New Approaches to Cryptococcal Meningitis
New Approaches to Cryptococcal Meningitis
The diagnosis of cryptococcal disease can be made by direct visualization, histopathology, culture, and or detection of cryptococcal antigens in blood, CSF, and urine. Cryptococcus exists in the blood, CSF, and tissues as a single-celled organism with a characteristic polysaccharide capsule. Microscopic identification of the organism can be done using India ink staining or observation with a phase-contrast microscope. India ink staining has been the traditional method for identifying Cryptococcus organisms, particularly in resource limited settings. India ink stains the surrounding material blue, but not the fungal capsule, giving a characteristic "starry night" appearance. The sensitivity and specificity of India ink staining can be highly variable and often operator dependent, as lysed leukocytes can be mistaken for fungal elements. The organism can readily be cultured from most sites, using routine and automated culture systems.
Histopathologic diagnosis of Cryptococcus can be done with several readily available staining techniques. The Giemsa stain only partially stains the organisms and is not typically utilized. Hematoxylin and eosin does not stain the capsule and the yeast is only weakly stained. Gomori methamine silver stain binds to fungal aldehydes and stains the yeast a characteristic black color, without staining the capsule. Staining of the capsule can be done with mucicarmine or Alcian blue stains. The capsule takes up the red color of mucicarmine or the blue color of Alcian to facilitate visualization. In tissues, yeast are typically contained within granulomas, particularly in immunocompetent hosts. In immunocompetent individuals, the Cryptococci are taken up by macrophages and induce an inflammatory response with the formation of epitheloid granulomas with central giant cells and surrounding lymphocytes. These granulomas typically do not have caseous necrosis. In immunocompromised individuals, full granuloma formation may not occur, and pseudocysts develop that are filled with encapsulated yeasts and surrounded by macrophages and lymphocytes.
An evaluation of CSF parameters, such as cell counts, glucose, protein, and opening pressures, can aid in the diagnostic evaluation of cryptococcal meningoencephalopathy. In a case series of 40 patients with cryptococcal meningitis in the setting of diabetes, malignancies, sarcoidosis, and other rheumatologic disease, most patients presented with abnormal CSF parameters. Abnormal CSF cell counts were present in 97%. Cell counts ranged from 6 to 808 cells/mm, with lymphocytes accounting for 8 to 100% of CSF white cells. Protein elevation was present in 90%, and a low CSF glucose in 55%. The opening pressure was elevated in 64%. In patients presenting with cryptococcal meningoencephalitis in the setting of advanced HIV infection, the classic findings of an elevated CSF white cell count, elevated protein, and low glucose are not always evident (Table 2).
Detection of capsular antigen is the most reliable diagnostic tool for cryptococcosis. Cryptococcal antigen can be detected in serum, CSF, and urine specimens. Detection of capsular antigen can be done by latex agglutination (LA) assays, enzyme immunoassays (EIAs), or the novel lateral flow assay (LFA). The latex agglutination assay has been used for several decades for the detection of cryptococcal antigen and has a higher sensitivity and specificity than India ink staining. Latex particles are coated with anticryptococcal antibodies, and in the presence of cryptococcal antigen will agglutinate, forming visible clumps that are subjectively read on a predefined scale. The sensitivity and specificity of the LA tests vary with manufacturer and the use of pronase. A study comparing four commercially available LA tests and an EIA test to culture found a serum sensitivity of LA that ranged from 83 to 97%. The tests with the lowest sensitivity did not use pronase on serum specimens. The specificity of the LA on serum ranged from 93 to 100%. The CSF sensitivity and specificity of the LA test was high and ranged from 93 to 100%, while the specificity range was 93 to 98%. The sensitivity and specificity on EIA testing of CSF was 100% and 98%, respectively. The sensitivity and specificity of the EIA on serum was 93% and 96%, respectively. Although the LA performs well when compared with EIA and culture, its primary limitation is that it is a cumbersome manual test with subjectivity in the interpretation of the result. The test also requires laboratory equipment and refrigeration of reagents, making it unsuitable for use in remote resource limited settings.
