Quantitation of microsporidia in cultured cells by flow cytometry

Franzen, Caspar; Müller, Andreas; Hartmann, Pia; Salzberger, Bernd

doi:10.1002/cyto.a.20028

Cited by 8 publications

(12 citation statements)

References 24 publications

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“…Flow sitometri (FCM) kullanılarak mikrosporidiaların tanısı yapıla-bilmektedir (38). Franzen ve arkadaşları (38) kültürü yapılmış olan mikrosporidiaları FCM yöntemi ile tespit etmişler ve bu yöntemin kullanım kolaylığı ve yüksek bir duyarlılığa sahip olması nedeniyle araştırmalarda kullanılabileceğini vurgulamışlardır.…”

Section: Flow Sitometri Yöntemiunclassified

Mikrosporidialar ve Mikrosporidiyozis

Yazar¹,

Koru²,

Hamamcı³

et al. 2013

TurkiyeParazitolDerg

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ÖZETMikrosporidialar, zorunlu hücre içi parazitidirler ve konak hücresi dışında metabolik aktivasyon göstermemektedirler. Ökaryotik hücrelerdir, fakat bazı tipik ökaryotik özellikler eksiktir. Şimdiye kadar tanımlamış 144 cins içinde 1200'den fazla türü vardır. Mikrosporidiaların en çok bilinene evresi küçük ve oldukça dirençli olan sporlarıdır. Boyutları türe göre farklılık gösterir ama genellikle 1-10 µm arasındadır. Mikrosporodiaların hayatları üç fazda incelenebilir; enfektif veya çevresel faz, proliferatif veya üreme fazı ve sporogonik faz. Mikrosporidiaların tanısında Işık mikroskobu, Transmisyon Elektron Mikroskobu (TEM), Immünofloresan tekniği (IFA) ve Moleküler yöntemler gibi değişik metotlar kullanılmaktadır. Klinik semptomlar, enfeksiyonun lokalizasyonu ve konağın immün durumu ile yakından ilişkilidir. İnsanlarda görülen mikrosporidiosis önemli ve hızlı gelişen fırsatçı bir hastalıktır özellikle de AIDS'li immunocompromise hastalarda daha ciddi seyretmektedir. Mikrosporidiosis'in tedavisi genellikle ilaç ve destek tedavi ile sağlanmaktadır. Enfeksiyona ve türüne bağlı olarak farklı ilaçlar kullanılmaktadır. Tedavide en yaygın olarak albendazol ve fumagilin kullanıldığı belirtilmektedir. (Turkiye Parazitol Derg 2013; 37: 123-34) Anahtar Sözcükler: Mikrosporidia, mikrosporidiyozis, tanı yöntemleri, klinik Geliş Tarihi: 02.01.2013Kabul Tarihi: 19.02.2013 ABSTRACTAll microsporidia are obligate parasites and have no active stages outside their host cells. Microsporidia lack some typical eukaryotic characteristics. There are now over 1200 species identified in 144 genera. The most familiar stage of microsporidia is the small, highly resistant spore, the size of which differs according to the species and is often 1-10 µm. The general life cycle pattern of the microsporidia can be divided into three phases: the infective or environmental phase, the proliferative phase, and the sporogony or spore-forming phase. There are several methods for diagnosing microsporidia: light microscopic, transmission electron microscopy (TEM), immunofluorescence assays (IFA) and molecular methods. The clinical course of microsporidiosis depends on the immune status of the host and site of infection. Microsporidia can cause infections such as diarrhoea, keratitis, myositis, bronchitis and brochiolitis. Human microsporidiosis represents an important and rapidly emerging opportunistic disease, occurring mainly, but not exclusively, in severely immunocompromised patients with AIDS. The treatment of microsporidiosis is generally achieved with medications and supportive care. Depending on the site of infection and the microsporidia species involved, different medications are utilized. The most commonly used medications for microsporidiosis include albendazole and fumagillin. (Turkiye Parazitol Derg 2013; 37: 123-34)

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Section: Flow Sitometri Yöntemiunclassified

Mikrosporidialar ve Mikrosporidiyozis

Yazar¹,

Koru²,

Hamamcı³

et al. 2013

TurkiyeParazitolDerg

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“…Only a small number of cases of Encephalitozoon intestinalis infection have been documented (25); E. intestinalis (reclassified from Septata intestinalis) has been detected in AIDS patients in developed countries and in Africa, as well as in other immunocompromised individuals, including patients who have undergone transplantation (8,9,24).…”

mentioning

confidence: 99%

“…The clinical manifestations of E. intestinalis infections in immunocompetent patients range from asymptomatic infections to self-limited diarrhea. However, in immunocompromised patients, it causes chronic diarrhea, which tends to disseminate, with the kidney being the major organ affected (8,9). Spores may spread to the environment from infected patients via feces, urine, and/or other body fluids and tissues (9,24).…”

mentioning

confidence: 99%

“…During the last 20 years, several methods for the recovery and detection of microsporidial spores have been developed and improved; these include electron microscopy and histologic examination of tissues samples (25). Light and/or immunofluorescence microscopy with polyclonal or monoclonal antibodies directed against microsporidial spores is nevertheless the most commonly used procedure, especially for the diagnosis of infection in immunocompromised patients (8,9,16,25). Such methods are very time-consuming and are subject to human error (8,16).…”

mentioning

confidence: 99%

“…Light and/or immunofluorescence microscopy with polyclonal or monoclonal antibodies directed against microsporidial spores is nevertheless the most commonly used procedure, especially for the diagnosis of infection in immunocompromised patients (8,9,16,25). Such methods are very time-consuming and are subject to human error (8,16). Although molecular studies are highly sensitive and specific, they are too complex, too difficult for use for routine analysis, and expensive and are unable to assess spore viability (24).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Cytometric Approach for Detection of Encephalitozoon intestinalis , an Emergent Agent

Barbosa

Rodrigues

Pina-Vaz

2009

Clin Vaccine Immunol

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Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 g/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 ؋ 10 4 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 ؋ g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health.

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Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry

et al. 2013

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Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n 5 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods. V C 2013International Society for Advancement of Cytometry Key terms Nosema apis; viability; fluorescence microscopy; flow cytometry; microsporidia; SYTO 16; SYBR 14; Propidium Iodide; SYTOX Red UNDERSTANDING host-parasite interactions depends on experimental methods that can estimate life history traits of parasites related to fitness. Quantification of these is often crucial to gain detailed insights into the intimate interactions between the parasite and its host and consequently determine the virulence or the damage caused by a parasite. Pathogens can avoid detection or overcome attacks by the host's immune system, so their survival and viability within the host is an important factor to understand the dynamics of initial establishment and consequent progression of an infection. The ability to quantify the viability of specific parasites can have a range of experimental applications to assess the effect of host defences on parasite survival. However, such a technique has applied benefits as well, for example to quantify the effectiveness of treatments on parasites or as biosecurity tools to monitor and limit the spreading of infections. Flow cytometry (FCM) is a technique that has been used in the past to detect and monitor the viability of parasites, for example, to quantify Mycoplasm spore survival in goats and sheep after antibacterial treatment (1)....

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Quantitation of microsporidia in cultured cells by flow cytometry

Cited by 8 publications

References 24 publications

Mikrosporidialar ve Mikrosporidiyozis

Mikrosporidialar ve Mikrosporidiyozis

Cytometric Approach for Detection of Encephalitozoon intestinalis , an Emergent Agent

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry

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