Detection of Macrolide and/or Fluoroquinolone Resistance Genes in Mycoplasma genitalium Strains Isolated from Men in the Northwest Region of Croatia in 2018-2023.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.

Rhinovirus-A True Respiratory Threat or a Common Inconvenience of Childhood?

A decade-long neglect of rhinovirus as an important agent of disease in humans was primarily due to the fact that they were seen as less virulent and capable of causing only mild respiratory infections such as common cold. However, with an advent of molecular diagnostic methods, an increasing number of reports placed them among the pathogens found in the lower respiratory tract and recognized them as important risk factors for asthma-related pathology in childhood. As the spread of rhinovirus was not severely affected by the implementation of social distancing and other measures during the coronavirus disease 2019 (COVID-19) pandemic, its putative pathogenic role has become even more evident in recent years. By concentrating on children as the most vulnerable group, in this narrative review we first present classification and main traits of rhinovirus, followed by epidemiology and clinical presentation, risk factors for severe forms of the disease, long-term complications and the pathogenesis of asthma, as well as a snapshot of treatment trials and studies. Recent evidence suggests that the rhinovirus is a significant contributor to respiratory illness in both high-risk and low-risk populations of children.

Comparison of MT-PCR with Quantitative PCR for Human Bocavirus in Respiratory Samples with Multiple Respiratory Viruses Detection.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

Prevalence and Molecular Characterization of Human Bocavirus Detected in Croatian Children with Respiratory Infection.

Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs' genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10-4 and 10-5 substitutions per site and year. Recombination was not detected among sequences from this study.

Seasonal Coronaviruses and Other Neglected Respiratory Viruses: A Global Perspective and a Local Snapshot.

Respiratory viral infections are the leading cause of morbidity and mortality in the world; however, there are several groups of viruses that are insufficiently routinely sought for, and can thus be considered neglected from a diagnostic and clinical standpoint. Timely detection of seasonality of certain respiratory viruses (e.g., enveloped viruses such as seasonal coronaviruses) in the local context can aid substantially in targeted and cost-effective utilization of viral diagnostic approaches. For the other, non-enveloped and year-round viruses (i.e., rhinovirus, adenovirus, and bocavirus), a continuous virological diagnosis needs to be implemented in clinical laboratories to more effectively address the aetiology of respiratory infections, and assess the overall impact of these viruses on disease burden. While the coronavirus disease 2019 (COVID-19) pandemic is still actively unfolding, we aimed to emphasize the persistent role of seasonal coronaviruses, rhinoviruses, adenoviruses and bocaviruses in the aetiology of respiratory infections. Consequently, this paper concentrates on the burden and epidemiological trends of aforementioned viral groups on a global level, but also provides a snapshot of their prevalence patterns in Croatia in order to underscore the potential implications of viral seasonality. An overall global prevalence in respiratory tract infections was found to be between 0.5 and 18.4% for seasonal coronaviruses, between 13 and 59% for rhinoviruses, between 1 and 36% for human adenoviruses, and between 1 and 56.8% for human bocaviruses. A Croatian dataset on patients with respiratory tract infection and younger than 18 years of age has revealed a fairly high prevalence of rhinoviruses (33.4%), with much lower prevalence of adenoviruses (15.6%), seasonal coronaviruses (7.1%), and bocaviruses (5.3%). These insights represent a relevant discussion point in the context of the COVID-19 pandemic where the testing of non-SARS-CoV-2 viruses has been limited in many settings, making the monitoring of disease burden associated with other respiratory viruses rather difficult.

Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis.

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4-8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis.

Human Papillomavirus (HPV) Prevalence, Temporal Dynamics and Association with Abnormal Cervical Cytology Findings in Women from Croatia: Is there a Compounding Effect of Low-Risk/High-Risk HPV Co-Infection?

