Laboratory diagnostics of CNS fungal infection

1. Microbiology Workout for
CNS Fungal Infections
Siti Hawa binti Hamzah
Microbiology Unit
Pathology Dept, HSJ

2. Introduction
• CNS fungal infections are frequently
lethal, and their diagnosis and
therapeutic management are
challenging
• Clinical presentation of CNS fungal
infection varies from meningitis,
meningoencephalitis, stroke, cerebritis,
and vasculitis to venous sinus
thrombosis

3. • A diagnosis of any invasive fungal infection is commonly regarded as
definite in patients with positive cultures from sterile sites or through
demonstration of fungal tissue invasion, with few exceptions (such as
in patients with cryptococcal and coccidioidal meningitis).
Clin Infect Dis 2008; 46: 1813–21.
• Although still far from being perfect or even readily available,
molecular-based diagnostics and other non-culture based
approaches have aided in advancing such capabilities
Future Microbiol, (2016).2217/fmb.15.138

4. Estimation of fungal infection in Malaysia
• The estimated incidence and prevalence of certain serious fungal
infections in Malaysia is approximately 2% of the population, equating
to 590,214 of the total burden
• The actual cases of serious fungal infection may be higher than the
current estimation, very little data on allergic fungal sinusitis, tinea
capitis, histoplasmosis in non-AIDS patients, mucormycosis,
sporotrichosis, mycetoma, chromoblastomycosis and
phaeohyphomycosis
• A national reporting system on fungal infections and a fungal
surveillance system should be implemented and improved to
ascertain the impact of serious fungal infections

5. J. Fungi 2018, 4, 38

6. • In immunocompromised patients, 10% to 25% with pulmonary
aspergillosis later developed CNS involvement
• Among the 306 reports and 6514 cases about cryptococcal
meningitis, there were 21% patients with HIV infection, 39% with
other underlying diseases and 40% without identifiable underlying
diseases.
• Main underlying diseases in non-HIV/AIDS patients cryptococcal
meningitis were tuberculosis (10.46%), liver disease (9.83%), systemic
lupus erythematosus (7.00%), diabetes (5.26%), kidney disease
(3.16%), lung disease (2.97%), and cancer (2.01%)
Abstracts / International Journal of Infectious Diseases 73S (2018) 3–398

7. Lancet Neurol 2018; 17: 362–72

8. Clinical features of CNS fungal infection
• Symptoms are non-specific, and
even patients with disseminated
fungal infection with multi-organ
involvement may not present
with organ-specific changes or
clinical signs.
• Neurologic complications
develop 50% of all patients
diagnosed with a fungal CNS
infection
J Microbiol Exp. 2017;5(6):14‒12.

9. Curr Opin Infect Dis 2019, 32:277–284

10. J Microbiol Exp. 2017;5(6):14‒12.

11. J Microbiol Exp. 2017;5(6):14‒12.

12. Approach of CNS fungal infection laboratory diagnosis

15. CSF examination : CSF cell count
• The classical finding for fungal meningitis is that of
a lymphocytic or monocytic pleocytosis.
• Aspergillus spp. and Blastomyces typically cause a
neutrophilic pleocytosis
• An eosinophilic predominance, should raise
suspicion of Coccidioides immitis as pathogen,
though CSF eosinophilia has rarely been reported
• Intra-parenchymal lesions may be associated with
no CSF abnormalities.

16. AFP, VOLUME 68, NUMBER 6 / SEPTEMBER 15, 200

17. Am. J. Trop. Med. Hyg., 103(4), 2020

20. Microscopic examination
• Direct microscopy has been an
important diagnostic
compliment to obtaining a
fungal culture and can often be
used to make a preliminary
diagnosis
• Important to identify yeast or
molds [ mucorales] based on
hyphae, conidia shape and
arrangement

22. Gram stain/ Fungal stain
• The Gram's stain is insensitive in
most cases of fungal meningitis
• Staining methods for CSF are less
helpful in the setting of fungal
foreign body infection and
identification of a fungal
pathogen in the setting of a focal
CNS lesion

