Evaluation of acridine orange stain for detection of Trichomonas vaginalis in vaginal specimens.

Vaginal exudates from 105 patients were examined by direct wet-mount microscopy and acridine orange stain for the presence of Trichomonas vaginalis. Both procedures demonstrated greater than 90% agreement in both sensitivity and specificity. When specimens can be examined immediately after collection, it appears that wet-mount microscopy is almost as sensitive as acridine orange staining for detection of T. vaginalis.     

Use of an immunochromatographic assay for rapid detection of Trichomonas vaginalis in vaginal specimens

Trichomonas vaginalis infection is estimated to be the most widely prevalent nonviral sexually transmitted infection in the world. Wet-mount microscopy is the most common diagnostic method, although it is less sensitive than culture. The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, Mass.) (referred to here as OSOM) is a new point-of-care diagnostic assay for T. vaginalis that uses an immunochromatographic capillary flow (dipstick) assay and provides results in 10 min. The purpose of this study was to determine the test characteristics of OSOM compared to those of a composite reference standard (CRS) comprised of wet-mount microscopy and T. vaginalis culture. This multicenter cross-sectional study enrolled sexually active women > or =18 years of age who presented with symptoms of vaginitis, exposure to T. vaginalis, or multiple sexual partners. Vaginal-swab specimens were obtained for T. vaginalis culture, wet mount, and rapid testing. The prevalence of T. vaginalis in this sample was 23.4% (105 of 449) by the CRS. The sensitivity and specificity of OSOM vaginal-swab specimens were 83.3 and 98.8%, respectively, while wet mount had a sensitivity and specificity of 71.4 and 100%, respectively, compared to the CRS. OSOM performed significantly better than wet mount (P = 0.004) and detected T. vaginalis in samples that required 48 to 72 h of incubation prior to becoming culture positive. The performance of the rapid test was not affected by the presence of coinfections with chlamydia and gonorrhea. The OSOM Trichomonas Rapid Test is a simple, objective test that can be expected to improve the diagnosis of T. vaginalis, especially where microscopy and culture are unavailable.     

Evaluation of affirm VP Microbial Identification Test for Gardnerella vaginalis and Trichomonas vaginalis.

A commercial system (Affirm VP Microbial Identification Test; MicroProbe Corp.) for detection of vaginal pathogens was evaluated with 176 consecutive women attending a sexually transmitted disease clinic for genital complaints. Vaginal swab specimens were used for culture of Gardnerella vaginalis and Trichomonas vaginalis, preparation of a vaginal smear for Gram stain interpretation, and wet mount evaluation. An additional swab was used to evaluate the 30-min nonisotopic oligonucleotide probe test. The automated probe system detected G. vaginalis in 69 (95%) of 73 women having > 5 x 10(5) CFU of G. vaginalis per ml by culture, and 20 (43%) of 47 specimens with < or = 5 x 10(5) CFU of G. vaginalis per ml. There were three false positives and four false negatives for the Affirm VP test compared with > 5 x 10(5) CFU of G. vaginalis per ml. The probe system detected G. vaginalis in 57 (90%) of 63 vaginal specimens from women having clue cells on wet mount examination, and in only 3 (3%) of 113 women without clue cells, suggesting that the Affirm probe for G. vaginalis could be used as a surrogate for wet mount examination for clue cells. The T. vaginalis probe was positive for 12 of 12 specimens positive by wet mount and 12 of 15 specimens positive by culture. There were no false positives and three false negatives for the Affirm VP test compared with culture and/or wet mount for T. vaginalis. The Affirm VP Microbial Identification System is a rapid, objective, and automated test for the detection of T. vaginalis and clinically significant levels of G. vaginalis that is comparable to wet mount examination for clue cells and is superior to wet mount examination for the detection of trichomonads.     

Evaluation of the enhanced rapid identification method for Gardnerella vaginalis.

