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Association between Lactobacillus species and bacterial vaginosis-related bacteria, and bacterial vaginosis scores in small population of pregnant Latvian women

Natalija Berza1,2*, Jana Zodzika2, Juta Kroica1, Aigars Reinis1, Ingus Skadins1, Linda Piekuse1, Polina Zalizko1, Olita Melngaile2, Rita Pundure2, Irina Lukojanova2, Olga Vasina2
  1. Department of Biology and Microbiology, Riga Stradins University, Dzirciema Street 16, Riga, Latvia
  2. Department of Gynecology and Obstetrics of Riga Stradins University, Dzirciema Street 16, Riga, Latvia
Corresponding Author: Natalija Berza, Lokomotives Street 70a-27, Riga, LV-1057, Latvia Email: [email protected]
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Abstract

Background: One of the most common reasons why females attend doctor are vaginal infections. Vaginal flora is a dynamic environment where a great variety of microorganisms exist in homeostasis. The main normal flora inhabitants are Lactobacillus species who protect from pathogens. Still the majority of factors remain unclear about this genteel environment and its interaction. Aim & Objectives: to analyze vaginal microflora types and microbial species in pregnant women, who were at their first trimester, using PCR and Nugent score diagnostic methods. Methods: 65 pregnant women before their 12th week of pregnancy participated in this study from 06.08.2012 till 31.01.2013. All participants were divided in to 2 groups, group A (n=45) normal pH and group B (n=20) pH ( ≥4,5). Their vaginal fluid were analysed with Nugent score and PCR methods. Results: Genus Lactobacillus (any Lactobacillus) was detected by PCR in all women irrespective of Nugent score, most common species were L. crispatus, L. jensenii, and L. inners, less common were L. gasseri, L. plantaris, L. rhamnosus and L. reuteri. G. vaginalis was present in both patient groups divided by pH but it was significantly higher in bacterial vaginosis and intermediate flora group detected by Nugent score and group B (high pH group). Megasphaera, Leptotrichia/Sneathia were detected more common in pathogenic flora than normal flora. A. vaginae finding was associated with pathologic flora. Conclusion: The most common isolated species in pregnant women vaginal flora were L. crispatus, L. jensenii, and L. inners. L. gasseri and L. plantaris were second most often found species. L. jensenii detection was significantly higher in normal flora group. A. vaginae were mainly detected in patients with bacterial vaginosis. Megasphera and Leptotrichia/Sneathia are more common for patients with pathologic flora. PCR method is the most precise to identify microorganisms in vaginal flora but rather expensive and time consuming than vaginal fluid examination by Nugent score

Keywords

Vaginal microflora, PCR, Lactobacillus, Nugent score.

Introduction

One of the most common gynaecological problems is vaginal infections like bacterial vaginosis (BV), Candida vulvovaginitis, trichomonal infection and aerobic vaginitis (AV). BV is caused by an overgrowth of Gardnerella vaginalis (G.vaginalis), anaerobes, Mycoplasma hominis (M.hominis), Ureaplasma urealyticum (U.urealyticum) and clinically diagnosed by Amsel criteria: presence of thin, grayish homogenous discharge; vaginal pH greater than 4.5; presence of clue cells, positive whiff test (detection/enhancement of fishy odor on additions of potassium hydroxide to the vaginal specimen). AV is absence of lactobacilli, presence of cocci or coarse bacilli, parabasal epithelial cells, vaginal leucocytes. AV is associated with growth of group B streptococci (GBS), E.coli, Staphylococcus aureus (S.aureus), has different immunological inflammation reaction and clinical signs – such as red, inflamed vaginal mucosa, yellowish sticky discharge, high vaginal pH, “not fish-like” odor.1,2 Vaginal infection especially bacterial vaginosis and aerobic vaginitis can be the reason for such complications as preterm delivery, horioamnionitis and low birth weight. The studies with antibiotic treatment in high risk preterm delivery pregnancies have not proven to be effective except clindamycin, but more studies are required.3,4
Mostly these infections are asymptomatic, 60% of patients that have bacteria vaginosis (BV) had no complains.5 It is important to discover all cases of vaginal infections early, because such deviations before 14th week of delivery can be as risk factor for preterm delivery.3
There are different diagnostic methods for vaginal infections – pH measure, KOH test, visual vaginal discharge evaluation, dark field microscopy, Grams staining microscopy, cultivation on artificial media and polymerase chain reaction (PCR). There is still a great dilemma - which method is easier, cheaper and more widely available for the specialist. Vaginal pH level is important diagnostic criteria which can be helpful in asymptomatic cases of BV but how precise this method is comparing to other diagnostic methods? 3
Nowadays, molecular diagnostic methods are becoming more popular, because they provide precise information about species of normal and pathogenic vaginal flora inhabitants. It is not fully understood how microorganisms determined with molecular methods correlate with basic daily tests like pH and Nugent score.6 Studies show that vaginal microbial composition is dependent on a variety of factors, including geographical, for example, the normal female flora in African women is considered as pathogenic for other region women.7-10 The most common lactobacillus in India is considered L.reuteri, but in Finland L.crispatus.11,12 There were no study data about which Lactobacillus and pathogenic microflora species present in the vaginal flora of Latvian women. So it was quite important to maintain this information and compare it with world data and also find the correlation between molecular diagnostic methods and Nugent score.13
The goal of this study was to analyze different vaginal flora types by Grams staining and PCR in pregnant women during their first trimester of pregnancy with a special emphasis on lactobacilli and pathogenic bacteria species connected with bacterial vaginosis.