23. Gram stain versus Indian ink
• Cryptococcus neoformans meningitis in AIDS patients, relatively high
organism burden leads to a much higher concentration of yeast in the
CSF, making detection on Gram's stain or Indian ink possible
• Dunbar et al reported that the Gram stain had a high sensitivity of
85.2% on 27 culture positive cases of cryptoccocal meningitis.
• In addition, Sato et al confirmed that the Gram stain was more
sensitive than the India ink, with sensitivities of 81% and 44%
respectively in 16 culture positive cases of cryptococcal meningitis.
• India ink has a sensitivity of only 50%, but it is highly diagnostic if
positive.
South Afr J Infect Dis 2015;30(2)

24. Gram stain versus Indian ink

25. Fungal culture
• A diagnosis of any invasive fungal infection is
commonly regarded as definite in patients with
positive cultures from sterile sites or through
demonstration of fungal tissue invasion
• Cultures should be incubated for at least 5 days and
for up to 3 weeks as most fungi grow slowly
• CSF cultures are frequently non-diagnostic,
especially in patients with a fungal brain abscess,
and a biopsy might be required to establish the
diagnosis.

26. Fungal culture
• Fungal cultures are positive in more than 95% of Cryptococcus
neoformans cases and in 66% of candidal meningitis cases. Other
fungi are less likely to be culture positive
• Similar to tuberculous meningitis, culture yield in fungal meningitis
can be increased by obtaining large volumes of CSF via repeated
lumbar punctures
Infect Dis Clin North Am. 1990;4:789–808.
Infect Dis Clin North Am. 1990;4:769–87.

27. • O'Brien et al. (2011) reported 11 cases of Candida CSF infection after
neurosurgery over a 12-year period.
• In total, 73% of the isolates were C. albicans and 73% of the patients
had antecedent bacterial meningitis.
• All infections were associated with foreign intracranial material,
including EVD, VPS and lumbar drain with a mortality rate of 27%
Acta Neurochirurgica 153, 1347–1350

28. • Of 1,225 samples of CSF reviewed, 12 tested
positive for fungi, either by bacterial
culture, fungal culture, or the cryptococcal
antigen test.
• Of cultures from these 12 samples, 10
fungal cultures were positive and 8 bacterial
cultures were positive.
• Cryptococcus neoformans was found in 10
of the specimens, Candida albicans was
found in 1, and a Cladosporium sp. was
found in 1

29. Serological testing
• Serologic tests for the detection of antibodies have been useful for
non-culture-based diagnosis of fungal infection since the 1950s.
• Available technologies include immunodiffusion (ID), complement
fixation (CF), and enzyme immunoassay (EIA).
• Lateral flow devices for specific antibodies are being developed and
validated

31. Mannan
• Mannan is a major component of the yeast cell wall,
and tests developed specifically to detect Candida
infection
• There are two factors:
1. Candida spp. are common human commensals
and heavy colonisation can cause positive
results in serum
2. Candida mannan is rapidly cleared from the
circulation, so frequent testing is important.

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33. Mikulska et al. Critical Care 2010, 14:R222

34. Mannan and Anti-mannan
• The sensitivity of both Mn and A-Mn varied for different Candida
species, and it was the highest for C. albicans, followed by C. glabrata
and C. tropicalis
• A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI,
79-97) and DOR, 12 (95% CI 7-21).
• Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity,
86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122).
• Mn and A-Mn are useful for diagnosis of IC, the performance of
combined Mn/A-Mn testing is superior to either Mn or A-Mn testing
Mikulska et al. Critical Care 2010, 14:R222

36. Candida albicans germ tube antibodies (CAGTA)
• C. albicans germ tube antigen (CAGTA) test; the antigen was originally
found in C. albicans, but the test also detects candidiasis with
other Candida spp., although it detects some non-
albicans Candida spp., including C. parapsilosis, with lower sensitivity
• The experience with CAGTA is more limited than that
with Candida spp. mannan antigen and antimannan antibody and β-
D-glucan tests.
• The sensitivity ranges from 53.3% to 74.1%, and specificity ranges
from 56.5% to 92.0%

