Species-specific Real-time PCR Assay Research Articles

Detection of Schistosoma japonicum and Oncomelania hupensis quadrasi environmental DNA and its potential utility to schistosomiasis japonica surveillance in the Philippines.

In recent years, the prevalence and infection intensity of Schistosoma japonicum in endemic areas of the Philippines have significantly decreased due to yearly population-based treatment strategies, yet transmission rates remain high and uninterrupted. An important indicator of active disease transmission is the presence of Schistosoma japonicum and its snail intermediate host Oncomelania hupensis quadrasi in freshwater habitats. In this study, we sought to apply a species-specific real-time PCR (qPCR) assay for the detection of S. japonicum and O. hupensis quadrasi in freshwater samples using environmental DNA approach that can complement the commonly utilized malacological survey in determining potential transmission foci in order to have a more effective snail surveillance strategy for schistosomiasis japonica in endemic areas. The newly developed assay was specific to S. japonicum and O. hupensis quadrasi with no amplification detected against non-target trematode Fasciola spp. and snails such as Lymnaea spp., Pomacea canaliculata, and Melanoides spp. that typically co-exist in the same environment. The assay effectiveness was determined using 19 environmental water samples collected from Northern Samar (N = 5 sites), Leyte (N = 11 sites) and Compostela Valley (N = 3 sites) and compared to malacological survey for determining O. hupensis quadrasi snail colonies and snail crushing to visualize S. japonicum cercariae. TaqMan qPCR targeting a short fragment of the cytochrome c oxidase subunit 1 (cox1) gene was positive for S. japonicum in 9 sites, for O. hupensis quadrasi in 9 sites, and for both S. japonicum and O. hupensis quadrasi in 5 sampling sites. Moreover, it was able to detect O. hupensis quadrasi in 3 out of 12 sites found negative and 6 out of 7 sites found positive through malacological survey, and in 4 of the 5 snail sites positive for snails with cercariae. Overall, this method can complement malacological surveys for monitoring of schistosomes in endemic areas of the Philippines, especially those with high risk of human infection.

A glance into the black box: Novel species-specific quantitative real-time PCR assays to disentangle aquatic hyphomycete community composition

Abstract Aquatic hyphomycetes (AH) are ubiquitous fungi playing a key role in the decomposition of leaf litter in streams. Though their functional performance is modulated by their community composition, this ecological relationship remains poorly investigated due to a lack of suitable methods to identify the biomass-contribution of individual species to AH communities. We, therefore, designed and validated TaqMan® probe-based qPCR assays targeting ten AH species common in temperate regions, allowing detection and quantification of these species within complex communities. In a further step, we compared qPCR-obtained DNA levels to concentrations of the traditional fungal biomass proxy ergosterol. We demonstrate that the qPCR assays are valid for use and that DNA and ergosterol concentrations were significantly positively correlated, suggesting DNA levels as a suitable species-specific biomass proxy. Accordingly, the use of these assays may facilitate multi-species experiments to address major research issues in stress and community ecology including biodiversity-ecosystem functioning relationships.

Persistent transmission of Plasmodium malariae and Plasmodium ovale species in an area of declining Plasmodium falciparum transmission in eastern Tanzania.

A reduction in the global burden of malaria over the past two decades has encouraged efforts for regional malaria elimination. Despite the need to target all Plasmodium species, current focus is mainly directed towards Plasmodium falciparum, and to a lesser extent P. vivax. There is a substantial lack of data on both global and local transmission patterns of the neglected malaria parasites P. malariae and P. ovale spp. We used a species-specific real-time PCR assay targeting the Plasmodium 18s rRNA gene to evaluate temporal trends in the prevalence of all human malaria parasites over a 22-year period in a rural village in Tanzania.We tested 2897 blood samples collected in five cross-sectional surveys conducted between 1994 and 2016. Infections with P. falciparum, P. malariae, and P. ovale spp. were detected throughout the study period, while P. vivax was not detected. Between 1994 and 2010, we found a more than 90% reduction in the odds of infection with all detected species. The odds of P. falciparum infection was further reduced in 2016, while the odds of P. malariae and P. ovale spp. infection increased 2- and 6-fold, respectively, compared to 2010. In 2016, non-falciparum species occurred more often as mono-infections. The results demonstrate the persistent transmission of P. ovale spp., and to a lesser extent P. malariae despite a continued decline in P. falciparum transmission. This illustrates that the transmission patterns of the non-falciparum species do not necessarily follow those of P. falciparum, stressing the need for attention towards non-falciparum malaria in Africa. Malaria elimination will require a better understanding of the epidemiology of P. malariae and P. ovale spp. and improved tools for monitoring the transmission of all Plasmodium species, with a particular focus towards identifying asymptomatic carriers of infection and designing appropriate interventions to enhance malaria control.

