Challenges with co-amplification of microbial DNA in interpretation of STR profiles obtained from human skeletal remains.

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.

Identification of developmental disorders including autism spectrum disorder using salivary miRNAs in children from Bosnia and Herzegovina.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by major social, communication and behavioural challenges. The cause of ASD is still unclear and it is assumed that environmental, genetic and epigenetic factors influence the risk of ASD occurrence. MicroRNAs (miRNAs) are short 21-25 nucleotide long RNA molecules which post-transcriptionally regulate gene expression. MiRNAs play an important role in central nervous system development; therefore, dysregulation of miRNAs is connected to changes in behaviour and cognition observed in many disorders including ASD. Based on previously published work, on diagnosing ASD using miRNAs, we hypothesized that miRNAs can be used as biomarkers in children with suspected developmental disorders (DD) including ASD within Bosnian-Herzegovinian (B&H) population. 14 selected miRNAs were tested on saliva of children with suspected developmental disorders including ASD. The method of choice was qRT-PCR as a relatively cheap method available in most diagnostic laboratories in low to mid-income countries (LMIC). Out of 14 analysed miRNAs, 6 were differentially expressed between typically developing children and children with some type of developmental disorder including autism spectrum disorder. Using the most optimal logistic regression, we were able to distinguish between ASD and typically developing (TD) children. We have found 5 miRNAs as potential biomarkers. From those, 3 were differentially expressed within the ASD cohort. All 5 miRNAs had shown good chi-square statistics within the logistic regression performed on all 14 analysed miRNAs. The accuracy of 5-miRNAs model training set was 90.2%, while the validation set had a 90% accuracy. This study has shown that miRNAs may be considered as biomarkers for ASD detection and may be used to identify children with ASD along with standard developmental screening tests. By combining these methods we may be able to reach a reliable and accessible diagnostic model for children with ASD in LMIC such as B&H.

A novel flavivirus strain detected in phlebotomine sandflies in Bosnia and Herzegovina.

Aim Phlebotominae sandflies are primary vectors of phleboviruses, causing the sandfly fever disease. The aim of this study was to detect and report the presence of flaviviruses in Phlebotominae sandflies captured in Bosnia and Herzegovina. Methods After a microscopic and morphometric analysis, the final identification of collected Phlebotomus specimens was confirmed by PCR, using a hemi-nested polymerase chain reaction on extracted and reversely transcribed RNA. Results We obtained a 155 nt long fragment of the viral non-structural protein 5 (NS5) gene (GenBank accession no. MN090154). The acquired nucleotide sequence, provisionally named as Dreznica, showed a maximum of 70-80% identity in 70-88% (110-137 nucleotides) of the query coverage with several Anopheles, Sabethes, Calbertado and Culex flaviviruses. Maximum likelihood phylogenetic analysis showed that the new flavivirus Dreznica clusters together with the flavivirus isolated from Culiseta annulata mosquitos. Conclusion We report the presence of flavivirus in Phlebotominae sandflies, captured in Dreznica, Herzegovina for the first time. The next phase of research will be directed towards virus cultivation, obtaining a longer or complete virus sequence and clarifying the medical and epidemiological importance of the Dreznica virus.

Development of a novel biofilm classification tool and comparative analysis of result interpretation methodologies for the evaluation of biofilm forming capacity of bacteria using tissue culture plate method.

Aim To develop an online biofilm calculation tool (Biofilm Classifier), which calculates the optical density cut off value and accordingly determines the biofilm forming categories for the tested isolates by standardized formulas, as well as to compare the results obtained by Biofilm Classifier to manual calculations and the use of predefined values. Methods The biofilm forming capacity of tested strains was evaluated using tissue culture plate method in 96 well plates, and optical density (OD) value of the formed biofilm was measured on an ELISA Microplate reader at 595 nm on a total of 551 bacterial isolates from clinical specimen. Results Comparative analysis indicated that the manual calculation was 100% in accordance with results obtained by the designed software as opposed to the results obtained by use of predefined values for biofilm categorization. When using predefined values compared to manual biofilm categorization for the determination of biofilm positive and biofilm negative strains the specificity was 100%, sensitivity 97.81%, positive predictive value 100%, negative predictive value 96.04% and accuracy 98.57%. Conclusion Considering obtained results, the use of the designed online calculator would simplify the interpretation of biofilm forming capacity of bacteria using tissue culture plate method.