Re-Emerging foci of visceral leishmaniasis in Armenia - first molecular diagnosis of clinical samples.

Visceral leishmaniasis (VL) was firstly reported in Armenia in 1913. Following a considerable increase of the number of cases until the mid 1950s, the disease disappeared after 1969 and re-emerged in 1999. Scientific literature about VL in Armenia is available only in Russian or Armenian. This paper presents a historical overview about leishmaniasis in Armenia based on this literature as well as an epidemiological update since the re-emergence of the disease. In 1999-2016, 116 indigenous VL cases were recorded mainly in children in 8 of the 11 districts, however, VL is underreported because of lack of trained medical personal and diagnostic facilities. The aim of this work was to apply for the first time molecular diagnosis of VL in Armenia. Out of 25 VL suspected patients, 22 were positive by microscopy and polymerase chain reaction (PCR). Genotyping using internal transcribed spacer 1-PCR-restriction fragment length polymorphism and sequencing identified the causative agent of VL in Armenia as Leishmania infantum. The present work is an important step towards the inclusion of molecular techniques in the current diagnosis of VL in Armenia and the establishment of local molecular diagnostic facilities.

Distribution of vectors and arboviruses, and healthcare workers' knowledge of vector-borne diseases in Armenia.

INTRODUCTION: Armenia's favorable geographical and climatic conditions support mosquitoes, sandflies, and ticks that can transmit various diseases. This study aimed to determine the prevalence of these vectors and circulating arboviruses in Armenia and assess healthcare workers` knowledge of arboviral diseases. METHODOLOGY: In 2021, we conducted fieldwork, combining morphological identification of vectors with polymerase chain reaction (PCR) analysis of pathogens to map the distribution of potential arbovirus vectors across Armenia. RESULTS: Our entomological surveys identified four mosquito genera-Anopheles, Aedes, Culex, and Culiseta-comprising 20 species; and 11 species of Ixodidae ticks. Culex pipiens was found in all 11 regions, while Culiseta spp. was absent in Ararat Province. PCR testing of mosquito and tick samples revealed Crimean-Congo hemorrhagic fever virus (CCHFV) in 13 tick samples, but West Nile virus (WNV) was not detected in mosquitoes. Specifically, 13 out of 525 Ixodes tick pools tested positive for CCHFV; the positive samples originated from Hyalomma marginatum ticks in Syunik's Sisian region. None of the 11 pools that contained 473 Cx. pipiens mosquitoes tested positive for WNV. Analysis of questionnaires from 499 healthcare workers showed that epidemiologists, infectious disease specialists, and family doctors had greater awareness of arboviral diseases than other specialists. However, there was a low rate of sample submission for laboratory diagnosis and confirmation. CONCLUSIONS: The extensive presence of vectors combined with limited knowledge of arboviral diseases complicates disease understanding in Armenia. Strengthening the surveillance system through training and improved sample collection is essential for disease monitoring and public health interventions.

Adaptation to mountain γ-background: bacteria speciation.

PURPOSE: To study the adaptation of bacteria to the natural γ-background of mountains and anthropogenic emissions from nuclear power plants; to establish the main factors of variability and speciation of bacteria. METHOD: Analysis of materials on the radiation background and its impact on living organisms in the landscape of Armenia, calculation of the absorbed dose by microbes due to rock radiation. RESULTS: The review shows the death, reproduction, radioresistance and speciation of bacteria in changing conditions of low variable natural and anthropogenic γ-background. CONCLUSION: We assume that γ-rays from rocks activate cellular epigenetic mechanisms that regulate genome expression, signaling and, ultimately, variability of bacteria. Some of them have already been studied, others require research.

Gamasid Ticks as Vectors of Tularemia in the Southeast of Armenia.

Background: The natural environment of southeastern Armenia, which includes the Syunik and Vayots Dzor regions, provides a high biodiversity of flora and fauna, including ectoparasites. Currently, the fauna and ecology of gamasid ticks and their role in the circulation of tularemia in this area are unclear and incomplete. To better understand the persistence of tularemia in Armenia, an assessment of specific hosts and their vectors is needed to evaluate their role in perpetuating tularemia. Materials and Methods: Utilizing data and samples collected from 1970 to 2020, we have evaluated the species composition of gamasid ticks found on the common vole and in their nests and burrows, and identified the presence of tularemia over time. We evaluated five different geographical landscapes: semidesert, dry mountain steppe, mountain steppe, mountain forest, and high mountain in the communities and open areas of Kapan, Goris, Sisian, Meghri, and Jermuk. Results: We determined the density of gamasid ticks in southeastern Armenia over the 50-year period and isolated 20 cultures of tularemia in 12 separate years. Conclusions: It is important to regularly monitor gamasid ticks in southeastern Armenia to clarify the risk factors for the occurrence of tularemia epizootics, among both carriers and vectors, to better understand the full epidemiological picture.