BACKGROUND: Human papillomavirus (HPV) is a major risk factor for cervical dysplasia and invasive cervical cancer; therefore, regular screening by cervical smear cytology or HPV testing is recommended. We aimed to determine the overall and risk group-specific HPV prevalence, age distribution, and temporal trends and to appraise the correlation of HPV positivity with abnormal cervical cytological findings. METHODS: This retrospective, single-center study involved a total of 751 women (aged 18 - 67) concurrently subjected to HPV DNA testing and cervical cytology evaluation over a 10-year period in Zagreb, Croatia. Digene HC2 HPV DNA test (Qiagen Corporation, USA) was employed in screening specimens for both low-risk and high-risk HPV risk groups. The cytology was reported using the Bethesda system and in accordance with uniform classification of uterine cervix cytological findings in Croatia "Zagreb 2002". Statistical significance was set at p < 0.05. RESULTS: The overall HPV prevalence in our study population was 48.6%, and the 18 - 30 age group presented with the highest infection burden (p = 0.046). A decrease in low-risk and high-risk mono-positivity has been observed over the 10-year period; conversely, there was a significant increase in low-risk/high-risk co-positivity (p = 0.007). Low-risk/high-risk HPV co-infection resulted in a compounding effect which increased the occurrence of abnormal cells, HPV-associated changes and low grade squamous intraepithelial lesions (LSIL/cervical intraepithelial neoplasia grade I) in cervical cytology when compared to mono-infection with either low-risk or high-risk HPV. On the other hand, such effect has not been demonstrated for high grade squamous intraepithelial lesions (HSIL/ cervical intraepithelial neoplasia grades II and III). CONCLUSIONS: The overall HPV prevalence in female outpatients was high, underscored with rising co-positivity rates. Such co-infection with both low-risk and high-risk HPV (predominantly seen in women younger than 30) can exhibit a compounding effect in the occurrence of cytological abnormalities and low grade squamous intra-epithelial lesions (LSIL), which has to be considered in future diagnostic and screening algorithms.

The Emerging Role of Rhinoviruses in Lower Respiratory Tract Infections in Children - Clinical and Molecular Epidemiological Study From Croatia, 2017-2019.

Rhinoviruses (RVs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of RVs among the infected children. Therefore, we investigated the rhinovirus (RV) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of RV infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of Croatia. For respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex RT-PCR. To determine the RV species in a subset of positive children, 5'UTR in RV-positive samples has been sequenced. Nucleotide sequences of referent RV strains were retrieved by searching the database with Basic Local Alignment Tool, and used to construct alignments and phylogenetic trees using MAFFT multiple sequence alignment tool and the maximum likelihood method, respectively. In our study population RV was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. Median age of RV-infected children was 2.25 years, and more than half of children infected with RV (55.8%) presented with lower respiratory tract infections. Most RV cases were detected from September to December, and all three species co-circulated during the analyzed period (2017-2019). Sequence analysis based on 5'UTR region yielded 69 distinct strains; the most prevalent was RV-C (47.4%) followed by RV-A (44.7%) and RV-B (7.9%). Most of RV-A sequences formed a distinct phylogenetic group; only strains RI/HR409-18 (along with a reference strain MF978777) clustered with RV-C strains. Strains belonging to the group C were the most diverse (41.6% identity among strains), while group B was the most conserved (71.5% identity among strains). Despite such differences in strain groups (hitherto undescribed in Croatia), clinical presentation of infected children was rather similar. Our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where RVs should always be considered as potentially serious pathogens.

An Outbreak of Human Parainfluenza Virus 3 (Phylogenetic Subcluster C5) Infection among Adults at a Residential Care Facility for the Disabled in Croatia, 2018.

INTRODUCTION: Although highly pertinent for children, outbreaks of human parainfluenza virus (HPIV) may cause up to 15% of all respiratory illnesses in adults and predispose them to serious adverse outcomes, with HPIV serotype 3 (HPIV3) being the most common. This study represents the first report of an HPIV3 outbreak among adults at a long-term health-care facility in Croatia. METHODS: A retrospective study was conducted to investigate an outbreak of acute respiratory infection (ARI) at a single residential care facility for the disabled in Croatia. Demographic, epidemiological, and clinical data were collected for all residents, while hospitalized patients were appraised in detail by laboratory/radiological methods. Multiplex PCR for respiratory viruses and sequencing was performed. Partial HPIV3 HN 581 nt sequences were aligned with HPIV3 sequences from the GenBank database to conduct a phylogenetic analysis, where different bioinformatic approaches were employed. RESULTS: In late June 2018, 5 of the 10 units at the facility were affected by the outbreak. Among the 106 residents, 23 (21.7%) developed ARI, and 6 (26.1%) of them were hospitalized. HPIV3 was identified in 18 (73%) of the residents and 5 (83%) of the hospitalized individuals. Isolated HPIV3 strains were classified within the phylogenetic subcluster C5 but grouped on 2 separate branches of the phylogenetic tree. During the entire outbreak period, none of the institution's employees reported symptoms of ARI. CONCLUSIONS: Our study has shown that this health care-associated outbreak of HPIV3 infection could have been linked to multiple importation events. Preventive measures in curbing such incidents should be enforced vigorously.

Antimicrobial Resistance Screening in Chlamydia trachomatis by Optimized McCoy Cell Culture System and Direct qPCR-Based Monitoring of Chlamydial Growth.

