Identification of macrolide-resistant Mycoplasma genitalium using real-time PCR.

OBJECTIVES: Mycoplasma genitalium is a common cause of non-gonococcal urethritis (NGU) in Western Europe, but is not routinely tested for in all clinics. A high prevalence of macrolide-resistant M. genitalium has been reported. An easy to use test that can predict likely macrolide treatment failure is potentially very valuable. We report the development of a rapid and reliable real-time PCR-assay which detects all relevant resistance loci in the M. genitalium 23S rRNA gene. METHODS: Mycoplasma genitalium-positive clinical samples were collected between December 2012 and May 2013, from samples sent routinely to the laboratory for diagnostic testing for M. genitalium. The real-time PCR assay was designed using forward amplification primers complementary to all relevant commonly identified 23s rRNA gene mutations, a common reverse amplification primer and a common TaqMan Probe. RESULT: We report a Taqman assay for detection of common 23S rRNA genotypes at position 2058 and 2059 (Escherichia coli numbering) associated with macrolide resistance, directly from clinical samples. We validated the assay by comparison with DNA sequence determination. CONCLUSION: Our TaqMan assay detects common genotypes associated with macrolide-resistant M. genitalium, namely, A2058G, A2059G and A2058C. We show association between the presence of resistant M. genitalium and treatment failure, thereby confirming the validity of testing for these mutants to prevent further spread of antimicrobial resistance and to allow informed choice of antibiotics for treatment.

Anatomic distribution of Neisseria gonorrhoeae, Chlamydia trachomatis and Mycoplasma genitalium infections in men who have sex with men.

BACKGROUND: New cases of gonorrhoea (Neisseria gonorrhoeae) and chlamydia (Chlamydia trachomatis) infections have been steadily increasing in Scandinavian countries over the last decade. There is a particular urgency in reducing new infections as isolation of multiple drug resistant strains of gonorrhoea is becoming more frequent. The aim of this study was to determine the prevalence and sites of infection of common sexually transmissible infections (STIs) in men who have sex with men (MSM). METHODS: We have performed a retrospective analysis of the three major STIs, gonorrhoea, chlamydia and Mycoplasma genitalium in urogenital, anorectal and oropharyngeal samples from MSM that attended two STI clinics in Oslo. RESULTS: One hundred and thirty-six men (6.0%) out of 2289 MSM tested were found to be positive for gonorrhoea using a porA gene targeted nucleic acid amplification test (NAAT). Of these, 106 (77.9%) would not have been identified through testing first-void urine alone. Two hundred and twenty eight (10.0%) patients from 2289 tested were found to be positive for chlamydia, 164 (71.9%) of which were identified through anorectal specimens. Ninety-one (5.1%) patients from 1778 tested were found to be positive for M. genitalium, with 65 (71.4%) identified through testing of anorectal specimens. CONCLUSIONS: Our results supports the European findings that the MSM population carries a high burden of STIs and that testing the anorectum and oropharynx will identify a significantly higher percentage of infected patients and reservoirs of STIs.

Respiratory tract infections during the 2011 Mycoplasma pneumoniae epidemic.

In 2011, Norway experienced a surge in community acquired Mycoplasma pneumoniae infections. Norway also has one of the highest rates of reported Bordetella pertussis infections, despite high vaccine coverage. We aimed to determine the prevalence of upper respiratory tract pathogens in patients attending primary care physicians for respiratory illness during the 2011 M. pneumoniae epidemic period. A retrospective analysis of data from 26,039 patients that have had nasopharyngeal swabs analysed by nucleic acid amplification testing (NAAT) for M. pneumoniae, C. pneumoniae and B. pertussis was performed. Subsets of samples were tested for additional pathogenic bacteria, including B. parapertussis and B. holmesii, as well as influenza virus. M. pneumoniae, C. pneumoniae and B. pertussis were detected in 2,484 (9.5 %), 261 (1.0 %) and 821 (3.2 %) patients, respectively. Co-infection of M. pneumoniae and B. pertussis was found in 50 (0.19 %) patients, C. pneumoniae and B. pertussis in 4 (0.02 %). Influenza virus was found in 899 (24.5 %) of 3,661 nasopharyngeal swabs. Co-infection of influenza virus and bacterial pathogens was common, although influenza virus co-infection with B. pertussis occurred significantly more often than with C. pneumoniae and M. pneumoniae (20.4 % versus 2.9 % and 9.1 %, respectively; p<0.005). Testing for Bordetella species genes IS1001, IS1002 and recA showed that B. holmesii was most likely misdiagnosed as B. pertussis in 5.8 % of cases. The most prevalent respiratory tract pathogen in the general population in 2011 was M. pneumoniae. B. pertussis was also found frequently as was B. pertussis and influenza virus co-infections.