The enhanced rapid identification method (RIM; Austin Biological Laboratories), a micromethod for the identification of Gardnerella vaginalis, is based on starch and raffinose fermentation and hippurate hydrolysis. We tested 105 clinical isolates of G. vaginalis with both the RIM and standard biochemical tests. The RIM agreed with the standard biochemical methods for 96 (91.4%) of the strains; nine isolates which were hippurate hydrolysis positive by standard biochemical tests were hippurate hydrolysis negative in the RIM. RIM may serve as a useful adjunct to Gram stain and colony morphology for the identification of G. vaginalis.     

Evaluation of six media for the growth of Trichomonas vaginalis from vaginal secretions.

Many media have been formulated for the growth of Trichomonas vaginalis, but the relative sensitivities of these media have not been determined. We evaluated the ability of six media, including all five media commercially available in the United States, to grow Trichomonas vaginalis from vaginal secretions. In a first experiment, we evaluated the ability of five media to grow T. vaginalis from vaginal secretions of 375 women and determined the optimal days on which to read culture tubes, by inoculating aliquots of secretions into each medium and reading the tubes 1, 2, 3, 4, and 7 days later. Sixty-five patients (17%) had a positive wet-mount examination for T. vaginalis, and all the positive results were confirmed by growth in at least one medium. Of 310 wet-mount-negative specimens, 37 (12%) grew T. vaginalis; overall, 102 women (27%) had a positive culture. Diamond and modified Diamond media (the latter being the only medium not commercially available) detected 99 (97%) and 92 (90%) isolates, respectively, compared with three formulations of Kupferberg medium, which detected 77 (75%), 50 (49%), and 43 (42%) isolates. The optimal single day to read wet-mount-negative cultures was day 7, but 4 (11%) of the 37 positive specimens were positive only before day 7. In a second study, we compared the ability of modified Diamond medium with that of a sixth medium, Lash medium, to grow T. vaginalis from 48 wet-mount-positive specimens; modified Diamond medium supported growth in all cases, whereas Lash medium supported growth in only 26 (54%) cases. We conclude that formulations of Diamond medium are superior to formulations of Kupferberg or Lash medium for growth of t. vaginalis.     

Minimal criteria for the identification of Gardnerella vaginalis isolated from the vagina

Vaginal swabs were examined for the presence of Gardnerella vaginalis. Of 294 isolates with appropriate colonial and cellular morphology subjected to an identification procedure, 203 (69%) were identified as G vaginalis. The 91 isolates not identified as G vaginalis were differentiated by their inability to ferment starch, cause diffuse beta haemolysis on human blood agar or hydrolyse hippurate. Other tests, often used in the identification of G vaginalis, were found to be insufficiently specific. Failure to ferment starch coexisted with failure to cause beta haemolysis and/or hydrolyse hippurate. The starch fermentation test may therefore be omitted. The tests for beta haemolysis and hippurate hydrolysis, being relatively simple to perform and interpret, are considered indispensable for the accurate identification of G vaginalis in the service laboratory.     

Colorimetric one-tube nested PCR for detection of Trichomonas vaginalis in vaginal discharge.

A colorimetric one-tube nested PCR was developed for the detection of Trichomonas vaginalis in clinical vaginal discharge specimens. A family of 650-bp specific DNA repeats from the T. vaginalis genome was targeted. There was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas hominis and Giardia lamblia. The colorimetric assay was applied as an adjunct to nested PCR for semiquantitative determination of T. vaginalis DNA at levels corresponding to 1 to 1,000 parasites. PCR of samples prepared by a rapid boiling method was as sensitive and specific as PCR of samples prepared by the standard DNA extraction method: the equivalent of one T. vaginalis organism in 20 microliters of vaginal discharge could be detected. The colorimetric nested PCR was compared with wet mount and culture for the detection of T. vaginalis. A total of 378 clinical vaginal discharge specimens from symptomatic patients were examined; 31 patients were positive for T. vaginalis both by culture and by nested PCR. However, only 17 of these 31 patients were positive by wet mount examination. In addition, of 113 asymptomatic patients, 9 were positive for T. vaginalis by nested PCR. Of these nine PCR-positive patients, only two were also positive both by wet mount and by culture, four patients were positive by culture but negative by wet mount, and three patients were negative both by wet mount and by culture. No specimens negative by nested PCR were positive by wet mount or by culture. The three asymptomatic patients with PCR-positive but wet mount- and culture-negative samples were subsequently found to have T. vaginalis infection after repeated and prolonged culture was performed. This colorimetric nested PCR was very sensitive compared with culture for the diagnosis of vaginal trichomoniasis, especially asymptomatic T. vaginalis infection. It is also simple, specific, rapid, and semiquantitative.     

