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Background/Objective. Toxin-producing strains of Clostridioides difficile (C. diff) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for C. diff infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. Materials. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to C. diff. Enzyme immunoassay (EIA) tests for the detection of C. diff toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of C. diff GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of C. jejuni and C. coli, an E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. Results. A total of 40 toxigenic strains of C. diff were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for C. diff culture but positive for the following diarrheal agents: Salmonella enterica subsp. arizonae (1/33, LAMP, culture, API 20E); C. jejuni (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, E. coli RT-PCR detection kit, culture); C. perfringens (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic K. pneumonia (2/33) and enterotoxigenic P. mirabilis (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of C. diff was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. diff, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for C. diff toxins A and B, which are the most widely used laboratory tests for CDI. Conclusions. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends.

Campylobacter infection is a serious health problem worldwide. There is still insufficient information in Bulgaria about the spread and course of the infection. Data on campylobacteriosis in adults are particularly scarce. In our study, we performed a comparative analysis in patients in different age groups, including the resistance of isolated strains. In the period 2018-2020, a total of 1,120 patients hospitalised with acute diarrhoea syndrome, aged 0-97 years, were studied, and a total of 1,120 faecal samples were examined by culture techniques and multiplex polymerase chain reaction (PCR). The antimicrobial resistance of some of the isolates was determined. A total of 385 (34.37%) isolates belonged to Campylobacter spp.: 340 (88.31%) to Campylobacter jejuni and 45 (11.68%) C. coli, respectively. The highest incidence was observed in the youngest (< 5 years of age) and the oldest (≥ 65 years of age) patients. Elderly adults are most likely to present with poor and non-specific symptoms despite the severe course of the infection. We observed severe antimicrobial resistance to tetracycline in both young children and older adults (about 40%). In older adults, we observed high resistance to ciprofloxacin. Regarding the C. jejuni strains, the resistance was 19.07% in the youngest, but it reached 59.15% in older adults. The mimicry of the symptoms in older adults can be misleading for the clinician with regard both to the diagnosis and the severity of the infection.

Background Cystic echinococcosis (CE) is a neglected parasitic zoonosis prioritized by the WHO for control. Several studies have investigated potential risk factors for CE through questionnaires, mostly carried out on small samples, providing contrasting results. We present the analysis of risk factor questionnaires administered to participants to a large CE prevalence study conducted in Bulgaria, Romania and Turkey. Methods A semi-structured questionnaire was administered to 24,687 people from rural Bulgaria, Romania and Turkey. CE cases were defined as individuals with abdominal CE cysts detected by ultrasound. Variables associated with CE at P  < 0.20 in bivariate analysis were included into a multivariable logistic model, with a random effect to account for clustering at village level. Adjusted odds ratios (AOR) with 95% CI were used to describe the strength of associations. Data were weighted to reflect the relative distribution of the rural population in the study area by country, age group and sex. Results Valid records from 22,027 people were analyzed. According to the main occupation in the past 20 years, “housewife” (AOR: 3.11; 95% CI: 1.51–6.41) and “retired” (AOR: 2.88; 95% CI: 1.09–7.65) showed significantly higher odds of being infected compared to non-agricultural workers. “Having relatives with CE” (AOR: 4.18; 95% CI: 1.77–9.88) was also associated with higher odds of infection. Interestingly, dog-related and food/water-related factors were not associated with infection. Conclusions Our results point toward infection being acquired in a “domestic” rural environment and support the view that CE should be considered more a “soil-transmitted” than a “food-borne” infection. This result helps delineating the dynamics of infection transmission and has practical implications in the design of specific studies to shed light on actual sources of infection and inform control campaigns.

Visceral leishmaniasis (VL) is a severe endemic disease with a fatal outcome if left untreated. The symptoms of patients are diverse and atypical. Against the background of anemia and pancytopenia, the condition of the patients gradually worsens with marked cachexia. Through sharing our experience, we aim to draw attention to this deadly disease. Clinical and laboratory data for 58 patients with VL treated over a forty-five-year period are presented. The diagnosis was established within a duration of 1 to 28 months of illness. Continuous fever (38–42 °C), splenomegaly, hepatomegaly, severe anemia (decreased hemoglobin to lowest values of 31 g/L), leucopenia (lowest values of leucocytes et 0.5 g/L), and thrombocytopenia (reduced thrombocyte count to 29 g/L) were observed. The diagnosis was made on the basis of microscopic evidence of amastigote forms in bone marrow smears and serological tests. The patients were treated with Glucantime for 17 to 21 days. Relapses were observed in seven patients (12.1%) and fatal outcome was observed in two patients (3.5%) during treatment, who developed acute respiratory and cardiovascular failure. In Bulgaria, Visceral leishmaniasis is primarily endemic in the southern regions and should be suspected not only in patients who have returned from tropical and subtropical countries, but also in those who have not traveled abroad. The challenges associated with VL stem from delayed diagnosis of patients, as this disease remains unrecognized by physicians.

One of the most pressing public health problems is the misuse of antibiotics, especially in industrialized countries. In addition to antibiotic resistance, a disease associated with this abuse is Clostridioides difficile infection (CDI). It is also a serious problem in long-term hospital treatment, as it often causes intra-hospital infections. The coronavirus disease 2019 (COVID-19) pandemic has also led to widespread misuse of antibiotics and prolonged hospitalizations. The aim of our study was to compare two groups of patients who developed CDI before and during the COVID-19 pandemic, respectively. A total of 246 CDI patients with an average age of 58 years were studied for the period of March-August 2021, who also recovered from COVID-19. They were compared with another group of CDI patients who had become ill in the period before COVID-19. We found an increase in CDI patients for the period of the COVID-19 pandemic by 21.95%. Patients with CDI associated with COVID-19 were older, with higher rates of neurologic, tumor and cardiovascular disease, and with metabolic syndrome. In the group with COVID-19 CDI, there was higher use of proton pump inhibitors, use of antibiotics and significantly higher use of corticosteroids. All these factors lead to the conclusion that increased caution is needed in patients with COVID-19 and that mandatory testing should be done for C. difficile infection at the onset of the diarrheal syndrome.

Cystic hydatid disease is a parasitic disease caused by the larvae of the small tapeworm . It is still a serious public health problem in endemic regions such as the Mediterranean basin, especially in the Balkans. Usually, the complaints caused by the cysts are non-specific and there are rarely abnormalities in routine laboratory tests. The most common is the involvement of the liver. The frequency of isolated kidney involvement, especially in a child, is uncommon. We describe a rare pediatric case of an isolated renal hydatid cyst presenting with a urinary tract infection-like clinical presentation, leading to misdiagnosis and delayed treatment.

Objectives: In this study, we describe ocular dirofilariasis in Bulgaria. Materials and Methods: We studied 7 patients with a subconjunctival or periorbital form of Dirofilaria repens infection during 2010–2019. Morphological, serological, and paraclinical diagnostic methods were used. Results: The patients (6 females, 1 male) were aged between 23 and 72 years. In 3 patients, subcutaneous nodules were detected in the area of the upper eyelid, in 1 patient the location was suborbital. In 3 other patients, a subconjunctival location was found. All patients were cured definitively by removal of the larva, without etiologic treatment. Conclusion: The most reliable and easily accessible diagnostic method is morphological analysis by microscopy of histological preparations of the parasite. In dirofilariasis, ocular location is the most common in humans, and it deserves special attention of clinicians.

Infections caused by Campylobacter spp. continue to be a serious health burden worldwide. The importance of Campylobacter jejuni/coli for the development of acute gastroenteritis in Bulgaria has not been studied well yet, especially in early childhood. In this study, we report the incidence and the course of infection with C. jejuni/coli in early childhood and the antibiotic resistance of the clinical isolates tested. Bacteriological examination, followed by Multiplex polymerase chain reaction (PCR), was used. A total of 139 isolates of Campylobacter spp. were obtained from 368 fecal samples from children aged 0-5 years. The C. jejuni strains were 122 (87.8%), and C. coli strains 17 (12.2%). Antibiotic resistance was determined using disc diffusion tests. Resistance to tetracycline (38.1%) and ciprofloxacin (22.1%) was observed, as well as co-resistance to both antibiotics (15.1%). There is a relatively high incidence of campylobacteriosis in hospitalized children with diarrhea. The increasing resistance to quinolone and non-quinolone antibiotics is of concern.

Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods. To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay. Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli. The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests. The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

Cystic echinococcosis is a neglected zoonotic infection that is distributed worldwide and prioritised by WHO for control efforts. The burden of human cystic echinococcosis is poorly understood in most endemic regions, including eastern Europe. We aimed to estimate the prevalence of abdominal cystic echinococcosis in rural areas of Bulgaria, Romania, and Turkey. We did a cross-sectional ultrasound-based survey that recruited volunteers from 50 villages in rural areas of Bulgaria, Romania, and Turkey. These villages were in provinces with annual hospital incidence of cystic echinococcosis within the mid-range for the respective countries. All people who attended a session were allowed to participate if they agreed to be screened. Abdominal ultrasound screening sessions were hosted in public community structures such as community halls, primary health-care centres, schools, and mosques. Lesions were classified using an adapted WHO classification. We reported the prevalence of abdominal cystic echinococcosis adjusted by sex and age through direct standardisation, using the country's rural population as a reference. From July 1, 2014, to Aug 3, 2015, 24 693 individuals presented to screening sessions and 24 687 underwent ultrasound screening. We excluded a further six indivduals due to missing data, leaving 24 681 people in our analysis. Abdominal cystic echinococcosis was detected in 31 of 8602 people screened in Bulgaria, 35 of 7461 screened in Romania, and 53 of 8618 screened in Turkey. The age and sex adjusted prevalence of abdominal cystic echinococcosis was 0·41% (95% CI 0·29–0·58) in Bulgaria, 0·41% (0·26–0·65) in Romania, and 0·59% (0·19–1·85) in Turkey. Active cysts were found in people of all ages, including children, and in all investigated provinces. Our results provide population-based estimates of the prevalence of abdominal cystic echinococcosis. These findings should be useful to support the planning of cost-effective interventions, supporting the WHO roadmap for cystic echinococcosis control. European Union Seventh Framework Programme.