Molecular Study of Gardnerella vaginalis Isolated from Vaginal Discharge of Women Referring to Gynecology Clinics in Chaharmahal and Bakhtiari, Iran, in 2017

AUTHORS

Hossein Hashemi ORCID 1 , Mahshid Shakibapour 1 , Hossein Fazeli 2 , Jaleh Varshowsaz ORCID 3 , Zahra Aliyari 4 , Hossein Yousofi Darani 1 , *

1 Department of Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Drug Delivery Systems Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Parasitology and Mycology, Shahrekord University of Medical Sciences, Shahrekord, Iran

How to Cite: Hashemi H, Shakibapour M, Fazeli H, Varshowsaz J, Aliyari Z, et al. Molecular Study of Gardnerella vaginalis Isolated from Vaginal Discharge of Women Referring to Gynecology Clinics in Chaharmahal and Bakhtiari, Iran, in 2017. Arch Clin Infect Dis. 2021;16(2):e92537. doi: 10.5812/archcid.92537.

ARTICLE INFORMATION

Archives of Clinical Infectious Diseases: 16 (2); e92537
Published Online: June 28, 2021
Article Type: Research Article
Received: May 5, 2019
Revised: May 13, 2021
Accepted: May 16, 2021
Crossmark
Crossmark
CHECKING
READ FULL TEXT

Abstract

Background: vaginal infections are common among women referring to gynecological clinics worldwide, but treatment modalities cannot provide complete remission of the disease. Laboratory diagnosis of vaginal infections using more sensitive and specific methods is essential for the best treatment options.

Objectives: In this study, diagnosis of bacterial vaginosis (BV) using the polymerase chain reaction (PCR) method was investigated.

Methods: vaginal samples were collected from 635 symptomatic women referring to gynecology clinics in Chaharmahal and Bakhtiari, Iran, in 2017. All samples were then diagnosed using microscopy, culture, and PCR methods.

Results: Of 635 symptomatic women, 200 cases (31.4%) were diagnosed with BV according to the culture method using the PCR method. However, 3.9% of samples who were negative based on the culture method, were diagnosed to have BV based on PCR results.

Conclusions: PCR is more sensitive than culture and microscopy methods for the diagnosis of BV.

1. Background

Vaginal infections are common problems among women, with more than 10 million annual cases in the United States (1). Although the disease is rarely life-threatening, it is associated with high treatment costs, infertility (1), premature labor, and pelvic inflammatory disease (2). Bacterial vaginosis (BV) is the most prevalent vaginal disorder in women of reproductive age (3). It is also the most common cause of abnormal vaginal discharge in women of childbearing age and has been implicated as a cause of the pelvic inflammatory disease (4, 5).

Gardnerella vaginalis, as the main cause of BV, is an anaerobic, catalase, and oxidase-negative bacterium with a mean size of 0.5 to 1.5 mm (6). It is mainly considered as a part of the lower female genital tract microflora (7) and can also be routinely isolated from the male urogenital tract (8-10).

Diagnosis of BV is based on clinical Amsel criteria and direct Gram-stained smear of vaginal secretions. The BVBlue system (Gryphus Diagnostics, L.L.C.) is a chromogenic diagnostic test based on the presence of elevated sialidase enzyme in vaginal fluid samples. The molecular quantification of G. vaginalis (DNA level, ≥ 109 copies/mL) has the highest diagnostic value for the diagnosis of BV, with excellent sensitivity (95%) and specificity (99%) (11).

Although microscopy and culture associated with clinical symptoms are very useful for the diagnosis of bacterial vaginitis, these techniques are not able to differentiate G. vaginalis from other causative agents of vaginal infections.

2. Objectives

In this study, molecular diagnosis of G. vaginalis in vaginal samples of women referring to gynecology clinics in Chaharmahal and Bakhtiari, Iran, in 2017 was investigated.

