Cases Of Penicillium & Talaromyces Species Can Be Pathogenic
Although rare, certain Penicillium and Talaromyces species can be pathogenic. No clinical infections have yet been associated with P. rubens but some invasive and cutaneous infections have been associated with P. chrysogenum (Houbraken et al., 2011). P. chysogenum is also found in high volumes in dust in the UK which may be inhaled by CPA and ABPA patients, possibly leading to colonisation (Visagie et al., 2014).
P. amphipolaria originated close to the North and South Poles and shows poor growth at 370C (Visagie et al., 2016). No cases of P. amphipolaria being pathogenic in humans have yet been reported. Paecilomyces variotii has been isolated from indoor air and food. It can also cause opportunistic infections in immunocompromised hosts (Houbraken et al., 2010). These infections can affect almost any organ but are often cutaneous or catheter-related.
Hamigera species were originally found in soil on the Gran Canary Island in Spain (Visagie et al., 2016) but no cases of infection in humans have yet been reported. Rasamsonia species previously belonged to the Geosmithia genus. Rasamsonia species share morphological similarities with Paecilomyces variotii. R. argillacea has been reported as a cause of invasive mycosis in 2 cases of patients with chronic granulomatous disease (Houbraken et al., 2013). R. piperina has been reported to be pathogenic in a German Shepherd dog (Houbraken et al., 2013) but not yet pathogenic in humans.
A significant example of a pathogenic non-Aspergillus species would be Talaromyces marneffei (previously known as Penicillium marneffei). This organism can cause the fatal infection penicilliosis marneffei. The mortality from this disease is high amongst both treated and un-treated individuals (Hu et al., 2013). This fungus is thermally dimorphic, meaning that at 250C, it can grow as a mould whereas 7at 370C it can grow as yeast (Vanittanakom et al., 2006).
T. marneffei is most common in South-East Asia and amongst immunocompromised people already infected with HIV. The symptoms can be severe: anaemia, hepatosplenomegaly and lymphadenopathy (Vanittanakom et al., 2006). It is important that this infection is diagnosed early to improve patient prognosis. As T. marneffei is a Category 3 organism, it can only be cultured in a level 3 biosafety laboratory. If there was a method to test for the organism which is non-culture based, it could improve diagnostic rates.
Polymerase chain reaction (PCR) does not involve culturing an organism so this may be a suitable method to use in laboratories which cannot culture Category 3 organisms. If a PCR can detect Penicillium, Talaromyces or Thermomyces species and each species can easily be distinguished, an early diagnosis could be made. The presence of Aspergillus can be tested using PCR on a variety of respiratory and blood specimens. Respiratory samples (bronchoalveolar lavage, bronchial washings and sputum) are more clinically useful when detecting Aspergillus as the majority of cases of aspergillosis begin in the lungs.
Many positive Aspergillus PCR results can often be produced from blood samples, however these results are often considered to be less clinically significant (White and Barnes, 2010). This is because it is not yet clear whether free Aspergillus DNA circulates in the bloodstream and so the origin of the DNA signal cannot be accurately determined (Barton, 2013). PCR can be highly sensitive as a small, initial amount of Aspergillus DNA can be amplified and quantified. Specifically designed primers and probes would allow for the accurate detection of fungal DNA from a particular fungal species (Barton, 2013).
However, DNA can also be detected from species which have colonised the patient’s lungs but do not appear to be causing infection. PCR is currently the only non-culture-based assay that has the potential to be genus and species specific (White et al., 2015). However, the results of other clinical tests must also be considered before acting on a positive PCR result due to EORTC criteria. In certain cases, antifungal drugs may influence the result of the PCR (Reinwald et al., 2012). One of the main issues associated with the PCR procedure is false positive results.
False positive results can be caused by background contamination from exogenous DNA sources, possibly from PCR reagents, pipettes and lab surfaces (Yang and Rothman, 2004). This is particularly important in the case of Aspergillus because of its abundance in the environment. Due to the power of PCR, a small amount of DNA can serve as a substrate for the amplification process (Yang and Rothman, 2004). Some PCR assays can lack specificity due to poor design of the primers or probes and due to the conserved nucleotide sequences between closely related species. DNA may be lost due to an inefficient DNA extraction procedure prior to the PCR leading to a false negative result (Goncalves-de-Albuquerque et al. 2014).
To ensure efficiency, internal amplification controls (IAC) are used (these are often the human beta globin gene) to monitor presence of both purified DNA and potential PCR inhibition. If the PCR procedure has run successfully, the IAC will always produce a signal even though the target sequence may not be present in the sample (Hoorfar et al., 2003). However, if no IAC signal has been produced, this could indicate PCR inhibition and so the assay has failed.8Real time PCR (qPCR) is often used to detect for Aspergillus.
The advantage of using qPCR over end- point PCR is that qPCR can calculate the number of amplicons accumulated during each amplification cycle using fluorescence whereas end-point PCR only produces a result at the end of the reaction (Smith and Osborne, 2009). qPCR assays may require careful design to ensure that the probes are specific to a single species, however this is not always possible (Morton et al., 2017). Aspergillus qPCR often targets the ITS regions of the genome as they are often highly conserved amongst the genus (Libert et al., 2015).
For this investigation, an Aspergillus genus-specific kit was used, Aspergillus spp. ELITe MGB® Kit to test for cross-reactivity with non-Aspergillus species.This ELITech kit is designed to detect, amplify and quantify DNA from the Aspergillus genus found in respiratory samples such as bronchoalveolar lavage, sputum and bronchial washings. The kit can be used to detect A. fumigatus, A. niger, A. nidulans, A. terreus, A. flavus, A. versicolor and A. glaucus. The kit contains a probe which is labelled with FAM fluorophore.
This kit also contains a minor groove binder (MGB) which increases the melting temperature of single stranded DNA (ssDNA) by forming stable complexes with the DNA which allows for shorter probes to be used (Kutyavin et al., 2000). Another probe is specific for the 5’ promoter region of the human beta globin gene and is used as an internal control to check for PCR inhibition. The probe activates and fluoresces when bound to the product of the Aspergillus amplification reaction. Fluorescence increases as more amplification products are produced. The amount of fluorescence is measured and the amount of Aspergillus DNA in a sample is quantified (ELITechGroup, 2014).
