Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens.

Coccidioidomycosis (Valley fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

Evidence of Extensive Circulation of Yersinia enterocolitica in Rodents and Shrews in Natural Habitats from Retrospective and Perspective Studies in South Caucasus.

Yersinia enterocolitica culture-positive rodents and shrews were reported in different territories across Georgia during 14 of 17 years of investigations conducted for the period of 1981-1997. In total, Y. enterocolitica was isolated from 2052 rodents (15 species) and 33 shrews. Most isolates were obtained from Microtus arvalis, Rattus norvegicus, Mus musculus, and Apodemus spp. During the prospective study (2017-2019), isolates of Yersinia-like bacteria were cultured from 53 rodents collected in four parts of Georgia. All the Yersinia-like isolates were confirmed as Y. enterocolitica based on the API 20E and the BD Phenix50 tests. Whole-genome (WG) sequencing of five rodents and one shrew strain of Y. enterocolitica revealed that they possessed a set of virulence genes characteristic of the potentially pathogenic strains of biogroup 1A. All isolates lacked distinguished virulence determinants for YstA, Ail, TccC, VirF, and virulence plasmid pYV but carried the genes for YstB, YmoA, HemPR-HmuVSTU, YaxAB, PhlA, PldA, ArsCBR, and a flagellar apparatus. One strain contained a gene highly homologous to heat-labile enterotoxin, a chain of E. coli, a function not previously described for Y. enterocolitica. The WG single-nucleotide polymorphism-based typing placed the isolates in four distinct phylogenetic clusters.

Geographic distribution and genetic characterization of poxviruses from human infections in Georgia, 2009-2014.

Anthrax is endemic in Georgia, as are multiple zoonotic poxviruses. Poxvirus-associated infections share some clinical manifestations and exposure risks with anthrax, and so it is important to distinguish between the two. With this in mind, an archived collection of anthrax-negative DNA samples was retrospectively screened for poxviruses, and of the 148 human samples tested, 64 were positive. Sequence analysis confirmed the presence of orf virus, bovine papular stomatitis virus, and pseudocowpox virus. This study provides evidence of previously unrecognized poxvirus infections in Georgia and highlights the benefit of the timely identification of such infections by improving laboratory capacity.

Identification of a Novel Yersinia enterocolitica Strain from Bats in Association with a Bat Die-Off That Occurred in Georgia (Caucasus).

Yersinia entercolitica is a bacterial species within the genus Yersinia, mostly known as a human enteric pathogen, but also recognized as a zoonotic agent widespread in domestic pigs. Findings of this bacterium in wild animals are very limited. The current report presents results of the identification of cultures of Y. entercolitica from dead bats after a massive bat die-off in a cave in western Georgia. The growth of bacterial colonies morphologically suspected as Yersinia was observed from three intestine tissues of 11 bats belonging to the Miniopterus schreibersii species. These three isolates were identified as Y. enterocolitica based on the API29 assay. No growth of Brucella or Francisella bacteria was observed from tissues of dead bats. Full genomes (a size between 4.6-4.7 Mbp) of the Yersinia strains isolated from bats were analyzed. The phylogenetic sequence analyses of the genomes demonstrated that all strains were nearly identical and formed a distinct cluster with the closest similarity to the environmental isolate O:36/1A. The bat isolates represent low-pathogenicity Biotype 1A strains lacking the genes for the Ail, Yst-a, Ysa, and virulence plasmid pYV, while containing the genes for Inv, YstB, and MyfA. Further characterization of the novel strains cultured from bats can provide a clue for the determination of the pathogenic properties of those strains.

Janibacter species with evidence of genomic polymorphism isolated from resected heart valve in a patient with aortic stenosis.

The authors report isolation and identification of two strains of bacteria belonging to the genus Janibacter from a human patient with aortic stenosis from a rural area of the country of Georgia. The microorganisms were isolated from aortic heart valve. Two isolates with slightly distinct colony morphologies were harvested after sub-culturing from an original agar plate. Preliminary identification of the isolates is based on amplification and sequencing of a fragment of 16SrRNA. Whole genome sequencing was performed using the Illumina MiSeq instrument. Both isolates were identified as undistinguished strains of the genus Janibacter. Characterization of whole genome sequences of each culture has revealed a 15% difference in gene profile between the cultures and confirmed that both strains belong to the genus Janibacter with the closest match to J. terrae. Genomic comparison of cultures of Janibacter obtained from human cases and from environmental sources presents a promising direction for evaluating a role of these bacteria as human pathogens.

Bat Research Networks and Viral Surveillance: Gaps and Opportunities in Western Asia.

