Vaginal symptoms are commonly caused by yeast, Trichomonas vaginalis, and bacterial vaginosis. Staphylococcus aureus and Beta-hemolytic streptococcus Group A (GAS) may be associated with Toxic Shock Syndrome. Occasionally, Beta-hemolytic streptococci can cause purulent vaginitis.
Bacterial vaginosis (BV) is an unusual “ecologic” condition characterized by a large change in the bacterial flora in the vagina. The normal Lactobacillus sp. are replaced by various other species. This is not accompanied by inflammation which is the reason for calling it bacterial vaginOSIS rather than bacterial vaginitis. BV only occurs in women of child-bearing age which we arbitrarily define as age 12-55. If the typical microscopic changes of BV are present in girls less than 12 it may reflect early puberty and if present in women older than 55 it often is a result of hormone replacement therapy.
Examine for the presence of yeast, clue cells, organisms associated with bacterial vaginosis and polymorphonuclear leukocytes (PMNs).
Wet preparation (if Trichomonas vaginalis is requested and sample less than 24 hrs old)
To be set up immediately. Gently press the swab into a drop of sterile saline on a slide. Place a cover slip on the slide and examine under the microscope using the 40 X objective.
Gram Stain is the only routinely performed process. However, there are 3 circumstances that prompt culture:
- If Group B streptococcus is requested process as regular vaginal/rectal Group B streptococcal screen.
- Gram stain reveals 3 or 4 + WBCs and a predominance of Gram-positive cocci resembling streptococci.
- Toxic shock syndrome is indicated as a suspected diagnosis.
If 3 – 4 + WBCs and streptococci in smear set up:
|Selective Streptococcus Agar (SSA)||CO2, 35°C x 48 hours|
If toxic shock syndrome is suspected set up:
|Blood Agar (BA)||CO2, 35°C x 48 hours|
|Phenylethyl Alcohol Blood Agar (PEA)||O2, 35°C x 48 hours|
Scoring and Grading for Bacterial Vaginosis
Gram-stained slides are examined under oil immersion (×1000). Smears are observed and quantitated for the presence of the following morphotypes:
- Large Gram-positive bacilli (Lactobacillus morphotypes)
- Small Gram-variable bacilli (Gardnerella morphotypes)
- Curved Gram-negative or gram-variable bacilli (Mobiluncus morphotypes)
- Gram-negative cocco-bacilli (Bacteroides morphotypes)
The number of organisms seen are quantified according to the following scale:
- 1 + = <1 organism per field
- 2 + = 1–4 organisms per field
- 3 + = 5–30 organisms per field
- 4 + = >30 organisms per field
A total numerical score is calculated by summing the scores for the three components as indicated in the following table:
|Lactobacillus||Score||Gardnerella or Bacteroides||Score||Mobiluncus||Score|
|+||+||= Total Score|
Examine the SSA or BA or PEA plate for colonies suspicious of S. aureus, Group A streptococcus and Group B streptococcus.
Send S. aureus isolates to PHL for toxin testing and freeze all toxin-producing strains.
Wet Preparation (if requested)
Negative Report “No Trichomonas seen. Negative results may not be reliable if specimen was either refrigerated or delayed in transport to the laboratory. A fresh specimen is recommended for reliable trichomonas examination.”
Positive Report “Trichomonas vaginalis seen.”
Report PMNs and streptococci only if Gram stain reveals 3 or 4 + WBCs and a predominance of Gram-positive cocci resembling streptococci. (i.e. If a culture has been prompted report the Gram stain with quantitation)
Bacterial Vaginosis The total score is interpreted and reported as follows: 0 – 3 “Gram stain indicates normal bacterial vaginal flora” 4 – 6 “Gram stain reveals altered vaginal flora that is not diagnostic of Bacterial Vaginosis but not normal. This frequently represents a transitional stage. If signs and/or symptoms persist, repeat testing is warranted” 7 – 10 “Gram stain consistent with Bacterial Vaginosis”
Yeast Negative Report:
“No microscopic evidence of candidiasis” or “No microscopic evidence of bacterial vaginosis on candidiasis”
“Microscopic evidence of candidiasis”
If toxic shock syndrome requested: “No Staphylococcus aureus or beta hemolytic streptococcus isolated.”
If toxic shock syndrome requested:
Report all significant isolates with appropriate susceptibilities. Do not quantitate except S. aureus.
If Set up because of 3-4+ WBCs and Gram-positive cocci on smear:
Report Beta-hemolytic streptococcus Group A or B with comment “Gram stained smear showed large numbers of WBCs, Gram positive cocci (resembling streptococcus) and an absence of normal flora. While controversial, some experts believe Group B and A Streptococci are rare causes of purulent vaginitis. Consideration to systemic or topical antibiotic therapy may be given.”
Cumitech 17A, 1993. “Lab. Diagnosis of Female Genital Tract Infections, ASM Press.
LPTP Survey B-9412, Feb. 21, 1995. Microbiology Handling of Female Genital Specimens. A Pattern of Practice Survey.
Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis. A newly defined specific infection previously classified “nonspecific” vaginitis. Am J Obstet Gynecol 1955;69:962-976.
Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983;74(1):14-22.
Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991;29(2):297-301.
Spiegel CA, Amsel R, Holmes KK. Diagnosis of bacterial vaginosis by direct gram stain of vaginal fluid. J Clin Microbiol 1983;18(1):170-7.
Spiegel CA. Bacterial vaginosis. Clin Microbiol Rev 1991;4(4):485-502.
Spiegel CA. Bacterial vaginosis: changes in laboratory practice. Clin Microbiol Newsletter 1999;21:33-37.