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• W1532048386 abstract "Objective To describe and validate fluorescence in situ hybridization (FISH), a new method of Leishmania spp. identification. FISH allows for a rapid detection of target organisms by specific binding of fluorescently labelled oligonucleotide probes to ribosomal RNA. Methods Two genus-specific, fluorescently labelled Leishmania spp. FISH probes were designed and evaluated with a panel of 18 Leishmania spp. and six Trypanosoma spp. including well-defined strains and clinical isolates. In addition, the FISH probes were tested in comparison with Giemsa staining in formalin-fixed, paraffin-embedded tissues of five mice that had been artificially infected with Leishmania major strains, leading to concordant results. Finally, 11 tissue samples of patients with cutaneous leishmaniasis, four tissue samples of patients with visceral leishmaniasis, and one native bone marrow sample of a patient with visceral leishmaniasis were analysed with FISH and Giemsa staining. Results Concordant results were achieved by FISH and Giemsa staining in 15/16 specimens. Conclusion This analysis provides proof of principle that FISH is a suitable method for the rapid and easy detection of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples. Because of the good contrast of Leishmania spp. in tissue, FISH facilitates the identification of these organisms in tissue samples even by less experienced investigators. Objectif: Décrire et valider l’hybridation fluorescente in situ (FISH), une nouvelle méthode d’identification de Leishmania spp. La méthode FISH permet une détection rapide d’organismes cibles en liant spécifiquement des sondes oligonucléotidiques marquées par fluorescence à l’ARN ribosomal. Méthodes: Deux sondes FISH spécifiques du genre Leishmania spp, marquées par fluorescence ont été conçues et évaluées avec une série de 18 souches bien définies de Leishmania spp. et 6 de Trypanosoma spp. y compris des isolats cliniques. De plus, les sondes FISH ont été testées en comparaison avec la coloration de Giemsa dans des tissus fixés au formol et enrobés dans de la paraffine, provenant de 5 souris qui avaient été artificiellement infectées par des souches de Leishmania major, conduisant à des résultats concordants. Enfin, 11 échantillons de tissus de patients atteints de leishmaniose cutanée, 4 échantillons de tissus de patients atteints de leishmaniose viscérale et un échantillon natif de moelle osseuse d’un patient atteint la leishmaniose viscérale ont été analysés par FISH et la coloration de Giemsa. Résultats: Des résultats concordants ont été obtenus avec FISH et la coloration de Giemsa pour 15/16 spécimens. Conclusion: Cette analyse fournit la preuve de principe que l’hybridation fluorescente in situ est une méthode appropriée pour la détection rapide et facile de Leishmania spp. dans des échantillons de tissus fixés au formol et enrobés dans de la paraffine. En raison de l’excellent contraste de Leishmania spp. dans les tissus, FISH facilite l’identification de ces organismes dans des échantillons de tissus, même par des manipulateurs peu expérimentés. Objetivo: Describir y validar la hibridación fluorescente in situ (FISH), un nuevo método para la identificación de Leishmania spp. El FISH permite una detección rápida de los organismos objetivo mediante la anidación específica de sondas de oligonucleótidos marcadas con fluorescencia al ADN ribosomal. Métodos: Se diseñaron dos sondas FISH para Leishmania spp, específicas para el género, y marcadas con fluorescencia. Las sondas FISH fueron diseñadas y evaluadas con un panel de 18 Leishmania spp. y de 6 Trypanosoma spp., incluyendo cepas bien definidas y aislados clínicos. Adicionalmente, las sondas FISH se compararon con la tinción de Giemsa de tejidos fijados en formol e incluidos en parafina de 5 ratones artificialmente infectados con cepas de Leishmania major. Se obtuvieron resultados concordantes. Finalmente, utilizando FISH y tinción con Giemsa, se analizaron 11 muestras de tejido de pacientes con leishmaniasis cutánea, 4 muestras de tejido de pacientes con leishmaniasis visceral y una muestra de