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Diagnostic value of dual-fluorescence staining in bacterial vaginosis

Diagnostic value of dual-fluorescence staining in bacterial vaginosis

  • Categories:Newsroom
  • Author:Cui Li, Zhongliang Duan, Jing Zhang, Jing Gao, Chunmei Ying
  • Origin:
  • Time of issue:2023-08-03
  • Views:0

(Summary description)Dual-fluorescence staining for the detection of bacterial
diseases of the vagina exhibited acceptable consistency with Gram
staining and performed well with respect to dyeing time, stability, and
the interpretation of results. We argue that this method should be used
in outpatient services.

Diagnostic value of dual-fluorescence staining in bacterial vaginosis

(Summary description)Dual-fluorescence staining for the detection of bacterial
diseases of the vagina exhibited acceptable consistency with Gram
staining and performed well with respect to dyeing time, stability, and
the interpretation of results. We argue that this method should be used
in outpatient services.

  • Categories:Newsroom
  • Author:Cui Li, Zhongliang Duan, Jing Zhang, Jing Gao, Chunmei Ying
  • Origin:
  • Time of issue:2023-08-03
  • Views:0
Information
ABSTRACT
Objective: The aim of this study was to investigate the epidemiology of bacterial vaginosis (BV) in Shanghai, China, and to explore the value of a dual-fluorescence staining method in the diagnosis of BV.
Methods: Specimens were collected from women with vaginitis at the Obstetrics and Gynecology Hospital of Fudan University from January 2020 to December 2021, and the proportions of various vaginitis types (such as Candida vaginitis, Trichomonas, and bacterial vaginitis) were analyzed statistically. To explore the diagnostic value of dual fluorescence staining for BV, we first executed a dual-fluorescence staining method to analyze the vaginal secretions of 265 patients, then confirmed our diagnoses by consulting clinical physicians and by using Nugent scoring of Gram staining.
Results: There were 16,905 patients who were diagnosed with vaginitis over the previous 2 years, with a median age of 32 (minimum age of 9 years and maximum of 84 years). Of these patients, we noted 10,887 cases (64.40%) of BV. Our staining results revealed that the dual-fluorescence method was consistent with Gram staining in the diagnosis of BV, with a P value of less than .001 using a χ2 test and a consistency kappa value of 0.896. Compared with Gram staining, the dual-fluorescence staining method required an acceptable time (2.2 min vs 2.5 min, respectively) and exhibited different visual effects (green and yellow vs purple and red, respectively). 
Conclusion: Dual-fluorescence staining for the detection of bacterial diseases of the vagina exhibited acceptable consistency with Gram staining and performed well with respect to dyeing time, stability, and the interpretation of results. We argue that this method should be used in outpatient services.
 
 
Bacterial vaginosis (BV) encompasses disorders of vaginal bacterial communities or pathogen infections caused by a diminution in the abundance or the complete disappearance of Lactobacillus and an elevation in the abundance of anaerobic or facultative anaerobic bacteria.1 Common pathogens include facultative anaerobes (eg, Gardnerella vaginalis), anaerobes (Prevoella, Campylobacter, Bacteroidetes, Atopoupella vaginalis), Ureaplasma urealyticum, and Mycoplasma hominis. 2 Bacterial vaginosis is one of the most common infectious vaginal diseases in women of childbearing age, and it primarily occurs in sexually active women. There are many complications associated with BV, including pelvic inflammatory disease, gynecological infection, infertility, and abnormal pregnancy,3 and BV pathogens are complexed with many other pathogens. The disease has a high recurrence rate after treatment4 that can cause serious harm to female reproductive health.5 Symptoms of BV are often atypical, and its diagnosis is easily missed. The incidence of BV varies by countries and regions due to the diversity of patients, races, and diagnostic methods,6 and its incidence is high in part due to concurrence with immunodeficiency disease.7 Approximately 11% of women undergoing physical examinations are infected with BV, and 36%–60% of outpatients are infected with BV in China according to 1 report;8 however, epidemiological data regarding BV in China are lacking.
Rapid and accurate diagnosis of BV using vaginal secretions is an important prerequisite for appropriate clinical treatment, particularly with outpatients. The Gram staining method is relatively accurate but time-consuming. The stain colors (purple and red) are not easily distinguished for some technicians, and their identification partially depends on the technique used by each technician. Thus, Gram staining is not suitable for outpatient testing. Examination of vaginal microbial chips is another diagnostic method,9 but it is relatively expensive. A novel method therefore needs to be sought that is easy to perform, requires less time than the Gram staining method, and is inexpensive. Dual-fluorescence staining is one such newly developed method that applies direct microscopic examination using a fluorescence microscope that can store photomicrographs and allows collective reporting for direct clinical review.
There are currently few reports on the application and resulting data of this technique in the routine analysis of vaginal secretions. For this study, we therefore analyzed the advantages and disadvantages of a dual-fluorescence staining system in the detection of BV by comparing it with the classical Gram staining method for the detection of BV.
 
