Molecular Analysis of Dihydropteroate Synthase Gene Mutations in Pneumocystis jirovecii Isolates among Bulgarian Patients with Pneumocystis Pneumonia.

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.

Study on the Prevalence of Pneumocystis jirovecii as a Causative Agent of Lung Pathology in People with Different Immune Status.

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.

Unveiling the incidences and trends of the neglected zoonosis cystic echinococcosis in Europe: a systematic review from the MEmE project.

The neglected zoonosis cystic echinococcosis affects mainly pastoral and rural communities in both low-income and upper-middle-income countries. In Europe, it should be regarded as an orphan and rare disease. Although human cystic echinococcosis is a notifiable parasitic infectious disease in most European countries, in practice it is largely under-reported by national health systems. To fill this gap, we extracted data on the number, incidence, and trend of human cases in Europe through a systematic review approach, using both the scientific and grey literature and accounting for the period of publication from 1997 to 2021. The highest number of possible human cases at the national level was calculated from various data sources to generate a descriptive model of human cystic echinococcosis in Europe. We identified 64 745 human cystic echinococcosis cases from 40 European countries. The mean annual incidence from 1997 to 2020 throughout Europe was 0·64 cases per 100 000 people and in EU member states was 0·50 cases per 100 000 people. Based on incidence rates and trends detected in this study, the current epicentre of cystic echinococcosis in Europe is in the southeastern European countries, whereas historical endemic European Mediterranean countries have recorded a decrease in the number of cases over the time.

Epidemiological and clinical characteristics of imported malaria in Bulgaria: A retrospective study of а 21-year period.

BACKGROUND: Bulgaria, with a high endemicity for malaria in the past, was declared by the WHO as a malaria-free country in 1965. We intended to analyze the epidemiological and clinical implications of imported malaria cases in Bulgaria. METHODS: This is a retrospective cross-sectional analysis of all recorded cases of imported malaria in Bulgaria over a 21-year period (2000-2020). Patients' clinical records and information gathered from the epidemiological survey of each recorded malaria case were reviewed. RESULTS: A total of 232 cases of imported malaria were reported, 147 (63.4%) were Bulgarian citizens (BC) and 85 (36.6%) were foreign nationals (FN). Two thirds (66.4%) of cases were diagnosed from April to October. Most BCs had travelled for work (66.6%) to Africa (93.9%) and were infected with P. falciparum (83.3%), while most FNs were migrants (54.7%), exposed in Asia (63.5%) with P. vivax infection (62.4%). Clinical complications and a fatal outcome were noted in 14.7% (n = 34) and 3.5% (n = 8) of cases respectively. All complicated cases were in BNs with P. falciparum infection. CONCLUSIONS: Bulgaria experiences a steady import of malaria. Efforts to improve diagnosis, management and prevention of malaria, as well as maintenance of a high degree of epidemiological vigilance are needed.

First case of ocular dirofilariosis in Bulgaria caused by gravid female Dirofilaria repens.

Dirofilariosis caused by Dirofilaria (Nochtiella) repens is recorded sporadically among people in Europe, Asia and Africa. Still a worldwide controversy exist upon human parasite hosting. Herein, the first case of ocular dirofilariosis in Bulgaria caused by gravid female is presented. A single nematode was removed from the eye of the 76- year-old patient in the course of cataract surgery. Microscopic examination of the histological slides revealed microfilariae in the parasite's uterus, but not in the blood. Knott's method for detection of microfilariae in the peripheral blood was negative and the rest of laboratory blood and biochemical tests were within reference limits. A comprehensive review of the etiology, pathogenesis and clinical presentation of Dirofilaria repens infection is also highlighted.

Study of IgG avidity and the level of specific IgA antibodies and their significance in the diagnosis of human toxocarosis.

Toxocarosis is a zoonotic disease caused by migration and subsequent localization of nematode larvae of Toxocara spp. in human organs and tissues, which is manifested with development of various non-specific clinical symptoms. Main diagnostic methods are serological and consists in proving the presence of anti-Toxocara IgG antibodies in patient's sera. In humans, anti-Toxocara IgG has been shown to persist in the serum for a long time and cannot be used to distinguish between past and recent infection. Aim of the present work is to investigate the diagnostic significance of the specific IgG avidity level, determined by an immuno enzyme test developed by us, and the presence of anti-Toxocara IgA for distinguishing between acute and chronic toxocarosis. The study included 130 patients with positive results in routine serological ELISA and Western blot tests and with clinical symptoms of visceral and ocular toxocarosis. The results revealed low IgG avidity (≤40%) in nine (7.3%) and presence of anti-Toxocara IgA antibodies in 36 (26.2%) of the subjects. Low avidity of IgG antibodies was found only in the first tests, and a presence of specific IgA for up to 9 months. The results of our study give us reason to believe that determination of the IgG avidity in toxocarosis is of greater diagnostic value than the presence of IgA to establish the stage of the disease.

Extra-hepatopulmonary cystic echinococcosis in Bulgaria: frequency, management and outcome of the disease.

In the endemic countries, human cystic echinococcosis (CE) poses a serious medical and social problem. Because it most often affects the liver and lungs we aimed to define the proportion of cases with different organ localization, the diagnostic and therapeutic approaches in such cases, and the outcome of them. For a period 2010-2019, a total of 2863 cases of CE were registered in Bulgaria, of which 148 (5.17%) with organ localization other than liver and/or lung. The majority of patients with extra-hepatopulmonary localization of CE were adults. The distribution by gender showed predominance of female patients (57.43%) over those of males and primary cases (85.14%) exceeded the cases of recurrence. According to our study most common is the spleen involvement, followed by involvement of the abdominal cavity, kidneys and muscle/subcutaneous tissue. Other extra-hepatopulmonary organ localizations are significantly less common. This study shows that the extra-hepatopulmonary localization of CE is not so rare, and in most cases it is a primary disease. In respect of this, clinicians should consider hydatidosis in the differential diagnosis when cystic formation(s) is found, regardless of the organ involved.

Human Parasitic Diseases in Bulgaria in Between 2013-2014.

BACKGROUND: In Bulgaria, more than 20 autochthonous human parasitic infections have been described and some of them are widespread. Over 50 imported protozoan and helminthic infections represent diagnostic and therapeutic challenges and pose epidemiological risks due to the possibility of local transmission. AIMS: To establish the distribution of autochthonous and imported parasitic diseases among the population of the country over a 2-year period (2013-2014) and to evaluate their significance in the public health system. STUDY DESIGN: Cross sectional study. METHODS: We used the annual reports by regional health inspectorates and data from the National Reference Laboratory at the National Centre of Infectious and Parasitic Diseases on all individuals infected with parasitic diseases in the country. Prevalence was calculated for parasitic diseases with few or absent clinical manifestations (oligosymptomatic or asymptomatic infections). Incidence per 100.000 was calculated for diseases with an overt clinical picture or those that required hospitalisation and specialised medical interventions (e.g. surgery). RESULTS: During the research period, parasitological studies were conducted on 1441.244 persons, and parasitic infections were diagnosed in 22.039 individuals. Distribution of various parasitic pathogens among the population displayed statistically significant differences in prevalence for some intestinal parasites (enterobiasis 0.81%, giardiasis 0.34% and blastocystosis 0.22%). For certain zoonotic diseases such as cystic echinococcosis (average incidence of 3.99 per 100.000) and trichinellosis (average incidence of 0.8 per 100.000), the incidence exceeds several times the annual incidence recorded in the European Union. CONCLUSION: Parasitic diseases still pose a substantial problem with social and medical impacts on the residents of our country. Improved efficiency regarding autochthonous and imported parasitic diseases is essential in providing the public health system the tools it needs to combat these diseases. Attention should be focused on the various imported vector-borne parasitic diseases (e.g. malaria and cutaneous leishmaniasis) for which the country is potentially endemic.

Imported malaria in Bulgaria, status and prognosis after eradication in 1965.

BACKGROUND: This retrospective analysis assessed all recorded malaria cases in Bulgaria after 1965, when the country was certified as malaria-free by the World Health Organization (WHO), and evaluated the readiness of the public health system to interrupt an outbreak of local transmission in case of infection importation. METHODS: The cases were analyzed according to causative species; geographic origin of the imported case; and the citizenship, age, and gender of the infected individuals. RESULTS: In the 50-year study period (1966-2015), there were a total of 3011 cases of malaria imported to Bulgaria from different regions of the world. The majority of the cases originating in Africa were caused by Plasmodium falciparum (65.5%), while most of these originating in Asia were caused by P. vivax (80.9%). The potential season for malaria transmission in Bulgaria is from April to October, and 58.5% of the malaria cases were imported during that time of the year. CONCLUSIONS: The increasing movement of people to and from areas endemic for malaria requires the health authorities of countries with appropriate conditions for reintroduction to conduct enhanced measures for surveillance and control of this potentially deadly disease.

Molecular characterization of Echinococcus granulosus isolates from Bulgarian human cystic echinococcosis patients.

Although cystic echinococcosis (CE) is highly endemic in Bulgaria, there is still scarce information about species and/or genotypes of the Echinococcus granulosus complex that infect humans. Our study tackled the genetic diversity of E. granulosus complex in a cohort of 30 Bulgarian CE patients. Ten animal E. granulosus isolates from neighboring Greece were additionally included. Specimens were comparatively analyzed for partial sequences of five mitochondrial (mt) (cox I, nad I, rrnS, rrnL, and atp6) and three nuclear (nc) genes (act II, hbx 2, and ef-1α) using a PCR-sequencing approach. All 30 Bulgarian isolates were identified as E. granulosus sensu stricto (s.s.) and were showing identical sequences for each of the three examined partial nc gene markers. Based upon concatenated sequences from partial mtDNA markers, we detected 10 haplotypes: 6 haplotypes (H1-H6) clustering with E. granulosus s.s. (G1) and 4 haplotypes (H9-H13) grouping with E. granulosus s.s. (G3), with H1 and H10 being the most frequent in Bulgarian patients. The haplotypes H1, H4, and H11 were also present in Greek hydatid cyst samples of animal origin. In conclusion, E. granulosus s.s. (G1 and G3 genotypes) is the only causative agent found so far to cause human CE in Bulgaria. However, further studies including larger sample sizes and other additional geographic regions in Bulgaria will have to be performed to confirm our results.

Treatment of Hepatic Cystic Echinococcosis in Patients from the Southeastern Rhodope Region of Bulgaria in 2004-2013: Comparison of Current Practices with Expert Recommendations.

Cystic echinococcosis (CE) is a clinically complex chronic parasitic disease, management options for which include surgery, percutaneous treatments, and treatment with albendazole (ABZ) for active cysts, and the "Watch-and-Wait" approach for uncomplicated, inactive cysts. We examined, retrospectively, the clinical management of 334 patients with hepatic CE from the southeastern Rhodope region of Bulgaria between 2004 and 2013. Cysts were reclassified according to the World Health Organization Informal Working Group on Echinococcosis (WHO-IWGE) on the basis of ultrasound reports and images. The majority (62.3%) of uncomplicated cysts were CE1, 66% of which were treated surgically. Of all interventions, 5% were performed on inactive uncomplicated CE4-CE5 cysts. About half (47.6%) of these cysts were therefore treated inappropriately, exposing patients to unnecessary treatment-related risks and the health system to unnecessary costs. No management change was observed after the publication of the WHO-IWGE Expert Consensus recommendations in 2010. In Bulgaria, ABZ is still used in interrupted cycles as this is reimbursed, and peri-interventional chemoprophylaxis was not administered in the majority of surgical patients. Efforts are needed to introduce the WHO-IWGE classification and management recommendations and to encourage reception of state-of-the-art practices by public health regulatory bodies to improve patient quality of care and optimization of health resources.