Molecular exploration for Mycoplasma amphoriforme,Mycoplasma fermentans and Ureaplasma spp. in patient samples previously investigated for Mycoplasma pneumoniae infection

ObjectivesTo determine the presence and genotypic macrolide susceptibility of Mycoplasma amphoriforme, and the presence of Ureaplasma spp. and Mycoplasma fermentans among clinical samples from England previously investigated for Mycoplasma pneumoniae.MethodsQuantitative and conventional PCR methods were used to retrospectively screen a collection of 160 clinical samples previously submitted to Public Health England (PHE) for the detection of M. pneumoniae between October 2016 and December 2017. Samples which were positive for M. amphoriforme DNA were further investigated for mutations associated with genotypic macrolide resistance by sequencing domain V of the 23s rRNA.ResultsM. amphoriforme was detected in 10/160 samples (6.3%), Ureaplasma parvum was detected in 4/160 samples (2.5%), and M. fermentans was not detected in any samples (0/160). Of the nine individuals (two samples were from the same patient) in which M. amphoriforme was detected, eight were male (age range 10–60 years) and one was female (age range 30–40 years). One individual with cystic fibrosis was positive for both M. amphoriforme and U. parvum. All M. amphoriforme DNA was genotypically susceptible to macrolides.ConclusionsMycoplasma amphoriforme was found in clinical samples, including lower respiratory tract samples of patients with pneumonia. In the absence of other respiratory pathogens, these data suggest a potential role for this organism in human disease, with no evidence of acquired macrolide resistance. Ureaplasma parvum was detected in cerebrospinal fluid and respiratory tract samples. These data suggest that there is a need to consider these atypical respiratory pathogens in future diagnostic investigations.     

Activity of moxifloxacin against the urogenital mycoplasmas Ureaplasma spp., Mycoplasma hominis and Mycoplasma genitalium and Chlamydia trachomatis

The activity of moxifloxacin was compared with that of other antimicrobial agents against 54 strains of Ureaplasma spp., 54 strains of Mycoplasma hominis , 14 strains of Mycoplasma genitalium , and 44 strains of Chlamydia trachomatis . Moxifloxacin inhibited 90% of all isolates at a concentration ≤1 mg/L, being the most active compound against C. trachomatis and sharing the highest activity with garenoxacin and gemifloxacin against mycoplasmas. Moxifloxacin killed the 30 mycoplasma isolates tested at a concentration ≤1 mg/L, except those resistant to fluoroquinolone. Thus, moxifloxacin has attracted interest as a potential therapy for mycoplasmal or chlamydial urogenital infections.     

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.     

Use of a urine enzyme immunoassay as a diagnostic tool for Chlamydia trachomatis urethritis in men.

We collected first-voided urine specimens from 659 males attending a sexually transmitted disease clinic and performed both enzyme immunoassay (EIA) for detection of chlamydial antigen and leukocyte esterase testing on these urine samples. The overall prevalence of chlamydial urethritis in the study population as determined by culture of urethral swabs was 11%. However, 46% of all men in the study had no symptoms of urethritis. Compared with urethral cultures for chlamydiae, the urine EIA had a sensitivity of 42% and a specificity of 99%. The sensitivity of the EIA strongly correlated with the amount of antigen present in culture as assessed by numbers of inclusion-forming units. The sensitivity of the leukocyte esterase test compared with that of chlamydia culture was 88%. We conclude that in this population of men, which included many patients without symptoms of urethritis, the urine EIA was a relatively insensitive means of screening for chlamydial infection.     

Molecular detection of human papillomavirus in women with minor-grade cervical cytology abnormalities.