Methods

A total of 65 pregnant Latvian women were enrolled in this study during routine prenatal visits at SIA "Dzirciema Clinic" Ltd "Aura R" and the "Jugla Medical Center" from August 2012 to February 2013. Informed consent was obtained from all participants in verbal and written form. All necessary approvals were received from Riga Stradins University ethical comity. Including criteria in the study were pregnant women older than 18 years with no serious extragenital abnormalities in their 6-12 week of pregnancy. Estimated date of delivery was determined from the last menstrual period and early gestational fetal ultrasonographic measurements. Patients were divided into 2 groups: group A with normal pH (<4,5) and group B with elevated pH (≥4,5), pH set to Machery Nagel pH strips (measuring range 3.6-7.0). A sterile speculum was inserted into the vagina and two specimens of vaginal fluid were obtained by brushing the posterior vaginal fornix with a swab. A vaginal smear was prepared by rolling a swab onto a glass slide, which was then air-dried, heat-fixed, and Gram-stained. The smears were then assessed according to Nugent criteria (Table 3). Nugent score for the diagnosis of BV is ≥ 7 and it is considered as pathogenic flora, intermediate flora 4-6 and for normal flora ≤ 3. Overall, the Nugent scoring system for Gram –stained vaginal smears has shown high intracenter and intercenter reliability and reproducibility, however practitioners are not usually familiar with performing in-office Gram-stain-based diagnosis, Nugent’s criteria are widely applied in the absence of standardized pre-analytical and analytical conditions and interpretation, especially of the so called intermediate flora, is also a matter of concern.1,6,14 Second specimen was placed in Amies media and immediately transported for molecular investigation.
Swab from Amies media was placed 2 ml containers and the pellet was digested with proteinase K at 56°C for 60–90 min and the DNA was extracted and purified with a QIAmp DNA Investigator Kit (Qiagen, Germantown, MD) in accordance with the manufacturer's instructions, resulting in 100 μl of DNA solution. PCR mixtures consisted of PCR buffer with 1.5 mM of MgCl2, 10 pmol of each primer, 2.0 μM of each deoxyribonucleoside triphosphate, 0.1 μl of Taq DNA polymerase, and 1 μl of template DNA solution in a final volume of 25 μl.
Sequences and annealing temperatures for the various primer sets are listed in 1st table. All primers were located in the 16S rDNA region. PCR was carried out for 40 cycles. For the Lactobacillus genus and its four species, the denaturation was performed at 95°C for 15 sec followed by a 1-min annealing and extension step. For four BV-related bacteria, the denaturation step was set at 94°C for 30 sec, followed by the annealing step for 40 s, with extension at 72°C for 1 min for all reactions. A final extension step at 72°C for 7 min was added for all reactions. Aliquots of 8 μl of the PCR products were electrophoreses in agarose gels and visualized by ultraviolet transillumination after ethidium bromide staining.
The statistical analysis was made using SPSS, Chi square test and Pearson correlation was performed.