In 2009, a rapid point of care diagnostic test known as the lateral flow assay (LFA) was developed. The Cryptococcal antigen (CrAg) LFA is a capillary flow sandwich immunochromographic assay that can be done as a point of care test. Gold-conjugated anticryptococcal monoclonal antibodies and control goat IgG antibodies are deposited on a membrane on the test strip. Specimen diluent is added to a tube to which 40 μL of the patient specimen is added. The test strip is added to the tube and incubated at room temperature for 10 minutes before the result is read. The specimen migrates by capillary flow up the test strip, and in the presence of cryptococcal antigen will bind to the anticryptococcal monoclonal antibodies. The bound antibodies will continue to flow up the dipstick to the detection lines. The first detection line contains immobilized anticryptococcal monoclonal antibodies, and the distal line contains immobilized bovine antigoat IgG antibodies. A gold-conjugated anticryptococcal antibody that is bound to cryptococcal antigen will bind to the first detection line containing anticryptococcal antibody, creating a sandwich that is detected as a visible line at the test line site. The control goat IgG antibodies will continue to migrate up the test strip and bind to the bovine antigoat IgG antibodies and be detected as a visible line in the control test line. A positive test is indicated by the presence of two lines (the test line and the control line); a negative test is indicated by the presence of the control line only. An invalid test is indicated by the absence of a control line. The sensitivity and specificity of the lateral flow assay is very high, with high levels of concordance with EIA and latex agglutination assays. In a study using archived specimens in Thailand, the LFA was positive in all blood culture positive specimens and had strong concordance with the EIA on testing of serum. The LFA has also been shown to have high sensitivity in detection of all serotypes (A–D). The test has been approved by the Food and Drug Administration for use on serum and CSF.
The introduction of the lateral flow assay has the potential to revolutionize cryptococcal diagnosis in resource limited settings. It is as simple to perform as a urine pregnancy test, and therefore does not require trained laboratory personnel. It can be performed in the field and does not require sophisticated laboratory equipment, refrigeration and/or centrifugation.
Diagnosis of Cryptococcal Meningitis
The diagnosis of cryptococcal disease can be made by direct visualization, histopathology, culture, and or detection of cryptococcal antigens in blood, CSF, and urine. Cryptococcus exists in the blood, CSF, and tissues as a single-celled organism with a characteristic polysaccharide capsule. Microscopic identification of the organism can be done using India ink staining or observation with a phase-contrast microscope. India ink staining has been the traditional method for identifying Cryptococcus organisms, particularly in resource limited settings. India ink stains the surrounding material blue, but not the fungal capsule, giving a characteristic "starry night" appearance. The sensitivity and specificity of India ink staining can be highly variable and often operator dependent, as lysed leukocytes can be mistaken for fungal elements. The organism can readily be cultured from most sites, using routine and automated culture systems.
Histopathologic diagnosis of Cryptococcus can be done with several readily available staining techniques. The Giemsa stain only partially stains the organisms and is not typically utilized. Hematoxylin and eosin does not stain the capsule and the yeast is only weakly stained. Gomori methamine silver stain binds to fungal aldehydes and stains the yeast a characteristic black color, without staining the capsule. Staining of the capsule can be done with mucicarmine or Alcian blue stains. The capsule takes up the red color of mucicarmine or the blue color of Alcian to facilitate visualization. In tissues, yeast are typically contained within granulomas, particularly in immunocompetent hosts. In immunocompetent individuals, the Cryptococci are taken up by macrophages and induce an inflammatory response with the formation of epitheloid granulomas with central giant cells and surrounding lymphocytes. These granulomas typically do not have caseous necrosis. In immunocompromised individuals, full granuloma formation may not occur, and pseudocysts develop that are filled with encapsulated yeasts and surrounded by macrophages and lymphocytes.
