A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Comparison of multilocus sequence typing and multilocus typing microarray of Chlamydia trachomatis strains from Argentina and Chile.

This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C. trachomatis positive specimens were analyzed by ompA sequencing and MLST and of these 76 by MLT array. Obtained MLST sequence types (STs) were compared to sequences in the database http://mlstdb.uu.se. The resolution obtained for MLST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 1.3 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MLST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.

High Frequency of Chlamydia trachomatis Mixed Infections Detected by Microarray Assay in South American Samples.

Chlamydia trachomatis is one of the most common sexually transmitted infections worldwide. Based on sequence variation in the ompA gene encoding the major outer membrane protein, the genotyping scheme distinguishes 17 recognized genotypes, i.e. A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, K, L1, L2, and L3. Genotyping is an important tool for epidemiological tracking of C. trachomatis infections, including the revelation of transmission pathways and association with tissue tropism and pathogenicity. Moreover, genotyping can be useful for clinicians to establish the correct treatment when LGV strains are detected. Recently a microarray assay was described that offers several advantages, such as rapidity, ease of standardization and detection of mixed infections. The aim of this study was to evaluate the performance of the DNA microarray-based assay for C. trachomatis genotyping of clinical samples already typed by PCR-RFLP from South America. The agreement between both typing techniques was 90.05% and the overall genotype distribution obtained with both techniques was similar. Detection of mixed-genotype infections was significantly higher using the microarray assay (8.4% of cases) compared to PCR-RFLP (0.5%). Among 178 samples, the microarray assay identified 10 ompA genotypes, i.e. D, Da, E, F, G, H, I, J, K and L2. The most predominant type was genotype E, followed by D and F.

Detection of atypical Chlamydiaceae in roe deer (Capreolus capreolus).

Investigations on fecal samples, vaginal swabs and sera from roe deer (Capreolus capreolus) in south-western France led to the detection of a non-classified Chlamydiaceae strain. A total of 85 vaginal swabs were sampled from roe deer that had been captured in 2012 (n=42) and 2013 (n=43). Using a Chlamydiaceae family-specific real-time PCR, only one vaginal swab out of the 42 samples done in 2012 tested positive and was subsequently identified as Chlamydia (C.) psittaci. In contrast, 6/43 vaginal swab samples were positive in 2013. Four of these positive samples came from a single group of roe deer, captured in the Fabas plain. Fecal samples from this group of 9 females were subsequently analyzed, with 6 of them testing positive with the Chlamydiaceae-specific PCR. All positive samples collected in 2013 were negative when re-tested with C. abortus-, C. pecorum- and C. suis-specific real-time PCR assays. Sera from this group of 9 females were analyzed with two immunoassays (recomLine and ELISA). Whereas intense positive reactions with C. pneumoniae antigens were observed for all sera when tested with the recomLine test, none was positive with the C. abortus specific ELISA test. Comparative sequence analysis of the 16S, 23S rRNA and ompA gene sequences from 3 animals, as well as the MLST analysis from 2 animals, showed that this roe deer group likely harbored the same bacterium related to members of the family Chlamydiaceae. Notably, the roe deer strain formed a separate entity different from the currently recognized chlamydial species, with C. trachomatis, C. suis and C. muridarum appearing as its closest relatives.

Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

Rapid microarray-based genotyping of Chlamydia spp. strains from clinical tissue samples.

Pathogenic Chlamydia (C.) psittaci and C. trachomatis strains can be genotyped based on variations in the ompA genomic locus. In the present chapter, we describe rapid genotyping assays for both chlamydial agents using the ArrayStrip™ (AS) microarray platform. The test is targeting multiple discriminatory sites in the variable domains of the ompA gene by using 35 (C. psittaci) and 61 (C. trachomatis) oligonucleotide probes representing genotype-specific polymorphisms. In addition to discrimination among the established genotypes, this approach allows identification of atypical strains that were not accessible to typing using previously established techniques, such as PCR-RFLP or serotyping. The present DNA microarray assay can be conducted directly on clinical tissue samples and is suitable for tracing epidemiological chains and exploring the dissemination of particular genotypes. The procedure is easy to handle and economically affordable, and it allows genotyping of up to 32 clinical samples per day, thus lending itself for routine diagnosis as well.