Morphological Changes in the Oral Mucous Membrane in Viral Hepatitis C Patients: A Cross-Sectional Study.

Background: The objective was to reveal the most typical changes in oral mucosa in HCV patients and compare them with those in HCV negative patients. Methods: The study involved 96 HCV patients and 100 patients without HCV who applied to a dental clinic. The content of cytokines IL-2, IL-4, IL-10 and ɤ-INF in the oral fluid was determined by ELISA. Buccal mucosa and gums biopsies passed histological examination. An immunohistochemical study of mucous membrane biopsies was performed using monoclonal mouse antibodies to CD3+ and CD20+. Results: The HCV patients group included 96 (63.5% males), and the non-HCV group included 100 subjects (62.0% males) with lesions of the oral mucous membrane. The lesions of lips and oral mucosa were more frequent in HCV than in the non-HCV group-e.g., erosion (13.5% vs. 1%), cracks in the mouth corners (42.7% vs. 0%), changes in the oral mucosa surface (89.6% vs. 3.0%), hemorrhages (78.1% vs. 0%), etc. The pro-inflammatory IL-2 level was higher and anti-inflammatory IL-4 level was lower in HCV patients compared with those in the non-HCV group. Conclusions: Morphological changes developed in the microvasculature both worsen the tissue trophism and accelerate the healing with differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicated a decrease in local humoral immune response.

Molecular Analysis of SARS-CoV-2 Lineages in Armenia.

The sequencing of SARS-CoV-2 provides essential information on viral evolution, transmission, and epidemiology. In this paper, we performed the whole-genome sequencing of SARS-CoV-2 using nanopore and Illumina sequencing to describe the circulation of the virus lineages in Armenia. The analysis of 145 full genomes identified six clades (19A, 20A, 20B, 20I, 21J, and 21K) and considerable intra-clade PANGO lineage diversity. Phylodynamic and transmission analysis allowed to attribute specific clades as well as infer their importation routes. Thus, the first two waves of positive case increase were caused by the 20B clade, the third peak caused by the 20I (Alpha), while the last two peaks were caused by the 21J (Delta) and 21K (Omicron) variants. The functional analyses of mutations in sequences largely affected epitopes associated with protective HLA loci and did not cause the loss of the signal in PCR tests targeting ORF1ab and N genes as confirmed by RT-PCR. We also compared the performance of nanopore and Illumina short-read sequencing and showed the utility of nanopore sequencing as an efficient and affordable alternative for large-scale molecular epidemiology research. Thus, our paper describes new data on the genomic diversity of SARS-CoV-2 variants in Armenia in the global context of the virus molecular genomic surveillance.

Assessment of pathomorphological characteristics of the oral mucosa in patients with HBV, HCV and HIV.

INTRODUCTION: Oral clinical manifestations in HBV HCV and HIV patients indicate a deterioration in general health status. The aim of the study was to assess pathomorphologic features of oral mucosa observed in patients with these diseases. METHODOLOGY: The study was conducted in N1 Dental Clinic of YSMU after M. Heratsi. The total number of patients taking part in the research was 120, including HBV (n = 40), HCV (n = 40) and HIV (n = 40). After biopsy and subsequent histological examination of the oral mucosa, statistical analysis was carried out using Excel 2013 and R software. RESULTS: Pathomorphological examination revealed inflammatory infiltrations in all samples collected from HBV, HCV and HIV patients. These changes included microcirculatory disorders in 98.3% of samples: fibrinous-like deposits lining the surface of erosions and ulcers on the oral mucosa (1.67%), fibrosis of the mucous membrane (70%), dystrophy of squamous epithelium (93.3%) and bone sequestration (3.3%). Comparative analysis of pathomorphological characteristics revealed distinct content of infiltrates: lymphoplasmacytic infiltration in patients with HBV and HCV, while HIV patients showed neutrophils infiltration and lack of plasmocytes. CONCLUSIONS: There are common abnormal morphological changes in the oral mucosa typical of all patients with HBV, HCV and HIV, as well as liver diseases specific to each of them. Inflammation in the patients with HIV indicated impairment of the humoral immune system. Understanding the distinct characteristic of inflammation in the oral cavity could be useful for early differential diagnosis and management of patients with HIV, HBV and HCV.

Microsatellite based molecular epidemiology of Leishmania infantum from re-emerging foci of visceral leishmaniasis in Armenia and pilot risk assessment by ecological niche modeling.

