Phylogenetic Characterization of Orthohantavirus dobravaense (Dobrava Virus).

We report complete coding sequences of Orthohantavirus dobravaense (Dobrava virus) Igneada strains and phylogenetic characterization of all available complete coding sequences. Our analyses suggested separation of host-dependent lineages, followed by geographic clustering. Surveillance of orthohantaviruses using complete genomes would be useful for assessing public health threats from Dobrava virus.

Dobrava hantavirus variants found in Apodemus flavicollis mice in Kirklareli Province, Turkey.

Hantaviruses infect humans via inhalation of viral particles within secretions of infected rodents or rarely through direct contact with infected rodents. Determining the prevalence of hantavirus infections among rodent populations is of vital importance to obtain information on hantavirus-related cases and to predict possible outbreaks. We hypothesized that DOBV strains circulating in the Thrace Region in Turkey would be related to other Balkan DOBV strains. In this study, hantavirus infections in the rodent population of the Kirklareli-Igneada Region (north-western Turkey, near the Bulgarian border) were investigated. This region is of particular importance, as it is located in the south-eastern margin of the European continent and was used as an entrance point of Asian faunal elements into Europe. DOBV infection was detected in eight of 73 rodents; all were of the Apodemus flavicollis species. Partial sequences of the viral S-, M-, and L-genome segments were recovered and compared with previously reported DOBV sequences. The newly characterized Turkish strains were similar to other DOBV variants. Silent nucleotide mutations were dominant. The hantavirus prevalence in the Igneada region was similar to what has been reported in Greece and Bulgaria. For the first time, the M-segment sequences of DOBV from Turkey were recovered and genetic data of hantaviruses from Thrace region of Turkey were obtained.

[Optimization of ELISA and immunoblot methods for the detection of IgG antibodies against old world hantaviruses in wild rodents]. / Yabani kemiricilerde Eski Dünya hantavirus IgG antikorlarinin saptanmasi için ELISA ve immünoblot yöntemlerinin optimizasyonu.

Hantaviruses infect humans via inhalation of viral particles in infected rodents' secretions such as saliva, urine and faeces or via direct contact with infected rodents. The rodent species that are known as the carriers of Dobrava (DOBV), Puumala (PUUV), Saaremaa (SAAV), Tula (TULV) and Seoul (SEOV) viruses are found in our country. The presence of specific antibodies against hantaviruses have been demonstrated in rodents collected from Black Sea and Aegean Regions of Turkey in 2004 for the first time. The first hantavirus-related hemorrhagic fever with renal syndrome (HFRS) cases were reported in Black Sea region in 2009. The determination of the hantavirus prevalence in wild life and rodent populations in the field is crucial for the information about hantavirus-related cases and to clarify the state of risk. There is no commercial product optimized for the screening of rodent serum samples in terms of HFRS agents like DOBV and PUUV that are widely seen in Eurasia as well as Turkey. In this study, the antigens belonging to the commercial enzyme-linked immunoassay (ELISA) and immunoblot tests that are produced for the screening of human sera were used for the development of antibody screening tests against hantavirus in rodent sera and were optimized. The most appropriate serum and conjugate dilutions were determined for the optimization of ELISA (Anti-Hantavirus Pool ELISA; Euroimmun, Germany) and immunoblot (Euroline Anti-Hanta Profile 1 strips; Euroimmun, Germany) methods. Optimized ELISA method was used for the screening and optimized immunoblot method was used for the confirmation. A total of 84 wild rodent sera that belonged to Apodemus and Microtus species were evaluated with this procedure and the cut-off value, sensitivity and specificity of optimized ELISA method were determined. For the optimization of ELISA 1/50, 1/100 and 1/200 serum dilutions and 1/10.000, 1/20.000 and 1/40.000 conjugate dilutions were tested. For the optimization of immunoblot, 1/50 and 1/100 serum dilutions and 1/5.000 and 1/10.000 conjugate dilutions were tested. The horseradish peroxidase conjugated goat anti-mouse IgG for ELISA and the alkaline phosphatase conjugated goat anti-mouse IgG for immunoblot were used. We followed the manufacturer's recommendations for the incubation parameters, substrate and the number of washes. 1/50 serum dilution and 1/10.000 conjugate dilution for ELISA and 1/100 serum dilution and 1/5.000 conjugate dilution for immunoblot were determined as optimal concentrations. By using the optimized ELISA, 26.2% (22/84) of rodents were found positive for hantavirus antibodies according the determined cut-off value (OD(450/620): 0.325). By using immunoblot as a confirmatory test, 20 out of 22 ELISA positive samples could be studied because of the insufficient amount of sera and 17 of them was found positive in terms of DOBV antibodies. Of these rodents 11 were Apodemus flavicollis, three were Apodemus agrarius, two were Microtus guentheri and one was Apodemus sylvaticus. When the results of ELISA were compared to immunoblot results, the optimized ELISA's sensitivity and specificity were found as 100% and 95%, respectively. In this study, a method that can be used in the screening of rodent sera was constituted which uses commercial antigens that can be provided easily, gives fast and reliable results. Similar serological methods optimized for different types of rodents are of great importance for the realization of active follow-up and monitoring of the studies in the field.

