Diagnostic Peptide Discovery: Prioritization of Pathogen Diagnostic Markers Using Multiple Features

The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete “peptidomes”. Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked 10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes.     

Rapid Diagnostic Tests for Dengue Virus Infection in Febrile Cambodian Children: Diagnostic Accuracy and Incorporation into Diagnostic Algorithms

BackgroundDengue virus (DENV) infection is prevalent across tropical regions and may cause severe disease. Early diagnosis may improve supportive care. We prospectively assessed the Standard Diagnostics (Korea) BIOLINE Dengue Duo DENV rapid diagnostic test (RDT) to NS1 antigen and anti-DENV IgM (NS1 and IgM) in children in Cambodia, with the aim of improving the diagnosis of DENV infection.ConclusionsThe DENV RDT had low sensitivity for the diagnosis of DENV infection. The high co-prevalence of infections in our cohort indicates the need for a broad microbiological assessment of non-localised febrile illness in these children.     

Clinical Diagnostic Process: An Analysis

An analysis of observations made during 1,307 diagnoses by a total of 28 clinicians (503 diagnoses in real life, and 804 on simulated patients) concerned primarily the interview of patients suffering from abdominal pain. Interviews ranged from 10 to 35 questions, and from “stereotyped” procedures, in which identical (and often irrelevant) questions were asked to each patient, to “adaptive” interviews, in which specific relevant questions were put to each patient. Senior clinicians tended to ask fewer, more relevant questions than their junior counterparts; and urgent cases were dealt with in a more adaptive fashion than routine cases in outpatients. Disappointingly, there was considerable difference between real-life and simulated situations. From these results it is suggested (a) that the “diagnostic process” does not exist, (b) that any automated diagnostic system must be flexible to accommodate the wishes of a variety of clinicians, and (c) that studies based on artificial clinical situations should be treated with extreme caution.     

Sporotrichosis: Update on Diagnostic Techniques

Purpose of Review

Sporotrichosis, the disease caused by Sporothrix spp, ranges from subcutaneous infections to the severe disseminated or invasive diseases. The taxonomy of Sporothrix has been revised. The subcutaneous disease is suspected easily, but the extra-cutaneous disease is diagnosed by chance or with high suspicion. This review provides the overview of currently available diagnostic techniques.

Recent Finding

Enzyme-linked immunosorbent assay (ELISA) or latex agglutination test with partially purified antigens helps in the diagnosis of extra-cutaneous sporotrichosis. Molecular methods have been used for the identification and typing of the fungus. Calmodulin, beta tubulin, translation elongation factor and chitin synthase genes are targeted for species differentiation. MALDI-TOF MS has been standardized to identify the species.

Summary

PCR-based molecular techniques and matrix-assisted laser desorption ionisation time of flight mass spectroscopy (MALDI-TOF MS) help in the identification of Sporothrix species, whereas ELISA helps in diagnosing extra-cutaneous form. Utility of molecular techniques for detection of Sporothrix directly from clinical specimen needs to be evaluated.

     

Diagnostic cystocentesis: technique and considerations

Cystocentesis, the aspiration of urine from the urinary bladder, has both diagnostic and therapeutic uses. This month, we discuss the diagnostic reasons for using this technique and summarize the steps necessary to safely perform the procedure in laboratory animals.     

Perinatal HIV infection: Diagnostic issues

HIV diagnosis for the majority of at-risk individuals—children, adults, and pregnant women—can readily be accomplished by the standard approach of EIA and Western blot. Children under 18 to 24 months of age pose a special diagnostic problem due to the presence of passively acquired, maternal antibodies to HIV. It is becoming imperative that definitive diagnosis of infants be accomplished as early in life as possible because of current or anticipated therapeutic options and prophylactic measures. Diagnostic approaches other than EIA/Western blot need to be considered and/or developed for the young infant at risk for infection.The two most promising assays are PCR and viral culture. To date, very few newborns and young infants have been evaluated by these assays as evidenced by the published scientific literature. Several perinatal collaborative studies, both in the U.S. and abroad, hope to fill this knowledge gap in the near future. Almost all HIV-infected infants can be detected now by 3 months of age. It seems reasonable to expect that up to 25% of newborns will have undetectable infection utilizing any foreseeable technique. This subgroup apparently has very low levels of circulating virus, implying that HIV may reside in fixed tissue or privileged sites, such as the central nervous system. Although we have come a long way from what was state-of-the-art practice just a few years ago, it is clear that further research is warranted concerning the pathogenesis and diagnosis of HIV in the perinatal period.     

Diagnostic testing revisited: pathways through uncertainty

To aid physicians who may be having difficulty applying the principles of decision analysis to diagnostic data according to the methods published in the past several years, the authors of this paper set out a few principles and schemes for using and interpreting diagnostic data obtained from dichotomous tests. They also present a simple BASIC program for calculating post-test probabilities from likelihood ratios and pretest probabilities that a particular disease is present in a particular patient; the program can be adapted for use on microcomputers.     

Editorial: Diagnostic Pathology continues to rise

With the second anniversary of Diagnostic Pathology approaching, this Editorial looks at the journal's achievements so far and outlines the prospects and challenges.