Species-specific real-time PCR assay for enumeration of Anoxybacillus flavithermus and Geobacillus stearothermophilus spores in dairy products

The thermophilic spore-forming bacteria, Anoxybacillus flavithermus and Geobacillus stearothermophilus, grow readily, especially on milk-powder processing lines, and are important for processing-plant hygiene management. We developed a real-time PCR assay using SYBR Green I to monitor the spores of these species in dairy products. We designed new primer pairs specific for each species from the nucleotide sequences of the stage 0 sporulation gene A (spo0A). Ethidium monoazide treatment enabled us to quantify only spores. In dairy products with high protein content, treatment with proteinase K allowed precise quantification of spores. This assay was superior for counting spores, having high linearity (r2 = 0.99) and a wide quantification range (101 to 106 spores mL−1). This new method can be applied to quantification of spores in samples of milk, skimmed milk, and cream.

First molecular evidence of bovine hemoplasma species (Mycoplasma spp.) in water buffalo and dairy cattle herds in Cuba

BackgroundHemotropic mycoplasmas (aka hemoplasmas) are small bacteria which cause infectious anemia in several mammalian species including humans. Information on hemoplasma infections in Cuban bovines remains scarce and no studies applying molecular methods have been performed so far. The aim of the present study was to utilize real-time PCR and sequence analysis to investigate dairy cattle and buffalo from Cuba for the presence of bovine hemoplasma species.ResultsA total of 80 blood samples from 39 buffalo and 41 dairy cattle were investigated for the presence of Mycoplasma wenyonii and “Candidatus Mycoplasma haemobos” using two species-specific real-time TaqMan PCR assays. PCR results revealed overall 53 (66.2%; 95% CI: 55.3–75.7%) positive animals for M. wenyonii and 33 (41.2%; 95% CI: 31.1–52.2%) for “Ca. M. haemobos”; the latter were all co-infections with M. wenyonii. The sample prevalences were similar in cattle and buffalo. Based on the sequence analysis of the nearly full-length 16S rRNA gene from two cattle and two buffalo, the presence of M. wenyonii and “Ca. M. haemobos” was confirmed. Statistical analysis revealed that buffalo and cattle one year of age or older were more frequently infected with M. wenyonii or “Ca. M. haemobos” than younger animals. PCR-positivity was not associated with anemia; however, the infection stage was unknown (acute infection versus chronic carriers).ConclusionsThe high occurrence of bovine hemoplasma infections in buffalo and dairy cattle may have a significant impact on Cuban livestock production. To the best of our knowledge, this is the first molecular evidence of bovine hemoplasma species infection in dairy cattle and buffalo from Cuba and the Caribbean.

Development of a species-specific TaqMan-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet

Olsenella scatoligenes is the only skatole-producing bacterium isolated from the pig gut. Skatole, produced from microbial degradation of l-tryptophan, is the main contributor to boar taint, an off-odor and off-flavor taint, released upon heating meat from some entire male pigs. An appropriate method for quantifying O. scatoligenes would help investigating the relationship between O. scatoligenes abundance and skatole concentration in the pig gut. Thus, the present study aimed at developing a TaqMan-MGB probe-based, species-specific qPCR assay for rapid quantification of O. scatoligenes. The use of a MGB probe allowed discriminating O. scatoligenes from other closely related species. Moreover, the assay allowed quantifying down to three target gene copies per PCR reaction using genomic DNA-constructed standards, or 1.5 × 103 cells/g digesta, using O. scatoligenes-spiked digesta samples as reference standards. The developed assay was applied to assess the impact of dietary chicory roots on O. scatoligenes in the hindgut of pigs. Olsenella scatoligenes made up < 0.01% of the microbial population in the pig hindgut. Interestingly, the highest number of O. scatoligenes was found in young entire male pigs fed high levels of chicory roots. This indicates that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Accordingly, the abundance of O. scatoligenes in the hindgut does not seem to be an appropriate indicator of boar taint. The present study is the first to describe a TaqMan-MGB probe qPCR assay for detection and quantification of O. scatoligenes in pigs.