Phylogenetic analysis of HCV-4d in Turkey: the curious case of Kayseri province.

In Turkey, genotype 1, especially type 1b virus, causes approximately 90% of these infections, while types 2, 3, and 4 exist, albeit in low prevalences and are due to relatively recent and limited introductions. Two recent reports from Kayseri, a relatively large city in Central Anatolia, indicated unusually high prevalence for type 4 infections in the province reaching a 35% among patients admitted to hospitals for treatment of chronic hepatitis C. In this study, the origin, the demographic history, and the dynamic of the epidemic of unusual HCV genotype 4d in Turkey by using Bayesian coalescent-based method were investigated. A gene flow migration approach was also used to describe the synchronous geographical dispersal and genetic diversification of this unusual genotype in Kayseri province. The Turkish clade had a tMRCA of 44 years corresponding to the year 1967 and seems to have a different origin being completely segregated from the European one. Gene flow migration analysis indicated that Kayseri province appeared to be the epicenter of HCV-4d epidemic, exporting the infections. The demographic history of HCV-4d showed that the epidemic started in 1970s year then following a slow exponential growth until 1980s. The Turkish monophyletic clade suggests a segregate circulation of the epidemic in this region mostly due to unsafe parenteral medical procedures (with drug addiction playing a relatively negligible role).

Comprehensive evaluation of nucleic acid amplification methods widely used for generic detection of sandfly-borne phleboviruses.

Sandfly-borne phleboviruses (SBPs), which cause sandfly fever, aseptic meningitis, encephalitis, and meningoencephalitis, are emerging pathogens of major public health concern. Virus nucleic acid testing is essential for SBP diagnosis, especially in the early stages of infection, and for the discovery of novel SBPs. The efficacy of utilizing generic primers that target conserved nucleotide sequences for the detection of both known and novel SBPs has not been extensively evaluated. We aimed to compare and evaluate the performance of five generic primer sets, widely used to detect S- and L-segments of arthropod-borne phleboviruses and designed as singleplex (n = 3) and nested (n = 2) formats, including both well-known and recently characterized 15 Old World virus strains. Furthermore, we performed in silico analysis to assess the detection capabilities of these generic primer sets. The initial evaluation of previously published generic primer sets for SBP detection yielded two singleplex primer sets with the potential to be adapted for use in real-time or high-throughput detection settings. Studies are ongoing to develop and further optimize a preliminary assay and test various hosts and vectors to assess their capacity to detect known and novel viruses. IMPORTANCE: Virus nucleic acid testing is the primary diagnostic method, particularly in the early stages of illness. Virus-specific or syndromic tests are widely used for this purpose. The use of generic primers has had a considerable impact on the discovery, identification, and detection of Old World sandfly-borne phleboviruses (OWSBP). The study is significant because it is the first to carry out a comparative evaluation of all published OWSBP generic primer sets.

