Purpose. The routine diagnosis of enterobiasis consists of a microscopic examination of perianal imprints on a transparent scotch tape for the presence of parasitic eggs. However, this method has low sensitivity when the number of eggs is small. The development of highly sensitive and specific methods, such as PCR, will increase the possibilities of detecting the parasite and will enable its genetic characterization. Material/Methods. The study was conducted from May 2022 to September 2022 and comprised 24 patients infected with Enterobius vermicularis. DNA was extracted from parasite eggs/adult worms using PureLink Genomic DNA Mini Kit (INVITROGEN), according to the manufacturer's instructions. Nested polymerase chain reaction (PCR) for detection of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was applied with two sets of primers. Results. The developed and described method for genetic determination of Enterobius vermicularis in samples from infected individuals is suitable and applicable to improve the diagnosis of the disease, as well as for further phylogenetic research of the parasite, which is of great importance for its successful treatment and control. Conclusions. Regardless of existing morphological diagnostic methods, genotyping studies concerning Enterobius vermicularis need to be deepened. Molecular-biological analyses increase diagnostic sensitivity, provide valuable knowledge about the geographic distribution and diversity of helminths, and allow the monitoring of therapy.

Background: Pneumocystis pneumonia (PCP) commonly affects immunocompromised individuals, whereas in immunocompetent persons, it occurs relatively rarely, and in most cases, the Pneumocystis infection is detected as an asymptomatic colonization. The present study aimed to establish the prevalence of Pneumocystis jirovecii infection in human hosts with different immune status (immunocompromised and immunocompetent), using molecular diagnostic methods, and to compare their diagnostic value with that of classical staining methods. Methods: We used the collected-to-this-moment data from a prospective study on the prevalence of pneumocystosis among the Bulgarian population. Clinical specimens (including throat secretions, induced sputum, tracheal aspirates, and bronchoalveolar lavage) collected from 220 patients suspected of PCP (153 immunocompetent and 67 immunocompromised patients) were examined with microscopic staining methods and real-time PCR for detection of P. jirovecii. Results: DNA of the pathogen was detected in 38 (17%) specimens (32 immunocompromised patients and 6 immunocompetent subjects). From all 220 clinical samples examined by staining methods, only five (2%) P. jirovecii cysts were detected by the Gomori stain. All patients with PCP were treated with trimethoprim-sulfamethoxazole, but in ten of them (HIV-positive patients), the disease had a fatal outcome. Conclusions: This study is the first in Bulgaria including the main available laboratory methods for diagnosis of human pneumocystosis. Regarding the etiological diagnosis of PCP, in our study the sensitivity of real-time PCR was higher compared to the staining methods. The choice of a method for sample collection and examination has an important role in the efficiency of the laboratory diagnostics.

Pneumocystis jirovecii pneumonia (PCP) is a significant cause of morbidity and mortality in immunocompromised people. The widespread use of trimethoprim-sulfamethoxazole (TMP-SMZ) for the treatment and prophylaxis of opportunistic infections (including PCP) has led to an increased selection of TMP-SMZ-resistant microorganisms. Sulfa/sulfone resistance has been demonstrated to result from specific point mutations in the DHPS gene. This study aims to investigate the presence of DHPS gene mutations among P. jirovecii isolates from Bulgarian patients with PCP. A total of 326 patients were examined via real-time PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene and further at the DHPS locus. P. jirovecii DNA was detected in 50 (15.34%) specimens. A 370 bp DHPS locus fragment was successfully amplified in 21 samples from 19 PCP-positive patients, which was then purified, sequenced, and used for phylogenetic analysis. Based on the sequencing analysis, all (n = 21) P. jirovecii isolates showed DHPS genotype 1 (the wild type, with the nucleotide sequence ACA CGG CCT at codons 55, 56, and 57, respectively). In conclusion, infections caused by P. jirovecii mutants potentially resistant to sulfonamides are still rare events in Bulgaria. DHPS genotype 1 at codons 55 and 57 is the predominant P. jirovecii strain in the country.