médula ósea nativa de un paciente con leishmaniasis visceral. Resultados: Se obtuvieron resultados concordantes utilizando el FISH y la tinción con Giemsa en 15/16 muestras. Conclusión: Este análisis provee una demostración preliminar de que la hibridación fluorescente in situ es un método adecuado para la detección rápida y fácil de Leishmania spp. en muestras de tejido fijadas con formol e incluidas en parafina. Debido al buen contraste de Leishmania spp. en el tejido, el FISH facilita la identificación de estos organismos en muestras de tejido, incluso para los investigadores menos experimentados. Leishmania are persistent, intracellular, protozoic parasites with worldwide distribution in subtropical and tropical regions. They are transmitted by Phlebotomus or Lutzomyia mosquitoes: 20-μm-sized flagellated promastigote forms are taken up by macrophages. After phagocytosis, they persist as 2-μm-sized aflagellate amastigote forms and cause the clinical variants of visceral, cutaneous, or mucocutaneous leishmaniasis depending on the infecting Leishmania species (Grimaldi & Tesh 1993; Reithinger et al. 2007; Mondal et al. 2010). Leishmania comprises the subgenera Leishmania, Viannia, and Sauroleishmania. Species complexes of known pathogenic potential to humans are Leishmania donovani complex, Leishmania major complex, Leishmania tropica complex, Leishmania mexicana complex, Leishmania guyanensis complex, and Leishmania braziliensis complex. Phylogenetic approaches are based on multilocus enzyme electrophoresis or sequence typing with varying numbers of validated species depending on the chosen method (Fraga et al. 2009; Schönian et al. 2010). Symptoms of leishmaniasis are non-specific. Visceral leishmaniasis, caused, for example, by L. donovani and Leishmania infantum, is associated with fever, hepatosplenomegaly, anaemia, leukopenia, or thrombopenia (Mondal et al. 2010). These symptoms are equally common for many other infectious or malign diseases. Fatal courses of visceral leishmaniasis may occur because of disturbed coagulation or secondary infections in immunocompromised patients, despite therapy. Cutaneous leishmaniasis, caused for example by L. major or L. tropica complex, is associated with prominent ulceration but is rarely fatal. Infected macrophages are intracutaneously or subcutaneously located. The lesions can be confused with cutaneous mycobacterial lesions, cutaneous infections with tropical fungi, or other dermatologic or oncologic differential diagnoses (Reithinger et al. 2007). In cases of mucocutaneous leishmaniasis, caused, for example, by the New World Leishmania species L. braziliensis, ulceration involves mucous membranes. Because of the non-specific symptoms, a reliable and quick detection of Leishmania spp. is crucial for early and targeted therapeutic intervention. Diagnostic specimens comprise skin snips if cutaneous leishmaniasis is suspected, blood and bone marrow in case of suspected visceral leishmaniasis, or bioptic materials from localized manifestations of both cutaneous and visceral diseases, for example, from skin, lymphatic nodes, and even spleen or liver. Primary screening for Leishmania spp. is usually based on traditional microscopy. In that method, histopathological examination of skin biopsies provides better sensitivity than skin smears (Gazozai et al. 2010) in cases of suspected cutaneous leishmaniasis. Traditional staining methods comprise cell and tissue staining according to Giemsa and differential staining of DNA and RNA with acridine orange (Mbati et al. 1995; Arechavala et al. 2010; Gazozai et al. 2010; Meymandi et al. 2010). Microscopic analysis requires experience and skill to reliably discriminate parasites from tissue artefacts or staining artefacts. Therefore, preliminary microscopic analysis by Giemsa staining is confirmed by PCR in some laboratories. Positive PCR results may be achieved from Giemsa-stained slides (Pandey et al. 2010) even up to several decades after the sample was taken (Volpini et al. 2006). Nevertheless, the reliability of histological examination is reduced if pathologists have limited experience with leishmaniasis because of its rare