Evaluating climate-dependent distribution of orthohantaviruses with monitoring wild rodents: One Health Perspective†.

Orthohantaviruses, cause hemorrhagic fever with renal syndrome, nephropathia epidemica, and hantavirus pulmonary syndrome, are major public health problems all over the world. Wild rodent surveillance for orthohantaviruses is of great importance for the preparedness against these human infections and the prediction of possible outbreak regions. Thus, we aimed to screen orthohantaviruses in wild rodents in Southern Anatolia, where the area has some of the glacial period refugia in the Mediterranean Basin, and interpret their current epidemiology with climatic biovariables in comparison with previously positive regions.We trapped muroid rodents between 2015 and 2017, and screened for orthohantaviruses. Then, we evaluated the relationship between orthohantavirus infections and bioclimatic variables. In spite of the long-term and seasonal sampling, we found no evidence for Orthohantavirus infections. The probable absence of orthohantaviruses in the sampling area was further evaluated from the climatic perspective, and results led us suggest that Orthohantavirus epidemiology might be relatively dependent on precipitation levels in driest and warmest quarters, and temperature fluctuations.These initial data might provide necessary perspective on wild rodent surveillance for orthohantaviruses in other regions, and help to collect lacking data for a such habitat suitability study in a bigger scale in the future.

[Comparison of Immune Responses Elicited by Adjuvanted Tachyzoite Lysate Vaccines Developed from Two Different Toxoplasma gondii Strains Isolated in Turkey]. / Türkiye'de Izole Edilen Iki Farkli Toxoplasma gondii Susundan Üretilen Adjuvanli Takizoit Eriyik Protein Asilarinin Uyardigi Immün Yanitlarin Karsilastirilmasi

Toxoplasma gondii the causative agent of toxoplasmosis is an obligate intracellular parasite with a wide host range including all warm-blooded animals and birds. T.gondii infection causes congenital toxoplasmosis in newborns and this may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxoplasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. The pathogenesis of toxoplasmosis change due to differences in the specific immune response elicited by diverse T.gondii strains. The protective immunity against toxoplasmosis is conferred by cellular immune responses. In the present study, two different strains isolated from Turkey named T.gondii Ankara and Ege were used to evaluate the types of humoral and cellular immune responses elicited by adjuvanted tachyzoite protein vaccines in an animal model. In the study, 6-8 weeks old female BALB/c mice were used and six study groups (each contains three mice) were composed for vaccination. The first and second groups were vaccinated with T.gondii Ankara and Ege (TAnkPE and TEgePE, respectively) tacyhzoite lysates, the third and fourth groups were vaccinated by tacyhzoite lysates adjuvanted with Freund's adjuvant (TAnkPE-Freund; TEgePE-Freund, respectively). The fifth and sixth groups were vaccinated with PBS and Freund's adjuvant as controls. Immunization of the animals was performed two times at three weeks intervals. The serum samples were collected before vaccination and after each vaccination to determine the IgG response by Western blotting, and IgG1 and IgG2a responses by ELISA. To determine the cellular immune response, CD8/CD4 cell ratio, intracellular IFN-g and IL-4 levels were determined in stimulated spleen cells grown in cell culture systems by flow cytometry. Toxoplasma IgG antibodies were only detected in TAnkPE-Freund group. IgG1 and IgG2a responses did not increase in any vaccination groups and there was not any polarization towards IgG1 or IgG2a. There was no significant increase in CD8/CD4 ratio of stimulated spleen cells. IFN-g level was increased in only TAnkPE-Freund vaccination group, however IL-4 levels were increased in TAnkPE-Freund, TEgePE-Freund and TEgePE groups. Our data showed that TAnkPE-Freund vaccine led to increase in IgG and IFN-g responses in BALB/c mice, however, tachyzoite lysate vaccines developed in this study did not induce sufficient protective immune response against toxoplasmosis. Thus, use of specific immunogenic proteins must be taken into consideration in the future vaccine development studies against toxoplasmosis.

