Diagnostics of Sexually Transmitted Diseases

Prikazana je dijagnostika spolno prenosivih bolesti. Tradicionalni pristup dijagnostici C. trachomatis bazira se na staničnoj kulturi, što je tehnički vrlo zahtjevan postupak. Prednost testova baziranih na detekciji antigena i nukleinskih kiselina je u brzini (DFA, EIA, PACE 2) i u mogućnosti obrade velikog broja uzoraka (EIA, PACE 2). Metode bazirane na amplifikaciji nukleinskih kiselina (PCR, LCR) imaju iznimno visoku specifičnost i osjetljivost. U. urealyticum i M. hominis dobro rastu na posebno obogaćenim umjetnim hranjivim podlogama. Kultivacija M. genitalium traje 1-2 mjeseca te se za njezin dokaz rabe PCR-testovi. T. vaginalis se u sekretima iz genitalnog trakta dijagnosticira mikroskopiranjem nativnog preparata ili kulturom na različitim obogaćenim podlogama. Za dijagnostiku N. gonorrhoeae važan je mikroskopski pregled uzorka odmah nakon uzimanja. Uzorak se boji po Gramu ili metilenskim plavilom. Podloge za uzgoj N. gonorrhoeae sadržavaju bogatu hranjivu bazu s dodatkom krvi. Selektivne podloge sadržavaju i antibiotike koji omogućuju izolaciju gonokoka iz visokokontaminiranih uzoraka. Za dijagnostiku N. gonorrhoeae postoje komercijalni amplifikacijski testovi (PCR, LCR). Od molekularnih metoda DNA hibridizacijske metode (PACE 2, Digene Hybrid Capture II) osim odvojenih testova nude i paralelnu dijagnostiku N. gonorrhoeae i C. trachomatis. Za detekciju HPV-a najčešće se rabe molekularne metode bazirane na hibridizaciji nukleinskih kiselina (HCII) i lančane reakcije polimerazom (PCR). Testom je obuhvaćeno 13 genotipova visokog rizika i 5 genotipova niskog rizika.The article presents the diagnostics of sexually transmitted diseases. Traditional approach to the diagnostics of C. trachomatis is based on cell culture, what is technically a very demanding procedure. The advantage of tests founded on the antigen and nuclein acids detection is in their speed (DFA, EIA, PACE 2) and in the possibility of the analysis of large samples (EIA, PACE 2). Methods based on the nuclein acids amplification (PCR, LCR) have extremely high specificity and sensitivity. U. urealyticum and M. hominis grow well on separately artificial nutritive substrates. The cultivation of M. genitalium lasts 1-2 months, and is proved by PCR tests. T. vaginalis is diagnosed in genital tract secretions by microscopy of a native preparation or culture on various enriched substrates. For the diagnostics of N. gonorrhoeae, microscopy of the sample immediately after taking is of great importance. The sample is colored by Gram or with methylene blue. The substrates for the N. gonorrhoeae cultivation contain rich nutritive basis with addition of blood. Selective substrates also contain antibiotics enabling isolation of gonococcus from highly contaminated samples. For the diagnosis of N. gonorrhoeae commercial amplification tests (PCR, LCR) can be used. Out of molecular methods, DNA hybridization methods (PACE 2m Digene Hybrid Capture II), besides separated tests, offer parallel diagnostics of N. gonnorrhoeae and C. trachomatis as well. For the detection of HPV, molecular methods are mostly used, based on the nuclein acid hybridization (HCII) and polymerase chain reactions (PCR). The test encompasses 13 high risk and 5 low risk genotypes

Microbiological Diagnostics of Sexually Transmitted Infections

Spolno prenosive infekcije važan su problem u svijetu i u nas, kako zbog velike učestalosti tako i zbog mogućih trajnih posljedica za zdravlje kao što su neplodnost, zdjelična upalna bolest, izvanmaternična trudnoća, karcinom, kongenitalne infekcije, pa čak i smrt. Za razliku od drugih infekcija koje se prenose kontaktom, velik broj oboljelih nema jasnih simptoma, ili su simptomi odsutni, a višestruke su ("miješane") infekcije također česte. Za točnu etiološku dijagnozu uzročnika spolno prenosivih infekcija potrebni su osjetljivi i specifi čni testovi. Osim klasičnih dijagnostičkih metoda danas se dijagnostika spolno prenosivih infekcija sve više temelji na metodama molekularne biologije koje u odnosu na klasične metode imaju značajno veću osjetljivost i specifi čnost te postaju "zlatni standard" za većinu ovih infekcija. Na tržištu postoji velik broj komercijalnih testova koji se baziraju na hibridizaciji (hybrid capture) ili na jednoj od amplifi kacijskih metoda (PCR, TMA, SDA). Zbog visoke osjetljivosti ovih testova uzročnici se mogu detektirati u neinvazivnim uzorcima (urin) ili omogućavaju bolesnicama/bolesnicima da same/i uzmu uzorak (obrisak vagine, vulve ili analne regije). Na taj način postiže se bolja suradljivost ciljne populacije.Sexually transmitted infections (STIs) are an important global problem both due to their increasing rate and serious health consequences such as infertility, pelvic inflammatory disease, ectopic pregnancy, congenital infections, and even mortality. Many STIs are asymptomatic and therefore go undetected and untreated. This is of particular concern with the recognition that HIV transmission is increased with co-existent STIs. Therefore, accurate, cost-effective and reliable diagnostic assays are needed to obtain more precise data on the incidence of various STIs. Conventional diagnostic assays have a relatively low sensitivity. However, with the advent of molecular technologies, including target and signal amplifi cation methods, diagnostics of STIs have been revolutionised and allow the use of non-invasive or minimally invasive sampling techniques, some of which are self-collected by the patient, e.g. first-void urine and low vaginal swabs. Most studies evaluating such self-sampling with molecular diagnostic techniques have demonstrated an equivalent or superior detection of STIs as compared to conventional sampling and detection methods

Tinea Incognita in a Patient with Crest Syndrome: Case Report

Tinea incognita is a dermatophytic infection that is difficult to diagnose, usually modified by inappropriate topical or systemic corticosteroid therapy. We report an extensive case of tinea incognita caused by the zoophilic dermatophyte Trichophyton mentagrophytes (var. granulosa) in a 49-year-old female patient with CREST (Calcinosis; Raynaud phenomenon; Esophageal involvement; Sclerodactyly; Teleangiectasia) syndrome. Immunocompromised patients, as well as patients with keratinization disorders, seem to be especially susceptible to dermatophytic infections with atypical clinical presentation that is sometimes bizarre and difficult to recognize. Therefore, close monitoring and mycological skin examination is recommended in order to avoid misdiagnosis and to give the patient the best chance of recovery.</p

Kerion Celsi due to Microsporum canis with dermatophytide reaction

Microsporum (M.) canis is the most common fungus to cause tinea capitis in Europe, especially in the Mediterranean region and South and Central Europe. Fungal scalp infections caused by M. canis tend to be non-inflammatory. Recently, a growing number of cases of tinea capitis characterized by inflammatory infection caused by M. canis and M. gypseum have been registered. We present a case of highly inflammatory tinea capitis, also known as kerion celsi, caused by M. canis in a 6-year-old-patient. Scalp infections due to M. canis are a growing problem in dermatological practice. Changes in epidemiology, etiology, and clinical patterns of fungal infections due to M. canis are significant. Greater awareness of this problem is needed in order to establish proper diagnosis and successful treatment strategy for these patients. </p

Molecular Variants of Human Papilloma Viruses Type 16 and 6 in Women with Different Cytological Results Detected by RFLP Analysis

HPV infections are common and the presence of the same high-risk type in cervical specimens can be due to reinfection or persistence. Persistent infection is the most important predictor for development of cervical carcinoma. The aim of this study was to validate PCR-RFLP with two sets of primers: MY09/MY11 that amplify a fragment of L1 and P1/P2 that amplify a fragment of E1 ORF. PCR product of MY09/MY11 was digested with a set of 6 restriction enzimes (RE) and PCR product of P1/P2 with a set of 12 RE. Cervical samples from 110 women patients of the University Gynecologic Clinic CHC Zagreb were analyzed. There were 98 (89.1%) PCR positive samples detected with P1/P2 primers, and 94 (85.5%) PCR positive samples detected with MY09/MY11 primers. Seven HPV types were detected with P1/P2-RFLP technique and 17 with MY09/MY11-RFLP. PCR positive samples amplified with both primer pairs agreed with each other in 82 samples; 16 samples were only positive with P1/P2 and 12 samples were only positive by MY09/MY11. HPV 16 was detected in 39 samples with MY09/11-RFLP, out of these two variants (two different patterns) were found with P1/P2 using Dde I, Hae III and Eco I. HPV 6 was detected in 9 samples with MY09/11-RFLP, out of these two variants were found with P1/P2 using HinfI. Combining these two PCR-RFLP methods subtypes of HPV 16 and HPV 6 were detected

HPV Testing for Cervical Cancer Screening in Croatia

Opportunistic screening based on the Pap smear has been undertaken in Croatia since 1953. However, cervical cancer remains an important health problem in Croatia when compared to European countries with organised screening programmes. In Croatia, in addition to screening based on well established cytology, Human papillomavirus (HPV) testing is widely used as secondary test as a triage to borderline cytology and as a follow-up after treatment of severe cervical lesions. Many different approaches for HPV testing arose in Croatia over the last decade depending on the needs of each medical institution involved. Presently, there is an urgent need for better networking between the laboratories, the implementation of quality assessment and the adaptation of a uniform system of referring to and reporting of HPV testing. In conclusion, the best possible organisation for HPV testing would be essential for implementation of HPV testing as primary screening test in Croatia, an thus ultimately and hopefully, the more successful cervical cancer control

Value of Rapid Aetiological Diagnosis in Optimization of Antimicrobial Treatment in Bacterial Community Acquired Pneumonia

In 80 adult patients with community acquired pneumonia (CAP) conventional microbiological methods, polymerase chain reaction (PCR) and serum C-reactive protein (CRP) levels were performed and the appropriateness of the empirical antimicrobial treatment was evaluated according to bacterial pathogen detected. The aetiology was determined in 42 (52.5%) patients, with Streptococcus pneumoniae as the most common pathogen. PCR applied to bronchoalveolar lavage (BAL) provided 2 and PCR on sputum samples 1 additional aetiological diagnosis of CAP. The mean CRP values in the S. pneumoniae group were not significantly higher than in the group with other aetiological diagnoses (166.89 mg/L vs.160.11 mg/L, p=0.457). In 23.8% (10/42) of patients with determined aetiology, the empirical antimicrobial treatment was inappropriate. PCR tests need further investigation, particularly those for the atypical pathogens, as they are predominant in inappropriately treated patients. Our results do not support the use of CRP as a rapid test to guide the antimicrobial treatment in patients with CAP

Epidemic spread of OXA-48 beta-lactamase in Croatia

PURPOSE: A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. ----- METHODS: Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. ----- RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. ----- CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017