Materials and Methods
Sources of Specimens and Clinical Data
Specimens from outpatient vaginitis patients in the Department of Gynecology at the Obstetrics and Gynecology Hospital of Fudan University from January 2020 to December 2021 were collected for analysis, including the proportions of various vaginitis types—Candida vaginitis (VVC), Trichomonas vaginitis (TV), and BV. Bacterial vaginosis was diagnosed by exploiting a clinical and laboratory vaginal microbial chip and confirmed by gynecologists. We also collected derived data and clinical information. The microbial chip method, BD Affirm VPIII Microbial Identification Test (Affirm, BD), has been widely used in our hospital.
The main principle is based on the principle of nucleic acid hybridization. The specimens were cracked, combined with a probe analysis card including capture probe and chromogenic probe, and interpreted according to the chromogenic results. Bacterial vaginosis was associated with increase of Gardnerella vaginalis in this method.
Finally, 16,905 patients were enrolled in this study for clinical data analysis, and we selected 265 random specimens from them for method comparison.
Gram staining and dual-fluorescence staining were used to analyze vaginal secretions of patients. We subsequently compared the sensitivity, specificity, consistency, time, and visual effects of the novel staining technique with Gram staining to illustrate the diagnostic value of the novel method. For Gram staining, Nugent score was used to diagnose BV, with the Nugent score no less than 7. As dual-fluorescence staining is a morphological method, the Nugent score was also used to diagnose BV, with the Nugent score no less than 7.
This study complied with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University.
 
Principles of Dual-Fluorescence Staining
This modality uses fluorescent dyes to stain DNA, RNA, dextran, and peptidoglycan in vaginal secretion samples such that each target component displays a different color under a specific wavelength of fluorescence excitation. The combination of the fluorescent stains with methyl green as a counterstain significantly improved the coloration of cell structures.10 This compound staining method also clarified the morphologies of epithelial cells, leukocytes, bacilli, cocci, Candida, and Trichomonas under a fluorescence microscope, thus allowing convenient observation and evaluation.
 
Materials, Equipment, and Reagents
We used an OLYMPUS CX series microscope and fluorescence analysis system, an automatic dye machine (Minomics) and a dual-fluorescence staining solution (Medomics Medical Technology), ordinary glass slides, cover slips, 0.9% normal saline, and a thermostatically controlled heater.
 
Sample Testing Process Operation of the Microscopy System
We set the serial number and other relevant indices and connected the microscopy system to a laboratory information system (LIS).
 
Preparation of Patient Information
Patient information was input to the LIS.
 
Preparation of Specimens
We placed vaginal secretions onto a microslide, dried the specimen with a heater, cooled the specimen, and inserted it into the dyeing machine for automatic staining under green light. We subsequently removed the microslide when the green light flashed, coverslipped it, dried it with absorbent paper, and proceeded with our analysis.
 
Microscopic Examination
Vaginal secretions were stained and the morphological types of different bacteria were observed under a microscope (×1000, oil immersion, OLYMPUS CX23). We selected at least 2 images to provide for an appropriate field of vision and analyzed and stored them; a laboratory technician ultimately reviewed the report and transmitted it to the LIS.
 
Statistical Analysis
Continuous variables are presented as means ± SD or median and interquartile range according to their normality of distribution (as assessed with the Shapiro-Wilk test). We used the χ2 test for categorical data, and the Mann-Whitney U test was applied to compare data that did not follow a normal distribution. Data were analyzed using SPSS 19 software (SPSS) and GraphPad Prism 7 (GraphPad). The kappa consistency test and paired χ2 test were adopted to compare the difference between the 2 staining methods: a kappa greater than or equal to 0.75 was considered to exhibit acceptable consistency, a kappa greater than or equal to 0.4 and less than 0.75 was considered generally consistent, and a kappa less than 0.4 was considered to show poor consistency. A P value < .05 denoted statistical significance.
 
Results
Clinical Information
There were 16,905 patients in this study, with a median age (interquartile range) of 32 years (27, 41); the youngest was 9 years of age and the oldest was 84 years old. Among the total 16,905 patients with vaginitis, we noted only 3 with all 3 types of vaginitis, accounting for 0.02%, whereas there were 126 cases (0.75%) with both BV and TV, one case with TV and VVC (0.01%), and 2521 cases (14.91%) with BV and VVC. Regarding infection with only a single type of vaginitis, we observed 230 cases (1.36%) with TV, 3137 cases (18.56%) with VVC, and 10,887 cases (64.40%) with BV. Importantly, there were 5204 patients (30.78%) who were re-infected with vaginitis 6 months later. Thus, vaginitis is certainly worthy of serious consideration in the clinic, especially with respect to BV (TABLE 1).
 