Comprehensive Cross-Sectional Evaluation of Human Sandfly-Borne Phlebovirus Exposure in an Endemic Region.

Sandfly-borne phleboviruses are endemic in countries around the Mediterranean Basin and pose a significant health threat for populations, with symptoms spanning from febrile diseases to central nervous system involvement. We carried out a comprehensive cross-sectional screening via microneutralization (MN) assays for a quantitative assessment of neutralizing antibodies (NAs) to seven phleboviruses representing three distinct serocomplexes, using samples previously screened via immunofluorescence assays (IFAs) in Turkey, an endemic region with various phleboviruses in circulation. We detected NAs to three phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), and sandfly fever Sicilian virus (SFSV), while assays utilizing Adana virus, Punique virus, Massilia virus, and Zerdali virus remained negative. The most frequently observed virus exposure was due to TOSV, with a total prevalence of 22.6%, followed by SFNV (15.3%) and SFSV (12.1%). For each virus, IFA reactivity was significantly associated with NA detection, and further correlated with NA titers. TOSV and SFSV seroreactivities were co-detected, suggesting exposure to multiple pathogenic viruses presumably due to shared sandfly vectors. In 9.6% of the samples, multiple virus exposure was documented. In conclusion, our findings demonstrate widespread exposure to distinct pathogenic phleboviruses, for which diagnostic testing and serological screening efforts should be directed.

Healthcare-Related HCV Genotype 4d Infections in Kayseri, Turkey.

BACKGROUND: The frequency of genotype 4 hepatitis C virus infection is significantly higher in a city compared to other provinces in Turkey. In this study, we aimed to investigate the epidemiology and risk factors of hepatitis C virus genotype 4 infection in Kayseri province of Turkey. METHODS: A case-control study was conducted with 61 hepatitis C virus genotype 4-infected patients and 71 controls. A questionnaire was administered to the patients and controls, asking for information about the risk factors of hepatitis C virus transmission. Core/ E1 and NS5B regions of hepatitis C virus genome were amplified and sequenced by Sanger method. Phylogenetic analysis and molecular clock analysis were performed. The risk was determined by calculating the odds ratio and 95% CI. Logistic regression analysis was performed to determine the effect of risk factors by controlling for confounding variables. RESULTS: Kayseri isolates were closely related to type 4d sequences but formed a separate cluster. According to the molecular clock analysis, hepatitis C virus genotype 4d entered Kayseri province probably between 1941 and 1988. Blood transfusion and surgical intervention were found to be significant risk factors for the infection. CONCLUSION: Epidemiological data showed that hepatitis C virus genotype 4d infections are significantly associated with unsafe medical procedures.

Several Tick-Borne Pathogenic Viruses in Circulation in Anatolia, Turkey.