BACKGROUND: Human papillomavirus (HPV) has been shown to be the major risk factor for the development of cervical carcinoma, the second most common cancer among women worldwide. Cervical cytology has been the main screening tool for detection of premalignant lesions in last 50 years. OBJECTIVE: The utility of a molecular assay for detection of HPV in cervical smears was evaluated. STUDY DESIGN: A total of 466 women with minor-grade cervical cytology abnormality supposed to be produced by HPV were included. Patients were classified into three groups: Patients with reactive changes, patients with cervical intraepithelial neoplasia grade 1 (CIN 1), and patients with cervical intraepithelial neoplasia grade 2 (CIN 2). In all patients, another cervical swab was obtained and tested for the HPV genome using the Digene Hybrid Capture II. This assay is able to distinguish between high-risk and low-risk HPV types. RESULTS: Based on cytology results, 44 patients showed reactive changes, 250 patients displayed CIN 1, and 172 patients displayed CIN 2. With the molecular assay, HPV was detected in 289/466 (62%) patients. The high-risk HPV type was present in 263 (56.4%) patients and the low-risk type in 26 (5,5%) patients. In 25% of patients with reactive changes, the HPV genome was detected. Corresponding rates for patients with CIN 1 and CIN 2 were 55 and 81%, respectively. CONCLUSION: Molecular detection of HPV should additionally be used to cytology in patients whose cervical smears display reactive changes, CIN 1, or CIN 2. The employed assay allows identification of patients who are at risk for development of high-grade cervical lesions.     

Rapid detection of Mycoplasma genitalium,Mycoplasma hominis,Ureaplasma parvum,and Ureaplasma urealyticum organisms in genitourinary samples by PCR-microtiter plate hybridization assay

We present a method for detecting the presence of Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.     

High prevalence of human papillomavirus infections in urine samples from human immunodeficiency virus-infected men

Infection with human immunodeficiency virus (HIV) and the resulting immunosuppression are associated with an increased risk for human papillomavirus (HPV) persistence and related malignancies. In the present study we investigated the prevalence of HPV in urine samples from 104 HIV-infected men with low CD4+ cell counts (<100 per mm(3)) and 115 urine samples from HIV-negative men. A high prevalence of HPV DNA (39.4%) was found in the HIV patients. Most of the HPV types were high risk (81.4%), with HPV 52 as the most prevalent type (12.5%), followed by HPV 18 (6.7%), HPV 35 (5.8%), and HPV 70 (4.8%). Multiple HPV genotypes were observed in 17 (41%) of the 41 HPV- and HIV-positive men. In contrast, only 11 (9.6%) HPV DNA-positive cases were observed among the 115 HIV-uninfected men, and 3 (27.3%) contained multiple genotypes. Quantitative analyses indicated that the HPV viral load, as measured in urine samples, is significantly higher in HIV-positive men compared to HIV-negative men. In the present study we show that urine samples are useful for detecting HPV DNA, there is a high prevalence of HPV in HIV-positive men, and the HPV viral load is substantially higher in HIV-positive than in HIV-negative men. More studies are needed to evaluate the risk and natural development of HPV-related malignancies in HIV-positive men.     

Comparison of physician- and self-collected genital specimens for detection of human papillomavirus in men

There is currently no consensus regarding the most appropriate methods of sampling for the detection of genital human papillomavirus (HPV) in men. We employed a recently developed collection method involving abrasion and moistened swabbing of the genital skin surface for the detection of HPV in a cohort of 136 university-affiliated males in Hawaii. Genital specimens collected by physicians using this method were compared with self-collected specimens from the same individuals obtained 24 h later. Self-collected specimens yielded a greater proportion of sufficient specimens than physician-collected specimens. HPV detection was comparable in physician- and self-collected specimens; detection was highest in the penile shaft (51.2% and 51.5%, respectively, P = 0.96), followed by the scrotum (41.2% and 46.2%, P = 0.43), the glans/coronal sulcus (31.9% and 33.1%, P = 0.84), and the foreskin (33.3% and 28.6%, P = 0.74). Site-specific agreement in HPV detection between paired physician- and self-collected samples ranged from 67.2% (kappa = 0.34) for the penile shaft to 95.0% (kappa = 0.89) for the foreskin. There was a high degree of concordance in HPV genotypes in HPV-positive pairs. The most common type was HPV type 84, which comprised approximately 15% of the specimens. The emery paper-swab method offers an efficient sampling method for genital HPV DNA detection in men that could be used both within and outside of the clinical setting.     

