Monkeypox in Bulgaria: Significance of Various Clinical Samples, Clinical Manifestation, and Molecular Detection.

Background/Objectives: Monkeypox (mpox) is currently the most common orthopoxvirus (OPXV) zoonotic disease, and, since 2022, there has been atypical person-to-person transmission observed in non-endemic countries. The present study aimed to investigate the frequency of monkeypox virus (MPXV) and OPXV DNA detection in recommended and alternative clinical materials taken during the acute and convalescent phases of infection in Bulgarian patients. Methods: The study included laboratory investigation by real time PCR of 181 clinical samples from 42 Bulgarian patients with possible mpox infections. Results: MPXV DNA was detected in 23/181 (12.71%), and OPXV DNA in 20/181 (11.05%) clinical samples. There were six mpox-confirmed patients aged 23 to 44. At the highest frequency, MPXV and OPXV DNA were detected in samples of vesicular contents (6/6) and nasal/oropharyngeal secretions (5/6 and 4/6) during the first three days from the appearance of clinical symptoms. We demonstrated MPXV and OPXV DNA in alternative samples (urine, feces, ejaculate, and saliva), and in follow-up patient samples, taken two weeks after mpox confirmation in the convalescent phase (vesicular contentsand urine). Conclusions: Our findings suggested that MPXV may be detected in a larger set of clinical materials, including alternatives, where the virus can persist for more than two weeks.

Detection of measles virus in Bulgaria from 2012 to 2018.

AIM: To determine the circulation patterns of measles virus in Bulgaria from 2012 to 2018 after a large measles outbreak in the country (2009-2011). METHODS: Three types of clinical material were collected: serum samples, urine samples, and nasal swabs. Enzyme-linked immunosorbent assay (ELISA) was used to detect specific viral immunoglobulin (Ig) M/IgG antibodies. Viral RNA was extracted from all urine and nasal swabs. RESULTS: In the investigated period, 102 patients were confirmed to have measles (age range: two months to 55 years). A total of 101 samples (99%) were measles-IgM positive. Most of them were detected in 2017 (73%, 74/101), when a measles outbreak in the country was reported. The majority of patients were unvaccinated children aged under 13 months. Out of 101 measles serum samples confirmed by ELISA, 18 (20.45%) were measles-IgG positive and 15 (17.05%) were borderline. Thirty-three positive PCR products were sequenced and genotyped. In 2013, 2016, 2017, and 2018, three different measles viral genotypes were detected: D8, H1, and B3. Most patients were unvaccinated or insufficiently vaccinated. CONCLUSION: Preventive measures are indispensable to limit the infection in different regions of Bulgaria and its spread to other countries. As vaccination coverage against measles and other vaccine-preventable infections, including SARS-Co2, is low, it is necessary to perform molecular identification of viruses to monitor their circulation and pathogenicity.

Measles Virus and Subacute Sclerosing Panencephalitis.

BACKGROUND: Subacute Sclerosing Panencephalitis (SSPE) mainly affects children and young people. It is a rare, chronic progressive degenerative form of cerebral inflammation with various infectious noxa, which develops for years after a primary, uncomplicated infection, and the highest percentage can be caused by measles virus and in rare cases by rubella. The aim of the present study is to investigate in the laboratory the role of measles virus in the development of neurological symptoms and diseases of the CNS. METHODS: A total of 46 clinical materials (23 sera samples and 23 CSF) obtained from 23 patients with neurological symptoms and diagnoses: "SSPE" (in 10 patients) and "Encephalitis" (in 13 patients), in the period January 2011 - December 2020 were tested in the National Reference Laboratory (NRL) "Measles, mumps and rubella" at National Centre of Infectious and Parasitic Diseases (NCIPD), Sofia, Bulgaria. Serological (indirect ELISA test for the detection of specific measles IgG/IgM antibodies in serum samples and cerebrospinal fluid) and molecular (RT-PCR for the demonstration of viral RNA) methods were used. RESULTS: The study was performed by parallel testing of serum samples and CSF from each patient. Positive results for measles IgG antibodies in sera were found in 21 patients. Presence of measles IgG antibodies in CSF was demonstrated in four children with diagnosis SSPE (two children at 4 years, one child at 4 years and 6 months, and one at 11 years old). All children with positive laboratory results for SSPE had evidence of MeV infection before 2 years of age. The patients with SSPE had high antibody titers (CSF > 230 U/mL) in their CSF. Patients with positive anti-Measles IgG in the CSF were also found to have positive results for protective measles IgG in the serum samples and their IgG titers were nearly twice as high compared to other patients' sera. The presence of specific measles IgM antibodies was not demonstrated in the tested specimens. RT-PCR test was performed for all samples, and the presence of viral RNA was not detected. CONCLUSIONS: The measles infection can be a reason for developing serious complications affecting CNS in all age groups. SSPE itself is extremely difficult to diagnose, which is why laboratory confirmation of any clinical case is a necessary condition for effective disease surveillance.

Primate Erythroparvovirus 1 Infection in Patients with Hematological Disorders.

Primate erythroparvovirus 1, commonly referred to as Parvovirus B19 (B19V), is a DNA virus that normally results in a mild childhood infection called "erythema infectiosum". Besides respiratory spread, B19V can also be transmitted through transfusions, which may result in persistent anemia in immunodeficient hosts. Dialysis patients often face acute or chronic anemia after infection with B19V. Here, we describe the laboratory investigation of 21 patients with hematological disorders for B19V infections. B19V DNA was detected in 13 (62%) of them, with specific IgM antibodies in three of the DNA positives. All 13 patients received treatment and were laboratory-monitored over a period of one year. In only two patients (a 14-year-old child with a kidney transplantation and a 39-year-old patient with aplastic anemia), markers of recent B19V infection were still detectable in follow-up samples. For four B19V DNA positive samples, short sequences could be obtained, which clustered with genotype 1a reference strains. Our findings suggest that all cases of hematological disorders should be examined for specific B19V antibodies and DNA for accurate diagnosis and appropriate patient management.