Results

There were 65 pregnant women included in the study 45 with pH < 4,5 and 20 with pH ≥ 4,5. The mean age in both groups were 28 ± 5.2. Minimal age of participant was 18 and maximal– 43.
Comparing both pH and Nugent score diagnostic methods statistically significant difference was found (X 2= 6,607; p=0,01), high pH was measured only in 17% of participants, but Nugent score shoed pathogenic flora in 37% of participants, (diagram 1).
Based on our data pH test sensitivity compared with Grams staining method was 54% and specificity 88%.
Genus Lactobacillus (any Lactobacillus) was detected by PCR in all women irrespective of Nugent score, the most common species were L. crispatus, L. jensenii, and L. inners, less common were L. gasseri, L. plantaris, but least likely was L. rhamnosus and L. reuteri species which were found only in two patients specimen (diagram 2).
L. jensenii was detected in normal vaginal flora significantly more frequently (p<0,01) than in pathogenic flora, but in L. inners detection frequency was no significant difference both in normal and pathogenic flora.
Gardnerella vaginalis was detected in both normal and pathogenic group, but in BV and middle flora by Nugent score and high pH group it was detected more often (85-90% of cases) compared with normal pH and 0-3 Nugent scores (67-73%). Megasphaera, Leptotrichia were found less frequent in 0-3 Nugent scores, but Leptotrichia detection in both pH groups did not differ statistically. An A. vagina was detected mainly in pathogenic flora group.
The incidence of microorganisms, detected with PCR, in vaginal flora, depending on the diagnostic method are displayed in 2nd table.
Performing Pearson correlation test on all summarised data some significant correlations was discovered. The correlation between Nugent and pH test was moderate (r=0,453; p<0,01) also there was found moderate correlation between presence of Megasphera spp. with Leptotrichia spp. and G. vaginalis in vaginal flora (r=0,367;p<0,01 and r=0,324;p<0,01). Finding A. vaginae in vaginal flora closely correlated with the pathogenic findings of flora (r = 0,7, p <0,01) .

Discussion

Vaginal flora is delicate and dynamic system, with dominating inhabitant Lactobacillus species in the majority of women.10,11,17,18
This study included only small female population – first trimester pregnant women. In this study was confirmed by species-specific 16S rDNA gene PCR that L. crispatus, L. inners and L. jensenii are the most common species in Latvian pregnant women normal flora, that does not differ from the Finnish and Japanese data.6,14,19 The incidence of L. gasseri in other similar studies match our data.19,20
L. jensenii detection in the normal flora group was higher while L. inners frequency did not differ between both groups, which was also confirmed in other similar studies.14
A. vaginae, G. vaginalis, Megasphera mainly prevailed in abnormal vaginal flora that did not differ from the global data. Interesting was the fact that one pathogenic bacteria correlated with each other, but there was no similar data in literature. 14, 21, 22 A. vaginae finding significantly correlated with pathogenic flora that is important because the literature describes a microorganism high resistance to metronidazole and susceptibility to clarithromycin. It means that in bacterial vaginosis treatment the use of metronidazole alone, which is effective against G. vaginalis, may not give the expected result.4,14
Comparing Grams staining method with pH measurements we found moderate correlation, but the vaginal pH measurements did not show all abnormal flora cases. However the pH test is specific enough, but with a low sensitivity, because not in all cases of abnormal flora pH is increased (≥ 4.5), so if only pH diagnostic method is used there may be many undiagnosed abnormal vaginal microflora cases, this makes it necessary to supplement this method of investigation with vaginal discharge microscopy.1,3,17,18 Microscopy data were also compared with PCR. Although, the PCR can accurately identify the composition of bacteria in the vagina, but it is expensive and it has some drawbacks. There is no precise criteria for interpreting the normal or pathogenic, and using conventional polymerase chain method it can only prove the presence of microorganism in the vagina, but not the number of colony forming units, while Gram staining microscopy is simple vaginal flora diagnostic techniques that have established criteria for diagnostic the pathology.1,3,17,18
Unfortunately, in our country, such studies using PCR have not been performed so far. Our study group was relatively small, and analyzed cases only in Riga, therefore there is no clear vision on our region vaginal microflora nuances. During the study, we confronted with several problems and one of them was number of participants, although patient involvement lasted 6 months, the normal pH group included 45 of the expected 50 and a high pH group, only 20 of the 50 samples, this can be explained by the small population of our country. Patient involvement will continue to reach the necessary number of respondents. The other problem of the study was the impossibility of using real-time PCR, because of study costs.
Overall this study gave a deeper insight into the occurring lactobacilli species in the Latvia. This is the root of future studies that will allow us to gain a deeper understanding of the vaginal flora in the Baltic region.

Conclusion

The most often isolated lactic acid bacteria in the vagina of pregnant Latvian women are L. crispatus, L. jensenii and L. inners less common are L. gasseri and L. plantaris. L. jensenii is the most common isolated lactic acid producing bacteria in normal flora of pregnant Latvian women. A. vaginae, G. vaginalis, Megasphera are the most common microbes found in pathogenic flora of vagina. Grams’ staining method and vaginal pH measurements correlate with each other, but the vaginal pH measurements does not show all abnormal flora cases. In order to evaluate the vaginal environment for pregnant women, in addition to vaginal pH measurement is necessary for additional vaginal microflora diagnostics. Although the PCR can accurately identify the composition of bacteria in the vagina it is expensive, while the Grams staining microscopy is a simple vaginal flora change diagnostic methods. In order to better conclude on normal and abnormal vaginal microflora composition in Latvian women the further research are necessary.