Symptomatic candidiasis: Using self sampled vaginal smears to establish the presence of Candida, lactobacilli, and Gardnerella vaginalis

In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis ("clue cells") and neutrophils.All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle.Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus-predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida.     

Evaluation of the OSOM Trichomonas rapid test versus wet preparation examination for detection of Trichomonas vaginalis vaginitis in specimens from women with a low prevalence of infection

The OSOM Trichomonas rapid test (OSOM Trich) was compared to the wet preparation examination (WP) for the detection of Trichomonas vaginalis vaginitis in women with a low prevalence of infection. A total of 19/1,009 (2%) women had T. vaginalis infection. OSOM Trich had very good performance, with sensitivity, specificity, efficiency, positive predictive value, and negative predictive value of 94.7, 100, 99.9, 100, and 99.9%, respectively. The implementation of OSOM Trich would decrease labor costs.     

A study of the susceptibility of three species of primate to vaginal colonization with Gardnerella vaginalis

In an attempt to develop an animal model of Gardnerella-associated vaginitis, several strains of Gardnerella vaginalis were inoculated into the lower genital tract of female pig-tailed macaques, tamarins and chimpanzees. G. vaginalis was not recovered from either tamarins or chimpanzees, but was recovered from each of 1O pig-tailed macaques inoculated with either of two freshly isolated Gardnerella strains, colonization persisting for 11-39 days. Examination of Gram-stained vaginal smears obtained from infected pig-tailed macaques failed to demonstrate clue cells, a feature which is pathognomonic of Gardnerella-associated vaginitis in humans. Other features characteristic of non-specific vaginitis, namely an increase in vaginal pH, and an increase in the ratio of succinate to lactate (S/L ratio) in vaginal fluid were not found. However, the physiology of the macaque vagina was found to be different from that of the human, the vaginal pH and S/L ratio of uninfected macaques both being higher than that seen in humans. The physiological differences between the macaque and human vagina may be due, in part, to a difference in their anaerobic vaginal flora. While these inter-species differences in vaginal physiology and microbiology limit the relevance of the pig-tailed macaque as a model of Gardnerella-associated vaginitis, the ease with which macaques are colonized with G. vaginalis may prove useful in studying bacterial adhesion and local immunity.     

Evaluation of CandiSelect4, a new chromogenic medium for isolation and presumptive identification of Candida species from clinical specimens

In clinical laboratories, isolation of Candida species is generally based on the culture of specimens on Sabouraud dextrose agar. This strategy does not allow species identification on primary culture and makes it difficult to detect mixed cultures. Chromogenic media contain substrates that react specifically with different Candida species, and partly overcome these difficulties.ObjectivesThe aim of this study was to compare two chromogenic media: (i) CandiSelect4 (C4), a new medium developed for direct identification of C. albicans and presumptive identification of C. krusei, C. tropicalis and C. glabrata (ii) CHROMagar Candida (CH), a medium licensed for direct identification of C. albicans, and presumptive identification of C. tropicalis and C. krusei, and employed in our laboratory since 2002.Materials and methodsAltogether, 533 clinical specimens were seeded on both media. Identification on C4 was compared to that achieved on CH, API 32 C test or an in-house C. glabrata rapid identification test.ResultsDiscrepant positive cultures occurred in 16 samples and were shared equally between C4 and CH. The ability of both media to detect associations was equivalent, with 34 associations identified with C4 and CH. No discrepancy was observed with respect to identification of C. albicans. C4 identified six of six C. krusei, but false-negative identification of C. tropicalis occurred in 2/11 cases and false-positive identification of C. glabrata occurred in 2/34 cases.ConclusionsThe overall performance of C4 and CH appeared almost identical during a two-month comparison in the clinical mycology setting.     