occurrence, for example, in European or US laboratories. In the authors’ experience at the German National Reference Center for Tropical Diseases, new leishmaniasis cases in travellers returning from endemic countries are often diagnosed with delays of several weeks or even months. As an advance in microscopic analysis, in situ hybridization (ISH), a histochemical staining procedure based on the hybridization of predominantly kinetoplast DNA probes (Kennedy 1984; Barker 1987, 1989; van Eys et al. 1987; Schoone et al. 1991), has been used for decades for the differentiation of Leishmania spp. beyond the genus level. More recently, an ISH procedure based on the specific binding of a short labelled DNA-oligonucleotide probe to 5.8S RNA has been shown to allow the identification of Leishmania spp. without further species differentiation even in difficult materials such as paraffin-embedded tissue samples (Dinhopl et al. 2011). Nevertheless, ISH is a laborious and time-consuming procedure, and particularly, the ISH-based species identification has widely been replaced by amplification- or antigen-based diagnostic procedures in many laboratories. Sophisticated amplification-based diagnostic procedures have been described including DNA hybridization, real-time PCR with and without quantification, high-resolution melt analysis, sequencing, DNA microarrays and antigen-based procedures including protein microarrays (Rodríguez et al. 1994; Nicolas et al. 2002; Tavares et al. 2003; Foulet et al. 2007; Castilho et al. 2008; van der Meide et al. 2008; Antinori et al. 2009; Meymandi et al. 2010; Talmi-Frank et al. 2010; Roelfsema et al. 2011). Modern typing methods such as multilocus enzyme typing (MLEE), multilocus sequence typing (MLST), and multilocus microsatellite typing (MLMT) are used for discrimination beyond the species level (Schönian et al. 2011). None of these more or less complex diagnostic procedures has replaced microscopy as the method of choice for primary screening for Leishmania spp. in clinical samples. An easy-to-perform, rapid and reliable microscopic identification of Leishmania spp. in tissue is desirable to avoid incorrect diagnoses and to allow the start of a targeted, appropriate therapy. Fluorescence in situ hybridization (FISH) is a rapid diagnostic staining procedure based on the specific binding of fluorescently labelled probes to ribosomal target RNA. It has been broadly evaluated for the identification of various microorganisms in clinical samples by fluorescence microscopy (Hogardt et al. 2000; Kempf et al. 2000; Poppert et al. 2002; Poppert et al. 2005, 2010a,b; Wellinghausen et al. 2005). FISH-based differentiation of pathogens is even possible from more difficult specimens such as blood culture materials (Hogardt et al. 2000; Kempf et al. 2000; Poppert et al. 2010a,b) or formalin-fixed, paraffin-embedded tissue samples (Rüssmann et al. 2003; Yilmaz et al. 2007; Hagen et al. 2011). We developed and evaluated a rapid FISH-based microscopic screening procedure for Leishmania spp. in tissue samples under standard conditions (Hogardt et al. 2000; Kempf et al. 2000) as a useful tool for endemic areas and travel medicine clinics. It is based upon newly designed probes to optimize screening and to facilitate diagnosis. A panel of 18 Leishmania spp. strains, two Trypanosoma cruzei strains and a mixture of Trypanosoma brucei gambiense and rhodesiense from the strain collection and obtained from routine diagnostic procedures of the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany (Table 2) were grown in culture as positive and negative controls for the study. Leishmania spp. were grown in M199 medium (Sigma-Aldrich, Munich, Germany) in cell culture flasks at 25 °C. Growth was monitored by cell density analysis using a Schärfe System Casy Counter (Schärfe System Limited, Reutlingen, Germany). Trypanosoma spp. were grown in HMI-9 medium (Hirumi & Hirumi 1989) at 37 °C in a 5% CO2 atmosphere. Growth was assessed daily by microscopy (100× magnification). Two new probes for the detection of Leishmania spp. 5′-labelled with the red sulfoindocyanine dye Cy3 and bound to the 18S ribosomal subunit (Leish_18S_651: 5′-Cy3-GGC-GCC-ACA-CAC-CGA-ACC-3′ and Leish_18S_840: 5′-Cy3-AAA-GCG-GGC-GCG-GTG-CTG-3′) (Thermo Fisher Scientific, Ulm, Germany; Eurogentec Deutschland GmbH, Köln, Germany; biomers.net limited, Ulm, Germany; Eurofins MWG Operon, Ebersberg, Germany) were designed using the ARB software (http://www.arb-home.de) (Ludwig et al. 2004; Kumar et al. 2006) with sequence data extracted from the NCBI Nucleotide database. We used the new probes in concert with the green-fluorescing carboxyfluorescein-(FAM-) labelled probe staining virtually all eucaryotic cells EUK502 (5′-FAM-ACC-AGA-CTT-GCC-CTC-C-3′) (Amann et al. 1995) and a newly designed FAM-labelled derivate of this probe EUK_Leish499 (5′-FAM-GGC-ACC-AGA-CTT-GTC-CTC-C-3′) that was especially adapted to base-changes of Leishmania spp. and Trypanosoma spp. by a C–T exchange and three additional bases in the 5′-direction. In silico testing of all three developed FISH probes was based upon the software probeCheck (http://www.microbial-ecology.net/probecheck/) (Loy et al. 2008) using the sequence collection SILVA. Fluorescence in situ hybridization from cultured parasites was performed as described for bacteria (Hogardt et al. 2000; Kempf et al. 2000; Wellinghausen et al. 2005) in a multicentric approach at the Institute for Microbiology, Virology and Hygiene of the University Hospital Ulm, Germany and the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany. In short, 8-well Diagnostica slides (Menzel-Gläser, Braunschweig, Germany) with smears of cultured Leishmania spp. or Trypanosoma spp. were air-dried and fixed in 100% methanol. Hybridization was performed at 46 °C for 1 h in a moist chamber in an incubator. Afterwards, the slides were rinsed with preheated washing buffer and incubated for an additional 15 min at 46 °C in washing buffer. Subsequently, they were covered with the mounting medium ‘Vectashield with DAPI’ (Vector Laboratories, Burlingame, CA, USA) containing the non-intercalating DNA stain 4′,6-diamidino-2-phenylindole (DAPI). Prior to FISH from formalin-fixed, paraffin-embedded tissue slices, the specimens were deparaffinized as described for tissue samples containing Burkholderia pseudomallei (Hagen et al. 2011). In short, slides were washed in 100% Rotihistol® (Roth, Karlsruhe, Germany) twice for 10 min each and afterwards in 100% ethanol and 75% ethanol for 10 min each. Finally, fixation was performed in 100% methanol. The FISH-stained slides were analysed using an upright Leica DM5000B fluorescence microscope (Leica, Wetzlar, Germany) equipped with a Leica DFC 360 FX camera at 630× magnification. Images were acquired and processed using the Openlab 5.1 software (Improvision, Coventry, UK). Fluorescence intensity was subjectively evaluated by experienced investigators without automated measurement. All new probes were designed to allow hybridization at a formamide concentration of 30% in the hybridization buffer because most published DNA probes hybridize at this concentration (Hogardt et al. 2000; Kempf et al. 2000). Optimal hybridization conditions were identified in a direct comparison with different formamide concentrations from 10% to 50% as previously described (Moter & Gobel 2000) with a culture isolate of L. major (data not shown). Both newly developed Leishmania spp. specific FISH probes were evaluated with 18 cultured Leishmania spp. strains, one cultured T. brucei gambiense and rhodesiense mixture, two cultured T. cruzei strains, and one sample each with lyophilized T. brucei gambiense and T. brucei rhodesiense (donations from Antwerp) that no longer grew in culture (Table 2 of results chapter). All clinical Leishmania spp. isolates were sequence-typed as described (El Tai et al. 2000). The phylogenetically closely related (Stevens et al. 2001) and clinically highly relevant and morphologically similar Trypanosoma spp., the aetiologic agents of African sleeping sickness and South American Chagaz disease, were included in the testing to prove the probes’ specificity. Testing was also performed on formalin-fixed, paraffin-embedded tissue samples of laboratory mice that had been artificially infected with L. major (MHOM/72/SU/5ASKH). In