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39. Capsular polysaccharide antigen
(Cryptococcal antigen)
• Cryptococcus is a neurotropic fungus; polysaccharide serum antigen
titers in conjunction with host immune status are often used as a
diagnostic aid to determine need for lumbar puncture to evaluate
patient for CNS involvement.
• The baseline peak titer of polysaccharide antigen in serum or CSF has
demonstrated important prognostic significance with higher titer
(peak titer >1:1024) associated with antifungal therapy failure
• In a single report that examined patients >35 years of age with CNS
cryptococosis, an overall sensitivity and specificity of 93∼100% and
93∼98 %, respectively, were reported

40. • A total of 29 non-human immunodeficiency virus 1 cryptococcal meningitis cases, titer changes
in the latex agglutination test before and after therapy were reviewed along with clinical
manifestations, laboratory findings, and therapy regimens.
• The cryptococcal antigen titer decreased for every case after therapy and was correlated to
fungal clearance as defined by fungus smear and/or culture.
• However, cryptococcal antigen can remain at low titers for long periods of time after therapy,
even when fungus smears and/or cultures become negative.
JCM, June 2005, p. 2989–2990

42. J Clin Microbiol 57:e01238-18.

44. Galactomannan (GM)
• Galactomannan is a polysaccharide that is a major
constituent of Aspergillus cell walls.
• It is a soluble, heat stable antigen released by
especially Aspergillus species during active cell
replication (during hyphal growth).
• Circulating GM can be detected in serum or plasma
and may be present in BAL- fluid, cerebrospinal fluid
(CSF) or other body fluids

45. Galactomannan (GM)
• The GM EIA is performed with an optical read-out that is interpreted
as a ratio relative to the optical density (OD) of a threshold control
provided by the manufacturer; this ratio is called the OD index.
• The FDA has a suggested threshold OD index of 0.5 and an OD index
≥0.5 is generally considered to be a positive result.
• GM antigen has become an important diagnostic tool in the
management of patients at risk for IA.
• GM can be detected in the serum in some patients before the
presence of clinical signs or symptoms of IA.

46. Galactomannan (GM)
• A single positive GM index of ≥0.7 or two consecutive samples of ≥0.5
should prompt a diagnostic work-up
• Persistent GM antigenemia during therapy is a poor prognostic sign
and should prompt clinical reassessment.
• The sensitivity of GM testing varies in the range of 30–100%, while
specificity has been reported to be >75%
• A meta-analysis showed the efficacy of the GM test in case of proven
IA had an overall sensitivity and specifity of 71% and 89% respectively
J Immunol Sci. (2018); 2(5): 38-42

47. J Immunol Sci. (2018); 2(5): 38-42
• GM can also be detected in cerebrospinal fluid (CSF), pleural fluid, sputum
or urine, the experience with GM detection in these specimens are
reported to be insufficient so there are no enough specific
recommendations
Bone Marrow Transplantation. 2012; 47: 846–854.

48. J. Fungi 2016, 2, 22

49. False positivity of galactomannan
• False positivity of GM assay may occur by numerous factors.
• GM is found in many fungi and bacteria and can cause false positive
results.
• Cross-reactivity with some beta-lactam antibiotics (piperacillin-
tazobactam, amoxicillin- clavulanate) and with GM from other fungal
species (Fusarium, Penicillium, Cladosporium, Histoplasma,
Blastomyces, Paracoccidioides, Cryptococcus, Nigrospora,
Paecilomyces, Trichothecium, Lichtheimia ramosa, and Geotrichum)

50. False positivity of galactomannan
• False-positive results are more likely to occur during the first 100 days
following HCT and in patients with gastrointestinal tract mucositis
caused by chemotherapy or graft-versus-host disease (GVHD)
• In neonates and infants, immaturity of the intestinal mucosa may lead
to the translocation of lipoglycans of Bifidobacterium, resulting in
false-positive GM antigenemia

51. GM detection in CSF showed a good diagnostic performance when an ODI cutoff of 0.5 to
2.0 was used, and using GM in CSF, CA can be diagnosed or virtually ruled out without
the need for cerebral biopsy
J Clin Microbiol 54:428–431 (2016).