Obligate intracellular localization of Chlamydia trachomatis (C. trachomatis) complicates antimicrobial sensitivity testing efforts that we are so accustomed to in routine bacteriology. Cell culture systems with immunofluorescence staining, to identify cellular inclusions in the presence of various concentrations of antimicrobial drugs, are still the most pervasive techniques, but more specific and sensitive nucleic acid concentration measuring methods are increasingly being used. Here we describe how to approach antimicrobial susceptibility/resistance screening in C. trachomatis by using a McCoy cell culture system, optimized by a research group from Croatia, and direct qPCR-based monitoring of chlamydial growth, optimized by a research group from Hungary.

A 'pathogenic needle' in a 'commensal haystack': Genetic virulence signatures of Corynebacterium glucuronolyticum that may drive its infectious propensity for the male urogenital system.

The predominance of the genus Corynebacterium in the healthy male urogenital system contributes to the resident microbiome of not only the distal urethra, but potentially the proximal urethra and urinary bladder as well. However, for certain species in this genus, pathogenic potential was described, and the salient representative is Corynebacterium glucuronolyticum (C. glucuronolyticum) implicated in cases of urethritis and prostatitis in men. Nonetheless, some still question whether C. glucuronolyticum can actually be considered pathogenic or rather just a commensal species fortuitously isolated in patients with urogenital symptoms and/or syndromes. Although pathogen/commensal dichotomy is not always clear-cut, we hypothesize that specific genetic markers may expose C. glucuronolyticum as a convincingly pathogenic Corynebacterium. More specifically, characteristic pathogenic gene constellation inherent to this species (most notably the presence of specific sortase/SpaA-type pili gene clusters, but also the augmentative role of type VII secretion system) may significantly facilitate host tissue adhesion, with subsequent suppression/evasion of the immune response and acquisition of vitally important nutrients. Consequently, these genetic markers differentiate C. glucuronolyticum from its commensal counterparts, and give this species a pathogenic facet, which can be even further influenced by the Allee effect. In this paper we also propose a specific methodological approach on how to analyze C. glucuronolyticum epithelial colonization capacity and explore inceptive host cell-pathogen interactions that manipulate host environment and immune responses. This entails moving from approaches based primarily on overall homology of primary sequences towards specific structure-function studies to precisely evaluate all stakeholders involved in pili assemblage, cell adhesion and the expression of other virulence traits. In the era of high precision medicine, the hypothesized roles of C. glucuronolyticum adhesion systems in both virulence and nutrient acquisition may also reveal promising targets for future drug developments.

High Detection Rates of Human Bocavirus in Infants and Small Children with Lower Respiratory Tract Infection from Croatia.

BACKGROUND: Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. METHODS: During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. RESULTS: Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). CONCLUSIONS: Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease.

Viral pathogens associated with acute respiratory illness in hospitalized adults and elderly from Zagreb, Croatia, 2016 to 2018.

AIMS: To investigate the viral etiology of acute respiratory infection (ARI) in hospitalized adults and elderly patients in Croatia, compare the prevalence of detected viruses, and to determine clinical characteristics and seasonal occurrence of investigated infections. METHODS: From January 2016 to June 2018, a total of 182 adult patients presented with symptoms of ARI and admitted to the hospital were tested for 15 respiratory viruses by multiplex reverse-transcription polymerase chain reaction. Clinical data were collected by retrospective analysis of the patient's chart. RESULTS: A virus was identified in 106 (58.5%) of the patients. The most commonly detected virus was influenza virus (41.5%), followed by respiratory syncytial virus (13.8%), human metapneumovirus (13.0%), parainfluenza viruses (12.2%), rhinoviruses (11.4%), adenovirus and coronaviruses with equal frequencies (3.3%), and enterovirus (1.6%). The serum level of C-reactive protein and white blood cell count were significantly lower in patients with respiratory viruses identified when compared with those in whom no virus was detected (P < 0.001 and P = 0.007, respectively). There were no differences in clinical symptoms according to the type of the detected virus, except for more frequent illness exposure recall for influenza infection ( P = 0.010). Influenza, parainfluenza, and pneumoviruses were detected mostly in winter months, while rhinoviruses in autumn and spring. CONCLUSIONS: In addition to influenza, pneumoviruses, rhinoviruses, and parainfluenza viruses play an important role in etiology of ARIs in adults. Fast and accurate laboratory diagnosis for respiratory viruses in routine practice is needed for clinicians optimally manage patients with ARI and potentially avoid the unnecessary use of antimicrobial drugs.

Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene.

BACKGROUND: Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). Its coding region constitutes less than 50% of the complete gene length, enabling SH gene to be highly variable and the SH protein highly conserved. In standard HRSV molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (HVR2) are analyzed. To what extent do the strains identical in HVR2 differ elsewhere in genomes is rarely investigated. Our goal was to investigate whether diversity and inter-genotype differences observed for HVR2 are also present in the SH gene. METHODS: We sequenced 198 clinical samples collected within a limited area and time frame. In this HRSV collection, rapid and significant changes in HVR2 occurred. RESULTS: Over 20% of strains from this pool (containing HRSV genotypes NA1, ON1, GA5, BA9 and BA10) would be incorrectly assumed to be identical to another strain if only the HVR2 region was analysed. The majority of differences found in SH gene were located in the 5' untranslated region (UTR). Seven indels were detected, one was genotype GA5 specific. An in-frame deletion of 9 nucleotides (coding for amino acids 49-51) was observed in one of group A strains. Fifteen different SH protein sequences were detected; 68% of strains possessed the consensus sequence and most of others differed from the consensus in only one amino acid (only 4 strains differed in 2 amino acids). The majority of differing amino acids in group A viruses had the same identity as the corresponding amino acids in group B strains. When analysis was restricted to strains with identical HVR2 nucleotide sequences and differing SH protein sequences, 75% of differences observed in the SH ectodomain were located within region coding for amino acids 49-51. CONCLUSIONS: Basing HRSV molecular epidemiology studies solely on HVR2 largely underestimates the complexity of circulating virus populations. In strain identification, broadening of the genomic target sequence to SH gene would provide a more comprehensive insight into viral pool versatility and its evolutionary processes.

Molecular mechanisms of Chlamydia trachomatis resistance to antimicrobial drugs.

Chlamydia trachomatis (C. trachomatis) is a leading cause of bacterial sexually transmitted infections in developed and undeveloped countries, and therefore a global public health issue. In an era of increasing bacterial resistance to antibiotics, resistance has been an exceedingly rare phenomenon in C. trachomatis; however, clinical treatment failures attributed to multidrug-resistant C. trachomatis strains have been described on several occasions. Cell culture systems using McCoy cells and subsequent immunofluorescent staining are still the most common methodology used for antimicrobial susceptibility testing, but the presence of resistance markers should be appraised by further genetic analysis. Azithromycin resistance of C. trachomatis is often a result of the mutations in the peptidyl transferase region of 23S rRNA genes, tetracycline resistance is usually linked to the presence of foreign genomic islands integrated in chlamydial chromosome, whereas a predominant mechanism of fluoroquinolone resistance is a point mutation in the gyrA quinolone-resistance-determining region. A nucleotide substitution in rpoB gene is responsible for rifampin resistance, and different mechanisms have been involved in the development of resistance to aminoglycosides, lincomycin and sulphonamide/trimethoprim combinations.

Sociodemographic, Sexual Behavior, and Microbiological Profiles of Men Attending Public Health Laboratories for Testing for Sexually Transmitted Diseases.

In order to identify the groups at risk of sexually transmitted diseases (STDs), we assessed the sociodemographic profiles of men testing for STD, their sexual habits, and the results of microbiological analysis. During a three-year period, a total of 700 men older than 18 years of age completed the questionnaire regarding sociodemographic and sexual behavior. Urethral swabs were taken for microbiological analysis. Thirty-three percent of respondents reported not using condoms. Those that do not use condoms were predominantly less educated, unmarried but in steady relationships, employed, with children, and smokers. Alcohol or drug usage before sexual intercourse was disclosed by 21.4% of respondents, and 10.3% respondents reported sexual intercourses with commercial sex workers. Finally, 24.0% respondents reported sexual relations abroad. In 28.1% of subjects, one or more pathogens were observed in urethral swabs. The most commonly diagnosed microorganism was Ureaplasma urealyticum, followed by Chlamydia trachomatis, Mycoplasma hominis, Trichomonas vaginalis, and Neisseria gonorrhoeae. This study identified several factors that may contribute to the general risk of STD transmission, which will serve to better understand the transmission dynamics and implementation of adequate prevention programs.

Assessing the Need for Routine Screening for Mycoplasma genitalium in the Low-risk Female Population: A Prevalence and Co-infection Study on Women from Croatia.

