Validation of the Sysmex CS5100 coagulation analyzer and comparison to the Stago STA-R analyzer for routine coagulation parameters.

INTRODUCTION: The CS5100 analyzer (Sysmex) was validated for the determination of routine coagulation parameters. This fully automated coagulation analyzer uses multiple wavelength technology to perform coagulation (e.g., activated partial thromboplastin time - APTT, prothrombin time - PT, fibrinogen - FBG), chromogenic (e.g., antithrombin - AT) and immunological (e.g., D-dimers - DDi) assays. METHODS: A comparison with the currently used STA-R Evolution (Stago) was performed. Validation and verification of reference values of the CS5100 was performed in accordance to CLSI guidelines (H57-A, H47-A2, and C28-A3). RESULTS: As a different detection system and reagents were used, significant differences were observed (e.g. APTT). The within-day and between-day imprecision, accuracy and total error were all acceptable. The reference values defined by the manufacturer could be used except for APTT. In our settings, the therapeutic anti-Xa range of 0.3-0.7 IU/mL corresponded to an APTT range of 60-100 s (Dade actin FS reagent). The APTT reagent showed factor sensitivities between 46 and 72% for FVIII, IX, XI and XII while the PT reagent showed sensitivities between 34 and 52% for FII, FV, FXII, and FX. CONCLUSION: In conclusion, the CS5100 instrument is suitable for the determination of the APTT, PT, FBG, DDi and AT in routine analysis.

Chlamydophila psittaci zoonotic risk assessment in a chicken and turkey slaughterhouse.

Chlamydophila psittaci causes respiratory disease in poultry and can be transmitted to humans. We conducted a C. psittaci zoonotic risk assessment study of a chicken and turkey slaughterhouse. Eighty-five percent of the slaughtered chicken flocks tested positive by PCR and culture. Genotype D was discovered. Fifty-seven percent of the slaughtered turkey flocks tested positive by PCR and culture. Genotype D was present. For the chicken slaughterhouse employees, 7.5% and 6% tested positive for C. psittaci by PCR and culture, respectively. In the turkey slaughterhouse, 87% and 61% of the employees tested positive by PCR and culture, respectively. All genotyped human samples contained genotype D. Using stationary bioaerosol monitoring by means of an MAS-100 ecosampler and ChlamyTrap collection medium, chlamydial DNA, and viable organisms were detected in both the chicken and turkey slaughterhouses. Positive air samples were most frequently found in the animal reception area and evisceration room. Zoonotic transmissions were very common, especially from processed turkeys. Accurate diagnostic monitoring and reporting of C. psittaci infections should be promoted in poultry workers.

Key role of Chlamydophila psittaci on Belgian turkey farms in association with other respiratory pathogens.

Two hundred turkey sera from eight Belgian and two French farms were tested for the presence of antibodies against avian pneumovirus (APV), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum, Mycoplasma meleagridis and Chlamydophila psittaci. At slaughter, C. psittaci, APV and ORT antibodies were detected in 94, 34 and 6.5% of the turkeys, respectively. No antibodies against M. gallisepticum or M. meleagridis were present. Additionally, turkeys on three Belgian farms were examined from production onset until slaughter using both serology and antigen or gene detection. All farms experienced two C. psittaci infection waves, at 3-6 and 8-12 weeks of age. Each first infection wave was closely followed by an ORT infection starting at the age of 6-8 weeks, which was still detectable when the second C. psittaci infection waves started. Animals on farm A were not vaccinated against APV leading to an APV subtype B outbreak accompanying the first C. psittaci infection wave. Despite subtype A APV vaccination on farms B and C, the second C. psittaci infection waves were accompanied (farm B) or followed (farm C) by a subtype B APV infection. On all farms respiratory signs always appeared together with a proven C. psittaci, APV and/or ORT infection. This study suggests an association between C. psittaci, APV and ORT, and indicates the multi-factorial aetiology of respiratory infections in commercial turkeys. All three pathogens should be considered when developing prevention strategies for respiratory disease.

Evidence for a type III secretion system in Chlamydophila psittaci.

In order to examine the presence of a type III secretion system in Chlamydophila psittaci, we focused on the SctW (CopN) coding region in a locus containing four genes encoding putative products with similarity to chaperones (Sccl), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) formerly identified in the type III secretion system of other gram-negative bacteria. SctW regulates type III secretion in gram-negative bacteria. SctW expression was examined in C. psittaci infected HeLa cells using SctW-specific polyclonal antibodies in an indirect immunofluorescence staining. SctW expression was detected 29 hours post inoculation and was absent in uninfected control cells. Immunoblotting of whole HeLa cell lysate 72 hours post inoculation with the SctW-specific polyclonal antibody also revealed the presence of SctW. Results demonstrated SctW of C. psittaci to be expressed in at least one stage of the intracellular bacterial life cycle. The present finding may contribute to the future identification of a functional type III secretion apparatus in C. psittaci.

Serological and molecular characterization of Chlamydophila psittaci strains using serovar-specific monoclonal antibodies and ompA sequence analysis.

In the present study, 21 avian Chlamydophila psittaci field isolates from 4 different European countries (Belgium, Germany, Italy and The Netherlands) were characterized using serovar-specific monoclonal antibodies as well as ompA sequence analysis, enabling the comparison between the two characterization methods for future epidemiological studies. The 21 European isolates included 6 isolates from the order Psittaciformes, 6 isolates from the Anseriformes, 5 isolates from the order Columbiformes and 4 Galliformes respectively. Only 19 on 21 isolates could be serotyped while all isolates were successfully genotyped. In addition, genotyping revealed the presence of mixed infections in 5 on 21 isolates while serotyping could only detect one of these 5 mixed infections. Interestingly, genotyping indicated the existence of a new genotype designated E/B. The E/B genotype is closely related to the genotypes A, B and E but the ompA gene of E/B strains shows a guanosine on position 1006 and 1021 in combination with a cytidine on position 1022 to be unique. Genotype E/B isolates reacted with both the serovar E- and B-specific monoclonal antibody leading to the conclusion of a mixed infection while only one specific ompA sequence was present in the sample. For epidemiological studies genotyping by use of ompA sequence analysis is to be preferred as it is more sensitive than serotyping and can distinguish genotype B, E and E/B strains.