 4–6 | 41 | 7.4 |
| ≥7 | 47 | 8.5 |
| Flora select™ | Number | % |
| Relative dominance rate of Lactobacillus species | ||
| High (≥80%) | 350 | 63.0 |
| Medium (50%≤, <80%) | 49 | 8.8 |
| Low (0.1≤, <50%) | 95 | 17.1 |
| No detection (<0.1%) | 62 | 11.2 |
| Presence of BV-associated bacteria species | ||
| Gardnerella | 133 | 23.9 |
| Prevotella | 98 | 17.6 |
| Atopobium | 95 | 17.1 |
| Streptococcus | 39 | 7.0 |
| Miscarriage/preterm birth-associated bacteria species | ||
| Ureaplasma | 128 | 23.0 |
| Mycoplasma | 27 | 4.9 |
BV, bacterial vaginosis.
Table 2.
Results of Flora select™ compared with Nugent scoring.
| Nugent Score | Number | Lactobacillus Species <50% (%) | Presence of BV-Associated Bacteria Species (%) | Presence of ≥Two BV-Associated Bacteria Species (%) | BV-Associated Bacteria Species | Miscarriage/Preterm Birth-Associated Bacteria Species | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Gardnerella (%) | Prevotella (%) | Atopobium (%) | Streptococcus (%) | Ureaplasma (%) | Mycoplasma (%) | |||||
| 0–3 | 468 | 16.9 | 16.7 | 10.7 | 13.7 | 7.9 | 6.6 | 4.7 | 21.2 | 2.6 |
| 4–6 | 41 | 78.0 | 70.7 | 61.0 | 53.7 | 53.7 | 53.7 | 17.1 | 24.4 | 9.8 |
| ≥7 | 47 | 97.9 | 100.0 | 95.7 | 100.0 | 83.0 | 89.4 | 21.3 | 40.4 | 23.4 |
| Fisher’s exact test | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p = 0.014 | p < 0.01 | |
BV, bacterial vaginosis.
Table 3.
Nugent score and results of Flora select™ compared with relative dominance rates of Lactobacillus species.
Table 3.
Nugent score and results of Flora select™ compared with relative dominance rates of Lactobacillus species.
| Relative Dominance Rate of Lactobacillus Species | Number | Nugent Scores ≥7/Nugent Scores ≥4 (%) | Presence of BV-Associated Bacteria Species (%) | Presence of ≥Two BV-Associated Bacteria Species (%) | BV-Associated Bacteria Species | Miscarriage/Preterm Birth-Associated Bacteria Species | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Gardnerella (%) | Prevotella (%) | Atopobium (%) | Streptococcus (%) | Ureaplasma (%) | Mycoplasma (%) | |||||
| High (≥80%) | 350 | 0.0/2.0 | 3.1 | 2.0 | 2.3 | 1.7 | 1.4 | 0.9 | 18.3 | 1.4 |
| Medium (50%≤, <80%) | 49 | 2.0/6.1 | 44.9 | 22.5 | 36.7 | 20.4 | 18.4 | 8.2 | 28.6 | 10.2 |
| Low (0.1≤, <50%) | 95 | 30.5/45.3 | 84.2 | 72.6 | 76.8 | 57.9 | 63.2 | 17.9 | 33.7 | 13.7 |
| No detection (<0.1%) | 62 | 27.4/56.5 | 66.1 | 53.2 | 54.8 | 43.6 | 33.9 | 24.2 | 29.0 | 6.5 |
| Fisher’s exact test | p < 0.01/p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | p < 0.01 | |
BV, bacterial vaginosis.
Table 4.
BV-associated bacteria species detected by conventional bacterial culture and Flora select™.
Table 4.
BV-associated bacteria species detected by conventional bacterial culture and Flora select™.
| Gardnerella Species | Bacterial Culture | Total | Prevotella Species | Bacterial Culture | Total | ||
|---|---|---|---|---|---|---|---|
| Positive | Negative | Positive | Negative | ||||
| Flora select™ | Flora select™ | ||||||
| Positive | 46 | 87 | 133 | Positive | 4 | 94 | 98 |
| Negative | 5 | 418 | 423 | Negative | 0 | 458 | 458 |
| Total | 51 | 505 | 556 | Total | 4 | 552 | 556 |
| Atopobium Species | Bacterial Culture | Total | Streptococcus Species | Bacterial Culture | Total | ||
| Positive | Negative | Positive | Negative | ||||
| Flora select™ | Flora select™ | ||||||
| Positive | 0 | 95 | 95 | Positive | 16 | 23 | 39 |
| Negative | 0 | 461 | 461 | Negative | 55 | 462 | 517 |
| Total | 0 | 556 | 556 | Total | 71 | 485 | 556 |
BV, bacterial vaginosis.
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Yamada, H.; Shimada, S.; Ota, H.; Kobayashi, Y.; Fukushi, Y.; Wada, S.; Kataoka, S. A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy. Microorganisms 2024, 12, 2110. https://doi.org/10.3390/microorganisms12102110
AMA Style
Yamada H, Shimada S, Ota H, Kobayashi Y, Fukushi Y, Wada S, Kataoka S. A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy. Microorganisms. 2024; 12(10):2110. https://doi.org/10.3390/microorganisms12102110
Chicago/Turabian StyleYamada, Hideto, Shigeki Shimada, Hajime Ota, Yuta Kobayashi, Yoshiyuki Fukushi, Shinichiro Wada, and Soromon Kataoka. 2024. "A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy" Microorganisms 12, no. 10: 2110. https://doi.org/10.3390/microorganisms12102110
APA StyleYamada, H., Shimada, S., Ota, H., Kobayashi, Y., Fukushi, Y., Wada, S., & Kataoka, S. (2024). A New Real-Time PCR Test (Flora Select™) and Nugent Score for the Diagnosis of Bacterial Vaginosis During Pregnancy. Microorganisms, 12(10), 2110. https://doi.org/10.3390/microorganisms12102110
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