Performance of seven serological assays for diagnosing tularemia.

BACKGROUND: Tularemia is a rare zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Serology is frequently the preferred diagnostic approach, because the pathogen is highly infectious and difficult to cultivate. The aim of this retrospective study was to determine the diagnostic accuracy of tularemia specific tests. METHODS: The Serazym®Anti-Francisella tularensis ELISA, Serion ELISA classic Francisella tularensis IgG/IgM, an in-house ELISA, the VIRapid® Tularemia immunochromatographic test, an in-house antigen microarray, and a Western Blot (WB) assay were evaluated. The diagnosis tularemia was established using a standard micro-agglutination assay. In total, 135 sera from a series of 110 consecutive tularemia patients were tested. RESULTS: The diagnostic sensitivity and diagnostic specificity of the tests were VIRapid (97.0% and 84.0%), Serion IgG (96.3% and 96.8%), Serion IgM (94.8% and 96.8%), Serazym (97.0% and 91.5%), in-house ELISA (95.6% and 76.6%), WB (93.3% and 83.0%), microarray (91.1% and 97.9%). CONCLUSIONS: The diagnostic value of the commercial assays was proven, because the diagnostic accuracy was >90%. The diagnostic sensitivity of the in-house ELISA and the WB were acceptable, but the diagnostic accuracy was <90%. Interestingly, the antigen microarray test was very specific and had a very good positive predictive value.

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves. In view of the evidence obtained from the analyses, we propose the addition of two new species to the current classification: Chlamydia avium sp. nov. comprising strains from pigeons and psittacine birds (type strain 10DC88(T); DSMZ: DSM27005(T), CSUR: P3508(T)) and Chlamydia gallinacea sp. nov. comprising strains from poultry (type strain 08-1274/3(T); DSMZ: DSM27451(T), CSUR: P3509(T)).

Zoonotic Chlamydiaceae species associated with trachoma, Nepal.

Trachoma is the leading cause of preventable blindness. Commercial assays do not discriminate among all Chlamydiaceae species that might be involved in trachoma. We investigated whether a commercial Micro-ArrayTube could discriminate Chlamydiaceae species in DNA extracted directly from conjunctival samples from 101 trachoma patients in Nepal. To evaluate organism viability, we extracted RNA, reverse transcribed it, and subjected it to quantitative real-time PCR. We found that 71 (70.3%) villagers were infected. ArrayTube sensitivity was 91.7% and specificity was 100% compared with that of real-time PCR. Concordance between genotypes detected by microarray and ompA genotyping was 100%. Species distribution included 54 (76%) single infections with Chlamydia trachomatis, C. psittaci, C. suis, or C. pecorum, and 17 (24%) mixed infections that includied C. pneumoniae. Ocular infections were caused by 5 Chlamydiaceae species. Additional studies of trachoma pathogenesis involving Chlamydiaceae species other than C. trachomatis and their zoonotic origins are needed.

Rapid spoligotyping of Mycobacterium tuberculosis complex bacteria by use of a microarray system with automatic data processing and assignment.

Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability.

More than classical Chlamydia psittaci in urban pigeons.

In the literature, studies of Chlamydia infection in birds have usually been confined to the search for Chlamydia (C., formerly Chlamydophila) psittaci, so that little is known about the presence of other chlamydial agents. In the present study, cloacal swabs and faeces samples of urban pigeons have been examined by real-time PCR, DNA microarray assays and partial ompA sequencing. Whilst C. psittaci was the predominant chlamydial agent in this pigeon population (75.8% of all Chlamydiaceae positives), the combined use of highly specific and sensitive molecular assays facilitated the detection of atypical serovars of C. psittaci, as well as other species of Chlamydia, such as C. abortus. Detection of C. pecorum and C. trachomatis from an avian host is reported here for the first time. Rather unexpectedly, 19.5% of all Chlamydiaceae-positive cases turned out to be infected with non-classified organisms. The considerable prevalence of these novel agents raises the question of their epidemiological importance and possible role as pathogens. Future surveys in domestic and wild birds will have to take the extended variety of chlamydial organisms into account.

High-resolution genotyping of Chlamydia trachomatis by use of a novel multilocus typing DNA microarray.