A study of the susceptibility of three species of primate to vaginal colonization with Gardnerella vaginalis.

In an attempt to develop an animal model of Gardnerella-associated vaginitis, several strains of Gardnerella vaginalis were inoculated into the lower genital tract of female pig-tailed macaques, tamarins and chimpanzees. G. vaginalis was not recovered from either tamarins or chimpanzees, but was recovered from each of 1O pig-tailed macaques inoculated with either of two freshly isolated Gardnerella strains, colonization persisting for 11-39 days. Examination of Gram-stained vaginal smears obtained from infected pig-tailed macaques failed to demonstrate clue cells, a feature which is pathognomonic of Gardnerella-associated vaginitis in humans. Other features characteristic of non-specific vaginitis, namely an increase in vaginal pH, and an increase in the ratio of succinate to lactate (S/L ratio) in vaginal fluid were not found. However, the physiology of the macaque vagina was found to be different from that of the human, the vaginal pH and S/L ratio of uninfected macaques both being higher than that seen in humans. The physiological differences between the macaque and human vagina may be due, in part, to a difference in their anaerobic vaginal flora. While these inter-species differences in vaginal physiology and microbiology limit the relevance of the pig-tailed macaque as a model of Gardnerella-associated vaginitis, the ease with which macaques are colonized with G. vaginalis may prove useful in studying bacterial adhesion and local immunity.     

Determination of immunoglobulin A against Gardnerella vaginalis hemolysin,sialidase, and prolidase activities in vaginal fluid: implications for adverse pregnancy outcomes

A nested case-control study of low birth weight and preterm delivery was performed with singleton women. Immunoglobulin A (IgA) against the Gardnerella vaginalis hemolysin (anti-Gvh IgA) and sialidase and prolidase activities were determined in vaginal fluid at 17 weeks of gestation. Sialidase positivity and bacterial vaginosis with high prolidase activity were associated with 2- and 11-fold increased risks for low birth weight, respectively. No woman with bacterial vaginosis plus a strong anti-Gvh IgA response had an adverse outcome.     

Use of a sodium polyanetholesulfonate disk for the identification of Gardnerella vaginalis.

Several methods have been previously suggested for the presumptive identification of Gardnerella vaginalis in clinical laboratories, but none is entirely satisfactory. We previously found that sodium polyanetholesulfonate (SPS) inhibits G. vaginalis in blood culture media. We compared susceptibility to an SPS-containing paper disk with beta-hemolysis on human blood agar, hippurate hydrolysis, and inhibition by alpha-hemolytic streptococci for identification of 62 previously confirmed G. vaginalis strains. All strains were positive by SPS disk and alpha-hemolytic streptococcus inhibition, 78% were positive by beta-hemolysis, and 81% were positive by hippurate hydrolysis. Although positive reactions occurred with SPS disk and alpha-hemolytic streptococcus tests for 5 and 9 of 84 other bacteria tested, respectively, none of these bacteria were positive for both tests. We conclude that a combination of SPS disk susceptibility and alpha-hemolytic streptococcus inhibition provides excellent identification of G. vaginalis when performed by the methods suggested.     