detail, tissue from one lymphatic node and five feet of different infected mice that had been sacrificed 9 years prior to the experiment in a previous study (Jacobs et al. 2005) were analysed. Parallel Giemsa staining was performed (Table 3). Finally, stored formalin-fixed, paraffin-embedded residual bioptic materials from patients with clinically diagnosed cutaneous or visceral leishmaniasis were retrospectively stained by FISH and, in parallel, by Giemsa staining. The samples were between 5 and 12 years old at the time of the analysis. In total, 10 human skin snips of Syrian cutaneous leishmaniasis patients from the Department of Dermatology of the University Hospital Aleppo (Syria) were included in the analysis. The diagnosis of cutaneous leishmaniasis had been made clinically in Syria, but no molecular discrimination of the endemic L. major and L. tropica had been performed. Five tissue samples from travellers returning to Germany, including one skin snip containing L. major and four samples with L. donovani, that is, one liver sample, one spleen sample and two lymph nodes, from the tissue collection at the Bernhard Nocht Institute of Tropical Medicine in Hamburg (Germany), were analysed as well. One fresh bone marrow sample of a 1.5-year-old Armenian child with visceral leishmaniasis caused by L. infantum (see Table 4 of results section) was analysed by FISH and Giemsa staining without prior paraffin embedding. For all tissue samples, both Leishmania-specific probes were used combined in one assay to provide higher fluorescence intensities. Negative results of staining were accepted only after at least three negative slides. In silico evaluation of the FISH probes Leish_18S_651 and Leish_18S_840 with the software probeCheck based on sequences from the SILVA database in September 2011 confirmed specificity for ribosomal RNA of Leishmania spp. Matches within the relevant one-base-mismatch range with Trypanosomatida spp. such as Blastocrithidia spp., Endotrypanum spp., and Leptomonas spp. were considered non-critical, because these organisms play hardly any aetiologic role in infected humans. Outside this critical annealing range, matches with ribosomal RNA of other non-pathogenic organisms including Trypanosomatida spp. such as Asparagopsis spp. or Crithidia spp. and even bacteria were observed in the two-base-mismatch range (Table 1). There were no cross-matches with ribosomal RNA of pathogenic Trypanosoma spp. even in the four-base-mismatch range. As intended during the probe design, optimal formamide concentration in the hybridization buffer for use with the newly designed probes was determined at 30% formamide. Higher formamide concentrations led to a loss in fluorescence intensity as determined with the reference strain L. major MHOM/SU/72/5ASKH. An initially designed shorter Leish_18S_840a (5′-Cy3-AAG-CGG-GCG-CGG-TGC-T-3′) probe showed too weak binding irrespective of the formamide concentration. Thus the length had to be increased by three bases to ensure a sufficiently high fluorescence intensity. Among the reference strains and clinical isolates, both Leish_18S-651 and Leish_18S_840 correctly identified 18/18 Leishmania spp. (Figure 1) and were negative for 6/6 Trypanosoma spp. (Table 2). Cross-bindings to the slightly autofluorescing Trypanosoma spp. were weak and therefore well distinguishable from specific binding. (a–c) Fluorescence in situ hybridization (FISH) from cultured promastigote Leishmania peruviana at 630× magnification. (d–f) FISH from amastigote Leishmania donovani in formalin-fixed, paraffin-embedded human lymphatic node tissue. (a, d) Staining with the Cy3-labelled Leishmania-specific probe Leish_18S_651. (b, e) Staining with the FAM-labelled probes EUK502 and EUK_Leish499, staining virtually all eucaryotic cells. (c, f) DAPI staining of DNA. Prior to FISH from formalin-fixed, paraffin-embedded tissue samples, a deparaffinization procedure of about 50 min had to be performed. Autofluorescing erythrocytes were detected in all tissue samples but could be easily discriminated from parasites because of their size and the abundance of Leishmania spp. DNA. In parallel with