With the ever increasing population of immune-suppressed individuals, the last two decades have witnessed a considerable increase in the number of fungal central nervous system (CNS) infections which have high morbidity and mortality. Early diagnosis is the key in starting the appropriate therapy, which varies according to the fungal species identified therefore adding to the urgency of accurate identification. With the available conventional methods which include direct microscopy and culture based methods the turnaround time is long and contamination may be a problem especially from colonized sites. In addition the identification of fungal species associated with invasive infections is complicated, due to the astonishing variety of moulds and yeasts capable of causing infections. The isolates cultured from deep sites show pleomorphism and do not produce structures by which they can be identified. Identification also requires considerable expertise and exposure to certain may be hazardous to laboratory personnel. All these above mentioned factors clubbed together result in delay in the initiation of appropriate antifungal treatment in patients resulting in higher morbidity and mortality. Molecular methods have revolutionized the diagnosis of infectious diseases including fungal infections. We review the available molecular methods for CNS fungal infections.
CNS fungal infections: The presentation
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The clinical syndromes produced by fungi in the CNS are determined by the CNS pathology produced by them. The spread to the CNS is either hematogenous or direct. Morphology of fungi plays the key role when the spread is hematogenous. Small sized yeast forms (Blastomyces, coccidioides, candida, Cryptococcus etc) tend to reach the smaller arterioles and the capillaries producing meningitis and subpial ischemic lesions where as the larger morphological forms as seen in aspergillus, zygomecetes, large pseudohyphae of candida species etc. occlude the medium and large sized vessels producing tissue necrosis which subsequently evolve into abscesses. The direct extension of these fungal infections into the CNS may be from colonized paranasal sinuses and ear canal which include the skull base and rhino-orbito-cerebral syndromes. The spinal cord may rarely be involved.
With the introduction of the latest molecular methods in the field of clinical mycology there has been a significant improvement in the diagnosis of invasive fungal infections including those involving the central nervous system. These include methods which identify fungal nucleic acids either directly from clinical samples or from the culture, with or without amplification. In addition for the diagnosis of CNS fungal infections, detection of fungal metabolites and antigen may be of considerable assistance.
Fungal nucleic acid based methods include those like fluorescent in situ hybridization (FISH) and the commercially available Accuprobes which have been used for the direct detection from tissue specimens and cultures respectively. The polymerase chain reaction (PCR) and its modifications including the real-time PCR are one of the most useful tools in molecular mycology today. These not only allow direct identification of the organism from clinical samples but also help in reducing the turnaround time and are safe while dealing with hazardous fungal pathogens. DNA sequencing is increasingly being utilised by the clinical laboratories for the accurate identification of the fungal pathogen. Fungal metabolites like the galactomannan, beta-glucan, and cryptococal capsular antigen detection are one of the most important fungal antigens of diagnostic importance.
Nucleic Acid Hybridization Assays
Nucleic acid hybridization techniques have been available since the early 1990's when these were first used in identification of dimorphic fungi mostly from culture isolates with the exception of a few case reports.1 The hybridization probes require growth of the fungus in pure culture to achieve their reported sensitivities. Culture is required because there is no PCR step used that would amplify the target. Fluorescence in situ hybridisation (FISH) is an appropriate technique for the rapid and cultivation independent identification of fungal pathogens. 2
Hayden: In situ hybridization (ISH), most commonly used in infectious disease surgical pathology for the diagnosis of viral infections in tissue,3 has also proven useful for the identification of fungal agents, including Candida, Aspergillus, Mucor, Pneumocystis,4, 5 as well as dimorphic fungi.6 ISH offers rapid turnaround time, limited cost, and the potential for automation, together with a high degree of specificity. The sensitivity of these techniques has been markedly enhanced by the use of various signal amplification methods that can detect just a few copies of target sequence.7
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FISH has been successfully applied to the detection of C.neoformans cells in cerebrospinal samples from patients with clinical diagnosis of cryptococcosis.8 Martin et al.2 in their study described the development of fluorescently labelled rRNA-targeted oligonucleotides and a FISH assay to detect and identify Cryptococcus neoformans in culture and biological samples. Two 26S rRNA-targeted species-specific oligonucleotide probes were designed for C.neoformans, which are also complementary to the closely related C.gattii: Cne448 and Cne205.2 FISH has also been used to detect and identify fungal organisms, including clinically relevant yeasts such as Candida species.9
Polymerase Chain reaction (PCR)
Atkins review: The biological amplification, ie, growth in culture, has been replaced by enzymatic amplification of specific nucleic acid sequences. PCR in vitro amplification technique that was developed during the early 1980s and is a basic part of fungal molecular diagnostics. Conventional PCR is qualitative and has been used to detect fungi from a whole range of samples and is the core of fungal molecular diagnostics. Related technologies include methods that increase sensitivity such as nested PCR in which a second round using a separate primer set internal to the first round increases amplification of a specific region in the first PCR amplified target gene. This is more specific and sensitive allowing the detection of the target DNA several fold lower than conventional PCR. Reverse transcription PCR (rt PCR) exploits the use of the enzyme reverse transcriptase to convert RNA to cDNA before PCR amplification. This method enables gene activity to be investigated and is an important step forward in understanding gene function and activity. Detection of several different fungal isolates in the same PCR reaction can be achieved if the highly specific primers are designed to anneal at the same temperature and the PCR products are designed to be of different sizes to allow discrimination. This is known as multiplex PCR. A broad-range PCR uses conserved sequences within phylogenetically informative genetic targets to diagnose microbial infection.