Introduction: We screened host-collected ticks for tick-borne viruses, including those recently documented as human pathogens. Methods: During 2020-2021, ticks removed form cattle, sheep, dogs, and cats in 11 provinces in 5 geographically distinct regions of Anatolia were identified, pooled, and screened using pan-nairovirus, pan-flavivirus and individual assays for Jingmen tick virus (JMTV), and Tacheng tick virus 1 and 2 (TcTV-1 and TcTV-2). Results: A total of 901 tick specimens, comprising 6 species were included. Rhipicephalus sanguineus complex was the most abundant species (44.1%), followed by Rhipicephalus bursa (38.3%), Haemaphysalis parva (7.2%), and others. The specimens were screened in 158 pools with 12 pools (7.6%) being positive. Crimean-Congo hemorrhagic fever virus (CCHFV) lineage Europe 2 (genotype VI) sequences were detected in R. bursa in five (3.2%) of the pools, with similar prevalences in central and Mediterranean Anatolian provinces. JMTV was identified in four R. bursa and one Rhipicephalus turanicus pools, collected from Mediterranean and southeastern Anatolia, with a CCHFV and JMTV coinfected R. bursa pool. The JMTV segment 1 sequences formed a separate cluster with those from Turkey and the Balkan peninsula in the maximum likelihood analysis. TcTV-2 was detected in two Dermacentor marginatus specimens (1.3%) collected in central Anatolia, with nucleocapsid sequences forming a phylogenetically segregated group among viruses from humans and ticks from China and Kazakhstan. Discussion: CCHFV Europe 2 was initially documented in ticks from central Anatolian locations, where related orthonairoviruses had been previously recorded. Ongoing activity and a wider distribution of JMTV and TcTV-2 were observed. These viruses should be screened as potential etiological agents in human infections associated with tick bites.

Insights into the virologic and immunologic features of SARS-COV-2.

The host immunity is crucial in determining the clinical course and prognosis of coronavirus disease 2019, where some systemic and severe manifestations are associated with excessive or suboptimal responses. Several antigenic epitopes in spike, nucleocapsid and membrane proteins of severe acute respiratory syndrome coronavirus 2 are targeted by the immune system, and a robust response with innate and adaptive components develops in infected individuals. High titer neutralizing antibodies and a balanced T cell response appears to constitute the optimal immune response to severe acute respiratory syndrome coronavirus 2, where innate and mucosal defenses also contribute significantly. Following exposure, immunological memory seems to develop and be maintained for substantial periods. Here, we provide an overview of the main aspects in antiviral immunity involving innate and adaptive responses with insights into virus structure, individual variations pertaining to disease severity as well as long-term protective immunity expected to be attained by vaccination.

Vector-borne viruses in Turkey: A systematic review and bibliography.

Turkey serves as a natural hub for the dissemination of vector-borne viruses and provides many suitable habitats with diverse ecologies for introduction and establishment of new pathogens. This manuscript provides an updated systematic review and meta-analysis of the vector-borne viruses documented in Turkey. Following web-based identification, screening and eligibility evaluation, 291 published reports were reviewed. The publications were categorized and listed as a supplementary bibliography accompanying the manuscript. In brief, Crimean-Congo hemorrhagic fever virus (CCHFV) and West Nile virus (WNV) are currently documented as prominent tick and mosquito-borne viral pathogens in Turkey. CCHFV produces a significant number of infections annually, with severe outcome or death in a portion of cases. WNV gained attention following the clustering of cases in 2010. Exposure and infections with sandfly-borne phleboviruses, such as Toscana virus, are indigenous and widespread. Epidemiology, risk factors, symptomatic infections in susceptible hosts, vectors and reservoirs for these pathogens have been explored in detail. Detection of novel viruses in mosquitoes, sandflies and ticks from several regions is of particular interest, despite scarce information on their epidemiology and pathogenicity in vertebrates. Introduction and emergence of viruses transmitted by invasive Aedes mosquitoes constitute a threat, albeit only imported infections have so far been documented. Detection of Rift valley fever virus exposure is also of concern, due to its detrimental effects on livestock and spillover infections in humans. Vigilance to identify and diagnose probable cases as well as vector surveillance for established and potential pathogens is therefore, imperative.

Investigation of Orthohantavirus Seroprevalence in Northern Rural Areas of Denizli Province, Turkey.