BACKGROUND: Visceral leishmaniasis (VL) is re-emerging in Armenia since 1999 with 167 cases recorded until 2019. The objectives of this study were (i) to determine for the first time the genetic diversity and population structure of the causative agent of VL in Armenia; (ii) to compare these genotypes with those from most endemic regions worldwide; (iii) to monitor the diversity of vectors in Armenia; (iv) to predict the distribution of the vectors and VL in time and space by ecological niche modeling. METHODOLOGY/PRINCIPAL FINDINGS: Human samples from different parts of Armenia previously identified by ITS-1-RFLP as L. infantum were studied by Multilocus Microsatellite Typing (MLMT). These data were combined with previously typed L. infantum strains from the main global endemic regions for population structure analysis. Within the 23 Armenian L. infantum strains 22 different genotypes were identified. The combined analysis revealed that all strains belong to the worldwide predominating MON1-population, however most closely related to a subpopulation from Southeastern Europe, Maghreb, Middle East and Central Asia. The three observed Armenian clusters grouped within this subpopulation with strains from Greece/Turkey, and from Central Asia, respectively. Ecological niche modeling based on VL cases and collected proven vectors (P. balcanicus, P. kandelakii) identified Yerevan and districts Lori, Tavush, Syunik, Armavir, Ararat bordering Georgia, Turkey, Iran and Azerbaijan as most suitable for the vectors and with the highest risk for VL transmission. Due to climate change the suitable habitat for VL transmission will expand in future all over Armenia. CONCLUSIONS: Genetic diversity and population structure of the causative agent of VL in Armenia were addressed for the first time. Further genotyping studies should be performed with samples from infected humans, animals and sand flies from all active foci including the neighboring countries to understand transmission cycles, re-emergence, spread, and epidemiology of VL in Armenia and the entire Transcaucasus enabling epidemiological monitoring.

SARS-CoV-2 detection by extraction-free qRT-PCR for massive and rapid COVID-19 diagnosis during a pandemic in Armenia.

COVID-19 pandemic severely impacted the healthcare and economy on a global scale. It is widely recognized that mass testing is an efficient way to contain the spread of SARS-CoV-2 infection as well as aid in the development of informed policies for disease management. However, the current COVID-19 worldwide infection rates increased the demand for rapid and reliable screening of infection. We compared the performance of qRT-PCR in direct heat-inactivated (H), heat-inactivated and pelleted (HC) samples against RNA in a group of 74 subjects (44 positive and 30 negative). Then we compared the sensitivity of HC in a larger group of 196 COVID-19 positive samples. Our study suggests that HC samples show higher accuracy for SARS-CoV-2 detection PCR assay compared to direct H (89 % vs 83 % of the detection in RNA). The sensitivity of detection using direct samples varied depending on the sample transport and storage media as well as the viral loads (as measured by qRT-PCR Ct levels). Altogether, all the data suggest that purified RNA provides more accurate results, however, direct sample testing with qRT-PCR may help to significantly increase testing capacity. Switching to the direct sample testing is justified if the number of tests is doubled at least.

Evaluation of IL-2, IL-10, IL-4 and ɤ-interferon levels in the oral fluids of patients with hepatitis C, B and HIV.

INTRODUCTION: Oral fluid cytokine levels can vary considerably during the onset of Inflammatory Periodontitis (IP) especially in people with hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV). Aim of our study was to evaluate levels of oral cytokines during the onset of IP among HCV, HBV and HIV negative and positive individuals in order to evaluate local immunity state during these infections. METHODOLOGY: This was a case control study with 3 groups of virally infected individuals and control group. All had IP including control group. RESULTS: 45 patients (51.7%) had HCV, 18 (20.7%) HBV and 24 (27.6%) HIV. For IL-2 we received significant difference for all groups compared with control -2.83; HBV-31.1 (p < 0.001), HCV-25.99 (p < 0.001) and HIV-24.57 (p < 0.001). For IL-10 significant difference was observed between control -0.94 and HCV-3.63 (p = 0.027), HBV-8.38 (15.51) groups (p < 0.001). IL-4 was significantly higher in control group 14.29 compared to HCV 0.2 (p < 0.001) and HIV 0.21 (p = 0.037) group. The adjusted analysis where we consider age as possible confounder revealed that only IL-2 significantly differs for all groups compared with control group: control vs HCV (p = 0.001); control vs HBV (p = 0.024); control vs HIV (p = 0.004). CONCLUSIONS: Evidence for significant differences when comparing oral fluid cytokines of individuals with HCV, HBV and HIV with non-viral individuals was more obvious for IL-2. IL-2 levels were significantly higher in all 3 groups vs non-viral group even when age is confounder.