Mycoplasma genitalium in women with lower genital tract inflammation.

OBJECTIVES: To examine the prevalence of Mycoplasma genitalium in a large number of female patients attending a sexually transmitted infections (STI) clinic and to determine if there is an association with signs or symptoms of lower genital tract inflammation (LGTI). METHODS: Altogether, 7646 female patients who had symptoms or microscopic signs of LGTI or were perceived to be at high risk of exposure to an STI were tested for both M genitalium and Chlamydia trachomatis. Urethral and cervical smears were examined quantitatively for polymorphic mononuclear leucocytes (PMNLs). RESULTS: The prevalence of C trachomatis and M genitalium was 10.1% and 4.5%, respectively. We found a clear association between detecting M genitalium in first void urine (FVU) of patients and signs of urethral inflammation. The strongest association was between detecting M genitalium in FVU and number of PMNLs in urethral smears (n = 6790; OR 2.1; 95 % CI 1.5 to 2.9). The association was less significant between detecting M genitalium in cervical swabs and the number of PMNLs in urethral smears (n = 6785; OR 1.4; 95% CI 1.1 to 1.9), although cervical swabs gave higher sensitivity than FVU in detecting M genitalium (86% vs 62%). C trachomatis detection in FVU and cervical swabs was highly concordant and both significantly associated with urethritis (n = 6790; OR 3.6; 95% CI 3.0 to 4.4). CONCLUSIONS: This data support the hypothesis that M genitalium causes urethritis in women and that M genitalium infection of the genitourinary tract leads to different clinical manifestations depending on whether the site of infection is the urethral or the cervical epithelium.

Mycoplasma genitalium is associated with symptomatic and asymptomatic non-gonococcal urethritis in men.

OBJECTIVES: To examine the prevalence of Mycoplasma genitalium in a large number of male patients attending a sexually transmitted infections (STI) clinic and to determine if there is an association with objective non-gonococcal urethritis (NGU) in patients with and without clinical symptoms. METHODS: Patients were tested for both M genitalium and Chlamydia trachomatis if they had symptoms or microscopic signs of NGU or if they were perceived to be at high-risk of exposure to a STI (n = 8468). Urethral smears were examined for polymorphic mononuclear leucocytes. RESULTS: We found that M genitalium infection was associated with symptoms of non-chlamydial NGU (discharge and dysuria; OR 4.3; 95% CI 3.4 to 5.5). We also found that M genitalium infection was associated with signs of non-chlamydial NGU independently with or without symptoms of NGU (with symptoms: OR 4.7; 95% CI 3.2 to 6.7; without symptoms: OR 3.1; 95% CI 2.0 to 4.6). Prevalence of M genitalium was also associated with severity of urethritis as quantified by microscopic examination of urethral smears. CONCLUSIONS: These data add further evidence to the association of M genitalium infection with NGU and should allow better risk analysis of recent recommendations of not performing urethral smears in asymptomatic men attending STI clinics.

A rapid real-time PCR assay for determination of hepatitis C virus genotypes 1, 2 and 3a.

The genotypes of hepatitis C virus (HCV) in serum of patients have been described as independent predictors of success of antiviral therapy. Therefore, different antiviral regimens have been proposed depending on the infecting HCV genotype. HCV strain is usually determined by polymerase chain reaction (PCR) amplification of genome followed by sequencing or by line-probe assays. We report a new one step real-time PCR assay for genotyping of HCV strains that are prevalent in patients in Norway. HCV types 1, 2 and 3a were genotyped unambiguously in 37 patient serum samples with 100% correlation to genotyping by nucleotide sequence analysis and line-probe assays. Genotyping could also be confirmed against an HCV genotype panel from the National Institute for Biological Standards and Control. This assay does not require manipulation of amplified PCR products, it involves very little hands on and analysis time. This assay can be used for rapid genotyping of HCV-RNA in infected patients to aid physicians decide suitability of patients for treatment and subsequent length of treatment.