FISH, DNA staining with DAPI allowed for a reliable differentiation of specific probe binding from autofluorescence in tissue. In all tissue specimens from mice that were artificially infected with L. major, that is, one lymphatic node and five foot pads, FISH and Giemsa staining led to concordant results. Leishmania spp. were visible at intermediate to high numbers per infected cell in the lymphatic node and four of five foot pads, while no parasites were detected in the fifth foot pad (Table 3). In addition, repeated staining of neighbouring slices from an opposite, non-infected foot pad that was used as negative control did not yield signals by FISH or by Giemsa staining. In the human specimens of patients with suspected visceral or cutaneous leishmaniasis, concordant results were achieved in 15 of 16 samples by Giemsa and FISH staining. In detail, both procedures led to the detection of Leishmania spp. in four of 11 skin snips, one of two lymphatic nodes, and one native bone marrow sample, while one spleen sample and one liver sample were negative (Figure 1, Table 4). A fifth skin snip was positive in Giemsa staining but not in FISH staining in subsequent slides. Giemsa staining was negative in subsequent slides of this respective specimen as well. While intermediate to high numbers of leishmania were detected in the slides that were positive in both Giemsa and FISH staining, the particular 7-year-old skin snip that was positive in Giemsa staining but negative in FISH staining contained only very few organisms. Leishmania parasites were always detected already in the first stained slide in Giemsa staining as well as in FISH staining in case of positive results. Specimens without visible Leishmania spp. in the first slide were stained three times but remained negative in the subsequent two slides as well. Requiring not more than standard laboratory equipment plus a fluorescence microscope, Leishmania FISH might be suitable for screening for Leishmania spp. in tissues, even after formalin fixation and paraffin embedding, as previously described for B. pseudomallei (Hagen et al. 2011). To ensure optimum fluorescence intensity in tissue, we recommend the combined use of both labelled genus-specific probes Leish_18S_651 and Leish_18S_840. The recently published short ISH probe with specificity to Leishmania-5.8S RNA (Dinhopl et al. 2011) was not designed for hybridization under standard FISH hybridization conditions (Hogardt et al. 2000; Kempf et al. 2000), so it could not be used in concert with our newly designed FISH probes. A hybridization-based differentiation to species level is theoretically possible, as was demonstrated for ISH with labelled kinetoplast DNA (Kennedy 1984; Barker 1987, 1989; van Eys et al. 1987; Schoone et al. 1991), but is laborious and time-consuming. Although it would be useful to rapidly identify Leishmania spp. with the potential to cause mucocutaneous leishmaniasis from skin lesions, there are hardly enough published Leishmania sequences to allow the design of reliable species-specific short oligonucleotide probes for FISH. Further, our limited strain and specimen collection would not have allowed a thorough evaluation. In our evaluation, the genus-specific Leishmania spp. probes correctly identified all tested Leishmania spp. and excluded the closely related Trypanosoma spp., the only other known microorganisms in the order Kinetoplastida with pathogenic potential for humans (Field et al. 2004). Results of Leishmania spp. detection in tissue by FISH and conventional Giemsa staining showed a high degree of concordance, with just one more positive sample in only one slide in Giemsa staining. The practical relevance of this latter finding remains doubtful, because only single Leishmania parasites were detected within this particular slide by Giemsa staining, while subsequent slides were negative in both Giemsa and FISH staining. Therefore, we cannot exclude that the neighbouring slices that were analysed by FISH may indeed have been free of Leishmania parasites. It is theoretically possible that the long storage of the sample may have contributed to the falsely negative FISH result. Although