53. β-D-glucan
• The diverse BDG tests detect 1,3 β-D-glucan, which is the major cell
wall component of most fungal species, with the exception of fungi
of the subdivision Mucoromycotina, Cryptococcus spp. and some
other Basidiomycota (e.g., Malassezia spp.) that contain less 1,3 β-
D-glucan in their cell wall and are usually not detected by these
tests.
• Thus, BDG assay is not specific for the diagnosis of invasive
aspergillosis and is also used for the diagnosis of invasive
candidiasis.

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55. β-D-glucan
• In a case series 78 of patients with iatrogenic meningitis caused by E.
rostratum, β-D-glucan testing of CSF from 41 patients with confirmed
fungal meningitis and 66 control patients had 100% sensitivity and
98% specificity
• β-D-glucan concentrations in CSF could be useful in monitoring
therapeutic response,in a patient with aspergillus ventriculitis
• The guidelines of the IDSA for the management of candidiasis and
aspergillosis recommend serum (1-3)-β-D-glucan testing to assist in
the assessment of patients with suspected deep-seated fungal
infections but offer no additional recommendations for the specific
use of the assay

56. Interpretation of β-D-glucan
• The Fungitell assay should be used in conjunction with other
diagnostic procedures, such as routine bacterial/fungal cultures,
histologic examination of biopsy material and radiologic studies.
• Positive: (1,3)-Beta-D-glucan detected. [>60pg/ml]
• A single positive result should be interpreted with caution and
correlated alongside consideration of patient risk for invasive fungal
disease, results of routine laboratory tests (eg, bacterial and fungal
culture, histopathologic evaluation) and radiologic findings.
• Repeat testing on a new sample (collected in 3-4 days) is
recommended as serially positive samples are associated with a
higher diagnostic odds ratio for invasive fungal infection compared to
a single positive result.

57. • False-positive results may occur in patients who have recently (in the
past 3-4 days) undergone hemodialysis, treatment with certain
fractionated blood products (eg, serum albumin, immunoglobulins),
or those who have had significant exposure to glucan-containing
gauze during surgery.
• Indeterminate: Repeat testing on a new sample is recommended in
patients at risk for an invasive fungal infection.
• Negative: No (1,3)-Beta-D-glucan detected. [<60 pg/mL]
Mayo Clinic Laboratories

58. JCM , April 2013 Volume 51 Number 4

60. Molecular method
• Molecular techniques such as PCR could facilitate diagnostic
confirmation, and sequencing of fungal DNA can enable both
identification of the infecting fungus and detection of drug
resistance
• Most molecular tests are not standardized and the fast growing
genomic knowledge about fungi requires regular curation of
sequence data
• In term of practice, PCR diagnosis either by direct detection from
clinical sample or identification of fungi from the fungal colony aid in
laboratory diagnosis

61. Molecular method
• The use of PCR-based assays, DNA sequencing, and other molecular
methods, including those incorporating proteomic approaches such
as matrix assisted laser desorption ionization time of flight mass
spectroscopy (MAL DI-TOF MS) have shown promising results to aid in
accurate species identification of fungal cultures
• Molecular-based fungal identification is particularly helpful for fungi
that lack distinguishing morphological features, e.g. Apophysomyces
elegans, or to distinguish between species of the Aspergillus
fumigatus complex
• Comparative sequence analysis is now the ‘gold standard’ for
identification of fungi

64. • Regardless of the genetic locus selected, accurate sequence-based
identification is dependent upon database accuracy and adequate
species representation.
• GenBank is well known to contain numerous errors in sequences and
the species names attributed to the sequences, which are rarely
corrected.
• Therefore caution must be used when interpreting sequencing
comparisons against this database, and the use of multiple sequence
databases is encouraged

65. J. Nat. Prod. 2017, 80, 756−770
Curation - the selection, organization, and presentation of
online content, merchandise, information, etc., typically using
professional or expert knowledge.