Conflict of Interest

None declared.
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References

  1. Hayes SN. Preventing Cardiovascular Disease in Women. American Family Practice. 2006;74:1331-1340.

  2. Welty FK. Women and Cardiovascular risk. Am J Cardiology .2001;88:48-52.

  3. Bello N and Mosca L. Epidemiology of Coronary Heart Disease in Women. Prog Cardiovasc Dis .2004;46:287-295.

  4. Wenger NK. You've come a long way baby: Cardiovascular health and disease in women: problem and prospects. Circulation. 2004;109:558-60.

  5. Donald ML-J, Martin GL, Alexa B and David L. Lifetime risk of developing coronary heart disease. The Lancet. 1999; 353:89-92.

  6. Khoo KL, Tan H, Sambhi SJ, Aljafri AM, Hatijah A. Screening for blood pressure,cholesterol and glucose during National Heart Weeks 1992-1994. Malaysia Med Journal. 1996;51:307-315

  7. Khoo KL, Tan H, Liew YM. Serum lipid and their relationship with other coronary risk factors in health subjects in a city clinic. Malaysia Med Journal .1997;52:38-52.

  8. Population distribution and basic demographic characteristic report population and housing census 2000. In: Department PM, editor. Putrajaya; 2001.

  9. Prevention of Cardiovascular disease in women. In: Health Mo, editor. first ed. Putrajaya;2008.

  10. Celentano A, Palmieri V, Arezzi E, Sabatella M, Guillaro B, Brancati C, et al. Cardiovascular secondary prevention: patients' knowledge of cardiovascular risk factors and their attitude to reduce the risk burden, and the practice of family doctors. The "Help Your Heart Stay Young" study. Italian Heart Journal. 2004;5(10):767-773.

  11. Oliver-McNeil S, Artinian NT. Women’s Perceptions of Personal Cardiovascular Risk and Their Risk-Reducing Behaviors. American Journal of Critical Care. 2002;11(3):221-227.

  12. Fishbein M. The role of theory in HIV prevention. AIDS Care .2000;12(3):273-278.

  13. Khattab MS, Abolfotouh MA, Alakija W, Al-Humaidi MA, Al-Wahat S. Risk factors of coronary heart disease:attitude and behaviour in family practice in Saudi Arabia. Eastern Mediterranean Health Journal. 1999;5(1):35-45.

  14. Khanam S, Costarelli V. Attitudes towards health and exercise of overweight women. The Journal of the Royal Society for the Promotion of Health 2008;128(1):26-30.

  15. Kannel WB. Silent myocardial ischemia and infarction: insights from the ramingham study. Cardiol clin. 1986;4:583-591.

  16. Department of Statistics Malaysia. In: Kelantan @ a glance .2011.

  17. Kelantan Health Department. List of Health Clinics under Kota District Health Office in Kelantan. Kota Bharu; 2011.

  18. Rosediani M, Ranimah Y, Harmy MY. Knowledge , attitude and practice on Cardiovascular Disease among Women in North-Eastcoast alaysia.International Journal of Collaborative Research on Internal Medicine and Public Health. 2012; 4(1):86-97

  19. Mosca L, Jones WK, King KB, Quyang P, Redberg RF, Hill MN.Awareness,perception, and knowledge of heart disease risk and prevention among women in the United states. American Heart Association Women’s Heart Disease and Stroke Campaign Task Force. Awareness, perception and knowledge of heart disease risk and prevention among women in the United States. Arch Fam Med. 2000;9:506-15.

  20. Chia Y, Keevil V. Trend of Cardiovascular Disease Risk Profile of Patients Treated for Hypertension Over A 10 Year Period in A Primary Care Clinic in Malaysia:. Journal of Hypertension.2010; 28:15.58

  21. Mafauzy M, Lekhraj Rampal GR, Abdul Rashid AR, Mustaffa BE. A Prelimenary Result Of The Cardiovascular Risk Factor Intervention Study (PIKOM STUDY): Diabetes Mellitus, Hypertension And Their Associated Factors. Malaysian Journal of Medical Science 2005;12(1):20-25.

  22. Roger VrL, Go AS, Lloyd-Jones DM, Adams RJ, Berry JD, Brown TM, et al. Heart Disease and Stroke Statistic Update. Circulation .2011;123(4):e18-e209.