Orthohantaviruses infect humans via inhalation of the viral particles in the excreta of infected rodents or direct contact with infected rodents. The infections caused by Puumala orthohantavirus (PUUV) and Dobrava-Belgrade orthohantavirus (DOBV) have been reported in Turkey. Serum samples of 346 healthy volunteers who are in the high-risk group of Orthohantavirus infections among the residents of Çal, Baklan, Çivril, and Bekilli counties, located in the northeast part of Denizli province, were used in this study. The samples were screened and confirmed using commercial ELISA and immunoblot tests, which detect IgG antibodies against DOBV, PUUV, and Hantaan orthohantavirus. IgG antibodies against PUUV were detected in the samples of 2 volunteers (2/346, 0.6%). One was a veterinarian and the other a farmer and they live in the Baklan and Çal counties, respectively. Both of them have a high probability of exposure to the virus, based on their occupation and living conditions. However, no symptoms were found in the clinical findings of both cases. This study is the first publication of reported PUUV seropositivities from the southwestern part of Turkey.

Characterization of Bartonella taylorii Strains in Small Mammals of the Turkish Thrace.

Rodents play role as a reservoir for some Bartonella species which cause different clinical manifestations in humans. Bartonella spp. existence in rodents of Turkish Thrace has been detected for the first time, and the risky habitat types were evaluated for the infection. Ninety individuals belonging to three small rodent species were screened by PCR, and the overall prevalence of Bartonella infection was 22.2%. The strains were characterized molecularly based on the phylogenetic analyses of two housekeeping genes, rpoB and gltA. They clustered with B. taylorii. The significant effects of habitat types and rodent species on Bartonella infections were observed. It was detected that B. taylorii prevalence was the highest in the swamp forest habitat and A. flavicollis species. The present study demonstrates that A. flavicollis is the reservoir of B. taylorii in the European part of Turkey.

A novel genetic lineage of Tula orthohantavirus in Altai voles (Microtus obscurus) from Turkey.

Orthohantaviruses (family Hantaviridae order Bunyavirales) are emerging pathogens with a significant impact on human health. They are transmitted via aerosolized excreta of rodents which also act as reservoir hosts, constituting a unique route for dispersion. Dobrava-Belgrade and Puumala orthohantaviruses have been previously reported from Anatolia, in rodents, case reports and occasional outbreaks. We have collected rodents at several locations during a surveillance study in eastern Anatolia. The specimens were morphologically-identified and various tissues were screened via a generic orthohantavirus reverse transcription polymerase chain reaction assay. DNA barcoding via mitochondrial cytochrome b sequencing was performed in rodents with detectable orthohantavirus sequences. High throughput sequencing was performed for viral genome characterization. Fifty rodents were collected and identified morphologically as Microtus spp. (96%) and Apodemus spp. (4%). Orthohantavirus sequences were detected in lung and spleen or liver tissues of 4 voles (8%), barcoded as Microtus obscurus. The virus sequences were identified as Tula orthohantavirus (TULV) and near-complete genomic segments of the prototype viral genome, tentatively named as the Tula orthohantavirus-Turkey (TULV-T), could be characterized. Putative open reading frames for viral nucleocapsid and a nonstructural protein on the S segment, glycoproteins G1 and G2 on the M segment and viral replicase on the L segment were identified on the TULV-T. Several minor sequence variants were further characterized. No evidence of recombination could be detected and pairwise comparisons displayed over 95% amino acid sequence identities to various Eurasian TULV strains. Phylogenetic analyses revealed distinct clustering of all genome segments from previously-characterized TULV strains via various approaches and models. Here, TULV-T constituted a novel lineage, forming an intermediate among Asian and European TULV lineages. This report describes the initial documentation of TULV circulation and its potential reservoir in Anatolia. The extent of virus dispersion, alternate hosts or outcomes of human exposure require elucidation.

A metagenomic survey identifies Tamdy orthonairovirus as well as divergent phlebo-, rhabdo-, chu- and flavi-like viruses in Anatolia, Turkey.

