4-Dimethylaminoantipyrine as a Broad Electrochemical Indicator for Immunosensors Platform.

Here, we describe 4-dimethylaminoantipyrine (4-DMAA)-mediated interfacing as a broad biochemical indicator to stabilize and promote the higher response of electrodes for immunological detection. We hypothesized that the improved biological interactions of 4-DMAA with electrodes and biological samples may be due to the interaction properties of the benzene and pyrazole chemical groups with graphite and proteins, respectively. In order to demonstrate that 4-DMAA could be used as a general indicator in electrochemical immunoassays, we used peptides as probes for the diagnosis of four neglected tropical infectious diseases Tegumentary leishmaniasis, Visceral leishmaniasis, Strongyloidiasis, and Leprosy on commercial graphite screen-printed electrodes. 4-DMAA oxidation was used to indicate specific biological recognition between the epitope-based peptide and serum immunoglobulin G (IgG) from infected patients. We demonstrated that 4-DMAA should be incorporated into the electrodes prior to serum application, which avoids interference with its sensitivity and specificity. In addition, 4-DMAA oxidizes at a low anodic potential, and the oxidation peak is useful for detecting proteins in biological fluids. In summary, we have successfully demonstrated the broad application of 4-DMAA as a general indicator for the specific diagnosis of four infectious diseases in electrochemical immunosensors. Such a strategy is quite advantageous for indirect detection of proteins that lack electrochemical activities or are spatially inaccessible on the electrode surface. This new indicator opens a new avenue for monitoring biological recognition, especially for immunosensors.

Primary neural leprosy: clinical, neurophysiological and pathological presentation and progression.

Disability in leprosy is a direct consequence of damage to the peripheral nervous system which is usually worse in patients with no skin manifestations, an underdiagnosed subtype of leprosy known as primary neural leprosy. We evaluated clinical, neurophysiological and laboratory findings of 164 patients with definite and probable primary neural leprosy diagnoses. To better understand the disease progression and to improve primary neural leprosy clinical recognition we compared the characteristics of patients with short (≤12 months) and long (>12 months) disease duration. Positive and negative symptoms mediated by small-fibres were frequent at presentation (∼95%), and symptoms tend to manifest first in the upper limbs (∼68%). There is a consistent phenotypic variability between the aforementioned groups. Deep sensory modalities were spared in patients evaluated within the first 12 months of the disease, and were only affected in patients with longer disease duration (∼12%). Deep tendon reflexes abnormalities were most frequent in patients with longer disease duration (P < 0.001), as well as motor deficits (P = 0.002). Damage to large fibres (sensory and motor) is a latter event in primary neural leprosy. Grade-2 disability and nerve thickening was also more frequent in cases with long disease duration (P < 0.001). Primary neural leprosy progresses over time and there is a marked difference in clinical phenotype between patients with short and long disease duration. Patients assessed within the first 12 months of symptom onset had a non-length-dependent predominant small-fibre sensory neuropathy, whilst patients with chronic disease presented an asymmetrical all diameter sensory-motor neuropathy and patchily decreased/absent deep tendon reflexes.

Identification of a systemic interferon-γ inducible antimicrobial gene signature in leprosy patients undergoing reversal reaction.

Reversal reactions (RRs) in leprosy are characterized by a reduction in the number of bacilli in lesions associated with an increase in cell-mediated immunity against the intracellular bacterium Mycobacterium leprae, the causative pathogen of leprosy. To identify the mechanisms that contribute to cell-mediated immunity in leprosy, we measured changes in the whole blood-derived transcriptome of patients with leprosy before, during and after RR. We identified an 'RR signature' of 1017 genes that were upregulated at the time of the clinical diagnosis of RR. Using weighted gene correlated network analysis (WGCNA), we detected a module of 794 genes, bisque4, that was significantly correlated with RR, of which 434 genes were part of the RR signature. An enrichment for both IFN-γ and IFN-ß downstream gene pathways was present in the RR signature as well as the RR upregulated genes in the bisque4 module, including those encoding proteins of the guanylate binding protein (GBP) family that contributes to antimicrobial responses against mycobacteria. Specifically, GBP1, GBP2, GBP3 and GBP5 mRNAs were upregulated in the RR peripheral blood transcriptome, with GBP1, GBP2 and GBP5 mRNAs also upregulated in the RR disease lesion transcriptome. These data indicate that RRs involve a systemic upregulation of IFN-γ downstream genes including GBP family members as part of the host antimicrobial response against mycobacteria.

Diagnosis of mycobacterial infections based on acid-fast bacilli test and bacterial growth time and implications on treatment and disease outcome.

BACKGROUND: The establishment of therapeutic regimens for mycobacteriosis depends on the accurate identification of Mycobacterium species, and misdiagnosis can result in inappropriate treatment and increased mortality of patients. Differential diagnosis among Mycobacterium species has been made by conventional phenotypic and biochemical tests after a long culture period. Specialized molecular diagnostics of mycobacteria allows rapid detection and species identification; however, such tests are not available in public health programs. Our aim was to demonstrate the clinical implications of erroneous diagnosis by performing molecular genotyping of mycobacterial infections in patients that were diagnosed based on symptoms, culture and bacilloscopy. METHODS: Culture samples of mycobacterial infections from 55 patients clinically diagnosed as tuberculosis in 2013 and 2014, based on conventional methods, were identified by PCR -RFLP and results are discussed. RESULTS: We have confirmed 35 (63.6%) positive samples as M. tuberculosis, but 18 (32.7%) were identified as non-tuberculous mycobacteria (M. avium type 1, M. avium type 2, M. kansasii type 1 type 1, M. mucogenicum, M. chelonae, M. terrae type 3, and 1 unknown RFLP pattern) and two were negative. Regarding clinical diagnosis, 61.8% (34/55) was classified as pulmonary tuberculosis. It is important to emphasize that 36.4% (20/55) of samples were misdiagnosed by conventional methods, and 11 (61.1%) of the HIV positive patients (18/55) were NTM-coinfected. CONCLUSION: The identification of species in mycobacterial infections is essential for correct diagnosis and choice of treatment regimen, and misdiagnosis by conventional tools can lead to chronic disease, increased resistance and death.

Oral lesion in leprosy: borderline tuberculoid diagnosis based on detection of Mycobacterium leprae DNA by qPCR.

Oral lesions are rarely reported in paucibacillary forms of leprosy. We report here a case with an erythematous hyposensitive lesion in the palate and no skin lesions. In addition to routine tests, biopsies of the lesion in the palate and of clinically normal surrounding areas were performed and subjected to real-time PCR for detection of Mycobacterium leprae DNA. The biopsy of the oral lesion was positive for bacilli DNA, followed by positive serum anti-PGL-1 and Mitsuda test, but with negative histopathology. The patient was diagnosed with a borderline tuberculoid form. After multidrug therapy the lesion had significantly regressed and the bacilli DNA detection in the former lesion was negative. The bacilli DNA detection in an oral lesion by real-time PCR not only improved leprosy diagnosis, but also helped in the classification of clinical form, and in the establishment of the appropriate therapeutic regime.

Biomarkers for serum diagnosis of infectious diseases and their potential application in novel sensor platforms.

Nanotechnological tools and biomarkers for diagnosis and prognosis, as well as strategies for disease control and monitoring populations at higher risk, are continuous worldwide challenges for infectious diseases. Phage display and monoclonal antibody combinatorial libraries are important sources for biomarker discovery and for improved diagnostic strategies. Mimetic peptides were selected against polyclonal antibodies from patients with dengue fever, leprosy, and leishmaniasis as model diseases, and from immunized chickens with total antigens from all three pathogens. Selected single or combined multi-epitope peptide biomarkers were further associated with four different sensor platforms, classified as affinity biosensors, that may be suitable as general protocols for field diagnosis. We have also developed two methods for nanoparticle agglutination assays (a particle gel agglutination test and a magnetic microparticle [MMP]-enzyme-linked immunosorbent assay [ELISA]) and two electrochemical biosensors (impedimetric and amperometric) for DNA and antibody detection. For the agglutination tests, micro- and nanoparticles were coupled with filamentous bacteriophages displaying the selected mimotopes on their surfaces, which has favored the formation of the antigen-antibody or peptide-protein complexes, amplifying the optical detection in ELISA assays or after the chromatographic separation of the microagglutinates. We have also demonstrated a proof-of-concept for the electrochemical biosensors by using electrodes modified with novel functionalized polymers. These electrochemical biosensors have proven to be fast, very sensitive, and specific for the detection of pathogen DNA and circulating antibodies of patients, which may become important in a wide range of diagnostic devices for many infectious agents.

Risk and protective factors for leprosy development determined by epidemiological surveillance of household contacts.

Household contacts of leprosy patients are the group with the highest risk of developing the disease, and although many risk or prevention factors have been identified, they have not been employed in leprosy-monitoring programs. This investigation aimed to establish the relative risks or the preventive effects of the presence of BCG vaccination, the Mitsuda test, and the ML-Flow assay. Household contacts (1,396) were monitored for a 5-year period. Twenty-eight contacts (2%) developed leprosy and had their clinical and operational classifications established. All immunological tests were performed, and intradermal BCG vaccination was given after the BCG scar count. Of the affected contacts, 75% developed the disease in the first year, and 71.4% were classified as having paucibacillary forms. Contacts of lepromatous leprosy patients presented a 3.8-fold-higher risk of developing leprosy. BCG vaccination and the Mitsuda test showed a protective effect against leprosy of 0.27 (at least one scar) and 0.16 (>7 mm), respectively, and the positive ML-Flow test indicated a relative risk approximately sixfold higher for occurrence of the disease. All unfavorable combinations of two and three assays generated significant risk values that ranged from 5.76 to 24.47, with the highest risk given by the combination of no BCG scar, negative Mitsuda test, and positive ML-Flow test. We suggest that the BCG vaccination may be given to stimulate Mitsuda test positivity, reducing the patient's risk of developing multibacillary forms. The high significance of these tests may have a great impact on programs to monitor contacts and should be used to improve early detection and treatment.