  23. Vanhecke TE, Miller WM, Franklin BA, Weber JE, McCullough PA. Awareness,

  24. knowledge, and perception of heart disease among adolescents. European Journal of Cardiovascular Prevention & Rehabilitation. 2006;13(5):718-723.

  25. Frost R. Cardiovascular risk modification in the college student. Journal of General Internal Medicine. 1992;7(3):317-320.

  26. Green JS, Grant M, Hill KL, Brizzolara J, Belrnont B. Heart Disease Risk Perception in College Men and Women. Journal of American College Health. 2003;51:207-211.

  27. Leete R. Kelantan’s Human Development Progress and Challenges. In: United Nation Development Program; 2004.

  28. Ribisl KM, , Winkleby MA, Fortmann SP, Flora JA. The Interplay of socioeconomic status and ethnicity on Hispanic and White men's cardiovascular disease risk and health communication pattern. Health Education Research .1998;13:407417.

  29. Percentage Distribution of Households by income class Malaysia, 1970-2009.

  30. Ninth Malaysia plan 2006-2010. Achieving Growth With Distribution. 2010:323-353.

  31. Abdulla K. Accesibility To Knowledge and Information On Risk Factors And Prevention Of Cardiovascular Disease and Preventive Behaviours Of Residents Above 30 Years in Male Maldives. Bangkok: Chulalongkorn University 2008.

  32. Morrow M, Barraclough S. Tobacco control and gender in Southeast Asia. Part I:Malaysia and the Philippines. Health Promotion International. 2003;18(3):255-264.

  33. Tiwari R. Tobacco use and cardiovascular disease: A knowledge, attitude and practice study in rural Kerala. Indian J Med Sci. 2006;60:271-276.

  34. He J, Allen K, Prerost MR, Hughes J and Paul K Whelton PK. Passive Smoking and The Risk of Coronary Heart Disease- A Meta- analysis of Epidemiologic Studies. New England Journal of Medicine .1999;340:920-926.

  35. Mohamed M. Diabetes Mellitus In Malaysia. Med J Malaysia. 2006;61(4).

  36. Mafauzy M, Mokhtar N, Wan Mohamad WB. Hypertension and associated cardiovascular risk factors in Kelantan Med J Malaysia .2003;58(4):556-564.

  37. Wan Mohamad WB, Mafauzy M, Mustaffa BE, Musalmah M. Prevalance of obesity and overweight in northeastern peninsular Malaysia and their relationship with cardiovascular risk factors. Southeast Asian J Trop Med Public Health. 1996;27:339-342.

  38. Noraza AR, Harmy MY, Rosediani M. Optimal Cardiovascular Screening Activities:Prevalence and its associated factors among apparently healthy school teachers in Kota Bharu Malaysia: VDM Verlag Dr. Muller GmbH & Co. KG; 2010.

  39. Mosca L, Mochari-Greenberger H, Dolor RJ, Newby LK, Robb KJ. Twelve-Year Follow-Up of American Women: Awareness of Cardiovascular Disease Risk and Barriers to Heart Health. Circulation .2010 ;3(2):120-127.

  40. Mosca L, Ferris A, Fabunmi R, Robertson RM. Tracking women's awareness of heart disease: an American Heart Association national study. Circulation. 2004;109(5):573-579.

  41. Yarnall KSH, Pollak KI, Ostbye T, Krause KM, Michener JL. Primary Care: Is There Enough Time for Prevention? Am J Public Health. 2003;93(4):635-641.

  42. Smalley SE, Wittler RR and Oliverson RH. Adolescent assessment of ardiovascular heart disease risk factor attitudes and habits. The Journal of Adolescent Health.2004;35(5):374-379.

  43. Jafary F, Aslam F, Mahmud H, Waheed A, Shakir M, Afzal A, et al. Cardiovascular health knowledge and behavior in patient attendants at four tertiary care hospitals in Pakistan - a cause for concern. BMC Public Health .2005;5(1):124.

  44. Wilt S, Hubbard A andThomas A . Knowledge, Attittude, Treatment Practices and Health Behaviors of Nurses regarding Blood Cholesterol and Cardiovascular Disease.Preventive Medicine 1990;19:466-475.

  45. Avis NE, McKinlay JB, Smith KW. Is cardiovascular risk factor knowledge sufficient to influence behavior? American Journal of Preventive Medicine 1990;6(3):137-144.

  46. Robinson JG, Fox KM, Grandy S, Bays H, Bazata DD, Gavin Iii JR, et al. Attitudes about health and health-related behaviors in patients with cardiovascular disease or at elevated risk for cardiovascular disease. Preventive Cardiology .2009;12(3):136-143.

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