Phlebotomus halepensis (Diptera: Psychodidae) Vectorial Capacity in Afyon and Nigde Province, Turkey.

Leishmaniasis is a one of the vector-borne diseases and has two clinical forms in Turkey: cutaneous and visceral. The aim of this study was to determine the sand fly fauna in Afyon and Nigde provinces where endemic foci of cutaneous leishmaniasis (CL) in Turkey. In Afyon, 2,259 sand flies were collected in 73 locations in August 2009 and August 2010, using CDC light traps. In total, eight Phlebotomus species were identified; Phlebotomus halepensis (47.41%), Phlebotomus papatasi (31.42%), Phlebotomus neglectus/syriacus (9.38%), Phlebotomus balcanicus (7.48%), Phlebotomus simici (2.12%), Phlebotomus perfiliewi (1.90%), Phlebotomus sergenti (0.08%), Phlebotomus similis (0.13%), and Sergentomyia dentata (0.04%). A total of 418 sand fly specimens were caught by CDC light traps in 40 stations in Nigde in September 2009 and September 2010. In total, seven Phlebotomus species were identified; P. halepensis (74.16%), P. simici (13.87%), P. papatasi (3.82%), P. neglectus/syriacus (2.87%), P. balcanicus (2.63%), P. sergenti (2.39%), and Phlebotomus tobbi (0.23%). Collected sand flies were examined by microscope, and no promastigotes were found in their midguts. We categorized and pooled female specimens (1,031 females, 73 pools of 2-33 individuals). Leishmania species-specific ITS1 real-time PCR assay was performed for detection and identification of parasites. We detected 6 of the 73 pools with Leishmania tropica (Ross, 1903), (Trypanosomatidae). In conclusion, P. halepensis was found to be dominant species in both areas. We are in opinion that our findings support P. halepensis vectorial role for L. tropica in nature and it could be responsible for the transmission of L. tropica in these endemic areas.

Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples

Opisthorchiasis, which can lead to cholangiocarcinoma in cases of chronic infection, is a major public health problem in Southeast Asian countries. The trematode, Opisthorchis viverrini, is the causative agent of the disease. Accurate and rapid monitoring of O. viverrini is crucial for disease prevention and containment. Therefore, in this study we sought to develop a novel species-specific real-time PCR assay for detecting O. viverrini using environmental DNA (eDNA). The diagnostic sensitivity of the newly developed real-time PCR assay was similar to that of the traditional PCR assay for 50 fecal samples collected in Lao PDR (21 and 19 samples were positive by real-time PCR and traditional PCR, respectively). The efficacy of eDNA analysis and its applicability in the field were tested using a total of 94 environmental water samples collected from 44 sites in Savannakhet, Lao PDR during May and October 2015 and February 2016. O. viverrini eDNA was detected in five samples by real-time PCR, indicating the presence of the fluke in the area and the risk of infection for individuals consuming fish from these water sources. The application of eDNA analysis would facilitate the identification of O. viverrini endemic hotspots and contribute to the ecological control of opisthorchiasis, and this strategy can be applied to other eukaryotic water pathogens.

Molecular Detection of Zoonotic Rickettsiae and Anaplasma spp. in Domestic Dogs and Their Ectoparasites in Bushbuckridge, South Africa.

Members of the order Rickettsiales are small, obligate intracellular bacteria that are vector-borne and can cause mild to fatal diseases in humans worldwide. There is little information on the zoonotic rickettsial pathogens that may be harbored by dogs from rural localities in South Africa. To characterize rickettsial pathogens infecting dogs, we screened 141 blood samples, 103 ticks, and 43 fleas collected from domestic dogs in Bushbuckridge Municipality, Mpumalanga Province of South Africa, between October 2011 and May 2012 using the reverse line blot (RLB) and Rickettsia genus and species-specific quantitative real-time PCR (qPCR) assays. Results from RLB showed that 49% of blood samples and 30% of tick pools were positive for the genus-specific probes for Ehrlichia/Anaplasma; 16% of the blood samples were positive for Ehrlichia canis. Hemoparasite DNA could not be detected in 36% of blood samples and 30% of tick pools screened. Seven (70%) tick pools and both flea pools were positive for Rickettsia spp; three (30%) tick pools were positive for Rickettsia africae; and both flea pools (100%) were positive for Rickettsia felis. Sequencing confirmed infection with R. africae and Candidatus Rickettsia asemboensis; an R. felis-like organism from one of the R. felis-positive flea pools. Anaplasma sp. South Africa dog strain (closely related to Anaplasma phagocytophilum), A. phagocytophilum, and an Orientia tsutsugamushi-like sequence were identified from blood samples. The detection of emerging zoonotic agents from domestic dogs and their ectoparasites in a rural community in South Africa highlights the potential risk of human infection that may occur with these pathogens.

Species-specific real-time PCR assay for the detection of Streptococcus suis from clinical specimens

A real-time polymerase chain reaction was developed to detect all known strains of Streptococcus suis. The assay was highly specific, and sensitivity was <10 copies/assay for S. suis detection from clinical samples.

HRM analysis targeting ITS1 and matK loci as potential DNA mini-barcodes for the authentication of Hypericum perforatum and Hypericum androsaemum in herbal infusions

Hypericum species are among the medicinal plants that are being increasingly used due to their reported health benefits. Hypericum perforatum (St. John's wort) is well known for its anti-inflammatory, anti-depressive and anti-viral properties, as well as a healing agent. In Portuguese folk medicine, Hypericum androsaemum (Hipericão do Gerês) is largely used for its hepatic protective and diuretic properties. This work aimed at searching the potential use of two DNA mini-barcode candidates (ITS1 and matK) for the authentication of H. perforatum and H. androsaemum in herbal infusions. The sequencing results from ITS1 and matK regions were the basis for the development of species-specific PCR and real-time PCR assays coupled to High Resolution Melting (HRM) analysis, as simple approaches for the reliable discrimination of both species. The barcode regions were successful in the species-specific PCR identification of each target. ITS1 region revealed some intra-species variability from sequencing results, which compromises HRM analysis, while matK showed to be an adequate mini-barcode for the differentiation of both species by real-time PCR coupled to HRM analysis. The assays were effectively applied to commercial herbal infusions, enabling the consistent identification of two labels with non-declared Hypericum species.

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

First Report of Bacterial Fruit Blotch on Melon Caused by Acidovorax citrulli in California.

In July 2013, a melon plant sample (Cucumis melo cv. Saski) with disease symptoms resembling bacterial fruit blotch (BFB), was collected from a 10-acre field located in Yolo County, California, and submitted to the Plant Pest Diagnostics Center of the CDFA. Melon leaves had small (5 to 10 mm in diameter), tan to dark reddish-brown, angular lesions surrounded by yellow halos, and larger V-shaped lesions that extended from the leaf margins to the midrib. Bacterial streaming was observed at 400× magnification. The bacterium isolated from a leaf tissue wet mount formed smooth, round, cream-colored, non-fluorescent colonies on Pseudomonas F agar, was gram-negative, rod-shaped, aerobic, and oxidase-positive. The strain grew at 41°C and produced a strong hypersensitive response on tobacco (Nicotiana tabacum) 24 h after tissue infiltration. Based on a positive immunoassay test for Acidovorax citrulli (Eurofins STA Lab, Inc., Longmont, CO) and positive real-time PCR assays using species-specific primer sets, BX-L1/BX-S-R2 (1) and ZUP2436/2437 (4), the strain was identified as A. citrulli. A 360-bp fragment of the 16S ribosomal RNA gene was amplified using conventional PCR with primers WFB1 and WFB2 (3). The fragment, GenBank Accession No. KJ531595, showed 100% identity with the corresponding regions of A. citrulli (CP000512) strain AAC00-1 by BLAST query. Pathogenicity tests were performed by injecting 0.5 to 1 ml suspensions of the bacteria (106 CFU/ml) under the rind of three mature honeydew fruit (Cucumis melo var. indorus), three watermelon fruit (Citrullus lanatus cv. Sugar Baby), and into the cotyledons of ten, 10-day-old watermelon seedlings (cv. Sugar Baby). The fruit and seedlings were incubated in plastic bags at 30°C and similar treatments with sterile water served as negative controls. After 4 days, the seedlings inoculated with the suspect strain exhibited dark brown necrotic lesions with yellow halos that later coalesced, causing the cotyledons to collapse. Seven days after inoculation, the honeydew fruit exhibited dry, rotten gray cavities (4 to 6 cm in diameter) in the pericarp tissue below the rind. In contrast, the watermelon fruit had completely collapsed in a watery rot after 7 days. No symptoms were observed on the negative control fruits and seedlings treated with water. The pathogen was re-isolated from the inoculated fruit and seedlings and confirmed as A. citrulli by species-specific PCR and immunoassay as described above. The melon seed lot used to plant the field in Yolo County, CA, also tested positive for A. citrulli using species-specific real-time PCR assays (1,4). DNA fingerprinting by pulse field gel electrophoresis of Spe I-digested whole cell genomic DNA showed that all of the California A. citrulli strains were members of subgroup II (haplotype C strain) (3). These haplotypes normally occur on watermelon. BFB is a seed-transmitted disease of cucurbits and a major concern for national and global seed trade. First observed in United States commercial watermelon fields in 1989, BFB can cause economic losses up to 90% for commercial watermelon, cantaloupe, and honeydew growers (1,2). While BFB routinely occurs in the southeastern United States, this is the first official record of the disease in California.

Risks of suffering tick-borne diseases in sheep translocated to a tick infested area: A laboratory approach for the investigation of an outbreak

This study was designed to investigate an outbreak of high mortality that occurred in naïve Assaf sheep introduced into a Latxa sheep flock in the Basque Country, a region where piroplasmosis is endemic. To identify the causes of this outbreak, a panel of different methods, including traditional pathological, biopathological and parasitological analyses combined with recently developed molecular methods, was used. These novel molecular methods included a multiplex real-time PCR assay to screen for the presence of the most important tick-borne pathogens (piroplasms and anaplasmas), followed by a second species-specific multiplex real-time PCR assay for the identification of Anaplasma-positive samples. The identification of piroplasm-positive samples was carried out by a multiplexed microsphere-based suspension array using a Luminex® xMAP technology-based procedure. Anaplasmas and/or piroplasms were detected in 7/10 lambs and 11/13 ewes, with Babesia ovis being detected in 12 of the 23 animals, Theileria ovis in 6 and Anaplasma ovis in 4, both as single and mixed infections. Most of the animals infected with B. ovis had a marked decrease in the values of the red blood cell parameters. Ticks collected from the animals were identified as Riphicephalus bursa, recognised vector of B. ovis. Other haemolytic pathologies (clostridial disease, copper poisoning and leptospirosis) were ruled out and, considering all clinical, laboratory and epidemiological data, babesiosis by B. ovis was diagnosed. A detailed description of the clinical outcome, with ca. 60% of mortality, laboratory results and epidemiological findings are provided. The implications of the introduction of naïve animals into a piroplasmosis endemic area are discussed.

Prevalence of bloodstream pathogens is higher in neonatal encephalopathy cases vs. controls using a novel panel of real-time PCR assays.

BackgroundIn neonatal encephalopathy (NE), infectious co-morbidity is difficult to diagnose accurately, but may increase the vulnerability of the developing brain to hypoxia-ischemia. We developed a novel panel of species-specific real-time PCR assays to identify bloodstream pathogens amongst newborns with and without NE in Uganda.MethodologyMultiplex real-time PCR assays for important neonatal bloodstream pathogens (gram positive and gram negative bacteria, cytomegalovirus (CMV), herpes simplex virus(HSV) and P. falciparum) were performed on whole blood taken from 202 encephalopathic and 101 control infants. Automated blood culture (BACTEC) was performed for all cases and unwell controls.Principal FindingsPrevalence of pathogenic bacterial species amongst infants with NE was 3.6%, 6.9% and 8.9%, with culture, PCR and both tests in combination, respectively. More encephalopathic infants than controls had pathogenic bacterial species detected (8.9%vs2.0%, p = 0.028) using culture and PCR in combination. PCR detected bacteremia in 11 culture negative encephalopathic infants (3 Group B Streptococcus, 1 Group A Streptococcus, 1 Staphylococcus aureus and 6 Enterobacteriacae). Coagulase negative staphylococcus, frequently detected by PCR amongst case and control infants, was considered a contaminant. Prevalence of CMV, HSV and malaria amongst cases was low (1.5%, 0.5% and 0.5%, respectively).Conclusion/SignificanceThis real-time PCR panel detected more bacteremia than culture alone and provides a novel tool for detection of neonatal bloodstream pathogens that may be applied across a range of clinical situations and settings. Significantly more encephalopathic infants than controls had pathogenic bacterial species detected suggesting that infection may be an important risk factor for NE in this setting.