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expression.

By using 5' RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIalpha subunit of cAMP-dependent protein kinase that differed in their 5' untranslated regions. Two of these 5'-regions showed similarity with the human RIalpha exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIalpha 1b and RIalpha 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIalpha 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIalpha protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIalpha 1c haploid-specific mRNA, therefore, may be important for the regulation of RIalpha expression in sperm.

A novel isoform of human cyclic 3',5'-adenosine monophosphate-dependent protein kinase, c alpha-s, localizes to sperm midpiece.

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C alpha catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C alpha gene. Sequence comparison revealed similarity to an ovine C alpha variant characterized by protein purification and micropeptide sequencing, C alpha-s, identifying the cloned human cDNA as the C alpha-s isoform. The C alpha-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C alpha-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C alpha-s-specific antibody indicated that C alpha-s was localized in the midpiece region of the spermatozoon. The majority of C alpha-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C alpha-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI alpha, RII alpha, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C alpha-s. This novel C alpha-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s).

The gene encoding the C gamma catalytic subunit of cAMP-dependent protein kinase is a transcribed retroposon.

Three different catalytic isoforms of cAMP-dependent protein kinase have been identified (C alpha, C beta, and C gamma). We report the cloning and characterization of the human and rhesus monkey genes encoding the testis-specific C gamma subunit. The human C gamma gene is intronless with an open reading frame similar to the previously published cDNA sequence. The 3' and 5' flanking regions share high similarity with the C alpha nontranslated regions (82%) also outside the regions corresponding to the C gamma cDNA. The human gene is flanked by an Alu-related sequence in the 5'-end and there are insertions of two Alu-related sequences in the 3' nontranslated region. The observation that the C gamma gene is intronless and colinear with C alpha mRNA, together with the presence of remnants of a poly(A) tail and flanking direct repeats, indicates that the C gamma gene is a C alpha-derived retroposon. The 5' flanking region of this gene has a high G/C content and a putative TATA box situated at -138 compared to the translation initiation codon. Cloning and sequencing of a partial C gamma rhesus monkey gene demonstrate conservation of the sequence in primates. Northern analysis on isolated and fractionated human germ cells of testes from normal and estrogen-treated individuals demonstrates that the C gamma gene is expressed only in germ cells in the human testis. Our results indicate that the C gamma gene is a retroposon specifically transcribed in primate testicular germ cells.

Structure, function, and regulation of human cAMP-dependent protein kinases.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Regulation of protein kinase A subunits by cyclic adenosine 3',5'-monophosphate in a mouse Sertoli cell line (MSC-1): induction of RII beta messenger ribonucleic acid is independent of continuous protein synthesis.

We report the basal and cAMP-regulated expression of protein kinase A (PKA) subunits in a mouse Sertoli cell line (MSC-1). Of the PKA subunits expressed by these cells (RI alpha, RII alpha, RII beta, C alpha, C beta), only RII beta was regulated by cAMP. An approximately 8-fold induction of RII beta mRNA and a 3-fold induction of RII beta protein was observed during 48 h of cAMP-stimulation. This cAMP-mediated RII beta mRNA induction, reaching maximal levels after approximately 12 h, did not require ongoing protein synthesis. Fairly rapid decay of maximally induced RII beta mRNA was observed after removal of cAMP (t1/2 approximately 5 h). Further, ongoing transcription and translation were necessary for rapid degradation of RII beta mRNA. Thus, the MSC-1 cells expressed all the PKA subunits present in primary cultures of Sertoli cells and responded to cAMP with increased levels of RII beta at both mRNA and protein levels. Although the nature of some of these responses distinguished the observations in MSC-1 cells from previously described responses in primary cultures, these cells may prove to be useful in future studies addressing cAMP-mediated gene regulation.