Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin.

The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity1-5, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic 'dotty death' phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane6. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving7, or migrate and traverse cells to progress through the host tissues7-9 at the eventual expense of their own life.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri†3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

Hypervulnerability to Sound Exposure through Impaired Adaptive Proliferation of Peroxisomes.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

Development and evaluation of a dipstick diagnostic test for Neisseria meningitidis serogroup X.

The emergence of Neisseria meningitidis serogroup X (NmX) in the African meningitis belt has urged the development of diagnostic tools and vaccines for this serogroup, especially following the introduction of a conjugate vaccine against N. meningitidis serogroup A (NmA). We have developed and evaluated a new rapid diagnostic test (RDT) for detecting the capsular polysaccharide (cps) antigen of this emerging serogroup. Whole inactivated NmX bacteria were used to immunize rabbits. Following purification by affinity chromatography, the cpsX-specific IgG antibodies were utilized to develop an NmX-specific immunochromatography dipstick RDT. The test was validated against purified cpsX and meningococcal strains of different serogroups. Its performance was evaluated against that of PCR on a collection of 369 cerebrospinal fluid (CSF) samples obtained from patients living in countries within the meningitis belt (Cameroon, Côte d'Ivoire, and Niger) or in France. The RDT was highly specific for NmX strains. Cutoffs of 10(5) CFU/ml and 1 ng/ml were observed for the reference NmX strain and purified cpsX, respectively. Sensitivity and specificity were 100% and 94%, respectively. A high agreement between PCR and RDT (Kappa coefficient, 0.98) was observed. The RDT gave a high positive likelihood ratio and a low negative likelihood (0.07), indicating almost 100% probability of declaring disease or not when the test is positive or negative, respectively. This unique NmX-specific test could be added to the available set of RDT for the detection of meningococcal meningitis in Africa as a major tool to reinforce epidemiological surveillance after the introduction of the NmA conjugate vaccine.

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.

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.

New rapid diagnostic tests for Neisseria meningitidis serogroups A, W135, C, and Y.

BACKGROUND: Outbreaks of meningococcal meningitis (meningitis caused by Neisseria meningitidis) are a major public health concern in the African "meningitis belt," which includes 21 countries from Senegal to Ethiopia. Of the several species that can cause meningitis, N. meningitidis is the most important cause of epidemics in this region. In choosing the appropriate vaccine, accurate N. meningitidis serogroup determination is key. To this end, we developed and evaluated two duplex rapid diagnostic tests (RDTs) for detecting N. meningitidis polysaccharide (PS) antigens of several important serogroups. METHODS AND FINDINGS: Mouse monoclonal IgG antibodies against N. meningitidis PS A, W135/Y, Y, and C were used to develop two immunochromatography duplex RDTs, RDT1 (to detect serogroups A and W135/Y) and RDT2 (to detect serogroups C and Y). Standards for Reporting of Diagnostic Accuracy criteria were used to determine diagnostic accuracy of RDTs on reference strains and cerebrospinal fluid (CSF) samples using culture and PCR, respectively, as reference tests. The cutoffs were 10(5) cfu/ml for reference strains and 1 ng/ml for PS. Sensitivities and specificities were 100% for reference strains, and 93.8%-100% for CSF serogroups A, W135, and Y in CSF. For CSF serogroup A, the positive and negative likelihood ratios (+/- 95% confidence intervals [CIs]) were 31.867 (16.1-63.1) and 0.065 (0.04-0.104), respectively, and the diagnostic odds ratio (+/- 95% CI) was 492.9 (207.2-1,172.5). For CSF serogroups W135 and Y, the positive likelihood ratio was 159.6 (51.7-493.3) Both RDTs were equally reliable at 25 degrees C and 45 degrees C. CONCLUSIONS: These RDTs are important new bedside diagnostic tools for surveillance of meningococcus serogroups A and W135, the two serogroups that are responsible for major epidemics in Africa.