A less time consuming process involves the use of real-time PCR. During real-time PCR, the accumulation of PCR products is measured automatically during each cycle in a closed tube format using an integrated cycler/fluorimeter. Direct measurement of the accumulated PCR product allows the phases of the reaction to be monitored. The initial amount of target DNA in the reaction can be related to a cycle threshold (ct) defined as the cycle number at which there isa statistically significant increase in fluorescence. Target DNA can then be quantified by construction of a calibration curve that relates ct to known amounts of template DNA. The real time PCR has short turnaround time.
Cryptococcus spp.10, 11
For the diagnosis of neurocryptococcosis the application of more sensitive and specific laboratorial techniques are necessary in order to introduce early and specific antifungal therapy. PCR offers a good alternative11. It constitutes a method of choice for early alternative diagnosis to the conventional ones and contributes to supply important subsidies to the diagnosis of this pathology mainly when there is clinical suspicion of the disease12.
PCR assays for the detection of C. neoformans DNA in clinical specimens have been described, targeting 18S, 28S, or the ITS and 5.8S ribosomal DNA (rDNA) (5, 9, 11, 12). The detection limits reported are of 1 to 10 cells mlâˆ’1 or per volume used for DNA extraction (5, 11, 12). A number of DNA extraction procedures have been published for efficient disruption of cryptococcal cells, including enzyme digestion or glass beads (17). The PCR assays show cross-reactions with some fungal species related to C. neoformans . Some of these can be pathogenic, and their detection is beneficial. Multiplex PCR has been standardised by Leal et al to help differentiate between various species of Cryptococcus.12
Nested PCR is a sensitive, specific, and reproducible technique and represents a promising method to be used in the analysis of CSF samples from patients suspected of having cryptococcal meningitis. Moreover, since in the course of treatment of cryptococcosis the duration of therapy is still controversial, usually depending on the time needed for clearance of cryptococcal antigen from the CSF as demonstrated by the latex agglutination test, the nested PCR may be a useful tool not only for the rapid diagnosis of acute cryptococcosis but also for monitoring during therapy the clearance of the parasite from patients in follow-up exams.13
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To obtain a positive PCR result for aspergillus in csf, either fungal cells or fungal DNA should be present in the CSF sample. The number of cells present in a CSF specimen is extremely low, and the rate of clearance of DNA from the CSF is unknown. Therefore, even if the PCR assay is very sensitive, the assay may not be positive because fungal cells or fungal DNA is not present in the CSF sample.
In a study by Hummel et al15 Aspergillus DNA was detected in samples from six of six patients with cerebral aspergillosis by nested PCR assay. In all patients, samples that were obtained at the initial manifestation of cerebral aspergillosis showed a positive PCR result. In patients with serial sampling of CSF, follow-up samples obtained during antifungal treatment and after clinical improvement were negative. In five patients, detection of Aspergillus DNA from CSF samples was the only positive microbiologic finding in the CSF. Culture and direct microscopy, as well as galactomannan ELISA, were negative for any pathogen. With the exception ofCryptococcus neoformans, fungi are rarely detected in CSF obtained from patients having or suspected of having fungal meningitis (18).
So far, reports of Aspergillus PCR with CSF samples are few(10, 11, 19, 25) and consist mostly of single-case reports. Kamiet al. investigated one CSF sample each in five patients withcerebral aspergillosis and found positive Aspergillus PCR results(10). Verweij et al. investigated 26 serial CSF samples from a patient with proven cerebral aspergillosis and detectedAspergillus DNA in 4 of 26 samples (25)
Candida and other yeasts
Determination by polymerase chain reaction (PCR) of Candida genetic material in CSF does seem to be effective (Ralph & Hussain, 1996).
DNA sequencing based identification of fungi
Within the past decade, the amplification and sequencing of specific fungal nucleic acid targets has evolved from being primarily a research application to become a valuable clinical diagnostic tool. 73Whenattempting to identify microorganisms using DNA sequencing, the gene targeted must contain highly conserved regions that can serve as primer binding sites, and these conserved regions should flank regions with enough sequence variability to allow for discrimination to the genus or species level. Furthermore, the target gene should be present at high copy numbers whenever possible to increase the sensitivity of the PCR amplification prior to sequence analysis.