gyrA和parE基因检测在脲原体基因分型中的应用

目的 研究gyrA和parE基因检测在脲原体基因分型中的作用.方法 脲原体培养与药敏分析用Mycoplasma IST检测试剂盒;在脲原体培养阳性者中选取对喹诺酬耐药标本60份,PCR扩增gyrA和parE基因,扩增产物经测序分析后与基因库中的脲原体各血清型进行比对.结果 gyrA扩增片段在血清型1、3、6、14之间核苷酸序列相似性为100%,在血清型2,4、5、7～13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性91%.parE扩增片段在血清型1、3、6、14之间的核苷酸序列相似性为98%～99%;pare扩增片段在血清犁2、5、7、8、11之间核苷酸序列相似性100%,在血清型4、12、13之间核苷酸序列相似性100%,两组之间核苷酸序列相似性为90%.60份标本中微小脲原体(Ureaplasma parvum,Up)占68.3%(41/60),解脲脲原体(Ureaplasma urealyticum,Uu)占21.7%(13/60),两型混合感染占10%(6/60).在Up中,血清型3占48.8%(20/41).结论 gyrA检测可把脲原体分为微小脲原体和解脲脲原体两个基因型,parE检测可把微小脲原体分为4个亚型,分别与血清型1、3、6、14完全一致,其中血清型3感染最常见.     

Optimization of PCR based detection of human papillomavirus DNA from urine specimens.

BACKGROUND: Human papillomavirus (HPV) causes cervical cancer. Current screening requires a yearly pelvic exam and Pap smear. However, these procedures are impractical for screening all women at risk for disease. Urine sampling has been successfully utilized to screen for Chlamydia trachomatis (CT) and Neisseria gonorrhoreae (NG) infections and has been considered for HPV DNA detection by several investigators. However, no study to date has been performed to specifically optimize HPV detection in urine. OBJECTIVES: To compare handling and extraction techniques in order to optimize the HPV specific PCR system in urine specimens. STUDY DESIGN: Examination of 10 characteristics that may contribute to PCR inhibition in urine was performed utilizing 10SG mulitstixs. Five different DNA extraction methods were compared in spiked specimens and in 10 clinical specimens. After the optimal extraction technique was identified, concentration of the sample with and without prior dilution was compared to the original protocol. Lastly, specimen handling was compared between immediate processing, refrigerating overnight, or freezing overnight. RESULTS AND CONCLUSIONS: the presence of protein in urine enhanced amplification while nitrites decreased amplification. Of the extraction methods tested, the QIAamp DNA Mini Kit demonstrated the best amplification from urine samples spiked with HPV DNA and clinical specimens. The addition of a dilution step and a concentration step before applying the Qiagen protocol further increased amplification of beta-globin (from 50 to 63%) and the HPV L1 gene (from 13 to 33%). Lastly, refrigerating the specimens at 4 degrees C overnight appears to produce better amplification (62% beta-globin and 17% HPV positive) than either immediate processing (46% beta-globin and 13% HPV+) or freezing the specimen for 24h prior to processing (46% beta-globin and 10% HPV+). In these studies, amplification was low despite optimization. Additional improvements are required prior to clinical application of a urine-based HPV DNA detection system.     

Prevalence of Mycoplasma hominis and Ureaplasma urealyticum (T strains) in urine of adolescents.

Adolescent children were surveyed for colonization with Mycoplasma hominis and Ureaplasma urealyticum by culturing urine specimens. Rates were compared between three study groups: (i) 397 children attending parochial schools, (ii) 293 children attending an adolescent clinic specializing in adjustment problems, and (iii) 86 children attending a renal clinic. The recovery rate was higher among postpubertal girls attending the renal clinic (33%) and the adolescent clinic (26%) than among students attending parochial high school (males 2%, females 8%). Girls had approximately eightfold higher rates than boys of the same age. Isolation of Mycoplasmataceae was associated with certain sociological determinants, such as dating, cigarette smoking, and coming from a broken home, but also with abnormal findings (protein, leucocytes) in urine.     

Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR

We developed a TaqMan-based real-time PCR assay for quantifying Mycoplasma genitalium. This assay is able to specifically quantify concentrations of the M. genitalium 16S rRNA gene ranging from 10(7) to 10 copies/reaction. Using the TaqMan assay, we quantified the M. genitalium 16S rRNA gene in first-pass urine of men with urethritis and asymptomatic men who were positive for M. genitalium by PCR- and phylogeny-based assay. Of 130 men with gonococcal urethritis (GU), five were positive for M. genitalium. The mycoplasma load for each specimen was <5 x 10 copies/ml. Of 84 men with chlamydial non-GU (CNGU), seven were positive for M. genitalium. One man had an M. genitalium load of <5 x 10 copies/ml, and six men had loads ranging from 1.1 x 10(7) to 2.7 x 10(2) copies/ml. Of 86 men with nonchlamydial NGU (NCNGU), 17 were positive for M. genitalium. The mycoplasma loads for these men ranged from 3.3 x 10(6) to 2.3 x 10(2) copies/ml. Of 76 asymptomatic men, only two were positive for M. genitalium. For these men, the loads were 2 x 10(2) and <5 x 10 copies/ml. The patients with NGU had significantly higher concentrations of M. genitalium in their first-pass urine than did men with GU (P < 0.01) or asymptomatic men (P < 0.05). In addition, M. genitalium loads were significantly higher in men with NCNGU than those in asymptomatic men (P < 0.05). The quantitative assessment of M. genitalium loads by the TaqMan assay will provide useful information for understanding the pathogenicity of this mycoplasma in the urogenital tract.     

