Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

BACKGROUND: Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1-3 days subculture step currently required before any therapeutic adjustments can be made. METHODOLOGY/PRINCIPAL FINDINGS: Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000-7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. CONCLUSIONS/SIGNIFICANCE: Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia

Evaluation of the chagas western blot igg assay for the diagnosis of chagas disease

Chagas disease is a debilitating and often fatal pathology resulting from infection by the protozoan parasite Trypanosoma cruzi. In its recommendations, the World Health Organization states that the diagnosis of T. cruzi infection is usually based on the detection of antibodies against T. cruzi antigens and performed with two methodologically different assays. An inconclusive result can be resolved with a third “confirmatory” assay. The objective of this article is to evaluate the effectiveness of the Chagas Western Blot IgG assay (LDBio Diagnostics, Lyon, France) as a confirmatory serologic test. The Chagas Western Blot IgG assay was performed with native antigens derived from a T. cruzi strain of the TcVI genotype. Retrospective sera were provided by two parasitology laboratories (France and Argentina). The sensitivity, specificity, positive predictive value and negative predictive value of the Chagas blot were all 100% in our sera collection. The Chagas blot is an easy and qualitative method for the diagnosis of Chagas disease, with results in less than 2 h. This immunoblot has potential as a supplemental test for the confirmation of the presence of antibodies against T. cruzi in serum specimens. Nonetheless, the very good initial results presented here will need to be confirmed in larger studies.Fil: Brossas, Jean Yves. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Ballering, Griselda Edith. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Bisio, Margarita María Catalina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Guihenneuc, Jeremy. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Gulin, Julián Ernesto Nicolás. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Jauréguiberry, S.. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Lescure, François Xavier. Hôpitaux Universitaires Paris Nord val de Seine; FranciaFil: Fekkar, Arnaud. Sorbonne Université Hôpital Pitié-Salpêtrière; FranciaFil: Mazier, Dominique. Sorbonne University; FranciaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Paris, Luc. Sorbonne Université Hôpital Pitié-Salpêtrière; Franci

Aspergillus PCR in Bronchoalveolar Lavage Fluid for the Diagnosis and Prognosis of Aspergillosis in Patients With Hematological and Non-hematological Conditions

Objectives: We evaluated the usefulness of an Aspergillus fumigatus quantitative PCR assay performed in bronchoalveolar lavage fluid (BAL) for the diagnosis and prognosis of both invasive and non-invasive aspergillosis.Methods: This 4-year retrospective study involved 613 at-risk patients who had either hematological disorders or other immunosuppressive conditions, notably solid organ transplants. Thirty-five patients had proven/probable aspergillosis and thirteen had chronic non-invasive aspergillosis. We compared PCR, galactomannan index and mycological analysis of BAL.Results: For invasive aspergillosis (IA), PCR performed in BAL yielded 88.6% sensitivity and 95.5% specificity. Comparatively, galactomannan index and mycological examination yielded only 56.3 and 63.6% sensitivity and 97.6 and 94.5% specificity, respectively. Considering the 13 chronic aspergillosis cases, PCR, galactomannan index and mycological examination yielded 76.9, 15.4, and 84.6% sensitivity and 92.2, 94.9, and 93% specificity, respectively. Fungal load in BAL evaluated by PCR was able to discriminate between aspergillosis and contamination, but not between invasive and non-invasive forms. Finally, fungal load was predictive of 90-day mortality, with 23.1% mortality for patients with less than 500 copies/mL versus 68.4% for patients above that cut-off (p < 0.05).Conclusion: Our results indicate that Aspergillus PCR in BAL is of particular interest for both the diagnosis and the prognosis of IA. It is likewise an interesting tool for the diagnosis of non-invasive forms

New 8-nitroquinolinone derivative displaying submicromolar in vitro activities against both Trypanosoma brucei and cruzi

International audienceAn antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 μM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = −0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds

New diagnostic approaches to American Trypanosomiasis

La maladie de Chagas affecte six à huit personnes dans le monde. Depuis quelques décennies, cette pathologie se diffuse hors de sa zone d’endémie, l’Amérique latine. Cette infection présente de grandes difficultés tant pour le diagnostic que pour le traitement. Malgré les avancées importantes de ces dernières années, l'amélioration du diagnostic reste un défi majeur dans la prise en charge des patients atteints de Trypanosomiase Américaine. Dans une première partie, nous présentons nos études sur le développement d'un nouveau test de diagnostic basé sur l'identification directe d'antigènes parasitaires présents dans le sérum. Nous avons analysé le surnageant de culture de cellules infectées par T. cruzi par spectrométrie de masse et nous avons identifié une protéase parasitaire, la Chagasin, qui pourrait être un marqueur sérique de l’infection à T. cruzi. Dans une deuxième partie, nous présentons une étude permettant d’interpréter et d’évaluer un nouveau test diagnostic "Western Chagas IgG" fabriqué, à partir d’extraits protéique de T. cruzi, conjointement par la société LDBIO Diagnostics (Lyon, France) et le laboratoire de parasitologie et de mycologie de l'hôpital Pitié Salpétrière. Les résultats de cette étude montrent que les valeurs de sensibilité et de spécificité de ce test sont proches de 100%. Nous considérons que l'immunoblot "Western Chagas IgG"est un excellent test diagnostic de confirmation de la maladie de Chagas et destiné à la vérification de résultats équivoques obtenus par le biais de tests de dépistage plus classiques.Six to eight people are affected by Chagas disease worldwide. Most of the patients are found in South America; however, more and more countries in the north are confronted with this infection that presents great difficulties both in the diagnosis and in the treatment. Despite significant technological advances in recent years, improved diagnosis is a major challenge in the management of patients with Chagas disease. In a first part, we present our studies about the development of a new diagnostic test based on the direct identification of parasitic antigens present in the serum. We analyzed the culture supernatant of cells infected with T.cruzi by mass spectrometry and we identified soluble parasitic proteins. Subsequently, we showed that Chagasin, a parasitic protease, which could be good serum marker of T. cruzi infection.In second part, we present a study that allow the interpretation and evaluation of a new immunoblot assay "Western Chagas IgG" (CE mark) by LDBIO Diagnostics (Lyon, France). This assay based on trypomastigote and amastigote extract manufactured by the laboratory of parasitology and mycology of Pitié Salpétrière hospital. These results show that this assay has sensitivity and specificity values close to 100% and is considered an excellent serological assay intended for confirmatory testing of a positive or equivocal result obtained through classic screening tests

Performance of Aspergillus PCR in cerebrospinal fluid for the diagnosis of cerebral aspergillosis

International audienceObjectives: cerebral aspergillosis is a rare but often fatal form of invasive aspergillosis that remains difficult to diagnose. The literature has shown the value of Aspergillus PCR in blood-derived samples for the diagnosis of invasive aspergillosis but provides far less information for cerebrospinal fluid (CSF) in cerebral aspergillosis. Here, we evaluated the usefulness of an Aspergillus PCR assay performed on CSF for the diagnosis of cerebral aspergillosis.Methods: this retrospective study involved 72 patients with suspected cerebral aspergillosis for a total of 88 CSF samples in whom CSF Aspergillus PCR was performed.Results: seventeen patients had proven/probable invasive aspergillosis according to the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, including twelve cases of proven/probable cerebral aspergillosis. AspergillusPCR in CSF was positive in nine of the twelve patients with cerebral aspergillosis, i.e. 75% sensitivity. In contrast, CSF culture was positive for Aspergillus in only two patients. In the non cerebral aspergillosis group (60 patients), PCR was positive in one patient, i.e., 98.3% specificity. In this particular population of high-risk patients with suspicion of cerebral aspergillosis, the disease incidence was 16.7%. Therefore, the positive and negative predictive values of PCR were 90% and 95.2% respectively.Conclusion: the results of this study indicate that Aspergillus PCR in CSF is an interesting tool that may eliminate the need for cerebral biopsy in patients with suspected cerebral aspergillosis

Sustained versus transient ERK1/2 signaling underlies the anti- and proapoptotic effects of oxidative stress

PURPOSE. Oxidative stress is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which involves retinal pigmented epithelial (RPE) cell death. However, signaling pathways involved in the oxidative-stress-induced RPE cell death are poorly understood. This study was conducted to investigate the involvement of the MAP kinase pathways during the induction of RPE cell death by oxidative stress. METHODS. ARPE-19 cells were exposed to the oxidant tert-butyl hydroperoxide (t-BHP). Cell viability was assessed by cell counting and MTT-staining, and apoptosis was quantified by TUNEL and flow cytometry. Activation of JNK1/3, p38 ␣␤ MAPKs and ERK1/2 and their potential targets was detected by Western blot analysis and immunochemistry with specific antiphospho protein antibodies. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in cell death of RPE cells exposed to t-BHP. RESULTS. Exposure of RPE cells to t-BHP, associated with increase in reactive oxygen species and intracellular glutathione depletion, induced time-and concentration-dependent apoptosis, which was associated with the accumulation of inactive ERK1/2 in cell nuclei and a transient and weak ERK1/2 activation. This activation was accompanied by a deactivation of P90 RSK , the major target of ERK1/2 and consequently by the delayed activation of its transcription factor CREB. MEK1/2 inhibition completely suppressed the transient activation of ERK1/2 and completely blocked apoptosis, demonstrating the role of the MEK-ERK module in mediating oxidative-stressinduced RPE cell death. In contrast, neither JNKs nor p38 ␣␤ MAPKs were involved in mediating t-BHP-induced apoptotic signaling in RPE cells. CONCLUSIONS. The results suggest that inhibiting the MEK-ERK module may allow the development of selective methods for treating oxidative-stress-induced RPE degeneration, such as AMD. (Invest Ophthalmol Vis Sci. 2006;47:4614 -4623

Prediction of malaria transmission drivers in Anopheles mosquitoes using artificial intelligence coupled to MALDI-TOF mass spectrometry

International audienc