A retrospective study on nested PCR detection of syphilis treponemes in clinical samples: PCR detection contributes to the diagnosis of syphilis in patients with seronegative and serodiscrepant results.

Syphilis, caused by Treponema pallidum ssp. pallidum (TPA), is a persisting global health problem. Although syphilis diagnostics relies mainly on serology, serological tests have some limitations, and it is recommended that the final diagnosis be supported by additional tests. The purpose of this study was to analyze the relationship between serology and PCR in syphilis diagnostics. From the year 2004 to May 2019, a total of 941 samples were taken from 833 patients suspected of having syphilis, in Czech Republic. In all these samples, both nested PCR detection of TPA and serology testing were performed. Of the 941 samples, 126 were seronegative, 651 were seropositive, and 164 were serodiscrepant. Among seronegative samples (n = 126), 11 were PCR-positive (8.7%). Among seropositive samples (n = 651; i.e., samples positive for both non-treponemal and treponemal serology tests), 368 samples were PCR-positive (56.5%). The remaining 164 serodiscrepant samples included RPR negative and treponemal serological test-positive samples (n = 154) and a set of 10 RPR-positive samples negative in treponemal serological tests. While the first group revealed 73 PCR-positive samples (47.4%), the second revealed 5 PCR positive samples (50.0%). PCR detection rates were highest in primary syphilis, with lower rates in the secondary and undetermined syphilis stages. As shown here, the nested PCR can improve diagnostics of syphilis, especially in seronegative patients and in patients with discrepant serology.

MLST typing of Treponema pallidum subsp. pallidum in the Czech Republic during 2004-2017: Clinical isolates belonged to 25 allelic profiles and harbored 8 novel allelic variants.

A recently introduced Multilocus Sequence Typing scheme for Treponema pallidum subsp. pallidum was applied to clinical samples collected from 2004 to 2017 from the two largest cities (Prague and Brno) in the Czech Republic. Altogether, a total of 675 samples were tested in this study and 281 of them were found PCR-positive for treponemal DNA and typeable. Most of the typed samples (n = 281) were swabs from primary or secondary syphilis lesions (n = 231), and only a minority were whole blood or tissue samples (n = 50). Swab samples from patients with rapid plasma regain (RPR) values of 1-1024 were more frequently PCR-positive (84.6%) compared to samples from patients with non-reactive RPR test (46.5%; p-value = 0.0001). Out of 281 typeable samples, 136 were fully-typed at all TP0136, TP0548, and TP0705 loci. Among the fully and partially typed samples, 25 different allelic profiles were identified. Altogether, eight novel allelic variants were found among fully (n = 5) and partially (n = 3) typed samples. The distribution of TPA allelic profiles identified in the Czech Republic from 2004 to 2017 revealed a dynamic character with allelic profiles disappearing and emerging over time. While the number of samples with the A2058G mutation was seen to increase (86.7% in 2016/2017), the number of samples harboring the A2059G mutation was found to have decreased over time (3.3% in 2016/2017). In addition, we found several allelic profile associations with macrolide resistance or susceptibility, the gender of patients, as well as patient residence.

Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae co-infections among patients with newly diagnosed syphilis: a single-centre, cross-sectional study.

OBJECTIVES: The aim of the study was to determine the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae co-infections among patients with newly diagnosed syphilis. METHODS: In patients with any stage of newly diagnosed syphilis swabs were performed from urethra, rectum, pharynx and cervix according to the gender and type of sexual intercourse. From these smears standard validated nucleic acid amplification tests (NAATs) for Chlamydia trachomatis and Neisseria gonorrhoeae infections were done. RESULTS: From 548 (488 men, 60 women) screened patients co-infection was detected in 15.9% of the cases. The majority of the co-infections (86.2%) were asymptomatic. The overall prevalence of chlamydial infection was 11.1% and 8.8% for gonococcal infections. In men who have sex with men (MSM) the prevalence of co-infections was significantly higher (20.0%) than in heterosexual men and women (4.2%) (p < 0.001). In MSM patients the presence of co-infection was significantly associated with HIV infection (p < 0.001). Among MSM 9.6% of the tests detected infection in anorectal site, while prevalence in urethral (2.8%) and pharyngeal (2.4%) localization was significantly lower. In heterosexual patients prevalence was less than 2.0% in all anatomic sites. CONCLUSIONS: The implementation of screening tests in case of sexually transmitted infections in patients with newly diagnosed syphilis is an important part in the management of this disease. These results suggest that screening of asymptomatic heterosexual patients leads to detection of minimum co-infections, but in MSM (especially HIV positive) should always be performed at least in anorectal site, where asymptomatic co-infections are common.

Steady increase of lymphogranuloma venereum cases, Czech Republic, 2010 to 2015.

Since the notification of the first case of lymphogranuloma venereum (LGV) in the Czech Republic in 2010, the numbers of LGV cases have steadily increased in the country. In 2015, 40 LGV cases were diagnosed, bringing the total for 2010-2015, to 88 cases. The profile of the most affected group, HIV-positive men who have sex with men with a previous sexually transmitted infection, matches that of those described in LGV outbreaks in western Europe.

Molecular typing of Treponema pallidum in the Czech Republic during 2011 to 2013: increased prevalence of identified genotypes and of isolates with macrolide resistance.

From January 2011 to December 2013, a total of 262 samples, from 188 patients suspected of having syphilis were tested for the presence of treponemal DNA by PCR amplification of five chromosomal loci, including the polA (TP0105), tmpC (TP0319), TP0136, TP0548, and 23S rRNA genes. Altogether, 146 samples from 103 patients were PCR positive for treponemal DNA. A set of 81 samples from 62 PCR-positive patients were typeable, and among them, nine different genotypes were identified. Compared to a previous study in the Czech Republic during 2004 to 2010, the number of genotypes detected among syphilis patients in a particular year increased to six in both 2012 and 2013, although they were not the same six. The proportion of macrolide-resistant clinical isolates in this 3-year study was 66.7%.

Syphilis-causing strains belong to separate SS14-like or Nichols-like groups as defined by multilocus analysis of 19 Treponema pallidum strains.

Treponema pallidum strains are closely related at the genome level but cause distinct diseases. Subspecies pallidum (TPA) is the causative agent of syphilis, subspecies pertenue (TPE) causes yaws while subspecies endemicum (TEN) causes bejel (endemic syphilis). Compared to the majority of treponemal genomic regions, several chromosomal loci were found to be more diverse. To assess genetic variability in diverse genomic positions, we have selected (based on published genomic data) and sequenced five variable loci, TP0304, TP0346, TP0488, TP0515 and TP0558, in 19 reference Treponema pallidum strains including all T. pallidum subspecies (TPA, TPE and TEN). Results of this multilocus analysis divided syphilitic isolates into two groups: SS14-like and Nichols-like. The SS14-like group is comprised of SS14, Grady, Mexico A and Philadelphia 1 strains. The Nichols-like group consisted of strains Nichols, Bal 73-1, DAL-1, MN-3, Philadelphia 2, Haiti B and Madras. The TP0558 locus was selected for further studies because it clearly distinguished between the SS14- and Nichols-like groups and because the phylogenetic tree derived from the TP0558 locus showed the same clustering pattern as the tree constructed from whole genome sequences. In addition, TP0558 was shown as the only tested locus that evolved under negative selection within TPA strains. Sequencing of a short fragment (573bp) of the TP0558 locus in a set of 25 clinical isolates from 22 patients collected in the Czech Republic during 2012-2013 revealed that clinical isolates follow the SS14- and Nichols-like distribution.

Juvenile generalized pustular psoriasis treated with etanercept.

An 8-year-old boy with general pustular psoriasis (GPP) and iatrogenic secondary Cushing syndrome was treated successfully with etanercept after he had failed on acitretin, methotrexate, and methylprednisolone therapy. GPP is a severe and very rare variant of psoriasis in children often accompanied by life-threatening complications. Retinoids, cyclosporine, methotrexate, or dapsone used in a small number of case series and case reports were effective. Etanercept is a recombinant human tumor necrosis factor-alpha (TNF-alpha) receptor protein fused with Fc portion of IgG1 that binds to TNF-alpha, approved by Food and Drug Administration for the treatment of moderate-to-severe plaque psoriasis in children and teens who have not responded to other psoriasis treatments. In our patient, etanercept demonstrated significant clinical response associated with long-term efficacy without acute exacerbation, excellent tolerability, and good safety profile.

Pemphigus vulgaris treated with adalimumab: case study.

The study describes the case of a patient with a clinical and histopathological diagnosis of pemphigus vulgaris accompanied by severe side-effects of combined immunosuppressive therapy, who achieved a remission of the disease with adalimumab. Pemphigus vulgaris is a chronic blistering disease of the skin and mucous membranes. Before corticosteroids were introduced, mortality was high. Corticosteroids are currently used as first-line therapy. To reduce the dose of corticosteroids, therapeutic combinations with corticosteroid-sparing immunosuppressive agents are used. The therapy brings a number of complications due to its side effects. To achieve a remission of the disease by treating our patient with combined immunosuppressives, we administered adalimumab and achieved a very good clinical response.

[Lymphogranuloma venereum]. / Lymphogranuloma venereum.

Lymphogranuloma venereum is a sexually transmitted disease caused by serovars L1-3 of Chlamydia trachomatis. This infection was originally endemic in tropics and transmitted predominantly by heterosexual contact but since the beginning of the century it spreads in industrialized countries mainly among men having sex with men causing them severe proctitis. In the Czech Republic the first case was diagnosed in 2011. Lymphogranuloma venereum can resemble other forms of anorectal disorders inclusive inflammatory bowel diseases and thus it must be included into differential diagnostic considerations. Definitive diagnosis is based on detection of specific serovars of Chlamydia trachomatis by polymerase chain reaction. In patients with lymphogranuloma venereum it is also necessary to exclude other sexually transmitted diseases, particularly syphilis, HIV and also hepatitis C. The therapy of choice is doxycycline administered for three weeks.

[Detection of borrelia DNA from patients with neuroborreliosis and erythema migrans]. / Prukaz boreliové DNA u pacientu s neuroboreliózou.

AIM: Assessment of PCR procedure for proving of the Borrelia burgdorferi sensu lato DNA in nerve and skin forms of Lyme borreliosis. METHODS: DNA from plasma, urine and CSF was isolated by QIAamp DNA mini kit. PCR was designed as two-step amplification (nested-PCR). Each sample was tested in PCR for five target sequences: two were specific for plasmide genes encoding OspA and OspC proteins and three correlated with genes for 16SrDNA, flagellin and p66 protein. RESULTS: Borrelial DNA was proved in 41 patients suffering from neuroborreliosis out of 56 (77.4 %), among 48 patients with erythema migrans (EM) were found 26 positive (54.2 %). After treatment the specific DNA was detected in 22 patients with neuroborreliosis (41.5 %) and 16 patients with EM (38.1 %). Three months after the treatment 23 patients were positive in both of groups (28.7 %) and next 3 months later the specific DNA was found in 6 (9.5 %). The highest rate of positive results was manifested by 16SrDNA target, lower and comparable results were obtained by OspA, C and flagellin primers, the lowest rate was in p66 system. CONCLUSION: The tested PCR proved specific DNA in all tested biological fluids in both of the clinical forms of Lyme borreliosis with a relatively high sensitivity. The proving of DNA can not be used for the assessment of the effect of treatment due to the long persistence of PCR positivity after antibiotic treatment. To achieve a sufficient diagnostic sensitivity of PCR it is desirable to use minimally two amplification systems in parallel.

Examination of specific DNA by PCR in patients with different forms of Lyme borreliosis.

BACKGROUND: Borrelial specific DNA was examined in a group of 62 patients with different forms of Lyme borreliosis (LB) (32 patients suffered from neuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement). METHODS: Nested-PCR system with five newly derived primers was used in parallel. The study was organized prospectively, the presence of DNA was tested for plasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid and urine were examined after treatment. RESULTS: Before therapy, 36 patients (58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were found in the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement, and 7 (63.6%) were positive in arthritis. After treatment, 11 patients (36.7%) were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) in joint form of LB. Among 97 positive amplifications the most frequent target was found in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but very similar results were obtained with primers for OspA (18 positive amplifications; 18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positive amplifications; 13.4%). There were 11 patients in whom only DNA and no specific antibodies were found. CONCLUSIONS: Specific DNA was found in all clinical groups of LB with similar sensitivity. Examination of the borrelial DNA in urine displayed the same sensitivity as in CSF and had a two times higher sensitivity than in plasma.

Lyme borreliosis treatment.

Lyme borreliosis is the most common human tick-borne illness in the Northern Hemisphere. The causative agent is the spirochete Borrelia burgdorferi species complex, and the hard-shell ticks of the genus Ixodes is responsible for pathogen transmission from animals to humans. The incidence of the disease is increasing year by year and although lyme disease is not fatal, it can affect the skin, heart, nervous, and musculoskeletal system with an impairment of quality of life. The appropriate diagnosis of lyme disease should be promptly treated by antibiotics to prevent late stage of the disease. The choice of antibiotics depends on many factors such as the stage of the disease, the drug efficacy, adverse effects, type of delivery, duration of treatment, and cost. Treatment failure occurs as a result of many reasons, re-infection is possible. The recommended treatment schedule in the Czech Republic is presented.