Surveillance of Vittaforma corneae in hot springs by a small-volume procedure
Graphical abstract
Introduction
The Microsporidia are a diverse group of obligate intracellular fungi that infect insects, animals and humans. Currently there are over one hundred microsporidial genera known and almost 1500 species; many are found at high frequency in aquatic environments (Stentiford et al., 2013). The first recognized human microsporidial infection was described by Nageli 1857 and involved silkworms (Nageli, 1857). From 1985 onwards, various studies have reported opportunistic microsporidial infections in AIDS patients, with the symptoms including diarrhea and weight loss. Improvements in diagnostic methods have led to an increased awareness of the Microsporidia and more recent studies have shown that Microsporidia also are able to cause diseases in immunocompetent individuals (Bryan et al., 1991, Desportes et al., 1985, Didier et al., 2004, Weber et al., 2000). To date, seven genera of Microsporidia have been associated with human diseases (Enterocytozoon spp., Encephalitozoon spp., Trachipleistophora spp., Tubulinosema acridophagus, Anncaliia algerae, Pleistophora sp., and Vittaforma corneae), and clinical manifestations of human pathogenic microsporidia have been described in detail by US Center of Disease Control (Didier, 2005).
A case of corneal microsporidiosis was reported in 1993 that affected an immunocompetent patient and the pathogen was named Nosema corneum (Silveira et al., 1993). N. corneum was later renamed Vittaforma corneae because of the morphological characteristics of the fungi's spores in the infected cornea of mice. Various studies have confirmed that V. corneae is able to cause ocular microsporidiosis in both immunocompetent and immunocompromised patients (Deplazes et al., 1998, Shadduck et al., 1990, Sharma et al., 2011). Evidence from these studies suggests that V. corneae may present a significant risk of ocular microsporidiosis. In India, thirty cases of ocular microsporidiosis were reported between 2006 and 2008, and the number of infections was found to be increasing year by year (Reddy et al., 2011b). In Singapore, there were 124 confirmed cases of microsporidial keratitis reported between 2004 and 2009, and many of these reported cases were HIV-negative patients (Loh et al., 2009). Both studies showed that the risk of microsporidial keratitis (MK) was associated with seasonal rainfall. In the Singaporean study, 62 (50%) of the 124 patients had soil/mud exposure prior to infection. The first confirmed outbreak of microsporidial keratoconjunctivitis was reported in 2012 in Singapore. The outbreak was caused by V. corneae, and most of the patients had had prolonged soil or muddy water exposure during rugby games (Kwok et al., 2013, Lam et al., 2013, Tan et al., 2013). The above studies suggest that the disease occurs as a result of environmental exposure. In Taiwan, 23 microsporidial keratitis cases were reported between 2006 and 2011, and 14 (60.9%) had had prior exposure to hot springs (Fan et al., 2012). The same study also found that in four patients, the lapse time between hot spring exposure and symptom onset was less than 3 days. Microsporidia spores are heat resistant and therefore it is likely that spore exposure may be the major route of transmission when there is human infection with V. corneae.
Studies of Microsporidia in the aquatic environmental have mostly focused on Enterocytozoon bieneusi (Hu et al., 2014, Ma et al., 2015), and only two studies have reported evidence indicating the presence of V. corneae in aquatic environments (Dowd et al., 1998, Fournier et al., 2000). The amount of V. corneae in surface water is usually low, and this will often affects detection accuracy. The two studies targeting V. corneae were carried out by filtering large volumes of water (more than 10 L) and detection was via single-step PCR. Collection of large volumes of water as a sample is both time-consuming and costly, and the approach differs dramatically regarding the sample volume normally used for detecting waterborne pathogens, which is typically less than 1 L. In addition, while a single step PCR method is commonly used for pan-microsporidial detection, if the ambient concentration of V. corneae is low, which is expected to be the case in most water bodies, this method will be a relatively unsuitable approach to detecting V. cornea in an aquatic environment. In this context, a clinical study has shown that the use of diagnostic PCR can improve the diagnostic rate for V. corneae infections by 25% compared with a standard microscopic examination (Bharathi et al., 2013).
Although a pan-microsporidial one-step PCR yielded a 1148bp fragment of the 18S rRNA gene had been developed, which could be used for the detecting V. corneae, a more specific one-step PCR amplified a 472bp fragment within the above region was established to improve the detection of V. corneae by Bharathi et al. (Bharathi et al., 2013, Raynaud et al., 1998). In a number of studies, nested PCR, which involves a combination of different PCR tests, has been successfully used for the detection of protozoan/fungal pathogens in water samples (Badiee et al., 2015, Coupe et al., 2005, Miller and Sterling, 2007). Nevertheless, up to the present, the nested PCR method has not been used for detecting V. corneae.
Little is known about the potential for exposure to V. corneae in hot springs in Taiwan or elsewhere. We hypothesize that the use of nested PCR for the detection of V. corneae will enhance the detection limit for this organism and result in a requirement for less than 1 L of water sample when carrying out surveillance for V. corneae in hot spring water systems.
Section snippets
Detection methods for V. corneae
Three methods were used in this study to detect V. corneae, namely two one-step PCR approaches and a modified nested PCR approach. The first method (the “one-step universal PCR”) used the universal primers described by Raynaud et al. (1998) and is primarily a method used for pan-microsporidia detection. The second method (the “one-step specific PCR”), as described by Bharathi et al. (2013)), consists of a primer set specific for V. corneae. For the third method (nested PCR), the primer sets
Testing of the methods
The LOD for V. corneae was 5 × 105 copies/reaction when analyzed using the one-step universal PCR method (1193 bp), but it was 5 × 104 copies/reaction when analyzed by the one-step specific PCR method (472 bp; Fig. 2 and the full gel pictures shown in Supplemental file, Fig. S1). When the nested PCR assay method, a combination of these two PCR methods, was employed, the LOD decreased to 5 copies/reaction. In other studies on environmental microsporidia, the filtration of a bulk water sample of
Conclusions
- (1)
We have established a small-volume procedure for detecting V. corneae in water samples and improved the detection limits for V. corneae by using a modified nested PCR approach.
- (2)
In this study, the detection of V. corneae was associated with HPCs and with outdoor/soil exposure. Our results, together with other studies, suggest that soil or rainfall may be risk factors associated with V. corneae infection.
- (3)
A wide variety of DNA sequences related to V. corneae were isolated from the hot springs.
Acknowledgements
This work was supported by a research grant from the Centers for Disease Control, Taiwan, R.O.C. (MOHW105-CDC-C-114-122109; MOHW105-CDC-C-114-112601), obtained by DDJ. We thank all the participants who took part in the study as well as the employees in the Taiwan Centers for Disease Control (Taiwan CDC). We acknowledge the crucial support of the Health Bureaus of Taipei, New Taipei, Yilan, Taichung, Pingtung and Taitung City and County Governments.
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