Driving Laboratory Standardization of Bacterial Culture and Antimicrobial Susceptibility Testing in Veterinary Clinical Microbiology in Europe and Beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level.

Detection of mycobacterial DNA by a specific and simple lateral flow assay incorporating cadmium selenide quantum dots.

Cadmium selenide quantum dots have been incorporated to a lateral flow assay for the specific and very simple detection of different mycobacterial DNA targets within only a few minutes, bypassing the complexity of conventional DNA hybridization assays. The method extends our previous work on protein detection using an identical procedure.

Evaluation of indirect immunofluorescence antibody test and enzyme-linked immunosorbent assay for the diagnosis of infection by Leishmania infantum in clinically normal and sick cats.

Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P < 0.001). The diagnostic sensitivity of all serological tests, using PCR as the gold standard, was very low, but ELISA and IFAT for anti-Leishmania IgM had 100% specificity. The diagnostic sensitivity of all serological tests could not be improved by changing the cut-off values. Seropositivity for Leishmania spp was not associated with signalment, living conditions, season of sampling and health status of the cats or with seropositivity to feline leukemia virus, feline immunodeficiency virus, feline coronavirus, Toxoplasma gondii and Bartonella henselae. In conclusion, because of their low sensitivity and very high specificity two of the evaluated serological tests (ELISA for anti-Leishmania IgG and IFAT for anti-Leishmania IgM) may be useless as population screening tests but valuable for diagnosing feline infection by L. infantum.

Investigation of comorbidities in dogs with leishmaniosis due to Leishmania infantum.

In endemic areas, dogs with leishmaniosis due to Leishmania infantum frequently have comorbidities, including mostly neoplastic, infectious, and parasitic diseases. The aim of this study was to compare the prevalence of comorbidities among dogs that are not infected by L. infantum, dogs that are infected but do not present leishmaniosis, and dogs with leishmaniosis, and to examine if certain comorbidities are independent risk factors for the infection by L. infantum and/or for the development of canine leishmaniosis (CanL). A total of 111 dogs, older than 1-year and non-vaccinated against CanL, were allocated into three groups: group A (n = 18) included dogs that were not infected by L. infantum, group B (n = 52) included dogs that were infected by L. infantum but did not present CanL, and group C (n = 41) included dogs with CanL. Signalment and historical data were obtained using a structured questionnaire. Laboratory examinations included complete blood count, serum biochemistry, urinalysis, fecal parasitology, modified Knott's test, microscopic examination of capillary blood, buffy coat, lymph node, bone marrow and conjunctival smears, qualitative serology for Dirofilaria immitis, Anaplasma phagocytophilum/A. platys, Borrelia burgdorferi and E. canis, IFAT for L. infantum, ELISA for Babesia spp. and Neospora caninum, and real-time PCR for L. infantum in bone marrow, skin biopsies and conjunctival swabs. A variety of comorbidities were found in all three groups. No independent risk factors for infection by L. infantum were found. On the contrary, among dogs infected by L. infantum, being a mongrel [odds ratio (OR): 11.2], not receiving prevention for dirofilariosis (OR: 26.5) and being seropositive to N. caninum (OR: 17.1) or to Babesia spp. (OR: 37.6), were independent risk factors for presenting CanL. Although no comorbidities influence the probability of canine infection by L. infantum, certain comorbidities may be precipitating factors for the transition from the subclinical infection by L. infantum to the overt CanL.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

Prevalence and Risk Factors for Bartonella spp. and Haemoplasma Infections in Cats from Greece.

Bartonellosis and haemoplasmosis are vector-borne diseases with global impact on the health of domestic cats and of zoonotic importance. The aim of this study was to describe the epidemiological aspects of various populations of cats infected with Bartonella spp. or haemoplasma species. The populations evaluated included client-owned cats, stray cats and cats that live in breeding catteries in Greece. A total of 452 cats were prospectively enrolled into the study. A commercially available indirect immunofluorescence antibody testkit was used for the detection of Bartonella henselae IgG antibodies in serum. PCRs for the detection of Bartonella spp. and haemoplasma species DNA in the blood were also performed in a subgroup of 242 of the 452 cats. Risk factors for B. henselae seropositivity and infection with the haemoplasma species were determined using multivariable analysis. Overall, 160 (35.4%) of the 452 cats were seropositive for B. henselae. Seven (2.9%) and 46 (19%) of the 242 cats were PCR-positive for Bartonella spp. and haemoplasma species, respectively. The factors associated with B. henselae seropositivity, based on multivariate analysis, included older age, outdoor access, living region and flea infestation. Non-administration of ectoparasiticides was associated with haemoplasma species infection. This study shows a high prevalence of seropositivity for B. henselae and a relatively high prevalence of infection with haemoplasma species. Therefore, it is necessary to establish optimal strategies for the prevention of Bartonella spp. and haemoplasma species infections, considering the high-risk groups of cats identified in this study.

Allele and genotype frequencies of the SOD1 gene polymorphism associated with canine degenerative myelopathy in Belgian Malinois dogs in Greece.

BACKGROUND AND AIM: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. MATERIALS AND METHODS: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. RESULTS: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy-Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. CONCLUSION: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

Development and characterization of oligonucleotide-tagged dye-encapsulating EPC/DPPG liposomes.

Liposomes applications in health care include meanly their ability to carry drugs and genes inside the human body for therapeutic purposes. Nevertheless their applicability can extend far beyond and could be used as analytical tools in order to perform rapid, low-cost, sensitive and specific analyses. Their physical characteristics, such as large internal volume and extended surface area, render them ideal for these applications and specifically for improving the specificity and sensitivity of the analytical assay. The purpose of this study was to develop a simple, stable and low-cost oligonucleotide-tagged liposomal formulation consisting of EggPC and DPPG with a simple to synthesize thiol-reactive conjugate (Mal-SA) incorporated into the lipid bilayer of liposomes. The prepared liposomes, having also the water soluble dye Sulforhodamine B encapsulated in their inner cavity, were characterized in terms of their physicochemical (size, size distribution, zeta-potential, lipid content) and mechanical (morphology, rigidity) properties. The results showed that the final liposomal formulation could be used in the future as analytical tool for detecting pathogen strains of microorganism in biological milieu.

Evaluation of clinicopathological abnormalities in sick cats naturally infected by Leishmania infantum.

Feline infection by Leishmania infantum (syn. L. chagasi) has been described in areas where canine leishmaniosis is endemic. A wide variety of clinicopathological abnormalities have been reported in cats presenting clinical signs of leishmaniosis but there is a paucity of information regarding cats infected by L. infantum that do not suffer from leishmaniosis but from other diseases. The aim of this study was to compare: a) the frequency of clinicopathological abnormalities and b) the values of hematology, serum biochemistry and urinalysis parameters, between non-infected sick cats and sick cats that were infected by L. infantum. A total of 50 cats with cutaneous, ocular and/or systemic clinical signs that lived in an endemic area and had been tested for infection by L. infantum using PCR from four different tissues, were included. Based on the results of PCR, 20/50 cats were found to be infected and 30/50 non-infected. The only difference between the two groups of cats was that the concentration of inorganic phosphorus (P = 0.043) was higher in infected cats. This finding may suggest an association between infection by L. infantum and feline kidney disease.

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.

CARD15/NOD2, CD14, and toll-like receptor 4 gene polymorphisms in Greek patients with sarcoidosis.