Bat research networks and viral surveillance are assumed to be at odds due to seemingly conflicting research priorities. Yet human threats that contribute to declines in bat populations globally also lead to increased transmission and spread of bat-associated viruses, which may pose a threat to global health and food security. In this review, we discuss the importance of and opportunities for multidisciplinary collaborations between bat research networks and infectious disease experts to tackle shared threats that jeopardize bat conservation as well as human and animal health. Moreover, we assess research effort on bats and bat-associated viruses globally, and demonstrate that Western Asia has limited published research and represents a gap for coordinated bat research. The lack of bat research in Western Asia severely limits our capacity to identify and mitigate region-specific threats to bat populations and detect interactions between bats and incidental hosts that promote virus spillover. We detail a regional initiative to establish the first bat research network in Western Asia (i.e., the Western Asia Bat Research Network, WAB-Net), with the aim of integrating ecological research on bats with virus surveillance to find "win-win" solutions that promote bat conservation and safeguard public and animal health across the region.

Prevalence, diversity, and host associations of Bartonella strains in bats from Georgia (Caucasus).

Bartonella infections were investigated in seven species of bats from four regions of the Republic of Georgia. Of the 236 bats that were captured, 212 (90%) specimens were tested for Bartonella infection. Colonies identified as Bartonella were isolated from 105 (49.5%) of 212 bats Phylogenetic analysis based on sequence variation of the gltA gene differentiated 22 unique Bartonella genogroups. Genetic distances between these diverse genogroups were at the level of those observed between different Bartonella species described previously. Twenty-one reference strains from 19 representative genogroups were characterized using four additional genetic markers. Host specificity to bat genera or families was reported for several Bartonella genogroups. Some Bartonella genotypes found in bats clustered with those identified in dogs from Thailand and humans from Poland.

The Fecal Microbial Community of Breast-fed Infants from Armenia and Georgia.

Multiple factors help shape the infant intestinal microbiota early in life. Environmental conditions such as the presence of bioactive molecules from breast milk dictate gut microbial growth and survival. Infants also receive distinct, personalized, bacterial exposures leading to differential colonization. Microbial exposures and gut environmental conditions differ between infants in different locations, as does the typical microbial community structure in an infant's gut. Here we evaluate potential influences on the infant gut microbiota through a longitudinal study on cohorts of breast-fed infants from the neighboring countries of Armenia and Georgia, an area of the world for which the infant microbiome has not been previously investigated. Marker gene sequencing of 16S ribosomal genes revealed that the gut microbial communities of infants from these countries were dominated by bifidobacteria, were different from each other, and were marginally influenced by their mother's secretor status. Species-level differences in the bifidobacterial communities of each country and birth method were also observed. These community differences suggest that environmental variation between individuals in different locations may influence the gut microbiota of infants.

Genome Sequences of Human and Livestock Isolates of Brucella melitensis and Brucella abortus from the Country of Georgia.

Brucellosis, which is among the most widespread global zoonotic diseases, is endemic in the nation of Georgia and causes substantial human morbidity and economic loss. Here, we report whole-genome sequences of three Brucella melitensis and seven Brucella abortus isolates from cattle, sheep, and humans that represent genetic groups discovered in Georgia.

Epidemiological and Clinical Features of Brucellosis in the Country of Georgia.

BACKGROUND: Brucellosis is an endemic disease in the country of Georgia. According to the National Center for Disease Control and Public Health of Georgia (NCDC), the average annual number of brucellosis cases was 161 during 2008-2012. However, the true number of cases is thought to be higher due to underreporting. The aim of this study was to provide current epidemiological and clinical information and evaluate diagnostic methods used for brucellosis in Georgia. METHODOLOGY: Adult patients were eligible for participation if they met the suspected or probable case definition for brucellosis. After consent participants were interviewed using a standardized questionnaire to collect information on socio-demographic characteristics, epidemiology, history of present illness, and clinical manifestation. For the diagnosis of brucellosis, culture and serological tests were used. RESULTS: A total of 81 participants were enrolled, of which 70 (86%) were from rural areas. Seventy-four percent of participants reported consuming unpasteurized milk products and 62% consuming undercooked meat products before symptom onset. Forty-one participants were positive by the Wright test and 33 (41%) were positive by blood culture. There was perfect agreement between the Huddelston and Wright tests (k = 1.0). Compared with blood culture (the diagnostic gold standard), ELISA IgG and total ELISA (IgG + IgM), the Wright test had fair (k = 0.12), fair (k = 0.24), and moderate (k = 0.52) agreement, respectively. CONCLUSIONS: Consumption of unpasteurized milk products and undercooked meat were among the most common risk factors in brucellosis cases. We found poor agreement between ELISA tests and culture results. This report also serves as an initial indication that the suspected case definition for brucellosis surveillance purposes needs revision. Further research is needed to characterize the epidemiology and evaluate the performance of the diagnostic methods for brucellosis in Georgia.

