Quantitative detection and phylogeny-based identification of mycoplasmas and ureaplasmas from human immunodeficiency virus type 1-positive patients

doi:10.1007/s10156-005-0418-7

Journal of Infection and Chemotherapy

Volume 12, Issue 1, 2006, Pages 25-30

https://doi.org/10.1007/s10156-005-0418-7 Get rights and content

Abstract

More than 20 years ago, Mycoplasma genitalium was first isolated from men with nongonococcal urethritis (NGU). Some strains of Mycoplasma and Ureaplasma are associated with NGU or other genitourinary infections. It is reasonable to suggest that mycoplasmas and ureaplasmas may increase the development of acquired immunodeficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected patients. This study investigated a rapid and reliable diagnostic method, based on a LightCycler SYBER green I real-time PCR assay and phylogenetic analysis, for detecting mycoplasmas and ureaplasmas in urine from HIV-1-infected patients. Primers were designed to amplify a 711-bp fragment of the 16S rRNA gene. Positive PCR-amplified products were further sequenced, and a phylogenetic tree was constructed by neighbor joining of the V4 and V5 sequences from 15 prototypes strains. PCR-positive samples were detected in 17 (9.1%) urine samples, their prototypes being as follows: 2/17 (11.2%) M. genitalium, 1/17 (5.9%) M. hominis, 12/17 (70.6%) Ureaplasma urealyticum, 1/17 (5.9%) mixed M. genitalium and M. hominis, and 1/17 (5.9%) M. hominis and U. urealyticum, respectively. Mycoplasma and Ureaplasma load for each specimen was 3.8 × 10 copies/ml to 7.2 × 10³ copies/ml. Overall, this study demonstrates that real-time PCR and phylogenetic analysis are the fastest available methods for the detection of mycoplasmas in the urogenital tract.

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Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium
2012, Journal of Microbiological Methods
Citation Excerpt :
M. genitalium qPCRs permit a more nuanced understanding of the association between quantitative M. genitalium infection and its sequelea and treatment outcomes. To date, qPCRs have successfully been used in a number of clinical studies, including to demonstrate the associations between M. genitalium and NGU, to monitor the growth of M. genitalium in Vero cell cultures, to show the effect of long-term storage of urine specimens before culture on isolation success, and to monitor the effect of antibiotic treatment on the growth of wild-type M. genitalium strains (Deguchi et al., 2002; Dupin et al., 2003; Hamasuna et al., 2009, 2005, 2007; Hashimoto et al., 2006; Jensen et al., 2004; Manhart et al., 2008; Svenstrup et al., 2005; Yoshida et al., 2002). The susceptibility of M. genitalium to antimicrobial treatment has also been investigated in a number of other studies using qPCR (Dupin et al., 2003; Hamasuna et al., 2009, 2005).
We developed and validated a real-time quantitative polymerase chain reaction (qPCR) assay to determine Mycoplasma genitalium bacterial load in endocervical swabs, based on amplification of the pdhD gene which encodes dihydrolipoamide dehydrogenase, using the Rotor-Gene platform. We first determined the qPCR assay sensitivity, limit of detection, reproducibility and specificity, and then determined the ability of the qPCR assay to quantify M. genitalium in stored endocervical specimens collected from Zimbabwean women participating in clinical research undertaken between 1999 and 2007. The qPCR assay had a detection limit of 300 genome copies/mL and demonstrated low intra- and inter-assay variability. The assay was specific for M. genitalium DNA and did not amplify the DNA from other mycoplasma and ureaplasma species. We quantified M. genitalium in 119 of 1600 endocervical swabs that tested positive for M. genitalium using the commercial Sacace M. genitalium real-time PCR, as well as 156 randomly selected swabs that were negative for M. genitalium by the same assay. The M. genitalium loads ranged between < 300 and 3,240,000 copies/mL. Overall, the qPCR assay demonstrated good range of detection, reproducibility and specificity and can be used for both qualitative and quantitative analyses of M. genitalium in endocervical specimens and potentially other genital specimens.
A cross-sectional study on the relationship of age, gestational age and HIV infection to bacterial vaginosis and genital mycoplasma infection
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Genital mycoplasma & Chlamydia trachomatis infections in treatment naïve HIV-1 infected adults
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Prevalence of Mycoplasma genitalium among HIV-infected men in São Paulo city detected by realtime polymerase chain reaction
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Journal of Infection and Chemotherapy

Abstract

References (28)

Protective activity of tetracycline analogs against the cytopathic effect of the human immunodeficiency viruses in CEM cells

Res Virol

Development and evaluation of the polymerase chain reaction to detect Mycoplasma genitalium

FEMS Microbiol Lett

Molecular features of Millicutes

Clin Infect Dis

Molecular biology and pathogenicity of mycoplasmas

Microbiol Mol Biol Rev

Etiology of nongonococcal nonchlamydial urethritis

Dermatol Clin

The Harrison Lecture. The history and role of Mycoplasma genitalium in sexually transmitted diseases

Genitourin Med

Genital mycoplasma infections

Wien Klin Wochenschr

Urogenital challenge of primate species with Mycoplasma genitalium and characteristics of infection induced in chimpanzees

J Infect Dis

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructual study

Am J Trop Med Hyg

Heat shock protein of Mycoplasma salivarium and Mycoplasma orale strains isolated from HIV-seropositive patients

Bull Tokyo Dent Coll

Detection of Mycoplasma fermentans in broncho-alveolar lavage fluid specimens from AIDS patients with lower respiratory tract infection

HIV Med

Higher prevalence of urogenital mycoplasmas in human immunodeficiency virus-positive patients as compared to patients with other sexually transmitted diseases

J Clin Anal

Increased frequency of detection of Ureaplasma urealyticum and Mycoplasma genitarium in AIDS patients without urethral symptoms

J Clin Microbiol

Mycoplasma penetrans and other mycoplasmas in urine of human immunodeficiency virus-positive children

J Clin Microbiol

Cited by (7)

Development of a rotor-gene real-time PCR assay for the detection and quantification of Mycoplasma genitalium

A cross-sectional study on the relationship of age, gestational age and HIV infection to bacterial vaginosis and genital mycoplasma infection

Genital mycoplasma & Chlamydia trachomatis infections in treatment naïve HIV-1 infected adults

Ureaplasmas: From commensal flora to serious infections

Prevalence of Mycoplasma genitalium among HIV-infected men in São Paulo city detected by realtime polymerase chain reaction

Association of genital mycoplasmas including Mycoplasma genitalium in HIV infected men with nongonococcal urethritis attending STD and HIV clinics