We employed a direct metagenomic approach via next-generation sequencing for a cross-sectional investigation of viruses in 10 tick pools, collected from Aegean, Mediterranean and central Anatolian locations in Turkey. Sequences from all genome segments of Tamdy orthonairovirus (family Nairoviridae) were characterized in ticks collected from a Meriones tristrami. We further obtained near-complete L and partial S segments of several tick-associated phleboviruses (family Phenuiviridae), including Tacheng tick virus 2 and a novel virus, tentatively named as the tick phlebovirus Anatolia. Partial NS5-coding region of recently-described flavi-like virus (Tacheng tick virus 8) was further detected. Moreover, near-complete and polymerase-coding regions of arthropod-associated rhabdoviruses as well as sequences closely-related to the members of the newly-proposed virus family, the Chuviridae, were characterized. Despite origins of the viral sequences could not be fully elucidated, the findings suggest the circulation of diverse arthropod and tick-associated viruses in Anatolia. Occurrence and outcome of vertebrate exposure and probable health impact of these viruses require further investigation. We also report the initial detection of Tamdy orthonairovirus, an established human pathogen, which should be included in the diagnostic workup of infections with unknown etiology.

Characterization of HCV genotype 4d infections in Kayseri, Turkey.

BACKGROUND/AIM: The frequency of genotype 4 hepatitis C virus (HCV) infections is significantly higher in Kayseri compared to other provinces in Turkey. We aimed to characterize genotype 4 infections in Kayseri by analyzing the demographic and laboratory data of218 HCV RNA-positive, treatment-naive patients admitted to the Kayseri Training and Research Hospital in 2010 and 2011. MATERIALS AND METHODS: The distributions of sex, age, and viral loads of these patients with respect to HCV genotypes were analyzed. We also characterized the type 4 sequences at the subtype level. Randomly selected sera from 32 of the 72 genotype 4 patients from this cohort were subjected to PCR amplification in the NS5B region and further characterized by sequencing and phylogenetic and molecular clock analysis. RESULTS: Distribution rates of HCV genotypes 1, 4, and 2 in the 218 patients were 62.4%, 33.0%, and 4.6%, respectively. Most of the patients infected with types 1 and 4 were over the age of 40 and female. The NS5B sequences of 32 Kayseri genotype 4 isolates were closely related with type 4d sequences but formed a separate cluster. CONCLUSION: The introduction of type 4d HCV into the Kayseri region probably took place 30-75 years ago, as predicted by molecular clock analysis.

Detection of Pneumocystis in the nasal swabs of immune-suppressed rats by use of PCR and microscopy.

BACKGROUND: Detection of Pneumocystis jiroveci colonization in lungs or oral samples due to high sensitivity of PCR methods results in undue treatment of patients without any symptoms of Pneumocystis pneumonia. The aim of the present study is to demonstrate Pneumocystis carinii in rats, immune suppressed by oral and subcutaneous administration of dexamethasone. MATERIAL/METHODS: Blood, oral, nasal and eye swabs were collected prior to immune suppression and 2, 6, 12 weeks after administration of dexamethasone. Also, samples were collected from lung, heart, liver, kidney, diaphragm, brain, spleen, tongue, muscle, eye, intestine, and feces. Cysts and trophozoites were investigated in stained slides and MSG gene was detected by PCR. RESULTS: The results showed that weight loss is significantly higher in rats administered oral dexamethasone (P<0.05). Microscopy was positive only in lungs of rats orally administered dexamethasone. PCR was positive in lungs and oral swabs of rats prior to the administration of dexamethasone. After the administration of dexamethasone, the MSG gene was detected in oral swabs, lungs, spleen, kidney and (for the first time) in nasal swabs. PCR was positive in nasal swabs during the second and sixth weeks of oral and subcutaneous administration of dexamethasone, respectively. CONCLUSIONS: Presence of P. jiroveci in nasopharyngeal aspirate, oropharyngeal wash, oral swab, induced sputum or BAL, and absence in nasal swab in a patient without symptoms of PCP may support clinician's decision regarding colonization. Overall, detection of P. carinii in nasal swabs of rats by PCR demonstrated that nasal sampling can be used for the diagnosis of Pneumocystis pneumonia.