Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Evaluation of the Histoplasma capsulatum 100-kilodalton antigen dot blot for the rapid diagnosis of progressive histoplasmosis in HIV/AIDS patients.

Among people living with HIV (PLHIV), progressive disseminated histoplasmosis (PDH) represents an important cause of mortality. Since antigen detection allows a rapid diagnosis and the instauration of a specific treatment this study aimed to evaluate the analytical performance of the Hcp100 dot blot, an in-house assay that detects the Histoplasma capsulatum 100-kilodalton antigen in urine and compare it with 2 commercially available assays the Histoplasma Urine Antigen Lateral Flow Assay (MVD-LFA) (MiraVista® Diagnostics) and the Clarus Histoplasma Galactomannan EIA (Clarus HGM) (IMMY). Urine specimens from 23 PLHIV with PDH, 13 patients with other infectious diseases, and 20 healthy individuals were tested. The Hcp100 dot blot showed higher sensitivity (87.0%), specificity (97.0%) and accuracy (92.9%) than the MVD-LFA (73.9%, 78.8%, and 76.8%, respectively) and the Clarus HGM (78.3%, 90.9%, and 85.7%, respectively). The Hcp100 dot blot had high analytical performance and would be a valuable screening tool for diagnosing PDH among PLHIV.

Mediastinal histoplasmosis with cardiac involvement in a cat.

Histoplasmosis is the second most common fungal infection reported among domestic felines in the United States. Dissemination of the organism after inoculation is common and affected organ systems include the respiratory tract, gastrointestinal tract, reticuloendothelial organs, skeletal system, integument, and ocular system. However, histoplasmosis presenting as a discrete granulomatous mass identified on echocardiogram has never been reported in the veterinary literature. Here, we describe the first case of feline histoplasmosis presenting as a granuloma with cardiac involvement. The patient, a 6-year-old male neutered domestic longhair feline, was referred for tachypnea and dyspnea. A mass in the cranial mediastinum abutting the heart was diagnosed via two-dimensional echocardiography. Cytology of fine needle aspirates from the mass revealed round yeast structures consistent with Histoplasma spp. The patient was treated with oral fluconazole therapy, and subsequent rechecks have shown marked improvement in clinical parameters, lesion size, and antigen concentrations.

Urine-Based Antigen (Protein) Detection Test for the Diagnosis of Visceral Leishmaniasis.

This review describes and appraises a novel protein-based antigen detection test for visceral leishmaniasis (VL). The test detects in patient's urine six proteins from Leishmania infantum (chagasi) and Leishmania donovani, the etiological agents of VL. The gold standard test for VL is microscopic observation of the parasites in aspirates from spleen, liver, or bone marrow (and lymph node for dogs). Culture of the parasites or detection of their DNA in these aspirates are also commonly used. Serological tests are available but they cannot distinguish patients with active VL from either healthy subjects exposed to the parasites or from subjects who had a successful VL treatment. An antigen detection test based on the agglutination of anti-leishmania carbohydrates antibody coated latex beads has been described. However, the results obtained with this carbohydrate-based test have been conflicting. Using mass spectrometry, we discovered six L. infantum/L. donovani proteins excreted in the urine of VL patients and used them as markers for the development of a robust mAb-based antigen (protein) detection test. The test is assembled in a multiplexed format to simultaneously detect all six markers. Its initial clinical validation showed a sensitivity of 93% and specificity of 100% for VL diagnosis.

Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis.

Identification of pathogen-specific biomarkers present in patients' serum or urine samples can be a useful diagnostic approach. In efforts to discover Mycobacterium tuberculosis (Mtb) biomarkers we identified by mass spectroscopy a unique 21-mer Mtb peptide sequence (VVLGLTVPGGVELLPGVALPR) present in the urines of TB patients from Zimbabwe. This peptide has 100% sequence homology with the protein TBCG_03312 from the C strain of Mtb (a clinical isolate identified in New York, NY, USA) and 95% sequence homology with Mtb oxidoreductase (MRGA423_21210) from the clinical isolate MTB423 (identified in Kerala, India). Alignment of the genes coding for these proteins show an insertion point mutation relative to Rv3368c of the reference H37Rv strain, which generated a unique C-terminus with no sequence homology with any other described protein. Phylogenetic analysis utilizing public sequence data shows that the insertion mutation is apparently a rare event. However, sera from TB patients from distinct geographical areas of the world (Peru, Vietnam, and South Africa) contain antibodies that recognize a purified recombinant C-terminus of the protein, thus suggesting a wider distribution of isolates that produce this protein.

Community acquired pneumonia by Legionella pneumophila: Study of 136 cases. / Neumonía comunitaria por Legionella pneumophila: estudio de 136 casos.

OBJECTIVE: Most of the data on Legionella pneumonia in our country come from the Mediterranean area, and there are few studies from the Northwest area. This study tries to assess the situation of this infection in this area. METHOD: Retrospective study of all patients with positive Legionella antigenuria treated at the University Hospital Lucus Augusti in Lugo (Spain) from 2001, the year in which this test was introduced in our centre, until 2015. We analysed epidemiological data, risk factors, clinical, radiological and biochemical findings, and clinical outcome. RESULTS: The sampled included 136 patients. When comparing the first five years of the study with the last five, the incidence increased from 10.9 to 64.5 cases/1,000,000; the number of antigenuria requests increased 3.4 times, and compared to other pneumonia aetiologies Legionella increased from 0.9% to 15%. The mean age was 64.1years and 84.6% were males; 74.3% had comorbidities. Males were significantly younger (62.7±16.6 vs 71.9±17.3) and consumed more alcohol (26.1% vs 0%) and tobacco (67.8% vs 14.3%). Diagnosis was established within the first 72hours in 88.9% of cases and most received levofloxacin (95.6%). Hospitalisation was needed in 85% of cases, 11.7% in ICU and 4.4% died. CONCLUSIONS: After the introduction of antigenuria there was an increase in the incidence of Legionella pneumonia recorded in our health area. Its rate in recent years has been one of the highest in our country. Despite the fact that the patients had advanced age and comorbidities, mortality was low.