3. Methods

In this descriptive research, the study population consisted of symptomatic women referring to gynecology clinics in Chaharmahal and Bakhtiari, Iran, in 2017. After obtaining informed consent, a swab from vaginal discharges of 635 women with symptoms of vaginal infections referring to gynecology clinics in Chaharmahal and Bakhtiari in 2017 was collected by a gynecologist. Each swab was then transferred to tubes containing 1 mL sterile saline. A drop of 1 mL of samples was used for microscopic examination. Another drop of each sample was transferred to the Colombia agar medium for culture. The remainder of each 1 mL sample (about 800 µL) was used for the molecular experiment. DNA from clinical samples was extracted using the QIAamp DNA Mini Kit (QIAGEN, USA) in combination with pretreatment with proteinase K and mechanical homogenization. Briefly, vaginal specimens were vortexed vigorously, and 600 mL of the aliquot of UTM-RT transport medium was added and combined with 60 mL proteinase K (Qiagen). GAACCCGTGGAATGGGCC was used as the forward primer and GGGCGGGCTAGAGTGCA as the reverse primer. DNA samples were then amplified using the polymerase chain reaction (PCR) method. Finally, sequences of the products of the PCR were then determined. Species delineation was based on the comparison of the sequences with relevant reference sequences in GenBank, using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast). Nucleotide sequence data generated in this study were deposited in GenBank under accession number “MH898665.1”.

Sequence contigs of the samples were aligned and edited visually in consensus positions, and compared with sequences from GenBank using the Sequencher software v.4.1.4 (Gene Codes Corporation, Ann Arbor, Michigan, USA).

4. Results

Of 635 cases, 200 patients (31.4%) were diagnosed with BV according to the microscopy and culture methods. All samples positive for BV based on the culture method were also positive according to the molecular method. However, 3.9% of the samples that were negative based on the culture method were positive using the molecular method (Table 1).

Table 1. Results of Microscopy, Culture, And PCR for the Diagnosis of Bacterial Vaginosis in 635 Symptomatic Women for Vaginal Infections Referring to Gynecology Clinics in Chaharmahal and Bakhtiari, Iran in 2017
Diagnostic TestPositive ResultsNegative Results
Microscopy98537
Culture200435
PCR224411

When PCR products of DNA of vaginal samples were subjected to electrophoresis a band of 530bp was detected (Figure 1).

Figure 1. Electrophoresis of PCR products of 16srRNA gene of Gardnerella vaginalis. M: ladders, N: normal saline, 1, 2, and 3: samples of patients with G. vaginalis infection, and N1-N3 samples of patients without G. vaginalis infection.

The product of PCR of samples with BV was then subjected to DNA sequencing (Figure 2). After a comparison of the sequences with the reliable sequences in GenBank, the isolated bacteria strain was identified as G. vaginalis.

Figure 2. Sequences results of the PCR products of 16srRNA gene of Gardnerella vaginalis.

5. Discussion

Vaginal infections are common diseases of women referring to gynecological clinics all over the world. BV is the most common identified cause of vaginitis and is a common disorder in women throughout the world. Also, G. vaginalis is the most commonly diagnosed agent in the samples collected from cases with vaginal infections. However, no reliable diagnostic tool is available for the detection of this bacterium. Thus, it is necessary to improve diagnostic tests for the diagnosis of this infection, especially for differential diagnosis of BV from other causes of vaginal infections. Molecular methods, such as PCR and real-time PCR are used for the diagnosis of different diseases. However, for developing a simpler method applicable in clinical laboratories, especially in developing countries, we preferred to select a simple PCR method for the diagnosis of G. vaginalis in vaginal samples. In this research, we used a molecular method, in which less equipment and facilities are needed. Our results showed that 31.4% of samples of symptomatic women for vaginal infection were diagnosed with BV. Also, it has been shown that the PCR method is more sensitive than culture for the diagnosis of BV.

Molecular methods have been used in different investigations for the diagnosis of vaginal infections. Cartwright et al. used nucleic acid amplification (NAA)-based assays for the diagnosis of vaginitis in 323 symptomatic women with vaginal infections. They showed that using the NAA-based method resulted in a significant increase in the accuracy of diagnosis of BV in systematic women (12). Beverly et al. showed that Amsel criteria had a poor predictive value for the diagnosis of BV. They also reported that the PCR method was significantly more sensitive than Amsel criteria for the diagnosis of this infection (13).

