We are grateful for the comments made by Badenoch and colleagues on the publication by Bagyalakshmi et al.: “Newer emerging pathogens of ocular non-sporulating molds (NSM) identified by polymerase chain reaction (PCR)-based DNA sequencing technique targeting internal transcribed spacer (ITS) region,” Curr Eye Res. 2008;33:139–147.
First, we would like to highlight the background which formed the basis for the study in our eye hospital in India. The analysis of fungal ocular isolates in seven years (1999–2005) from our center—a tertiary eye care center in South India—showed 12% ocular fungal isolates were NSM, which appeared to be more than generally appreciated. Our study is the first attempt by an ophthalmic research center to identify 50 ocular NSM isolates to the genus/species level. It was purely a retrospective study, with the aim to identify the NSM isolates preserved in the laboratory to genus or species level by application of PCR-based DNA sequencing technique.
Badenoch et al. accepted some of the identifications of NSM included in our study, while questioning others. Regarding amplification of the Stramenopile P. insidiosum, we would like to clarify that the 9 isolates included in our study phenotypically were filamentous fungi, which were non-sporulating. The panfungal primers targeting the internal transcribed spacer region amplified the DNA extract of these 9 fungal isolates. The resulting amplified products also were within the expected range (illustrated in the gel photographs of the publication, p. 142, Figure 2). Furthermore the DNA sequences matched with the published sequences deposited in the GenBank to prove that the genome is present in the DNA of the fungal isolates identified as P. insidiosum. The references citedCitation1, Citation2, Citation3, Citation4 illustrate the use of 18SrRNA and ITS region in development of oligonucleotide array targeting ITS region for identification of P. insidiosum. In addition, the antifungal agents in use have also been tested against P. insidiosum and were effective proving that P. insidiosum strains are susceptible to antifungal agents.Citation5, Citation6
The fungi were identified to genus and/or species level as per the identification criteria of Pounder et al.Citation7 These isolates were subjected to sequencing using both forward and reverse primers. Then identification was carried out using the BLAST program. The coverage varied from 70–99%. The identity of each fungus was assigned based on the percentage similarity. We have tried to match the sequence with the available sequence in the GenBank. This is the closest match we could get from the GenBank to match our sequences. We considered the 82% coverage with 98% identity as the closest identification possible for EF446288 Macrophomina phaseolina. We have taken the 90% coverage and 97% identity as the best match for identification of the NSM EF446285 as P. insidiosum. Again we would like to stress that our study was not prospective, and thus the type of NSM and the clinical outcome of the ocular infection were not evaluated. In fact, as mentioned in Materials and Methods, the ocular isolates of NSM preserved in the laboratory were used for the study. From the medical records of the patients' exposure to fresh water could not be detected as a potential route of infection, and thus this was not discussed in the paper.
We are very sorry for not including the reference of Badenoch et al., (2001) (Reference 4 of this reply) in the discussion, but this was omitted from the final manuscript by mistake. We agree with Badenoch and colleagues that one needs to be judicious when identifying molds from sequences in GenBank. The main interest of the publication was to create an awareness to the ophthalmologists and ocular microbiologists that 27% of the NSM are the emerging newer fungal pathogens, which were not known to cause ocular infections in humans so far.
Sincerely,
K. L. Therese, R. Bagyalakshmi, S. Prasanna
and H. N. Madhavan
Vision Research Foundation,
Sankara Nethrlaya, Tamilnadu, India.
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