Comparison of commercially available media for detection and isolation of Ureaplasma urealyticum and Mycoplasma hominis.

The Mycotrim Triphasic flask system (Irvine Scientific, Irvine, Calif.) was compared with a system composed of Mycotrim GU broth (Irvine Scientific) and A7 or A8 agar (Remel, Lenexa, Kans.) for the ability to detect Ureaplasma urealyticum and Mycoplasma hominis from 129 genital specimens. Of the 64 specimens positive for U. urealyticum, 25, 98, and 100% were detected on Mycotrim Triphasic agar and A7 and A8 agars, respectively. All 18 specimens that grew M. hominis were detected by A7 and A8 agars, and 94% grew on Mycotrim Triphasic agar. Mycotrim GU broth detected all of the positive specimens, and Mycotrim Triphasic broth detected all but one. Mycotrim GU broth inoculated simultaneously with either A7 or A8 agar was found to be more sensitive and cost-effective than the Mycotrim Triphasic flask system.     

Use of the same archival papanicolanou smears for detection of human papillomavirus by cytology and polymerase chain reaction.

An optimal method for the processing of archival cervical Papanicolaou (pap)-stained smears for the amplification of human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) was developed. This methodology was then applied to a series of 44 pap smears designated as HPV positive or negative (on the basis of both major and minor cytological criteria) or cervical intraepithelial neoplasia (CIN)-cancer. For the detection of HPV DNA, each sample was tested with the consensus GP5/6 primers, and when negative, with CPI-IIG primers. The HPV DNA was detected in 100% (8 of 8) of CIN-cancer smears using the GP5/6 primers. In smears with cytological evidence of HPV without CIN. the use of both sets of primers yielded positive results in 100% (19 of 19) of the samples. Direct sequence analysis of PCR products showed that 16 of the 27 HPV-positive samples contained more recently described HPV types. When tested with both primer combinations, all 17 cytologically negative smears were positive for beta-globin but negative for HPV DNA. The findings show the value of using archival pap smears for further investigations to address issues such as latency, but they indicate that cytological criteria and DNA technology will be critical factors in the reliability of the results.     

Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis,Mycoplasma hominis,Ureaplasma urealyticum,and Mycoplasma genitalium in infertile women

PurposeSexually Transmitted Diseases (STDs) can cause sterility and many other problems for women planning pregnancy. Currently, almost 340 million people worldwide suffer from Sexually Transmitted Infections (STIs). This study made attempts to quickly identify STDs' most critical infectious agents using dedicated primers and probes.MethodsThe present study was done on the cervical samples of 200 infertile women. After extracting the total DNA of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium, quantitative methods were employed to determine the rate of target bacteria using multiplex real-time PCR.ResultsThe multiplex qPCR showed the rates of 47%, 16%, 46%, and 16.5% for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Mycoplasma genitalium in infertile women, respectively. In some patients, there were co-infections with two or three bacteria. The diagnostic approach used in our research could be employed as an alternative detection tool to identify the four most common STD-associated bacterial agents while detecting mixed infections.ConclusionsInfertile women with no biological problems could have their genital tract checked using this newly designed identification technique and get proper treatment for their infections as quickly as possible.     