Menard et al. found that molecular quantification of G. vaginalis has 96% sensitivity and 99% specificity (11). Obata-Yasuoka et al. used the PCR method for the diagnosis of BV. They found that based on this method, 20.3% of the vaginal samples were diagnosed with BV. The sensitivity and specificity of multiplex PCR in comparison with Gram staining were 78.4 and 82.9, respectively (14). Nancy K. Lowe used PCR for the diagnosis of vaginal infections. They showed that according to this method, 42% of vaginal samples were diagnosed with BV (15).

Makarova et al. compared morphological, bacteriological, serological, and genetic methods for the diagnosis of G. vaginalis. They showed that PCR was more efficient for the diagnosis of G. vaginalis (16). Menard et al. reported a sensitivity of 95% and specificity of 99% for molecular diagnosis of G. vaginalis (11). Balashov et al. using quantitative PCR identified G. vaginalis infection in vaginal samples (17). Janulaitiene, et al. reported that PCR is an efficient method for the detection of G. vaginalis in vaginal specimens (18). Sehgal et al. also reported that qualitative PCR had high sensitivity and specificity for the detection of G. vaginalis and Atopobium vaginae in vaginal samples (19).

The results of these investigations are in agreement with our results, indicating that molecular methods are more sensitive and specific than microscopic methods for the diagnosis of BV. However, they are associated with some limitations to be used in clinical laboratories. Firstly, these methods are time-consuming, and secondly, they need special equipment to be performed. Efforts have been made to develop simple and rapid methods for the diagnosis of vaginal infections (20). Thus, more investigations are needed for finding a rapid and simple method with high sensitivity and specificity.

5.1. Conclusion

Our results showed that PCR is more sensitive than microscopy and culture methods for laboratory diagnosis of BV.

Acknowledgements

Footnotes

References

  • 1.

    Martius J, Roos T. The role of urogenital tract infections in the etiology of preterm birth: a review. Arch Gynecol Obstet. 1996;258(1):1-19. doi: 10.1007/BF01370927. [PubMed: 8789428].

  • 2.

    Morales WJ, Schorr S, Albritton J. Effect of metronidazole in patients with preterm birth in preceding pregnancy and bacterial vaginosis: A placebo-controlled, double-blind study. Am. J. Obstet. Gynecol. 1994;171(2):345-9. doi: 10.1016/s0002-9378(94)70033-8.

  • 3.

    Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK. Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes. Microbiology (Reading). 2010;156(Pt 2):392-9. doi: 10.1099/mic.0.034280-0. [PubMed: 19910411]. [PubMed Central: PMC2890091].

  • 4.

    Sweet RL. Role of bacterial vaginosis in pelvic inflammatory disease. Clin Infect Dis. 1995;20 Suppl 2:S271-5. doi: 10.1093/clinids/20.supplement_2.s271. [PubMed: 7548573].

  • 5.

    Hauth JC, Goldenberg RL, Andrews WW, DuBard MB, Copper RL. Reduced incidence of preterm delivery with metronidazole and erythromycin in women with bacterial vaginosis. N Engl J Med. 1995;333(26):1732-6. doi: 10.1056/NEJM199512283332603. [PubMed: 7491136].

  • 6.

    Greenwood JR, Pickett MJ. Transfer of Haemophilus vaginalis Gardner and Dukes to a New Genus, Gardnerella: G. vaginalis (Gardner and Dukes) comb. nov. Int J Syst Bacteriol. 1980;30(1):170-8. doi: 10.1099/00207713-30-1-170.

  • 7.

    Catlin BW. Gardnerella vaginalis: characteristics, clinical considerations, and controversies. Clin Microbiol Rev. 1992;5(3):213-37. doi: 10.1128/CMR.5.3.213. [PubMed: 1498765]. [PubMed Central: PMC358241].

  • 8.

    Briselden AM, Hillier SL. Evaluation of affirm VP microbial identification test for Gardnerella vaginalis and Trichomonas vaginalis. J Clin Microbiol. 1994;32(1):148-52. doi: 10.1128/jcm.32.1.148-152.1994. [PubMed: 8126171]. [PubMed Central: PMC262986].

  • 9.

