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.

Pneumocystis jirovecii is an opportunistic organism that inhabits predominantly the human pulmonary alveoli. The fully sequenced genome of P. jirovecii was first reported in 2012. According to some authors, P. jirovecii is an obligate pathogen because genes encoding virulence factors and most enzymes for amino acid biosynthesis were not found in the genome. This suggests that the microorganism normally colonises the human lungs but causes disease only in immunocompromised persons. Pneumocystis pneumonia (PCP) is one of the most common opportunistic infections in patients developing acquired immune deficiency syndrome (AIDS). The use of conventional microscopic methods in diagnosis is associated with considerable limitations. Therefore, detection of Pneumocystis DNA in clinical samples by PCR techniques leads to significant advances in the diagnosis of PCP. The aim of this study is to determine the importance of PCR-based methods in the diagnosis of human pneumocystosis and to evaluate their diagnostic value in comparison with conventional microscopy methods. For a period of 20 months in the National Reference Laboratory “Diagnosis of Parasitic Diseases” at the National Centre of Infectious and Parasitic Diseases 33 patients were tested by real-time PCR and 11 of them were found positive for the presence of P. jirovecii DNA. Eight of the patients (72.7%) were HIV-infected. Although limited in extent, this is the first real-time PCR study on the distribution of human pneumocystosis in Bulgaria. Our data shows that PCR techniques have higher sensitivity and specificity than microscopic methods and provide new opportunities for the diagnosis of Pneumocystis pneumonia.

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.

The present study examined the presence of concomitant intestinal parasites and bacteria in individuals infected with Enterobius vermicularis in Bulgaria, and analyzed its effects on the intestinal microbiome and the risk of co-infection. Fecal samples from people with and without (control group) enterobiasis were tested for the presence of concomitant bacterial infection. The results were compared to find out about the intestinal microbiome in these groups. The microbiological examination of the control group showed that 8.3% had only conditionally pathogenic flora. However, in individuals with enterobiasis, 56% had normal intestinal bacterial flora, but 46% had disturbed microbiota: 7% were carriers of pathogenic intestinal bacteria and 24% had opportunistic pathogens. The most prevalent were Klebsiella pneumoniae (49%), followed by Enterobacter spp. (22%), and Proteus mirabilis (12.2%). Our study is the first in the country to investigate the impact of E. vermicularis infection on the composition and quality of the gut microbiome, as well as the potential for co-infections with pathogenic gut bacteria. Although our findings are preliminary, they suggest that this nematode may significantly contribute to a predisposition for dysbiosis or the onset of secondary bacterial infections.