BACKGROUND: There is an ongoing debate regarding possible cost and benefits, but also harm of universal screening for the emerging sexually transmitted pathogen Mycoplasma genitalium. METHODS: From the initial pool of 8665 samples that were tested, a subset of Chlamydia trachomatis-positive and randomly selected C. trachomatis-negative cervical swabs were further interrogated for M. genitalium by real-time polymerase chain reaction, using a 224 bp long fragment of the glyceraldehyde-3-phosphate dehydrogenase gene. RESULTS: M. genitalium was detected in 4.8% of C. trachomatis-positive samples and none of C. trachomatis-negative samples. Accordingly, a significant association was shown between M. genitalium and C. trachomatis (P < 0.01), but also between M. genitalium and Mycoplasma hominis infection (P < 0.01). CONCLUSIONS: Based on the results, routine screening is recommended only for women with one or more identified risk factors. Moreover, younger age does not represent an appropriate inclusion/exclusion criterion for M. genitalium testing in the low-risk female population.

Genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in Croatia.

Human metapneumovirus (HMPV) is recognized as a global and frequent cause of acute respiratory tract infections among people of all ages. The objectives of this study were molecular epidemiology and evolutionary analysis of HMPV strains which produced moderate and severe acute respiratory tract infections in children in Croatia during four consecutive seasons (2011-2014). A total of 117 HMPV-positive samples collected from hospitalized pediatric patients presenting with acute respiratory tract infections and tested by direct immunofluorescence assay were first analyzed by amplifying a part of the F gene. Sixteen samples were further analyzed based on complete F, G, and SH gene sequences. HMPV genome was identified in 92 of 117 samples (78%) and the circulation of multiple lineages of HMPV was confirmed. In 2011, 2012, and 2014, subgroups A2 and B2 co-circulated, while B1 gained prevalence in 2013 and 2014. The study established the presence of a novel subcluster A2c in Croatia. This subcluster has only recently been detected in East and Southeast Asia. This study provides new insights into epidemiology and genetic diversity of HMPV in this part of Europe.

Genetic analysis of human parainfluenza virus type 3 obtained in Croatia, 2011-2015.

PURPOSE: This study investigated the HPIV3 circulating strains in Croatia and whether the other parts of HPIV3 genome (F gene and HN 582 nucleotides fragment) could be equally suitable for genetic and phylogenetic analysis. METHODOLOGY: Clinical materials were collected in period 2011-2015 from children suffering from respiratory illnesses. In positive HPIV3 samples viral genome was partially amplified and sequenced for HN and F genes. Obtained sequences were analysed by phylogenetic analysis and genetic characterization was performed. RESULTS: All samples from this study belonged to subcluster C and over a short period of time, genetic lineage C3a gained prevalence over the other C genetic lineages, from 39 % in 2011 to more than 90 % in 2013 and 2014. Phylogenetic classifications of HPIV3 based on the entire HN gene, HN 582 nt fragment and entire fusion (F) gene showed identical classification results for Croatian strains and the reference strains. Molecular analysis of the F and HN glycoproteins, showed their similar nucleotide diversity (Fcds P=0.0244 and HNcds P=0.0231) and similar Ka/Ks ratios (F Ka/Ks=0.0553 and HN Ka/Ks=0.0428). Potential N-glycosylation sites, cysteine residues and antigenic sites are generally strongly conserved in HPIV3 glycoproteins from both our and the reference samples. CONCLUSION: The HPIV3 subclaster C3 (genetic lineage C3a) became the most detected circulating HPIV3 strain in Croatia. The results indicated that the HN 582 nt and the entire F gene sequences were as good for phylogenetic analysis as the entire HN gene sequence.

Genetic Variability and Sequence Relatedness of Matrix Protein in Viruses of the Families Paramyxoviridae and Pneumoviridae.

BACKGROUND: The families Paramyxoviridae and Pneumoviridae comprise a broad spectrum of viral pathogens that affect human health. The matrix (M) protein of these viruses has a central role in their life cycle. In line with this, molecular characteristics of the M proteins from variable viruses that circulated in Croatia were investigated. METHODS: Sequences of the M proteins of human parainfluenza virus (HPIV) 1-3 within the family Paramyxoviridae, human metapneumovirus (HMPV), and human respiratory syncytial virus from the family Pneumoviridae were obtained and analyzed. RESULTS: M proteins were very diverse among HPIVs, but highly conserved within each virus. More variability was seen in nucleotide sequences of M proteins from the Pneumoviridae family. An insertion of 8 nucleotides in the 3' untranslated region in 1 HMPV M gene sequence was discovered (HR347-12). As there are no samples with such an insertion in the database, this insertion is of interest and requires further research. CONCLUSION: While we have confirmed that M proteins were conserved among individual viruses, any changes that are observed should be given attention and further researched. Of special interest is inclusion of HPIV2 M proteins in this analysis, as these proteins have not been studied to the same extent as other paramyxoviruses.