    Eren AM, Zozaya M, Taylor CM, Dowd SE, Martin DH, et al. Exploring the diversity of Gardnerella vaginalis in the genitourinary tract microbiota of monogamous couples through subtle nucleotide variation. PLoS One. 2011;6(10). e26732. doi: 10.1371/journal.pone.0026732. [PubMed: 22046340]. [PubMed Central: PMC3201972].

  • 10.

    Swidsinski A, Doerffel Y, Loening-Baucke V, Swidsinski S, Verstraelen H, Vaneechoutte M, et al. Gardnerella biofilm involves females and males and is transmitted sexually. Gynecol Obstet Invest. 2010;70(4):256-63. doi: 10.1159/000314015. [PubMed: 21051845].

  • 11.

    Menard JP, Fenollar F, Henry M, Bretelle F, Raoult D. Molecular quantification of Gardnerella vaginalis and Atopobium vaginae loads to predict bacterial vaginosis. Clin Infect Dis. 2008;47(1):33-43. doi: 10.1086/588661. [PubMed: 18513147].

  • 12.

    Cartwright CP, Lembke BD, Ramachandran K, Body BA, Nye MB, Rivers CA, et al. Comparison of nucleic acid amplification assays with BD affirm VPIII for diagnosis of vaginitis in symptomatic women. J Clin Microbiol. 2013;51(11):3694-9. doi: 10.1128/JCM.01537-13. [PubMed: 23985917]. [PubMed Central: PMC3889753].

  • 13.

    Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, Spear GT. Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women. J Clin Microbiol. 2005;43(9):4607-12. doi: 10.1128/JCM.43.9.4607-4612.2005. [PubMed: 16145114]. [PubMed Central: PMC1234056].

  • 14.

    Obata-Yasuoka M, Ba-Thein W, Hamada H, Hayashi H. A multiplex polymerase chain reaction-based diagnostic method for bacterial vaginosis. Obstet Gynecol. 2002;100(4):759-64. doi: 10.1016/s0029-7844(02)02201-9. [PubMed: 12383546].

  • 15.

    Lowe NK, Neal JL, Ryan-Wenger NA. Accuracy of the clinical diagnosis of vaginitis compared with a DNA probe laboratory standard. Obstet Gynecol. 2009;113(1):89-95. doi: 10.1097/AOG.0b013e3181909f63. [PubMed: 19104364]. [PubMed Central: PMC2745984].

  • 16.

    Makarova LN, Kravtsov EG, Vasil'eva EA, Dmitriev GA, Medvedeva EA, Kirillov MY, et al. Modern methods for diagnosis of Gardnerella infection. Bull Exp Biol Med. 2000;130(8):780-2. doi: 10.1007/BF02766094. [PubMed: 11177243].

  • 17.

    Balashov SV, Mordechai E, Adelson ME, Gygax SE. Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR. J Med Microbiol. 2014;63(Pt 2):162-75. doi: 10.1099/jmm.0.066407-0. [PubMed: 24200640].

  • 18.

    Janulaitiene M, Paliulyte V, Grinceviciene S, Zakareviciene J, Vladisauskiene A, Marcinkute A, et al. Prevalence and distribution of Gardnerella vaginalis subgroups in women with and without bacterial vaginosis. BMC Infect Dis. 2017;17(1):394. doi: 10.1186/s12879-017-2501-y. [PubMed: 28583109]. [PubMed Central: PMC5460423].

  • 19.

    Sehgal PG, Dadwal R, Sharma B, Sehgal A, Bagga R, Chopra S, et al. Detection of co-infection of Gardnerella vaginalis and Atopobium vaginae using qualitative PCR: A better predictor of bacterial vaginosis. Anaerobe. 2021;69:102343. doi: 10.1016/j.anaerobe.2021.102343. [PubMed: 33582302].

  • 20.

    Hashemi H, Varshosaz J, Fazeli H, Sharafi SM, Mirhendi H, Chadeganipour M, et al. Rapid differential diagnosis of vaginal infections using gold nanoparticles coated with specific antibodies. Med Microbiol Immunol. 2019;208(6):773-80. doi: 10.1007/s00430-019-00622-9. [PubMed: 31183547].

  • Copyright © 2021, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
    COMMENTS

    LEAVE A COMMENT HERE: