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.

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.

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.

Clinical features, diagnostic tools, and treatment regimens for visceral leishmaniasis in Bulgaria.

PURPOSE: Visceral leishmaniasis (VL), caused by the parasite Leishmania infantum, which was once largely confined to Southern Europe is now being diagnosed throughout Northern Europe, including Great Britain. In an effort to help EU clinicians improve their diagnosis and management of VL, this paper defines clinical features typical of the disease as it presents in Bulgaria, where VL is endemic. METHODS: The list of clinical symptoms presented here was gleaned from the medical records (patient histories, epidemiological survey cards, laboratory data) of 59 Bulgarian patients with VL. This study also includes microscopic, serological, and molecular laboratory techniques. RESULTS: Described and analyzed are the clinical features, diagnostic techniques, and therapeutic regimens of 59 cases--part of the total number of VL case histories (P = 120, 116 Bulgarian and 4 not Bulgarian) collected in Bulgaria over the past 24 years (1988-2011). Although all of the studied 59 cases presented with classical symptoms of VL, only in three occasions, the initial diagnosis was correct. CONCLUSIONS: Left untreated, zooanthroponotic VL leads to debilitating chronic disease and even death. Yet, because VL is hard to recognize and relatively new to Northern Europe, misdiagnosis is common and treatment too often inappropriate and delayed.

The identification of free-living environmental isolates of amoebae from Bulgaria.

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named "6" (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)--all exhibiting a highly efficient proliferation in axenic cultures--were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.