BACKGROUND: Sarcoidosis, similarly to Crohn's disease (CD), is a complex inflammatory disease of unknown etiology. The belief that a genetic susceptibility to the development of sarcoidosis exists was derived from observations of familial clustering of sarcoidosis cases and racial differences in disease prevalence. Taking into account the remarkable similarity in the immunopathophysiology of sarcoidosis and CD, and in further exploring the genetic background of sarcoidosis, we study gene polymorphisms known for their implication in CD. These polymorphisms are in the CARD15/NOD2 gene (R702W, G908R and 3020insC), as well as mutations in the promoter of the CD14 gene (T/C at position -159) and in the TLR4 gene (Asp299Gly and Thr399Ile). METHODS: DNA was obtained from 100 sarcoidosis patients and 150 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis. RESULTS: Although CARD 15/NOD2 mutations were more frequent in cases than in controls, the difference was significant only for the G908R polymorphism (p = 0.024). Interestingly, the same was recorded with reference to the T allele (p = 0.002) and TT genotype (p = 0.017) frequencies of the CD14 promoter. No differences were observed in the 299Gly and 399Ile allele frequencies between patients and controls. Finally, the co-existence of a mutation in the CARD15/NOD2 and the CD14 genes was associated with sarcoidosis at a higher level of significance than any of these mutations separately. CONCLUSION: Our results suggest that the G908R mutation of the CARD15/NOD2 gene, as well as the T allele and TT genotype of the CD14 promoter are associated with increased susceptibility for developing sarcoidosis.

Relationship between Crohn's disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients.

AIM: To study the association between Crohn's disease (CD), Mycobacterium avium subspecies paratuberculosis (MAP), and genetic factors by examining the role of natural resistance-associated macrophage protein 1 (NRAMP1) gene polymorphisms (now SLC11A1) in Sardinian patients with CD and controls. METHODS: Thirty-seven CD patients and 34 controls with no inflammatory bowel disease (IBD) were recruited at the University of Sassari after giving written consent. Six SCL11A1 polymorphisms previously reported to be the most significantly associated with IBD were searched. M. paratuberculosis was identified by IS900 PCR and sequencing. Logistic regression was used to calculate odds ratios (OR) for the associations among CD, presence of MAP, and 6 loci described above. RESULTS: For the first time, a strong association was observed between polymorphisms at NRAMP1 locus 823C/T and CD. While CD was strongly associated with both NRAMP1 and MAP, NRAMP1 polymorphisms and MAP themselves were not correlated. CONCLUSION: Combined with previous work on the NOD2/CARD15 gene, it is clear that the interplay of genetic, infectious, and immunologic factors in the etiology of CD is complex.

The infectivity of sarcoid clinical material and its bacterial content inoculated in CBA mice.

BACKGROUND: Sarcoidosis is a multisystemic disorder that is currently viewed as the consequence of chronic immunological response associating genetic susceptibility and specific environmental or transmissible agents. Relevant evidence, although conflicting, justifies a concern about the involvement of specific pathogens to disease causation. In this study we assessed the infectivity of sarcoid clinical material, and of the pathogens found in it, to normal CBA mice used as a model of an immuno-competent host. MATERIALS AND METHODS: One hundred and eleven mice were inoculated into their footpads with fresh, filtered, and autoclaved, sputum and bronchoalveolar lavage homogenates, collected from patients with sarcoidosis, and with the mycobacterial and propionibacterial pathogens isolated from this material. RESULTS: The total number of positive reactors of the animals that received raw clinical material and the pathogens it contained was statistically significant compared to those of the control groups. However, the number of affected mice per group was in most cases less than 50% and inflammation was almost always mild and local. CONCLUSION: Based on the evidence provided by inoculation of normal CBA mice, some of the material under study, although of mild potency, can be infectious to an immuno-competent host.

Detection of mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany.

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms. Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×10(4)Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR.

Detection of Leishmania-specific DNA and surface antigens using a combination of functionalized magnetic beads and cadmium selenite quantum dots.

Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/µl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings.

Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.

A real-time PCR assay for quantitative detection of Mycobacterium avium paratuberculosis has been developed. It targets and amplifies sequences from the IS900 insertion element which is specific for this bacterium, and includes an internal amplification control. The assay was tested against 18 isolates of M. avium paratuberculosis, 17 other mycobacterial strains, and 25 non-mycobacterial strains, and was fully selective. It is capable of detecting <3 genomic DNA copies with 99% probability or alternatively, using cells directly in the reaction, 12 cells can be detected with 99% probability. Using prior centrifugation, the assay was able to consistently and quantifiably detect 10(2) M. avium paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(2) M. avium paratuberculosis in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium paratuberculosis.

Association between polymorphisms in the Toll-like receptor 4, CD14, and CARD15/NOD2 and inflammatory bowel disease in the Greek population.

AIM: Crohn's disease (CD) and ulcerative colitis (UC) are multifactorial diseases with a significant genetic background. Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp, Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC. METHODS: DNA was obtained from 120 patients with CD, 85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis. RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequencies of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026<0.05; P = 0.0048<0.01 and P = 0.047<0.05 respectively). Concerning the NOD2/CARD15 mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls. Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD). CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15 is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.

Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) can cause a very serious, often-fatal disease, namely paratuberculosis, in several animal species, especially ruminants. Recently, it has also been implicated in the pathogenesis of Infectious Bowel Disease of man. The aim of this study was to develop a molecular method for the routine detection and identification of MAP, from tissue samples of animal origin. The proposed assay would have to combine optimum performance and cost, with high reproducibility. To this goal, three laboratories in Greece and the Czech Republic undertook different parts of a study that involved evaluation of DNA extraction procedures, and PCR assays, for MAP detection. For DNA extraction we used one in-house, and one commercial method, and for the PCR we assessed a number of different assays, starting with the evaluation of primer specificity with an extended GenBank database search. Based on these results, we chose to assess a one-tube nested, 2 two-tube nested, and a single PCR assay, targeted to different genomic regions of the IS900 element. These four methods were applied on positive and negative control samples, consisted of pure bacterial cultures and formalin-fixed paraffin-embedded (FFPE) tissue samples collected from cattle with paratuberculosis and chickens with M. avium subsp. avium infection. Based on the criteria of reliability and cost, the procedure that performed better was the one-tube nested PCR assay combined with the in-house DNA extraction method. The agreement of the results obtained by the three collaborating laboratories indicates the reliability of the proposed assay even under different laboratory conditions.

Quantum dots-bevacizumab complexes for in vivo imaging of tumors.

BACKGROUND/AIM: The basic role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab for first-line treatment of cancer patients. Recent anticancer therapeutics based on active tumor targeting by conjugating tumor-specific antibodies has become of great interest in oncology. Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers able to deliver specific molecule payloads in a selective manner to improve the efficacy and safety of cancer imaging and therapy. We herein aimed to determine the targeting ability of bevacizumab-conjugated quantum dots (QDs) in vitro and in vivo. MATERIALS AND METHODS: We used QDs labeled with bevacizumab, in various in vitro experiments using cell lines derived from colorectal cancer (CRC) and breast cancer (BC). For a competition study of QD-bevacizumab complex and bevacizumab, the cells were pre-treated with bevacizumab (100 nmol/L) for 24 h before exposure to the QD-bevacizumab complex. The breast cancer cells (MDA-MB-231) were injected to 9 nude mice to make the xenograft tumor model. The QD-bevacizumab complex was injected into the tumor model and fluorescence measurements were performed at 1, 12, and 24 h post-injection. RESULTS: Immunocytochemical data confirmed strong and specific binding of the QD-bevacizumab complex to the cell lines. The cells pre-treated with an excess of bevacizumab showed absence of QD binding. The in vivo fluorescence image disclosed that there was an increased signal of tumor after the injection of QDs. Ex vivo analysis showed 3.1 ± 0.8%, 28.6 ± 5.4% and 30.8 ± 4.2% injected dose/g accumulated in the tumors at 1, 12 and 24 h respectively. Tumor uptake was significantly decreased in the animals pretreated with excess of bevacizumab (p=0.001). CONCLUSION: In conclusion, we could successfully detect the VEGF-expressing tumors using QDs-bevacizumab nanoprobes in vitro and in vivo, opening new perspectives for VEGF-targeted non-invasive imaging in clinical practice.