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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 different brain regions from three ALS patients. PCR was carried out as described. A) PCR analysis of different brain regions and controls using primers 1 and 2a. Table shows fungal species detected. B) PCR analysis of different brain regions and controls using primers 1 and 2b. Table shows fungal species detected. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers. FC: Frontal cortex. C: Cerebellum. O: Occipital cortex. WM: White matter. GM: Grey matter.
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Figure 5: PCR analysis of different brain regions from three ALS patients. PCR was carried out as described. A) PCR analysis of different brain regions and controls using primers 1 and 2a. Table shows fungal species detected. B) PCR analysis of different brain regions and controls using primers 1 and 2b. Table shows fungal species detected. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers. FC: Frontal cortex. C: Cerebellum. O: Occipital cortex. WM: White matter. GM: Grey matter.

Mentions: To analyze the potential fungal species present in brain samples from ALS patients, DNA was extracted from different CNS regions as indicated in Figure 5, and nested PCR was carried out as described using primers 1 in the first round and primers 2A in the second round. Interestingly, several DNA products were amplified in the three ALS patients analyzed, whereas no amplification was found in control sample (Figure 3). DNA sequencing of the isolated products resulted in the identification of the species, depicted in the bottom of Figure 5. Notably, several species could be identified from one patient and these species varied according to the CNS region analyzed. Further, different fungal species were detected in patients, although some were present in all three ALS patients. Accordingly, Malassezia globosa was present in most of the samples tested, whereas Cryptococcus neoformans was only detected in one sample. In our opinion, the most striking finding was that C. albicans appeared in all three patients. This yeast was also present in the CSF of several ALS patients (Figure 2) and it is acknowledged that C. albicans can be highly pathogenic; this also applies to the presence of C. neoformans in one patient. Possibly, the existence of different fungal species may account for the differences in the severity and evolution of disease observed between ALS patients. Results from the second PCR using panfungal primers (2B), revealed that, as before, different fungal species could be detected. Trychoderma viridae was evident in all samples, whereas Cryptococcus magnus appeared in the four samples of patient 6. Collectively, these findings show that fungal infection of the CNS in ALS patients can be detected by PCR analysis, and different fungal species can also be determined. However, we do not know if the species identified by PCR are exactly the ones observed by immunofluorescence.


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 different brain regions from three ALS patients. PCR was carried out as described. A) PCR analysis of different brain regions and controls using primers 1 and 2a. Table shows fungal species detected. B) PCR analysis of different brain regions and controls using primers 1 and 2b. Table shows fungal species detected. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers. FC: Frontal cortex. C: Cerebellum. O: Occipital cortex. WM: White matter. GM: Grey matter.
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Figure 5: PCR analysis of different brain regions from three ALS patients. PCR was carried out as described. A) PCR analysis of different brain regions and controls using primers 1 and 2a. Table shows fungal species detected. B) PCR analysis of different brain regions and controls using primers 1 and 2b. Table shows fungal species detected. Control PCR: PCR without DNA. CE: Control of DNA extraction without CFS DNA. 1: external primers. 2A: internal primers. 2B: panfungal primers. FC: Frontal cortex. C: Cerebellum. O: Occipital cortex. WM: White matter. GM: Grey matter.
Mentions: To analyze the potential fungal species present in brain samples from ALS patients, DNA was extracted from different CNS regions as indicated in Figure 5, and nested PCR was carried out as described using primers 1 in the first round and primers 2A in the second round. Interestingly, several DNA products were amplified in the three ALS patients analyzed, whereas no amplification was found in control sample (Figure 3). DNA sequencing of the isolated products resulted in the identification of the species, depicted in the bottom of Figure 5. Notably, several species could be identified from one patient and these species varied according to the CNS region analyzed. Further, different fungal species were detected in patients, although some were present in all three ALS patients. Accordingly, Malassezia globosa was present in most of the samples tested, whereas Cryptococcus neoformans was only detected in one sample. In our opinion, the most striking finding was that C. albicans appeared in all three patients. This yeast was also present in the CSF of several ALS patients (Figure 2) and it is acknowledged that C. albicans can be highly pathogenic; this also applies to the presence of C. neoformans in one patient. Possibly, the existence of different fungal species may account for the differences in the severity and evolution of disease observed between ALS patients. Results from the second PCR using panfungal primers (2B), revealed that, as before, different fungal species could be detected. Trychoderma viridae was evident in all samples, whereas Cryptococcus magnus appeared in the four samples of patient 6. Collectively, these findings show that fungal infection of the CNS in ALS patients can be detected by PCR analysis, and different fungal species can also be determined. However, we do not know if the species identified by PCR are exactly the ones observed by immunofluorescence.

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