Point-of-care test for detection of urogenital chlamydia in women shows low sensitivity. A performance evaluation study in two clinics in Suriname

Background

In general, point-of-care (POC) tests for Chlamydia trachomatis (Ct) show disappointing test performance, especially disappointing sensitivity results. However, one study sponsored by the manufacturer (Diagnostics for the Real World) reported over 80% sensitivity with their Chlamydia Rapid Test (CRT). We evaluated the performance of this CRT in a non–manufacturer-sponsored trial.

Methods

Between July 2009 and February 2010, we included samples from 912 women in both high- and low-risk clinics for sexually transmitted infections (STIs) in Paramaribo, Suriname. Sensitivity, specificity, positive- and negative predictive values (PPV and NPV) for CRT compared to NAAT (Aptima, Gen-Probe) were determined. Quantitative Ct load and human cell load were determined in all CRT and/or NAAT positive samples.

Results

CRT compared to NAAT showed a sensitivity and specificity of 41.2% (95% CI, 31.9%–50.9%) and 96.4% (95% CI, 95.0%–97.5%), respectively. PPV and NPV were 59.2% (95% CI, 47.5%–70.1%) and 92.9% (95% CI, 91.0%–94.5%), respectively. Quantitative Ct bacterial load was 73 times higher in NAAT-positive/CRT-positive samples compared to NAAT-positive/CRT-negative samples (p<0.001). Human cell load did not differ between true-positive and false-negative CRT results (p = 0.835). Sensitivity of CRT in samples with low Ct load was 12.5% (95% CI, 5.2%–24.2%) and in samples with high Ct load 73.5% (95% CI, 59.9%–84.4%).

Conclusions

The sensitivity of CRT for detecting urogenital Ct in this non–manufacturer-sponsored study did not meet the expectations as described previously. The CRT missed samples with a low Ct load. Improved POC are needed as meaningful diagnostic to reduce the disease burden of Ct.     

The Effect of Criticism on Functional Brain Connectivity and Associations with Neuroticism

Neuroticism is a robust personality trait that constitutes a risk factor for psychopathology, especially anxiety disorders and depression. High neurotic individuals tend to be more self-critical and are overly sensitive to criticism by others. Hence, we used a novel resting-state paradigm to investigate the effect of criticism on functional brain connectivity and associations with neuroticism. Forty-eight participants completed the NEO Personality Inventory Revised (NEO-PI-R) to assess neuroticism. Next, we recorded resting state functional magnetic resonance imaging (rsfMRI) during two sessions. We manipulated the second session before scanning by presenting three standardized critical remarks through headphones, in which the subject was urged to please lie still in the scanner. A seed-based functional connectivity method and subsequent clustering were used to analyse the resting state data. Based on the reviewed literature related to criticism, we selected brain regions associated with self-reflective processing and stress-regulation as regions of interest. The findings showed enhanced functional connectivity between the clustered seed regions and brain areas involved in emotion processing and social cognition during the processing of criticism. Concurrently, functional connectivity was reduced between these clusters and brain structures related to the default mode network and higher-order cognitive control. Furthermore, individuals scoring higher on neuroticism showed altered functional connectivity between the clustered seed regions and brain areas involved in the appraisal, expression and regulation of negative emotions. These results may suggest that the criticized person is attempting to understand the beliefs, perceptions and feelings of the critic in order to facilitate flexible and adaptive social behavior. Furthermore, multiple aspects of emotion processing were found to be affected in individuals scoring higher on neuroticism during the processing of criticism, which may increase their sensitivity to negative social-evaluation.     

Interruption of CXCL13-CXCR5 Axis Increases Upper Genital Tract Pathology and Activation of NKT Cells following Chlamydial Genital Infection

Background

Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection.

Methodology and Principal Findings

Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5−/− mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5−/− mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d−/− mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922).

Conclusions/Significance

These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.     

Multi locus sequence typing of <Emphasis Type="Italic">Chlamydiales</Emphasis>: clonal groupings within the obligate intracellular bacteria <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

Background  

The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease.     

Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at a Dice similarity of 92.6% (+/- 1.6% standard deviation). The fingerprints of the C. trachomatis isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular, omp1 and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of C. pneumoniae is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.     