Evaluation of the polymerase chain reaction for the detection of human papillomavirus from urine

The ability to detect the presence of human pap-illomavirus (HPV)-DNA sequences in urine was evaluated using polymerase chain reaction (PCR). DMA was purified and extracted from urine samples, and subjected to 40 cycles of amplification using the consensus primer pair MY11 and MY09. Coamplification using the β-globin primers, GH20 and PC04, was performed as an internal reaction control. Following assay optimization, urine samples from 22 women undergoing examination for cervical dysplasia were tested for the presence of HPV-DNA. PCR assay results were correlated with cytologic and histo-logic findings as well as Vira Type(tm) assay results. Overall, HPV was detected by PCR in 16 (76%) of the interpretable samples. HPV sequences were detected in 13 (87%) of the 15 specimens from women showing evidence of condylomata, dysplasia, or invasive carcinoma. HPV was detected in 3 (50%) of the women whose cytologic or his-tologic results were either negative or showed benign atypia. Although the sample size in this study is small, our results show that HPV can be detected by PCR in a majority of individuals showing evidence of HPV infection. The method described provides a means for the clinical laboratory to detect a broad range of HPV types from using a sample obtained by noninvasive techniques. The ability to easily obtain urine would allow for increased numbers of individuals to be tested, and thus, aid in our understanding of HPV. © 1995 Wiley-Liss, Inc.     

Subclassifying atypical squamous cells in Thin-Prep cervical cytology correlates with detection of high-risk human papillomavirus DNA.

Recent studies have proposed subclassifying ASCUS into "favor reactive" (ASFR), "not otherwise specified" (ASNOS), and "favor squamous intraepithelial lesion (SIL)" (ASFS). This study explored the reproducibility of these diagnoses with Thin-Prep cytology and their association with high-risk human papillomavirus DNA (HRHPV). Three pathologists and 1 cytotechnologist with 2 to 25 years of experience reviewed 144 Thin-Prep (Cytyc, Boxborough, MA) specimens previously diagnosed as normal, ASFR, ASNOS, ASFS, and SIL. Interobserver reproducibility was computed with the kappa statistic. The original laboratory diagnosis was compared with the presence of HRHPV types. Interobserver reproducibility for a normal or SIL diagnosis was very good (kappa = .68 and .63). Reproducibility for ASFR, ASNOS, and ASFS ranged from poor to fair (kappa = .21, .19, and .32). In a weighted analysis, kappa values for ASFR/ASNOS and ASFS/SIL were .36 and .62, respectively. HRHPV-positivity for preparations originally diagnosed as N, ASFR, ASNOS, ASFS, and SIL were 5.7%, 8.8%, 17.4%, 47.8%, and 54.5%, respectively. The difference in index of HRHPV for either N or ASFR and ASFS or SIL was significant (P < .001). Reproducibility for ASCUS is generally poor, but better reproducibility is obtained by combining ASFS with SIL and, to a lesser degree, ASNOS with ASFR. ASFS and SIL confer a similar index of HRHPV and merit similar management. ASFR may be managed with cytologic follow-up; but this may depend upon the individual laboratory. HPV testing, in conjunction with cytologic and biopsy follow-up, appears useful for estimating the significance of ASCUS subgroups in laboratory practice.     

Development of an automated extraction procedure for detection of human papillomavirus DNA in liquid based cytology samples

Liquid based cytology samples are being used increasingly to improve cervical screening and have the advantage that residual cell suspension is available for other tests such as human papillomavirus (HPV) detection. However, as the transport medium is optimised for downstream cytology, problems can be experienced during extraction of nucleic acid. This study aimed to develop a robust protocol for automated extraction of HPV DNA from cervical, liquid based cytology samples using a high throughput robotic system. Considerable modification of existing clinical extraction protocols for swab specimens, together with optimisation of required sample input volume was required to reduce sample blockage during the extraction to acceptable levels. The blockage rate and optimal processing volume was assessed by extracting a fixed volume (1/4) of re-suspended material from the centrifuged pellets of 10, 5 and 1 ml aliquots of 200 specimens. Analysis revealed 17.5% blockage with specimens originating from 10 ml aliquots; 3% with 5 ml and no blockage with 1 ml aliquots of the same samples. A 3% blockage level is acceptable for an automatic well clearance procedure to be followed. HPV testing of the extracts by real-time PCR showed a 1.5% loss of sensitivity in extracts originating from 1 ml aliquots as compared with 5 ml aliquots with a consequent loss of detectable HPV genotypes after reverse hybridisation. In short, 5 ml of liquid based cytology specimen is recommended for nucleic acid extraction, to allow optimal detection of HPV types in clinical samples while retaining maximum efficiency of the robotic extraction procedure.