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Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis.

Alonso R, Pisa D, Marina AI, Morato E, Rábano A, Rodal I, Carrasco L - Int. J. Biol. Sci. (2015)

Bottom Line: ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown.Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients.Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons.

View Article: PubMed Central - PubMed

Affiliation: 1. Centro de Biología Molecular "Severo Ochoa". c/Nicolás Cabrera, 1. Universidad Autónoma de Madrid. Cantoblanco. 28049 Madrid. Spain.

ABSTRACT
Among neurogenerative diseases, amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by a progressive motor neuron dysfunction in the motor cortex, brainstem and spinal cord. ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown. In the present work, we have explored the possibility of fungal infection in cerebrospinal fluid (CSF) and in brain tissue from ALS patients. Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients. Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons. Fungal DNA was also detected in brain tissue using PCR analysis, uncovering the presence of several fungal species. Finally, proteomic analyses of brain tissue demonstrated the occurrence of several fungal peptides. Collectively, our observations provide compelling evidence of fungal infection in the ALS patients analyzed, suggesting that this infection may play a part in the etiology of the disease or may constitute a risk factor for these patients.

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PCR analysis of DNA obtained from CSF. A) Schematic representation of fungal rRNA genes and the ITS1 sequence. Location of the primers employed for the PCR: primers 1 employed in the first PCR; primers 2A employed in the second PCR; primers 2B employed in the second PCR and previously described as panfungal primers. B) PCR was carried out as described from DNA samples obtained from CSF of ALS patients or controls. The primers employed were primers 1 for the first round PCR and primers 2A for the second round. After PCR, the samples were separated on agarose gels and stained with ethidium bromide. DNA size markers are shown on the left. Fungal species detected after sequencing each product is shown on the right. C) The samples obtained after the first PCR (primers 1) were amplified using primers 2B. PCR products were separated on agarose gels, extracted and sequenced. Fungal species detected are depicted on the right. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers.
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Figure 2: PCR analysis of DNA obtained from CSF. A) Schematic representation of fungal rRNA genes and the ITS1 sequence. Location of the primers employed for the PCR: primers 1 employed in the first PCR; primers 2A employed in the second PCR; primers 2B employed in the second PCR and previously described as panfungal primers. B) PCR was carried out as described from DNA samples obtained from CSF of ALS patients or controls. The primers employed were primers 1 for the first round PCR and primers 2A for the second round. After PCR, the samples were separated on agarose gels and stained with ethidium bromide. DNA size markers are shown on the left. Fungal species detected after sequencing each product is shown on the right. C) The samples obtained after the first PCR (primers 1) were amplified using primers 2B. PCR products were separated on agarose gels, extracted and sequenced. Fungal species detected are depicted on the right. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers.

Mentions: An additional sensitive method to assess fungal infection is PCR 32, 33. This technique has the added advantage that, after amplification of variable internal transcribed spacer 1 (ITS1) present between the rRNA genes (see scheme Figure 2), followed by agarose electrophoresis, sequencing of the amplified products reveals the fungal species present. However, it must be considered that the bulk of the DNA obtained is from human origin and only a very small amount would be fungal. Due to the limited volume of CSF, DNA could not be obtained from one of the patient samples; nonetheless, DNA was obtained from four CSF samples and the ITS1 sequences were amplified by nested PCR. After the first PCR, the second amplification was carried out with two independent primer pairs, as detailed in materials and methods (see scheme, Figure 2). The first primer pair, 2A ITS1, has been previously employed successfully by us 19, 31. The second primer pair, 2B ITS1, have been described as panfungal primers 34, although they do not completely hybridize with Malassezia spp. The use of different primers may serve to a better coverage of several fungal species. Moreover, future studies in this regard may benefit from the use of a wider range of primers to perform PCR studies. Notably, several products were amplified using 2A ITS1 primers in DNA extracted from CSF of ALS patients (Figure 2). In contrast, no products were obtained in control CSF samples or in controls for DNA extraction or PCR amplification without DNA, indicating that these amplified products were not due to contamination during DNA extraction or PCR amplification. DNA sequencing of the amplified products revealed the fungal species listed in Figure 2. Of interest, 2A ITS1 primers amplifed Malassezia spp. (Figure 2). More importantly, C. albicans was also detected in two patients (2 and 3). The use of the panfungal primers 2B ITS1 also revealed the presence of C. albicans DNA in these two patients. Additionally, Rhodotorula mucilaginosa was detected in patients 1 and 4 using the 2B ITS1 primer pair, but Malassezia spp. was not amplified. The possibility that DNA from other fungal species was present in lower amounts is not excluded by these results. Moreover, it is possible that fungal cells from some species, which are present in brain tissue, are not released to the CSF. Taken together, these results strongly suggest that DNA from several fungi is present in CSF from ALS patients.


Evidence for fungal infection in cerebrospinal fluid and brain tissue from patients with amyotrophic lateral sclerosis.