Performance of a new, rapid assay for detection of Trichomonas vaginalis

Trichomonas vaginalis infection is highly prevalent, may have serious health consequence, and is readily treatable. However, screening has been limited by currently available tests, which tend to be insensitive, expensive, or require a delay before results are reported. The XenoStrip-Tv (Xenotope Diagnostics, Inc., San Antonio, Tex.) was evaluated on vaginal swab specimens from 936 women attending sexually transmitted disease clinics in Seattle, Wash. (n = 497), and Birmingham, Ala. (n = 439). T. vaginalis prevalence by culture (InPouch; Biomed) was 8.7% in Seattle and 21.0% in Birmingham. Compared to culture, the XenoStrip assay in Seattle was 76.7% (95% confidence interval [95% CI] = 61.4 to 88.2) sensitive and 99.8% (95% CI = 98.8 to 99.9) specific, and in Birmingham it was 79.4% (95% CI = 69.6 to 87.1) sensitive and 97.1% (95% CI = 94.8 to 98.6) specific. The positive predictive values were 97.1% in Seattle and 87.9% in Birmingham; the negative predictive values were 97.8 and 94.7%, respectively. Rapid test performance did not vary by vaginal symptoms or by the presence of other vaginal or cervical syndromes or infections. The sensitivity did vary by day of culture-positive result, with a 71% decline in XenoStrip sensitivity for every additional day delay until T. vaginalis was first detected in cultures (odds ratio = 0.29, 95% CI = 0.18 to 0.49). The rapid assay was more sensitive than wet preparation microscopy (78.5% versus 72.4% [P = 0.04]) but was less specific (98.6% versus 100% [P = 0.001]). The XenoStrip rapid assay is well suited for use in settings with a moderately high prevalence of T. vaginalis infection, particularly when microscopy is not practical.     

Evaluation of Xenostrip-Tv, a rapid diagnostic test for Trichomonas vaginalis infection

An immunochromatographic strip test, Xenostrip-Tv, was compared to wet mount and PCR for the diagnosis of Trichomonas vaginalis infection in women. Of 428 specimens tested, 54 (12.6%) were positive by an "expanded gold standard," defined as either a positive wet mount and PCR test with primers TVK3 and TVK7 and/or a positive PCR test confirmed by a second PCR assay with primers TVA5-1 and TVA6; 26 (6%) were positive by wet mount, and 36 (8.4%) were positive by Xenostrip-Tv test. Since the Xenostrip-Tv test is rapid and easy to perform and proved to be more sensitive than wet mount, it should be considered as an alternative to wet mount for point-of-care diagnosis of trichomoniasis, especially in settings where microscopy is impractical.     

Improved diagnosis of Trichomonas vaginalis infection by PCR using vaginal swabs and urine specimens compared to diagnosis by wet mount microscopy, culture, and fluorescent staining

Four vaginal cotton swab specimens were obtained from each of 804 women visiting the outpatient sexually transmitted disease clinic of the Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands, for validation of various forms of Trichomonas vaginalis diagnostic procedures. One swab specimen was immediately examined by wet mount microscopy, a second swab was placed in Kupferberg's Trichosel medium for cultivation, and two swabs were placed in phosphate-buffered saline (PBS), pH 7.2. The resulting PBS suspension was used for direct staining with acridine orange and fluorescence microscopy, inoculation of modified Diamond's culture medium, and a PCR specific for T. vaginalis. A total of 70 samples positive in one or more of the tests were identified: 31 (3.8%) infections were detected by wet mount microscopy, and 36 (4.4%) were identified by acridine orange staining, as opposed to 40 (4.9%) and 46 (5.7%) positives in modified Diamond's and Trichosel media, respectively. PCR was positive for 61 (7.5%) samples. Secondly, from each of 200 women were obtained a urine sample and a vaginal cotton swab specimen, and 200 urine samples were obtained from men. For the women, 15 (7.4%) of the samples showed a positive result for either the wet mount (n = 1), Trichosel culture (n = 6), PCR on the vaginal swab sample (n = 10), or PCR on the urine specimen (n = 11). Four men (2%) were diagnosed with a T. vaginalis infection. Thus, PCR appears to be the method of choice for the detection of genital infections with T. vaginalis.