Typing of Chlamydia trachomatis is important to understanding its epidemiology. Currently used methods such as DNA sequencing of the ompA gene and multilocus sequence typing (MLST) either offer limited epidemiological resolution or are laborious and expensive, or both. DNA microarray technology using the ArrayStrip format is an affordable alternative for genotyping. In this study, we developed a new multilocus typing (MLT) DNA microarray, based on the target regions of a high-resolution MLST system as well as software for easy analysis. Validation of the array was done by typing 80 previously MLST-typed clinical specimens from unselected adolescents in school. The MLT array showed 100% specificity and provided 2.4-times-higher resolution than ompA sequencing, separating the commonly predominating ompA E/Bour genotype into 7 MLT array genotypes. The MLT array reproduced epidemiological findings revealed by the MLST system and showed sufficient sensitivity to work with clinical specimens. Compared to MLST analysis, the expenses needed for testing a sample with the MLT array are considerably lower. Moreover, testing can be completed within 1 working day rather than 3 or 4 days, with data analysis not requiring highly specialized personnel. The present MLT array represents a powerful alternative in C. trachomatis genotyping.

Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA-based DNA microarray assay.

Current typing methods of Chlamydia (C.) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube™ format for individual samples and the ArrayStrip™ format for higher throughput. The new test is exploiting multiple discriminatory sites by involving a total of 61 oligonucleotide probes representing genotype-specific polymorphisms in variable domains 1, 2 and 4 of the ompA gene. After multiplex amplification of these domains using biotinylated primers, the sample is hybridized in the microarray vessel under highly stringent conditions. The resulting binding pattern is genotype specific, thus allowing direct identification. We were able to show that DNA from each of the currently accepted genotypes (serovars) yielded a unique, theoretically expected and distinct hybridization pattern. The assay was also shown to be highly sensitive as a dilution containing the equivalent of 1 inclusion-forming unit was still correctly genotyped. In addition, when 62 clinical samples were examined and compared to PCR-RFLP typing results, the genotype was correctly identified by the DNA microarray in all cases. The present test is easy to handle and economically affordable, and it allows genotyping of C. trachomatis to be accomplished within a working day, thus lending itself for epidemiological studies and routine diagnosis.

Comparison of different methods for preparation and characterization of total RNA from cartilage samples to uncover osteoarthritis in vivo.

BACKGROUND: The isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize, e.g. cartilage samples. Additionally, cartilaginous tissues are characterized by a low cellularity and an abundance of extracellular matrix (ECM) molecules. But given the growing interest in understanding pathogenesis of degenerative diseases, e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), studies have to consider expression pattern of cells in its natural environment. FINDINGS: We compared the current RNA isolation methods for the extraction of high-quality RNA of snap-frozen biopsies from limited amounts of hypocellular cartilaginous tissue. The focus of the study was to gather information about procedure-related differences in RNA quality and yield. Here, we describe two protocols, the phenol/chloroform-free filter-based method (RNAqueous kit) and the combined protocol (TRIzol(R)/RNeasy Mini kit), working in a reproducible and reliable manner. CONCLUSIONS: We conclude that preparation, storage, homogenization, and quality control are altogether critical steps for in-depth analysis of differential gene expression, especially in hypocellular tissues with highly crosslinked ECM like cartilage.

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes.

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.

Microplate assay for cathepsin detection in viable cells using derivatives of 4-methoxy-beta-naphthylamide.

This unit describes an assay for the direct and selective detection of the four cathepsins B, H, K, and L in adherently growing cells. Cells are incubated with substrates that are peptidic derivatives of 4-methoxy-beta-naphthylamine partially selective for each cathepsin, together with 5-nitrosalicylaldehyde. The protease reaction is performed in microtiter plates and the fluorescent hydrolysis products are detected using a plate reader. The selectivity of detection is enhanced by parallel assays containing inhibitors that are also partially selective for each of the cathepsins. Individual cathepsin activities can then be calculated by the difference between the uninhibited and the inhibited assays. Detection of cathepsin H activity differs from the other assays in that other nonlysosomal aminopeptidases are inhibited by bestatin. The most common application of these assays is to compare directly cells treated with different substances, e.g., pharmaceutically interesting cathepsin inhibitors.