The targets most commonly used for fungi are the ITS1 and ITS2 regions between the 18S and 28S ribosomal subunits and an w600 base-pair region of the D1-D2 region of the 25-28S large ribosomal subunit. In some instances, these regions may not provide sufficient variability between fungal genera or species, and alternate targets have been used, such as the elongationfactor-1a for Fusarium spp or b-tubulin for Phaeoacremonium spp.75,76 Continuously changing fungal taxonomy, limitations of the currently available fungal sequence libraries, and lack of a consensus agreement on the percent identity score needed to identify specimens to the genus or species level make fungal identification using sequence analysis somewhat less robust.
Detection of fungal metabolites and antigens in CNS infections
CRYPTOCOCCUS CAPSULAR ANTIGEN
The most commonly used of the fungal antigens is the cryptococcal capsular antigen for the diagnosis of cryptococcal meningitis. Glucuronoxylomannan detection in body fluids by rapid and simple latex agglutination tests or enzyme immunoassay has a sensitivity >90% and, at a titre of >1:4, is very specific. In addition to serum and CSF, urine and bronchoalveolar lavage fluid may be used. In asymptomatic HIV-infected patients, serum antigenaemia identifies early cryptococcal disease, requiring CSF examination and treatment.16 High initial CSF titres (â‰¥1:1024) correlate with a high organism burden by quantitative culture and is considered as a poor prognostic marker. CSF antigen titres fall with successful treatment, but are of little value in management.17 In general, the sensitivity and specificity of commercially available latex-agglutination tests is very high. False-positive cases related to rheumatoid factor,18 infections with Stomatococcus mucilaginosus,19 Â TrichosporonÂ spp.20 as this organism produces the same polysaccharide in its capsule as is produced by the Cryptococcus spp. Infection due toÂ Capnocytophaga canimorsusÂ (formerly known as DF-2).21 Contamination during pipetting in the laboratory. Hydroxyethyly starch (HES) for intravascular volume replacement (fluid resucitation).22
Galactomannan detection is used increasingly for rapid diagnosis of aspergillosis.23 Evaluation of diagnostic tests in aspergillosis has been difficult because of the insensitivity and nonspecificity of direct examination and culture of neural specimens. In the absence of autopsy findings, proof of the diagnosis by tissue biopsy is extremely difficult. The central nervous system (CNS) is often involved in patients with disseminated aspergillosis, and antigen may be detected in cerebral spinal fluid (CSF). However, antigen was also detected in CSF from antigenemic patients without CNS involvement, presumably due to entry of blood intothe CSF caused by a traumatic lumbar puncture. Detection of antigen in the CSF in the absence of antigenemia would provide compelling evidence of CNS aspergillosis in the appropriate clinical setting.14, 24-28
False-positive results may occur in patients receiving the antibiotics piperacillin, amoxicillin, or ticarcillin, alone or combined with a beta-lactamase inhibitor. These antibiotics are fermentation products of Penicillium spp., a mold that produces a cross-reactive galactomannan (GM). Intratracheal or intravenous administration of plasmalyte can also give false positive reaction. In patients who have infections caused by mold containing cross-reactive GM false positive results can occur e.g. Penicillium, Paecilomyces, Alternaria, Geotrichum, and a few others. False negative have been observed in patients on antifungal treatment.
(1/3)-b-D-Glucan (BG) is a cell wall component of Aspergillus and most other fungi.29 BG activates factor G of the horseshoe crab coagulation cascade, leading to production of a chromogenic substance. 30 Some investigators recommend requirement of consecutive positive results to improve specificity, at the expense of sensitivity. Some investigators
recommend requirement of consecutive positive results to improve specificity, at the expense of sensitivity.31 The sensitivity for diagnosis of aspergillosis has ranged from 50% to 100%, and specificity ranges from 44% to 98%.32 A few studies have compared the BG and GM test and have shown similar33 or reduced sensitivity32 for the BG assay. The BG assay is not specific for aspergillosis. Positive results also occur in patients who have candidiasis,34 endemic mycoses,35 cryptococcosis etc.
Petraitiene et al.29 experimentally determined the utility of beta glucan as a marker for invasive cns fungal infection in the csf. The beta-glucan assay in CSF was significantly more sensitive than quantitative cultures of CSF in the rabbit model of candidal meningoencephalitis; beta-glucan was highly positive in all 25 animals. These data suggest that the release of cell wall carbohydrate fragments occurs more readily than that of whole organisms from microabscesses. In most cases of cns fungal infections serum or plasma beta glucan detection is taken as a surrogate marker for invasive infections. A combination of beta glucan and galactomannan have also been utilized.36
The introduction of the molecular methods described above into the clinical mycology laboratory has allowed for the sensitive, specific, and rapid detection of fungal pathogens. However, many fungi still require culture- and morphology based identification, and therefore opportunities exist for continued advancement in the field of fungal molecular diagnostics. Future studies will likely be directed at adapting existing technologies, including liquid bead (Luminex)-based (Luminex Corporation, Austin, Texas) and microarray-based methods to further enhance the speed, throughput, and accuracy of fungal diagnostic testing. Luminex suspension assays and microarrays permit the detection and identification of multiple infectious agents in a single test and their use in the diagnosis of several fungal infections