The specificity of the life cycle of T. gondii, the causative agent of toxoplasmosis, determines its relevance in some patient groups; especially in women of childbearing age. The objective of this study was to ascertain the prevalence of this parasitic infection in Bulgaria between 2014 and 2023, focusing on pregnant women and those applying for an in vitro procedure or after an abortion. During the period, 115,053 individuals were tested for toxoplasmosis in the country, with an average seropositivity rate of 16.96%. At the NRL for the Diagnosis of Parasitic Diseases, ELISA tests were utilized to ascertain specific Toxoplasma IgG, IgM, and IgA antibodies, IgG avidity, and PCR to substantiate the DNA of the parasite. Between 2014 and 2023, the laboratory conducted tests on 631 pregnant women, and 161 women applying for in vitro fertilization or after an abortion. In 24.7% of pregnant women, data demonstrated the presence of IgG antibodies only, and Toxoplasma IgG and IgM or IgG, IgM, and IgA antibodies were found in 16.6% and 8% of women, respectively. In the subset of individuals examined after an abortion or for an in vitro procedure, IgG was detected in 28%, IgG and IgM in 13%, and IgG, IgM, and IgA antibodies in 3.7%. Seroprevalence rises with increasing age, but does not show any dynamic compared to our previous studies. Our results show a higher seropositivity for toxoplasmosis in pregnant women in Bulgaria than in neighboring countries.

Introduction: Toxocariasis is a zoonotic helminth infection with difficult diagnosis. Determination of specific IgG antibodies alone does not allow to establish the disease stage and to evaluate the treatment efficacy. Therefore it is necessary to identify additional markers that will assist the diagnosis. The purpose of our study was to identify and monitor eosinophil cationic protein (ECP) levels in patients with toxocariasis confirmed by serology and to compare our data with the literature to determine the relevance of this protein as an indicator for recent infection and the effectiveness of the therapy. Material and methods: ELISA (CUSABIO) commercial kit was used for determination of ECP concentration. Sixty serum samples were studied from individuals previously tested and confirmed for toxocariasis by the presence of specific anti-Toxocara IgG antibodies in ELISA (Toxocara IgG Rbiopharm) and the presence of specific bands in Western blot as confirmatory test (LD BIO). Twenty serum samples from clinically healthy blood donors were used as a control group. Results: The mean concentration of serum ECP in the patients with toxocariasis was significantly higher than in clinically healthy subjects. Seventy-two percent of patients affected by toxicariasis showed increased serum concentration of ECP. However, no statistically significant differences were observed in terms of age (p = 0.451) and sex (p = 0.682) of the patients or clinical form of the disease. ECP levels among patients with visceral toxocariasis were relatively higher (mean 22.99 ng / ml ± 13.16 SD) in comparison to those with ocular involvement (15.60 ng/ml ± 9.92 SD). Correlation between the presence of peripheral eosinophilia and the concentration of serum ECP was not also established. Conclusion: Data from our study give us reason to believe that serum levels of ECP could serve as an additional marker indicating recent infection, especially in patients without marked increase in the blood eosinophils.

Summary: Enterobiasis is one of the most common human parasitic infections worldwide and in Bulgaria. The objective of this study was to ascertain the levels of intestinal secretory IgA antibodies in patients with enterobiasis, to determine the local immune response in this helminthiasis, and to evaluate its influence on clinical manifestations during infection. Faecal samples from 102 enterobiasis patients and 40 clinically healthy controls were examined. In individuals infected with Enterobius vermicularis , the range of values for SIgA was higher (from 27.5 μg/ml to 13916 μg/ml). However, no statistically significant difference was found between them and those in persons without evidence of infection (from 27.5 to 8999 μg/ml). In both groups of individuals (infected and non-infected), we observed differences in the levels of SIgA, which appeared to be dependent on the age and gender of the subjects. Significantly, higher values were observed in children and adolescents, as well as in males. In individuals with enterobiasis, a higher level of SIgA was observed in those with pronounced clinical symptoms (mean value = 2198.74) compared to asymptomatic individuals (mean value = 1588.54). The highest levels were observed in patients presenting with perianal pruritus (mean value = 3559.54). Our study of the local humoral immune response in people with enterobiasis is the first of its kind in the country. The results clearly show a direct correlation between the presence of clinical symptoms in enterobiasis and elevated levels of secretory IgA in faeces.