Structural basis for the role of LYS220 as proton donor for nucleotidyl transfer in HIV-1 reverse transcriptase

Biochemical studies by Castro et al. have recently revealed a crucial role for a general acid in the catalysis of nucleic acid transfer in distinct classes of polymerases. For HIV-RT LYS220 was identified as proton donor. This was unanticipated from a structural point of view, since in all ternary crystal structures of HIV-RT LYS220 are too distant from the active site to fulfill this role. In this work molecular dynamics simulations were used to reveal the dynamics of HIV-RT and to provide structural evidence for the role of LYS220. During a 1μs molecular dynamics simulation LYS220 migrates toward the active site and occupies several positions enabling direct and water mediated proton transfer towards pyrophosphate. A combination of quantum mechanical and molecular mechanics methods was used to validate the different modes of interaction.     

Evaluation of the Siemens VERSANT® CT/GC DNA 1.0 assay (kPCR) for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

The Siemens VERSANT kPCR system is an automated system which combines extraction of nucleic acids from 96 samples with subsequent real-time PCR. The VERSANT CT/GC DNA 1.0 (kPCR) assay detects Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in a multiplex real-time PCR on this system. We compared this assay with the BD ProbeTe™ ET System (PT) and the Roche Cobas Amplicor (CA). Three different sets of samples were tested in the kPCR: PT pre-treated samples, prospectively collected urine samples during routine CT/GC testing and urine samples obtained in a blinded fashion by an external lab facility. Agreement of kPCR with the comparator tests was > 0.99 for sample set I and complete agreement was observed for sample set II and III. The kPCR assay demonstrated to be an easy to use robust diagnostic platform. A few modifications to the manufacturer's instructions are recommended to intercept false positivity. We advise to retest samples with Cq values above 35 cycles at least one time and we suggest checking the amplification curves.     

Polymorphisms in Toll-like receptors 2, 4, and 9 are highly associated with hearing loss in survivors of bacterial meningitis

Genetic variation in innate immune response genes contributes to inter-individual differences in disease manifestation and degree of complications upon infection. We recently described an association of single nucleotide polymorphisms (SNPs) in TLR9 with susceptibility to meningococcal meningitis (MM). In this study, we investigate the association of SNPs in multiple pathogen recognition and immune response genes with clinical features that determine severity and outcome (especially hearing loss) of childhood MM and pneumococcal meningitis (PM). Eleven SNPs in seven genes (TLR2, TLR4, TLR9, NOD1, NOD2, CASP1, and TRAIL) were genotyped in 393 survivors of childhood bacterial meningitis (BM) (327 MM patients and 66 PM patients). Genotype distributions of single SNPs and combination of SNPs were compared between thirteen clinical characteristics associated with severity of BM. After correction for multiple testing, TLR4+896 mutant alleles were highly associated with post-meningitis hearing loss, especially MM (p=?0.001, OR 4.0 for BM, p=?0.0004, OR 6.2 for MM). In a multigene analysis, combined carriership of the TLR2+2477 wild type (WT) with TLR4+896 mutant alleles increases the risk of hearing loss (p<0.0001, OR 5.7 in BM and p=?0.0001, OR 7.6 in MM). Carriage of one or both mutant alleles in TLR4+896 and TLR9 -1237 increases the risk for hearing loss (p?=?0.0006, OR 4.1 in BM). SNPs in immune response genes contribute to differences in clinical severity and outcome of BM. The TLR system seems to play an important role in the immune response to BM and subsequent neuronal damage as well as in cochlear inflammation. Genetic markers may be used for identification of high-risk patients by creating prediction rules for post-meningitis hearing loss and other sequelae, and provide more insight in the complex immune response in the CNS possibly resulting in new therapeutic interventions.     

Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine β-casein

Summary The action of the cell-envelope proteinase (P_III-type) from Lactococcus lactis ssp. cremoris AM₁ on bovine -casein was studied. The results were compared with those obtained earlier with (P_I-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15°C) of -casein made with the P_III-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in -casein by the P_III-type proteinase. In nine cases the primary cleavage site (P₁-P₁) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P₂-P₃ and/or P₂-P₃ regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by P_I-type proteinases, although the kinetics may be different. The P_III-type proteinase shows a broader specificity in its initial cleavage of -casein than does the P_I-type. Offprint requests to: S. Visser