Seroepidemiology and molecular diversity of Leishmania donovani complex in Georgia.

BACKGROUND: Leishmaniasis includes multiple clinical syndromes, most notably visceral, cutaneous, and mucosal forms. Visceral leishmaniasis (VL), also known as kala-azar, is a potentially fatal disease endemic to large parts of Africa and Asia, and in South-Eastern Europe (Greece, Turkey, Georgia). Visceral leishmaniasis is a parasitic zoonosis caused by species of the L. donovani complex. In the classical epidemiological model the main reservoir for VL are canines. METHODS: The study included a cohort of 513 individuals of both genders (190 males and 323 females) from the ages of 1 to 70 years that were screened in ten villages across two districts in Kakheti using the Kalazar Detect™ rK39 rapid diagnostic test. The phylogenetic diversity patterns of local strains, based on the rDNA internal transcribed spacer (ITS) sequences, were assessed for samples obtained from patients with suspected L. donovani infection, from canine reservoirs and from Phlebotomus sand flies obtained from different geographical areas of Georgia and from Azerbaijan. RESULTS: Out of a total of 600 domestic dog blood samples 95 (15.8 %) were positive by rK39 rapid diagnostic tests. For symptomatic domestic dogs, the testing of conjunctival swabs or bone marrow aspirates revealed a higher VL incidence in Kvareli District (Kvareli; 19.4 %, n = 329) compared with that observed for Sagarejo District (Sagarejo; 11.4 %, n = 271). A total of 231 sand flies of both genders were collected during the 2-month period; of the 114 females, 1.75 % were PCR positive for the presence of Leishmania spp. CONCLUSIONS: VL infection rates remain high in both canines and humans in Georgia, with disease in several known natural foci. The genetic relationships derived from rDNA internal transcribed spacer (ITS) sequence comparisons identified genetic subgroups, revealing preliminary insights into the genetic structure of L. donovani complex members currently circulating in the South Caucasus and demonstrates the utility of ITS-based genotyping in the resource-limited country of Georgia.

Expansion of brucellosis detection in the country of Georgia by screening household members of cases and neighboring community members.

BACKGROUND: Brucellosis is considered as endemic zoonotic disease in the country of Georgia. However, the burden of the disease on a household level is not known. Therefore, this study sought to determine the benefits of active surveillance coupled to serological screening for the early detection of brucellosis among close contacts of brucellosis cases. METHODS: We used an active surveillance approach to estimate the rate of seropositivity among household family members and neighboring community members of brucellosis index cases. All participants were screened using the serum tube agglutination test (SAT). Blood cultures were performed, obtained isolates were identified by a bacteriological algorithm, and confirmed as Brucella spp. using real-time PCR. Further confirmation of Brucella species was done using the AMOS PCR assay. RESULTS: A total of 141 participants enrolled. Of these, 27 were brucellosis index cases, 86 were household family members, and 28 were neighboring community members. The serological evidence of brucellosis in the household member group was 7% and the rate at the household level was 21%. No screened community members were Brucella seropositive. Majority of brucellosis cases were caused by B. melitensis; only one index case was linked to B. abortus. CONCLUSION: We found evidence of brucellosis infection among household family members of brucellosis index cases. B. melitensis was the most common species obtained. Findings of this active surveillance study highlight the importance of screening household family members of brucellosis cases and of the use of culture methods to identify Brucella species in the country of Georgia.

Molecular phylogeny of hantaviruses harbored by insectivorous bats in Côte d'Ivoire and Vietnam.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles prompted a further exploration of their host diversification by analyzing frozen, ethanol-fixed and RNAlater®-preserved archival tissues and fecal samples from 533 bats (representing seven families, 28 genera and 53 species in the order Chiroptera), captured in Asia, Africa and the Americas in 1981-2012, using RT-PCR. Hantavirus RNA was detected in Pomona roundleaf bats (Hipposideros pomona) (family Hipposideridae), captured in Vietnam in 1997 and 1999, and in banana pipistrelles (Neoromicia nanus) (family Vespertilionidae), captured in Côte d'Ivoire in 2011. Phylogenetic analysis, based on the full-length S- and partial M- and L-segment sequences using maximum likelihood and Bayesian methods, demonstrated that the newfound hantaviruses formed highly divergent lineages, comprising other recently recognized bat-borne hantaviruses in Sierra Leone and China. The detection of bat-associated hantaviruses opens a new era in hantavirology and provides insights into their evolutionary origins.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

Real-time PCR assay for identification of Blastomyces dermatitidis in culture and in tissue.

The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens.