PCR - RFLP for species diffrenciation
The ITS regions and chitin synthase 1 were used in previous research studies to differentiate yeasts to the species level by using PCR methods (Cafarchia et al., 2009; Jaya et al., 2009). This is highlighted by the study of Kim et al. (2011), which tested the length of the small-subunit rDNA and adjacent internal transcribed spacer (ITS) regions and chitin synthase 1 were amplified with primers ITS1 - ITS4, ITS 1-2 (for - rev) and CHS1S - CHS1R. Digested amplified ITS and chitin synthase 1 products with the restriction endonuclease Mva I produced unique and easily identifiable fragment patterns for a majority of species (Howell et al., 1999; Kim et al., 2011). This report describes the application of PCR-RFLP for the identification of species and varieties of common dermatophytes utilizing a primer pairs ITS1 - ITS4, ITS 1-2 (for - rev) and CHS1S - CHS1R. The results showed (Table 4.2.1; Figs. 4.2.7 - 4.2.24) that, the 132 culture positive cases, the organisms identified by phenotypic characterization were belonging to three genera and eight species viz., T. mentagrophytes 52 (39.4%), T. rubrum 30 (22.7%), T. violacium 18 (13.6%), T. verrucosum 11 (8.3%), E. floccosum 10 (7.6%), M. canis 6 (4.5%), T. tonsurans 3 (2.3%) and T. schoenleinii 2 (1.5%) were subjected by PCR-RFLP found same genus and species with 100% sensitivity and specificity causing dermatophytosis. Hae III restriction patterns were highly reproducible and consistent for several species, indicating that the ITS and chitin synthase 1 regions in dermatophytes are conserved.
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The RFLP product using the dermatophyte specific primers on the Hae III and Hinf I enzymes showed an identical band size consistently and with the Mva I enzyme, it showed no recognition site on the two dermatophytes T. verrucosum and M. canis, which were subjected for PCR-RFLP. Therefore, there was no difference in the band patterns observed. Eventually, using the dermatophyte specific primer, followed by RFLP, produced similar band profiles and this made the identification of the dermatophytic species. In case of the ITS1 - ITS4, ITS 1-2 (for - rev) and CHS1S - CHS1R fungal primer, using the Hae III enzyme based RFLP speciation, produced similar band patterns and therefore, the Hae III enzyme is also suitable for the identification of the dermatophytic species. As was described previously, using the Mva I and Hinf I restriction enzymes produced unique band profiles consistently and it was reproducible. Therefore, the application of the Mva I and the Hinf I enzymes by using the ITS and chitin synthase 1 amplicons helped in the easy identification of the dermatophytic species. However, by using the dermatophyte specific fungal primer with these three restriction enzymes, it was possible to detect any strain variations among the T. rubrum and the T. mentagrophytes strains. Therefore, in the identification of the strain variations by using RFLP analysis, the recognition site for dermatophytes was not found to be located in the ITS and the chitin synthase 1 regions. As was described in earlier studies, the strain variations can be identified by targeting the ribosomal DNA of the non-transcribed spacer (NTS) region.
DNA sequencing confirms the isolates as T. rubrum var. raubitschekii, which were identified phenotypically as urease positive. Since T. rubrum var. raubitschekii possessed minor morphological and physiological features, it is currently being considered as a synonym of T. rubrum. The previous report on T. interdigitale from India was made in 1996 and the present report is the second one from India.
To conclude, the dermatophyte specific primer based PCR which targets the ITS and chitin synthase 1 is useful in the direct identification of dermatophytosis from clinical specimens and it can be applied in the routine diagnostics wherever the laboratory facilities are adequate. The application of the Hae III and the Hinf I restriction enzymes by using the ITS and chitin synthase 1 amplicons was equally good, stable and reproducible in the identification of the dermatophytic species. The PCR-RFLP method, on using the dermatophyte specific primer and the fungal primer with Mva I, Hae III and Hinf I, showed no strain differentiation among the T. rubrum and the T. mentagrophytes isolates. Since direct microscopy and culture have limitations, performing a direct PCR on the clinical specimens can augment the diagnosis of more dermatophytic cases. However, species identification by PCR may not have a direct impact on the clinical treatment.
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PCR - using ITS and chitin synthase 1 primers - RFLP were applied on 100 isolates of M. canis and 42 isolates of E. floccosum, Mva I digestion was applied to discriminate M. canis from the other members in Microsporum sp. by Kim et al. (2011). By RFLP analysis of ITS regions the most closely related taxa M. ferrugineum and M. audouinii and also in case of E. floccosum, RFLP patterns of the regions for each of the members were easily anticipated these result were coincide with Mochizuki et al. (2003) and Sharma et al. (2007). Nine isolates of M. canis showed same result with PCR - using ITS and chitin synthase 1 primers - RFLP and phenotypic identification found to be A. benhamiae a perfect state of T. mentagrophytes complex. This result is in agreement with Fumaeax et al. (2004) who isolated A. benhamiae as a causative agent for human dermatophytosis. In this work the dermatophytes have been characterized by flat raised center with strong yellow reverse and fail to give colour on BCP - MSGA. PCR - ITS sequencing is superior than the other method for identification of dermatophytes. Then purification of amplified DNA sequencing by using ITS and chitin synthase 1 primers were applied. Dendrogram analysis also confirmed the species of dermatophytes (Fig. 4.2.25).
As we observed in the study, T. verrucosum was one of the most important zoophilic infections increasing day by day, so we planned to detect source of infection from (culture and PCR positive) patients house domestic animals by PCR - RFLP. From the same source (houses) 10 isolates from patients and 10 domestic animals were subjected to both PCR and RFLP. The pattern of agarose gel electrophoresis was found to be infected by same organisms T. verrucosum having sensitivity and specificity of 100% as depicted in Figs. 4.2.1 - 4.2.3. This result shows that T. verrucosum infection is from animal source.
The Hae III restricted enzymes are highly conserved which is sufficient for the strain characterization. Strain specific variations in T. verrucosum are located in non specific transcribed spacer (NTS) region rather than the ITS region. The chitin synthase 1 polymorphism has provided the first molecular technique for strain differentiation in the species. On the other hand, this technique of PCR and RFLP is rapid and we can obtain result within 8-9 h from DNA extraction to electrophoresis. This technique gives more accuracy, sensitivity, specificity, source detection and also for epidemiological identification of the infectious disease. However, it requires expert man power and also the cost is high.
Dermatophytic infections are one of the most common infectious diseases. Primitive diagnosis of dermatophytosis can be done by KOH mount and culture, which takes longer time to report and cannot differentiate at the level of genus and species level. Results in our study indicates that PCR-RFLP may be considered as gold standard for the diagnosis and confirmation of source of infection of dermatophytosis and can aid the clinician for initiating prompt and appropriate antifungal therapy.
In conclusion, the dermatophyte specific primer based PCR which targets the internal transcribed spacer and chitin synthase 1 primers are useful in the direct identification of dermatophytosis from clinical specimens and it can be applied in the routine diagnostics wherever the laboratory facilities are adequate. The application of the Hae III restriction enzymes by using the ITS and chitin synthase 1 amplicons was equally good, stable and reproducible in the identification of the dermatophyte at the species level. The PCR-RFLP method on using the dermatophyte specific primer with Hae III showed no strain differentiation among the T. verrucosum from man and T. verrucosum from animal isolates. Since direct microscopy and culture have limitations, performing a direct PCR on the clinical specimens can augment the diagnosis of more dermatophyte cases. However, species identification by PCR and antifungal susceptibility has a direct impact on the clinical treatment.
5.3 In vitro antifungal susceptibility test
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PCR confirmed 132 dermatophyte samples were taken for antifungal sensitivity test. Among these isolated 132 dermatophytes, T. mentagrophytes cultures were maintained for four days and five days for T. rubrum, M. canis and E. floccosum species when incubated at 28°C for antifungal susceptibility test by MIC. In the current study, among 132 isolates of dermatophytes some are sensitive and some are moving towards resistance, but high MIC value indicated that it has slowly acquired adaptation towards the drug. This indicates in near future it will develop drug resistance against the antifungal agents. Twenty three isolates (14.4%) were showing high MIC value (T. mentagrophytes - 8, T. rubrum - 5 and T. verrucosum - 10) for fluconazole and M. canis - 3 had MIC50 of 16 µg ml-1. Second most frequently used drug next to fluconazole is ketoconazole, which had MIC50 of 0.125 µg ml-1 for most of the isolates. Griseofulvin, itraconazole and terbinafine showed similar results of 0.03 - 0.06 µg ml-1 (Fig. 4.3.2).
The present investigation showed MIC50 and MIC90 for antifungal agents and their geometric mean of the drug against 132 isolates were identified (Tables 4.3.1B - 4.3.5B). MIC90 were not determined in samples less than 10 dermatophytic isolates (Tables 4.3.1A - 4.3.5A). The MIC ranges of fluconazole, itraconazole and ketoconazole for C. parapsilosis ATCC - 22019 were within the value standardized (Clayton, and Midgley, 1989; DelPalacio et al., 1998; Barry et al., 2000; Ghannoum et al., 2004, 2008; Barros et al., 2006; Carrillo-Munoz et al., 2006; Dragos and Lunder, 2007; CLSI, 2008).
Differences in MIC values cannot be attributed to the incubation temperature (28 or 35oC), as Santos and Hamdan (2005) demonstrated that single parameter alone do not significantly influence MIC determination. According to Norris et al. (1999), a logistical advantage of using 35oC is that dermatophyte plates can be incubated with plates set up for yeast testing, eliminating the need for a second incubator for susceptibility testing. The incubation period is another point of discrepancy among the studies mentioned. Incubation time for T. rubrum and T. mentagrophytes were seven days, as they do not grow well in shorter periods (Kaaman et al., 1981; Calderon, 1989; De Haan et al., 1989; Santos and Hamdan, 2005). In addition, visualization of growth inhibition could be confused with poor growth of the fungi in microdilution wells, indicating a false susceptibility profile for a given agent.
As antifungal tests is not so common in Asian countries like India, but it is very much essential to know the resistance patterns among microbes i.e., not only in bacteria but also in fungi, which help to assess interventional efforts and empirical therapy to the patients. This MIC data are also essential to obtain distribution profiles of MIC values for fungal correlations of MICs with clinical response. Gupta and Kohli, (2003) and Nir-Paz et al. (2003) advised the method of testing susceptibility of antifungal agents against yeast and also additional effort to adapt NCCLS broth microdilution method for molds.
MIC done by incubation at 28°C for seven days gives only microconidia in buffered RPMI 1640 allows adequate growth for the study. Differences in MIC values cannot be attributed to the incubation temperature (28 or 35°C) as Perea et al. (2001) shown that temperature can influence MIC. Seven days incubation for T. rubrum and T. mentagrophytes was followed as Gupta and Kohli (2003), these fungal growths were luxurious and growth was observed for five days. Terbinafine was most potent agent tested in our study but slow and steady increasing MIC of terbinafine in T. mentagrophytes is also a point of view (Harrison and Harrison, 1986; Ryder and Favre, 1997; Koga et al., 2003; Ghannoum et al., 2008; Haroon et al. 2009).
Fluconazole is the drug that had high MIC value in Trichophyton, Microsporum and Epidermophyton which is 16-32 µg ml-1, similar report was found in research done by Santos et al. (2006). Ketoconazole is another drug which is more frequently used, also showed MIC increasing 0.125µg ml-1 as compared to the previous studies (Kaaman et al., 1981; Weitzman and Summerbell, 1995; Ogawa et al., 1998; Mukherjee et al., 2003; Nweze and Okafor, 2005; Singh et al., 2007).
With respect to the inhibition end points, it is recommended in the literature to use 50% (Fernandez-Torres et al., 2000), 80% (Gupta and Kohli, 2003) and 100% (Fernandez-Torres et al., 2002) growth inhibition as end points. A value of 80% growth inhibition appears to be suitable for fungistatic agents and 100% is suitable for fungicidal drugs (Orray and Sinnet, 1998; Nir-Paz et al., 2003; Odds et al., 2004; Ohst et al., 2004).
Monitoring antimicrobial resistance is useful because apart from tracking and detection of resistance trends by microorganisms, it also gives clues to emerging threats of new resistance. Terbinafine is also found to show mild type of resistance but in our study most frequently used drugs by patients were ketoconazole and fluconazole for months. Even if it was not getting treated then few patients were taking one flustat tablet for five days direct from drug house without any doctor’s prescription. That may be one of the reasons for drug resistance among few isolates. This type of drug resistance was mostly observed in zoonotic infection.
Lastly we conclude that the parameters for testing the susceptibility of dermatophytes to antifungal agents adopted here appear to be suitable and reliable, and could contribute to the possible development of a standard protocol and to also see the resistance pattern among dermatophytes. Terbinafine is most active and has excellent in vitro potency and broad spectrum activity against all the tested species. This can be used to treat a majority of dermatophytic infections and also in those infections causing dermatophytosis.
The present study demonstrated that terbinafine and itraconazole should be preserved for the treatment of drug resistant cases. As we have seen that terbinafine has least MIC and next to itraconazole, whereas fluconazole and ketoconazole has high MIC. MIC need to correlate with clinical form of disease for break point development against the dermatophytes.
In over all, the conventional method of microscopy and culture technique is time consuming and lacks sufficient sensitivity; however it is an efficient screening technique. Culture on specific selective media for dermatophytes will ensure the diagnosis and we can reach to the species level. However, it may be time consuming, costly, as it needs different culture media for proper identification. In addition, it needs special skills and expertise as the morphological characteristics of some species is atypical.
The genotypic differentiation by PCR-RFLP provides a rapid and practical tool for identification of dermatophytic isolates that has independent morphology and biochemical characteristics thus, enhances lab diagnosis of the dermatophytes. Further investigation of a large number of isolates from different part of the country could shed light upon more kinds of dermatophytes and its infection in human and animals.
This study shows the standard NCCLS M38-A broth dilution technique with minor modification made in temperature and incubation time are convenient for antifungal susceptibility testing of dermatophytes. Among the antifungal tested terbinafine was the most potent antifungal drug. Minimum inhibitory concentration need to be correlated with clinical outcome to develop interpretive breakpoint, which may specify the cause for lack of clinical response and detection of resistance. This will help for treatment of not only patients but also for immunocompromised patients and children.