66. Journal of Microbiological Methods 103 (2014) 80–100

68. A Moldy Application of MALDI: MALDI-ToF Mass
Spectrometry for Fungal Identification
• As a result of its being inexpensive, easy to perform, fast and accurate,
matrix-assisted laser desorption ionization time-of-flight mass spectrometry
(MALDI-ToF MS) is quickly becoming the standard means of bacterial and
fungal identification from cultures in clinical microbiology laboratories
• Yeasts and filamentous fungi claimed by the FDA cleared/approved versions
of at least one commercial MALDI-ToF MS system
• The Bruker and bioMérieux systems are different not just in databases, but
also in database matching and relatedness reporting strategies.
• In most comparative studies, performance of the two has been similar,
though not identical, assuming that the specific species being studied are
represented in both databases

71. MALDI TOFF limitations
• Growth on some media may yield low scores/percentages , and small
or mucoid colonies may fail.
• Using experimental capsule size manipulation, it was demonstrated
that capsule size of C. neoformans and C. gattii can compromise
identification by the Bruker system
• Mistakes that may occur include testing mixed colonies, spreading
amongst spots, spotting into incorrect target plate positions, not
properly cleaning re-usable target plates

72. Molecular method
• In CNS aspergillosis, PCR testing of CSF had a sensitivity of 100% and
a specificity of 93%
• PCR positivity rates among patients with proven or probable invasive
candidiasis were 85% (78 to 91%), while blood cultures were positive
for 38% (29 to 46%).
• The main advantage is that PCR-based methods allow quicker and
earlier diagnosis, which can lead to an early initiation of therapy and
therefore lower mortality
• Further, taxa identification based on DNA-sequences is more accurate
than identification based on morphology

73. Pneumocystis jiroveci CNS infections
• Pneumocystis jiroveci infections may extend beyond the lungs, and
extrapulmonary pneumocystosis has been described in 0.5% to 2.5%
of HIV-positive patients
• A total of 109 HIV positive patients with confirmed extrapulmonary
pneumocystosis were identified, and 7 of these patients had central
nervous system (CNS) pneumocystosis.
• All 7 cases were diagnosed after death, during necropsy and three
had a history of PJ pneumonia
Journal of the International Association of Physicians
in AIDS Care 11(3) 169-171

74. • Due to difficulties with isolating and culturing this
pathogen, microscopic examination of respiratory
specimens using various staining methods is used
to visualise and identify the morphological
structures of P. jirovecii.
• Giemsa stains detect all life stages of P. jirovecii.
• Methenamine silver and toluidine blue
preparations stain only the cyst wall and do not
allow detection of trophozoites

75. • Direct and indirect immunofluorescent
assays (DFA, IFA) are specific for different
life stages, depending on the antibody used.
• Comparative studies have shown DFA and
IFA to be the most sensitive stains for P.
jirovecii in sputum and bronchoalveolar
lavage, with sensitivities of 97% and 90%
respectively
Internal Medicine Journal 44 (2014)

76. Pneumocystis jiroveci PCR
• 302 samples were included (182 bronchoalveolar lavage, 67 sputum,
53 tracheal aspirates).
• PJ-PCR was positive in 51 (16.9%) and IFA in 11 (3.6%) of the patients
with PJP.
• There were not IFA positive/PCR negative samples.
• Sensitivity and specificity, IFA were 26% (95%CI 15.9-39.6%) and
100% (95%CI 98.5-100%)
• Whereas, sensitivity and specificity for PCR was 92% (95%CI 81.2-
96.8%), 98% (95% CI 95.4-99.2

77. • PJ-PCR had sensitivity > 80% and specificity > 90% in all type of
samples included.
• A definitive diagnosis of PJP was considered in 50 (16.6%) patients,
including 4 (1.3%) cases with negative PJ-PCR.
• Five cases (9.8%) with positive PJ-PCR were considered as colonization

82. Conclusion
• The diagnostic methods for CNS fungal infections have lagged behind
those for other infective agents
• The relative rarity of fungal infections of the CNS and in part it is due
to the lack of a gold standard against which to calibrate or verify
studies of new tests
• It is anticipated that there will be further evolution of molecular
diagnostic methods, particularly PCR, leading to the more rapid and
more sensitive detection of fungal pathogens in the CNS.

83. Thank you