Performance of rapid immunochromatographic assay in the diagnosis of Trichomoniasis vaginalis

Trichomonas vaginalis accounts for nearly half of all curable sexually transmitted diseases worldwide with serious health consequences. Effort to increase the sensitivity of its diagnosis is critical to both control measures and epidemiologic studies. This study was conducted to evaluate the OSOM® Trichomonas Rapid Test (Sekisui Diagnostics, Framingham, MA, USA), a qualitative antigen-detection immunochromatographic (IC) assay in the diagnosis of vaginal trichomoniasis comparable to the conventional methods. The study enrolled 258 females aged 18–50 years classified into symptomatic (185) and asymptomatic (73) groups. Vaginal swab specimens were obtained for wet mount, stained preparation (Giemsa, acridine-orange), culture (InPouch TV™, modified Diamond's), and for rapid OSOM testing. Trichomonas vaginalis was detected in 67, 66, 71, 99, 96, and 97 using wet mount, acridine-orange stain, Giemsa stain, modified Diamond's, InPouch media, and OSOM test, respectively. In comparison to a composite reference standard (CRS) of wet mount microscopy and culture, OSOM test reported 97.98%, 99.37%, 98.98%, 98.75%, and 98.84% for sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy, respectively. The OSOM test proved to be a simple and objective test. This rapid point-of-care-test will contribute significantly in the diagnosis of vaginal trichomoniasis and will increase the understanding of its still vague epidemiology.     

Rapid detection of coinfections by Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum by a new multiplex polymerase chain reaction

We developed a multiplex polymerase chain reaction (M-PCR) assay to simultaneously detect Trichomonas vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum. The test is extremely specific and has a sensitivity of 10 cells for T. vaginalis and U. urealyticum and of 1 cell for M. hominis. The technique was validated on vaginal swabs from 240 women presenting symptoms of vaginitis, and results were compared with data obtained using microscopic and culture techniques on the same patients. The M-PCR revealed to be greatly more sensitive and specific than traditional techniques. It has been well demonstrated, in vitro, that T. vaginalis can establish a symbiosis with M. hominis; our data confirm in vivo this strict association: in fact, M. hominis has been detected in 78.6% of all samples positive for T. vaginalis, as compared to only 4.8% of women without trichomoniasis. The species specificity of this association has been confirmed by the absence of any significant correlation between T. vaginalis and U. urealyticum.     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

Evaluation of Seeplex® STD6 ACE Detection kit for the diagnosis of six bacterial sexually transmitted infections

Traditionally, the diagnosis of bacterial sexually transmitted infection (STI) has been dependent on the isolation of the causative pathogens by culturing endocervical or urethral swab specimens on selective media. While such procedures typically provide excellent diagnostic accuracy, they are often time-consuming and expensive. A multiplex polymerase chain reaction (PCR) assay, based on a semi-automated detection system, was evaluated for the detection of six STI causative organisms. The Seeplex^® STD6 ACE (auto-capillary electrophoresis) Detection assay employed six pairs of dual priming oligonucleotide (DPO?) primers specifically targeted to unique genes of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Mycoplasma hominis, and Trichomonas vaginalis. A total of 739 specimens (304 cervical swabs and 435 urine samples) collected for 4 months were tested, and results were compared to those obtained with a combined monoplex PCR. The concordance between the multiplex PCR and monoplex PCR assay was 100% for both sensitivity and specificity. We also tested for the presence of two pathogenic bacteria (C. trachomatis and N. gonorrhoeae) and compared the results obtained with the multiplex PCR and BD ProbeTec duplex strand displacement amplification (SDA). The results of the multiplex PCR and duplex SDA were 99.7% concordant for C. trachomatis and 100% concordant for N. gonorrhoeae. The multiplex PCR assay using the Seeplex^® STD6 ACE Detection kit proved to be a novel cost-effective and fast diagnostic tool with high sensitivity and specificity for the simultaneous detection of six STI pathogens.     

Validation of an immunologic diagnostic kit for infectious vaginitis by Trichomonas vaginalis, Candida spp., and Gardnerella vaginalis

FemPure is a kit for the rapid diagnosis of vaginitis by Trichomonas vaginalis, Candida spp., and Gardnerella vaginalis, based on aggregation of latex particles joined to specific antibodies. The validation of the method involved the parameters specificity, detection limit, robustness, clinical sensitivity, and clinical specificity. Also, samples analyzed in parallel by the validated test and other recognized tests conducted by external laboratory were included. The method was specific for the 3 infectious agents, and no cross-reaction with other microorganisms usually present in vaginal exudates. The detection limit ≥1 × 10⁶ CFU/mL for Candida albicans and G. vaginalis avoids the detection of concentrations considered normal flora, whereas T. vaginalis was detected until 1 × 10⁵ cells/mL. Values of clinical sensitivity ≥80% and clinical specificity ≥90% and concordance ≥90% were found between samples evaluated in parallel by different methods. Robustness showed that the test can be used in laboratories with different management systems; its simple implementation without equipment allows the use in primary health care areas.     

Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis

The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen.     

Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene

Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In this study, we developed a real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp. Using Leptospira interrogans serovar Icterohaemorrhagiae DNA, the lower limit of detection was found to be 20 genomic equivalents/reaction with a 95% cutoff value. The assay detected pathogenic Leptospira strains, but not intermediately pathogenic or nonpathogenic strains. When testing the assay on spiked clinical specimens, whole blood and plasma were better specimens for detecting the same initial number of leptospires compared with serum from clotted and centrifuged blood. Leptospira spiked at the same concentration was better detected in centrifuged urine. This real-time PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis.     

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting.     

Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis

Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.     

Modified Field's staining—a rapid stain for Trichomonas vaginalis

Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.     

Anti-Trichomonas vaginalis activity of betulinic acid derivatives

Caused by Trichomonas vaginalis, trichomoniasis is the most common non-viral STD worldwide. Currently, metronidazole and tinidazole are the only drugs approved for treatment of the condition. However, problems such as metronidazole-resistant T. vaginalis isolates and allergic reactions have been reported. Based on data previously published by our group, structural changes in betulinic acid (1) were performed, generating three new compounds that were tested for in vitro anti-T.vaginalis activity in this study. Whereas derivative 2 did not demonstrate anti-T. vaginalis activity, derivatives 3 and 4 reduced trophozoite viability by 100%, with MIC values of 50 μM. The structural difference of two compounds was performed only on the C-28 position. Derivative 3 showed low cytotoxicity against Vero cells in 24 h; however, derivative 4 was highly cytotoxic, but efficient when associated with metronidazole in the synergism assay. ROS production by neutrophils was reduced, and derivative 3 showed anti-inflammatory effect. Collectively, the results of this study provide in vitro evidence that betulinic acid derivatives 3 and 4 are potential compounds with anti-T. vaginalis activity.     

Rapid detection of vancomycin-resistant enterococci from rectal swabs by the Cepheid Xpert vanA/vanB assay

The detection of vancomycin-resistant enterococci using a novel commercial multiplex real-time polymerase chain reaction assay (Xpert™ vanA/vanB, Cepheid, Sunnyvale, CA) was evaluated on 804 rectal swab specimens. Compared to enriched culture, sensitivity and negative predictive value of this method were 100%. Many false-positive results were recovered (sensitivity, 85.4%; positive predictive value, 8.7%), especially for the vanB gene.     

Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli

Lactobacilli play a key role in promoting vaginal health. Depletion of these bacteria is associated with bacterial vaginosis (BV), the most common vaginal disorder. Here we describe the development and laboratory validation of a novel single-tube multiplex TaqMan quantitative polymerase chain reaction (qPCR) assay for the identification and quantitative assessment of the four major vaginal Lactobacillus species: L. crispatus, L. jensenii, L. gasseri, and L. iners. The assay utility was evaluated by the analysis of lactobacilli in non-cultured clinical vaginal swab specimens collected from BV patients and healthy individuals. As confirmed by the assay, L. crispatus, L. jensenii, and to a lesser extent L. gasseri, are common in the vagina of healthy women, whereas L. iners dominance is associated with BV. The major assay limitation was preferential detection of dominant Lactobacillus species in samples with mixed lactobacilli resulting in lower sensitivity for minor species. The multiplex qPCR assay described here is an advance in the detection and quantitation of the major vaginal lactobacilli, potentially facilitating the molecular diagnosis of BV and post-therapy restoration of the vaginal microflora.     

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a κ coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.     

Application of loop-mediated isothermal amplification assay for the sensitive and rapid diagnosis of visceral leishmaniasis and post-kala-azar dermal leishmaniasis

Loop-mediated isothermal amplification (LAMP) is at the forefront in the search for innovative diagnostics for rapid and specific amplification of target DNA under isothermal conditions. We have applied LAMP assay using SYBR Green for clear-cut naked eye detection of Leishmania (Leishmania) donovani in 200 clinical samples of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). The assay was positive in 53/55 VL blood samples (sensitivity, 96.4%; 95% confidence interval [CI], 87.7–99%), 15/15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI, 79.6–100%), 60/62 PKDL tissue biopsy samples (sensitivity, 96.8%; 95% CI, 88.9–99.1%), and 1/68 control samples (specificity, 98.5%; 95% CI, 92.1–99.7%). The assay was specific for L. (L.) donovani, the causative species for VL and negative for L. (L.) infantum, L. (L.) tropica, and L. (L.) major. This is the first comprehensive clinical study demonstrating the applicability of the LAMP assay for a rapid and reliable molecular diagnosis of VL and PKDL.     

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.