Rapid diagnosis of diarrhea caused by Shigella sonnei using dipsticks; comparison of rectal swabs, direct stool and stool culture.

BACKGROUND: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6) CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%-98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6%) and 100 %, respectively. CONCLUSION: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.

Sensitivity and Specificity of a New Vertical Flow Rapid Diagnostic Test for the Serodiagnosis of Human Leptospirosis.

Background : Leptospirosis is a growing public health concern in many tropical and subtropical countries. However, its diagnosis is difficult because of non-specific symptoms and concurrent other endemic febrile diseases. In many regions, the laboratory diagnosis is not available due to a lack of preparedness and simple diagnostic assay or difficult access to reference laboratories. Yet, an early antibiotic treatment is decisive to the outcome. The need for Rapid Diagnostic Tests (RDTs) for bedside diagnosis of leptospirosis has been recognized. We developed a vertical flow immunochromatography strip RDT detecting anti-Leptospira human IgM and evaluated it in patients from New Caledonia, France, and French West Indies. Methodology/Principal Findings : Whole killed Leptospira fainei cells were used as antigen for the test line and purified human IgM as the control line. The mobile phase was made of gold particles conjugated with goat anti-human IgM. Standards for Reporting of Diagnostic Accuracy criteria were used to assess the performance of this RDT. The Microscopic Agglutination Test (MAT) was used as the gold standard with a cut-off titer of ≥400. The sensitivity was 89.8% and the specificity 93.7%. Positive and negative Likelihood Ratios of 14.18 and 0.108 respectively, and a Diagnostic Odds Ratio of 130.737 confirmed its usefulness. This RDT had satisfactory reproducibility, repeatability, thermal tolerance and shelf-life. The comparison with MAT evidenced the earliness of the RDT to detect seroconversion. When compared with other RDT, the Vertical Flow RDT developed displayed good diagnostic performances. CONCLUSIONS/SIGNIFICANCE: This RDT might be used as a point of care diagnostic tool in limited resources countries. An evaluation in field conditions and in other epidemiological contexts should be considered to assess its validity over a wider range of serogroups or when facing different endemic pathogens. It might prove useful in endemic contexts or outbreak situations.

Penicillin resistance compromises Nod1-dependent proinflammatory activity and virulence fitness of neisseria meningitidis.

Neisseria meningitidis is a life-threatening human bacterial pathogen responsible for pneumonia, sepsis, and meningitis. Meningococcal strains with reduced susceptibility to penicillin G (Pen(I)) carry a mutated penicillin-binding protein (PBP2) resulting in a modified peptidoglycan structure. Despite their antibiotic resistance, Pen(I) strains have failed to expand clonally. We analyzed the biological consequences of PBP2 alteration among clinical meningococcal strains and found that peptidoglycan modifications of the Pen(I) strain resulted in diminished in vitro Nod1-dependent proinflammatory activity. In an influenza virus-meningococcal sequential mouse model mimicking human disease, wild-type meningococci induced a Nod1-dependent inflammatory response, colonizing the lungs and surviving in the blood. In contrast, isogenic Pen(I) strains were attenuated for such response and were out-competed by meningococci sensitive to penicillin G. Our results suggest that antibiotic resistance imposes a cost to the success of the pathogen and may potentially explain the lack of clonal expansion of Pen(I) strains.

Dipstick test for rapid diagnosis of Shigella dysenteriae 1 in bacterial cultures and its potential use on stool samples.

BACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.

Development and evaluation of two simple, rapid immunochromatographic tests for the detection of Yersinia pestis antibodies in humans and reservoirs.

BACKGROUND: Tools for plague diagnosis and surveillance are not always available and affordable in most of the countries affected by the disease. Yersinia pestis isolation for confirmation is time-consuming and difficult to perform under field conditions. Serologic tests like ELISA require specific equipments not always available in developing countries. In addition to the existing rapid test for antigen detection, a rapid serodiagnostic assay may be useful for plague control. METHODS/PRINCIPAL FINDINGS: We developed two rapid immunochromatography-based tests for the detection of antibodies directed against F1 antigen of Y. pestis. The first test, SIgT, which detects total Ig (IgT) anti-F1 in several species (S) (human and reservoirs), was developed in order to have for the field use an alternative method to ELISA. The performance of the SIgT test was evaluated with samples from humans and animals for which ELISA was used to determine the presumptive diagnosis of plague. SIgT test detected anti-F1 Ig antibodies in humans with a sensitivity of 84.6% (95% CI: 0.76-0.94) and a specificity of 98% (95% CI: 0.96-1). In evaluation of samples from rodents and other small mammals, the SlgT test had a sensitivity of 87.8% (95% CI: 0.80-0.94) and a specificity of 90.3% (95% CI: 0.86-0.93). Improved performance was obtained with samples from dogs, a sentinel animal, with a sensitivity of 93% (95% CI: 0.82-1) and a specificity of 98% (95% CI: 0.95-1.01). The second test, HIgM, which detects human (H) IgM anti-F1, was developed in order to have another method for plague diagnosis. Its sensitivity was 83% (95% CI: 0.75-0.90) and its specificity about 100%. CONCLUSION/SIGNIFICANCE: The SIgT test is of importance for surveillance because it can detect Ig antibodies in a range of reservoir species. The HIgM test could facilitate the diagnosis of plague during outbreaks, particularly when only a single serum sample is available.

Dipstick for rapid diagnosis of Shigella flexneri 2a in stool.

BACKGROUND: Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS) using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5x10(7) CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. CONCLUSION: This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys.

Defective planar cell polarity in polycystic kidney disease.

Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

Direct recognition by alphabeta cytolytic T cells of Hfe, a MHC class Ib molecule without antigen-presenting function.

Crystallographic analysis of human Hfe has documented an overall structure similar to classical (class Ia) MHC molecules with a peptide binding groove deprived of ligand. Thus, to address the question of whether alphabeta T cells could recognize MHC molecules independently of bound ligands, we studied human and mouse Hfe interactions with T lymphocytes. We provide formal evidence of direct cytolytic recognition of human Hfe by mouse alphabeta T cell receptors (TCR) in HLA-A*0201 transgenic mice and that this interaction results in ZAP-70 phosphorylation. Furthermore, direct recognition of mouse Hfe molecules by cytotoxic T lymphocytes (CTLs) was demonstrated in DBA/2 Hfe knockout mice. These CTLs express predominantly two T cell antigen receptor alpha variable gene segments (AV6.1 and AV6.6). Interestingly, in wild-type mice we identified a subset of CD8+ T cells positively selected by Hfe that expresses the AV6.1/AV6.6 gene segments. T cell antigen receptor recognition of MHC molecules independently of bound ligand has potential general implications in alloreactivity and identifies in the Hfe case a cognitive link supporting the concept that the immune system could be involved in the control of iron metabolism.