 
Comparisons of Diagnostic Value Between Gram Staining and Dual- Fluorescence Staining Methods
To compare the dual-fluorescence staining method with Gram staining, we evaluated 265 specimens that were additionally diagnosed via clinical and laboratory examinations of vaginal microbial chips and confirmed by gynecologists. Representative characteristics and results are shown in FIGURE 1 and TABLE 2. Dual-fluorescence staining revealed 215 cases of BV, whereas there were 206 cases of BV detected by the Gram-staining method; our 
analysis showed a P value of less than .001 by χ2 test and a kappa value of 0.896 for consistency. Our results thus reflected favorable consistency between double-fluorescence staining and Gram staining in the diagnosis of BV. Comparison of the 2 methods regarding acceptable time (2.2 min vs 2.5 min, respectively), different visual effects (distinguishing green from yellow vs purple from red, respectively) and other characteristics are shown in TABLE 3.
 
 
 
 
Discussion
With the increasing attention paid to women’s health in the modern society, the concept of involvement of female reproductive tract microecology in the onset of disease has achieved heightened recognition. Pathogen infection or an imbalance in local flora may precipitate related gynecological and pregnancy-related diseases.11,12 Examination of vaginal secretions is currently the most routine clinical detection method used to observe the vaginal environment. Patients with BV often show significant changes in the dominant flora of the vaginal environment. As shown in our study, over 64.40% of vaginitis patientsmanifested BV, with the youngest at 9  years of age and the oldest at 84 years. Thus, BV is certainly worthy of receiving greater attention in the clinic.
The Amsel standard is the clinical gold standard for the diagnosis of BV. Diagnostic BV is defined as positivity for at least 3 of the following 4 clinical features: primary positivity for clue cells (ie, the number of clue cells over 20% of the total number of vaginal epithelial cells); a positive reaction on the amine test; a pH value of vaginal secretions >4.5; and vaginal secretions that are homogeneous, thin, and grayish white, and have positivity for clue cells.13 Nugent scoring of Gram staining is the laboratory gold standard for the diagnosis of BV. To an extent, our novel method combined the evidence of positive clue cells within the Amsel criteria with the characteristics of microflora distribution using the Nugent score.
In addition to the above diagnostic criteria, there are other methods used for the diagnosis of BV as a clinical reference in China and elsewhere. For example, Hay-Ison scoring has been implemented, as have graded Gram-stain smears of vaginal secretions.14,15 In terms of molecular diagnosis, nucleic acid detection of Gardnerella has been implemented in BV, including the Affirm VPIII and Aptima BV assays, which revealed a sensitivity of 78%–100%. There are also some functional assays used for the detection of BV. For example, regarding the detection of the anaerobic bacterial metabolite sialidase, we suggest that functional detection be combined with morphological detection results, and when the functional and morphological results are inconsistent, we suggest that the morphological detection results prevail.
Our novel dual-fluorescence staining method allowed clear differentiation among epithelial cells, leukocytes, bacilli, cocci, Candida, and Trichomonas under the fluorescence microscope; this is convenient for microscopic observation and evaluation. Moreover, dyeing and electronic reporting can be completed within 2 min after mastery of the technique, showing robust operational capability for outpatient departments. This method has the advantages of simple operation, rapid detection, and acceptable observation and identification of pathogenic microorganisms after staining. In addition, we were able to circumvent the effects of interference from contamination in samples such as blood, semen, and basal-layer cells on detection results; accuracy and detection rates were thus effectively improved. However, after comparative analysis and practical operational experience, we also found that there were certain interfering factors that affected this method; for example, specimens need to be sent for inspection immediately after being collected. If delayed (such as by over 2  h), abnormal results could beobtained. The location of specimen collection is also critical to obtaining appropriate samples, and a sufficient number of specimens need to be collected. The uniformity of the vaginal smear also has an impact on the results, as the smear cannot be too thick.
This method was first used in an outpatient setting for quick testing; however, it could be widely used in the clinical laboratory, pathology department, and molecular laboratory. According to its principle, this method could also be used to examine many other materials, such as peritoneal fluid.
To summarize, BV accounts for 64.40% of vaginitis in China. Dualfluorescence staining for the detection of BV manifested good consistency with the Gram-staining method while showing advantages in dyeing time, stability, and the interpretation of results. Thus, we postulate that dual-fluorescence staining as a novel modality is worth pursuing in outpatient gynecology departments and other clinical laboratories.
 
Conclusion
Vaginitis is certainly worthy of serious consideration in the clinic, especially with respect to BV. Dual-fluorescence staining for the detection of bacterial vaginal diseases showed acceptable consistency with Gram staining and performed well with respect to dyeing time, stability, and interpretation of results. We therefore argue that this modality should be used by outpatient services.
 
Acknowledgments
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
 
Funding
This work was supported by grants from the National Natural Science Foundation of China (No. 81873970 to C.Y. and No. 82102469 to Z.D.).
 
Conflict of Interest Disclosure
The authors have nothing to disclose.
 
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