Alonso R, Pisa D, Marina AI, Morato E, Rábano A, Rodal I, Carrasco L - Int. J. Biol. Sci. (2015)

PCR analysis of DNA obtained from CSF. A) Schematic representation of fungal rRNA genes and the ITS1 sequence. Location of the primers employed for the PCR: primers 1 employed in the first PCR; primers 2A employed in the second PCR; primers 2B employed in the second PCR and previously described as panfungal primers. B) PCR was carried out as described from DNA samples obtained from CSF of ALS patients or controls. The primers employed were primers 1 for the first round PCR and primers 2A for the second round. After PCR, the samples were separated on agarose gels and stained with ethidium bromide. DNA size markers are shown on the left. Fungal species detected after sequencing each product is shown on the right. C) The samples obtained after the first PCR (primers 1) were amplified using primers 2B. PCR products were separated on agarose gels, extracted and sequenced. Fungal species detected are depicted on the right. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers.
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Figure 2: PCR analysis of DNA obtained from CSF. A) Schematic representation of fungal rRNA genes and the ITS1 sequence. Location of the primers employed for the PCR: primers 1 employed in the first PCR; primers 2A employed in the second PCR; primers 2B employed in the second PCR and previously described as panfungal primers. B) PCR was carried out as described from DNA samples obtained from CSF of ALS patients or controls. The primers employed were primers 1 for the first round PCR and primers 2A for the second round. After PCR, the samples were separated on agarose gels and stained with ethidium bromide. DNA size markers are shown on the left. Fungal species detected after sequencing each product is shown on the right. C) The samples obtained after the first PCR (primers 1) were amplified using primers 2B. PCR products were separated on agarose gels, extracted and sequenced. Fungal species detected are depicted on the right. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers.
Mentions: An additional sensitive method to assess fungal infection is PCR 32, 33. This technique has the added advantage that, after amplification of variable internal transcribed spacer 1 (ITS1) present between the rRNA genes (see scheme Figure 2), followed by agarose electrophoresis, sequencing of the amplified products reveals the fungal species present. However, it must be considered that the bulk of the DNA obtained is from human origin and only a very small amount would be fungal. Due to the limited volume of CSF, DNA could not be obtained from one of the patient samples; nonetheless, DNA was obtained from four CSF samples and the ITS1 sequences were amplified by nested PCR. After the first PCR, the second amplification was carried out with two independent primer pairs, as detailed in materials and methods (see scheme, Figure 2). The first primer pair, 2A ITS1, has been previously employed successfully by us 19, 31. The second primer pair, 2B ITS1, have been described as panfungal primers 34, although they do not completely hybridize with Malassezia spp. The use of different primers may serve to a better coverage of several fungal species. Moreover, future studies in this regard may benefit from the use of a wider range of primers to perform PCR studies. Notably, several products were amplified using 2A ITS1 primers in DNA extracted from CSF of ALS patients (Figure 2). In contrast, no products were obtained in control CSF samples or in controls for DNA extraction or PCR amplification without DNA, indicating that these amplified products were not due to contamination during DNA extraction or PCR amplification. DNA sequencing of the amplified products revealed the fungal species listed in Figure 2. Of interest, 2A ITS1 primers amplifed Malassezia spp. (Figure 2). More importantly, C. albicans was also detected in two patients (2 and 3). The use of the panfungal primers 2B ITS1 also revealed the presence of C. albicans DNA in these two patients. Additionally, Rhodotorula mucilaginosa was detected in patients 1 and 4 using the 2B ITS1 primer pair, but Malassezia spp. was not amplified. The possibility that DNA from other fungal species was present in lower amounts is not excluded by these results. Moreover, it is possible that fungal cells from some species, which are present in brain tissue, are not released to the CSF. Taken together, these results strongly suggest that DNA from several fungi is present in CSF from ALS patients.

Bottom Line: ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown.Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients.Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons.

View Article: PubMed Central - PubMed

Affiliation: 1. Centro de Biología Molecular "Severo Ochoa". c/Nicolás Cabrera, 1. Universidad Autónoma de Madrid. Cantoblanco. 28049 Madrid. Spain.

ABSTRACT
Among neurogenerative diseases, amyotrophic lateral sclerosis (ALS) is a fatal illness characterized by a progressive motor neuron dysfunction in the motor cortex, brainstem and spinal cord. ALS is the most common form of motor neuron disease; yet, to date, the exact etiology of ALS remains unknown. In the present work, we have explored the possibility of fungal infection in cerebrospinal fluid (CSF) and in brain tissue from ALS patients. Fungal antigens, as well as DNA from several fungi, were detected in CSF from ALS patients. Additionally, examination of brain sections from the frontal cortex of ALS patients revealed the existence of immunopositive fungal antigens comprising punctate bodies in the cytoplasm of some neurons. Fungal DNA was also detected in brain tissue using PCR analysis, uncovering the presence of several fungal species. Finally, proteomic analyses of brain tissue demonstrated the occurrence of several fungal peptides. Collectively, our observations provide compelling evidence of fungal infection in the ALS patients analyzed, suggesting that this infection may play a part in the etiology of the disease or may constitute a risk factor for these patients.

Show MeSH
Related in: MedlinePlus