other samples that showed intensive specific FISH signals were as old or even older, RNA degradation in just a few cells may lead to falsely negative results if the density of the target organisms is very low. Autofluorescence is a typical obstacle to be overcome in tissue FISH (Hagen et al. 2011). The deparaffinizing agent Rotihistol® contributed to a reduction of background staining in comparison with xylole as assessed subjectively by us (data not shown). Further, autofluorescing DNA-free erythrocytes were reliably discriminated from specifically stained Leishmania spp. by DAPI counterstaining. The use of the non-intercalating DNA stain DAPI, which also allows screening for the nucleic and kinetoplast DNA of Leishmania spp. in tissue (Pérez-Cordero et al. 2011), is indicated in combination with FISH in tissue to prove specific binding (Swidsinski 2006). As internal reaction control, differently labelled FISH probes staining all eucaryotic cells were used in combination with the specific probes, as suggested elsewhere (Swidsinski 2006). Although successful amplification of Leishmania DNA from formalin-fixed, paraffin-embedded tissue specimens, even on Giemsa-stained slides, to confirm the histological detection of Leishmania spp. has been described (Volpini et al. 2006; Pandey et al. 2010), we failed to confirm positive FISH or Giemsa results with long-fragment PCRs and consecutive sequencing (El Tai et al. 2000) even from thick cuts of the paraffin-embedded, formalin-fixed materials (data not shown). This failure may be attributed to cross-linking between the DNA strands because of fixation with formalin as described for longer DNA segments that are required for reliable sequencing results (Hagen et al. 2002). In addition to ease of performance, FISH allows for rapid detection or at least confirmation of Leishmania spp. in tissue, requiring no more than 1.5 h without deparaffinization and <2.5 h including deparaffinization. Although time-to-result is not critical in chronic diseases like leishmaniasis, a rapid procedure can be easily implemented in routine diagnostic procedures. In addition, with material costs of about 1 USD per reaction (Hagen et al. 2011), DNA-FISH is also considerably cheaper than most alternative molecular methods (Rodríguez et al. 1994; Nicolas et al. 2002; Tavares et al. 2003; Foulet et al. 2007; Castilho et al. 2008; van der Meide et al. 2008; Antinori et al. 2009; Meymandi et al. 2010; Talmi-Frank et al. 2010; Roelfsema et al. 2011). Although yet in early stage of development, reliability of Leishmania FISH in tissue is presumably as low as that of Giemsa staining. Infections with very low numbers of parasites may be missed by either technique. Leishmania FISH hardly makes sense for an experienced pathologist for diagnostic purposes, but the high contrast between fluorescence-stained Leishmania spp. and background allows for a rapid screening by less experienced investigators. FISH staining should always be supplemented by Giemsa staining or comparable histopathological staining procedures, because the latter provide additional information on the composition of inflammatory cells in tissue (Mbati et al. 1995; Arechavala et al. 2010; Gazozai et al. 2010; Meymandi et al. 2010) that can give hints on the origin of inflammation even if parasites are absent. Thus, Leishmania FISH has the potential not to replace but to supplement existing staining procedures for the preliminary detection of Leishmania spp. even in formalin-fixed, paraffin-embedded tissue. Broader evaluation studies in endemic areas with more than the few available materials in our study might address its sensitivity and specificity. We are grateful to Laurent Vergnes for excellent technical assistance." @default.
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• W1532048386 title "Rapid identification of<i>Leishmania</i>spp. in formalin-fixed, paraffin-embedded tissue samples by fluorescence<i>in situ</i>hybridization" @default.
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• W1532048386 doi "https://doi.org/10.1111/j.1365-3156.2012.03024.x" @default.
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