Species-specific multiplex real-time PCR assay for identification of deer and common domestic animals

A multiplex real-time PCR method for discriminating deer and common domestic species, including cattle, goat, horse, donkey, pig, and chicken was developed. Species-specific primer pairs were designed and used to produce different size DNA fragments with diverse melting temperature (T m ) values. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. Multiplex real-time PCR analysis was performed using combined primers, yielding distinct melting curve profiles for each species. The sensitivity limit of the multiplex PCR method was evaluated. Trace DNA of other species in deer DNA could be identified. Common deer products, including blood, meat, and antler were tested using this multiplex PCR method and different species of deer and domestic animals were identified. The rapid multiplex real-time PCR assay described herein is a specific, sensitive, and reliable method for high-throughput authentication of deer and domestic animal products.

Broad-Range PCR-Electrospray Ionization Mass Spectrometry for Detection and Typing of Adenovirus and Other Opportunistic Viruses in Stem Cell Transplant Patients

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log10 copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.

The invasive ash dieback pathogen Hymenoscyphus pseudoalbidus exerts maximal infection pressure prior to the onset of host leaf senescence

Shoot dieback disease of European ash caused by the ascomycete Hymenoscyphus pseudoalbidus threatens ash on a continental scale. A spore sampler placed in a diseased ash forest in Southern Norway, coupled with microscopy and DNA-based fungal species-specific real-time PCR assays, was employed to profile diurnal and within-season variation in infection pressure by ascospores of H. pseudoalbidus and the potentially co-existing non-pathogenic Hymenoscyphus albidus. Hymenoscyphus pseudoalbidus was found to be predominant in the stand. Massive simultaneous liberation, by active discharge of pathogen ascospores in the morning, peaked in mid-Jul. to mid-Aug. Accumulation of pathogen DNA on leaflets of current-year leaves reached a high level plateau phase before appearance of autumn coloration, suggesting that pathogen establishment in leaves is terminated before the onset of leaf senescence.

Prevalence of Francisella noatunensis subsp. orientalis in Cultured Tilapia on the Island of Oahu, Hawaii

Francisellosis is an emergent disease in cultured and wild aquatic animals. The causative agent, Francisella noatunensis subsp. orientalis (Fno), is a gram-negative bacterium recognized as one of the most virulent pathogens of warmwater fish. The main objective of this project was to investigate the prevalence of Fno in cultured tilapia (specifically, Mozambique Tilapia Oreochromis mossambicus, Koilapia [also known as Wami Tilapia] O. hornorum, Blue Tilapia O. aureus, and Nile Tilapia O. niloticus hybrids) on the island of Oahu, Hawaii, using conventional and real-time PCR assays followed by statistical modeling to compare the different diagnostic methods and identify potential risk factors. During 2010 and 2012, 827 fish were collected from different geographical locations throughout the island of Oahu. Upon collection of fish, the water temperature in the rearing system and the length of individual fish were measured. Extraction of DNA from different tissues collected aseptically during necropsy served as a template for molecular diagnosis. High correlation between both molecular methods was observed. Moreover, the bacterium was isolated from infected tilapia on selective media and confirmed to be Fno utilizing a species-specific Taqman-based real-time PCR assay. Although a direct comparison of the prevalence of Fno between the different geographical areas was not possible, the results indicate a high prevalence of Fno DNA in cultured tilapia throughout the farm sites located on Oahu. Of the different tilapia species and hybrids currently cultured in Hawaii, Mozambique Tilapia were more susceptible to infection than Koilapia. Water temperature in the rearing systems and fish size also had a strong effect on the predicted level of infection, with fish held at lower temperatures and smaller fish being more susceptible to piscine francisellosis.

A Real-Time PCR Assay for Rapid Detection and Quantification of Exserohilum rostratum, a Causative Pathogen of Fungal Meningitis Associated with Injection of Contaminated Methylprednisolone

A species-specific molecular beacon real-time PCR assay was developed for rapid diagnosis of Exserohilum rostratum infection. As low as 100 fg of E. rostratum DNA can be reliably detected in the presence of 50 ng of human DNA, with a dynamic linear quantification range from 20 ng to 200 fg.