An evaluation of CSF parameters, such as cell counts, glucose, protein, and opening pressures, can aid in the diagnostic evaluation of cryptococcal meningoencephalopathy. In a case series of 40 patients with cryptococcal meningitis in the setting of diabetes, malignancies, sarcoidosis, and other rheumatologic disease, most patients presented with abnormal CSF parameters. Abnormal CSF cell counts were present in 97%. Cell counts ranged from 6 to 808 cells/mm, with lymphocytes accounting for 8 to 100% of CSF white cells. Protein elevation was present in 90%, and a low CSF glucose in 55%. The opening pressure was elevated in 64%. In patients presenting with cryptococcal meningoencephalitis in the setting of advanced HIV infection, the classic findings of an elevated CSF white cell count, elevated protein, and low glucose are not always evident (Table 2).
Detection of capsular antigen is the most reliable diagnostic tool for cryptococcosis. Cryptococcal antigen can be detected in serum, CSF, and urine specimens. Detection of capsular antigen can be done by latex agglutination (LA) assays, enzyme immunoassays (EIAs), or the novel lateral flow assay (LFA). The latex agglutination assay has been used for several decades for the detection of cryptococcal antigen and has a higher sensitivity and specificity than India ink staining. Latex particles are coated with anticryptococcal antibodies, and in the presence of cryptococcal antigen will agglutinate, forming visible clumps that are subjectively read on a predefined scale. The sensitivity and specificity of the LA tests vary with manufacturer and the use of pronase. A study comparing four commercially available LA tests and an EIA test to culture found a serum sensitivity of LA that ranged from 83 to 97%. The tests with the lowest sensitivity did not use pronase on serum specimens. The specificity of the LA on serum ranged from 93 to 100%. The CSF sensitivity and specificity of the LA test was high and ranged from 93 to 100%, while the specificity range was 93 to 98%. The sensitivity and specificity on EIA testing of CSF was 100% and 98%, respectively. The sensitivity and specificity of the EIA on serum was 93% and 96%, respectively. Although the LA performs well when compared with EIA and culture, its primary limitation is that it is a cumbersome manual test with subjectivity in the interpretation of the result. The test also requires laboratory equipment and refrigeration of reagents, making it unsuitable for use in remote resource limited settings.
In 2009, a rapid point of care diagnostic test known as the lateral flow assay (LFA) was developed. The Cryptococcal antigen (CrAg) LFA is a capillary flow sandwich immunochromographic assay that can be done as a point of care test. Gold-conjugated anticryptococcal monoclonal antibodies and control goat IgG antibodies are deposited on a membrane on the test strip. Specimen diluent is added to a tube to which 40 μL of the patient specimen is added. The test strip is added to the tube and incubated at room temperature for 10 minutes before the result is read. The specimen migrates by capillary flow up the test strip, and in the presence of cryptococcal antigen will bind to the anticryptococcal monoclonal antibodies. The bound antibodies will continue to flow up the dipstick to the detection lines. The first detection line contains immobilized anticryptococcal monoclonal antibodies, and the distal line contains immobilized bovine antigoat IgG antibodies. A gold-conjugated anticryptococcal antibody that is bound to cryptococcal antigen will bind to the first detection line containing anticryptococcal antibody, creating a sandwich that is detected as a visible line at the test line site. The control goat IgG antibodies will continue to migrate up the test strip and bind to the bovine antigoat IgG antibodies and be detected as a visible line in the control test line. A positive test is indicated by the presence of two lines (the test line and the control line); a negative test is indicated by the presence of the control line only. An invalid test is indicated by the absence of a control line. The sensitivity and specificity of the lateral flow assay is very high, with high levels of concordance with EIA and latex agglutination assays. In a study using archived specimens in Thailand, the LFA was positive in all blood culture positive specimens and had strong concordance with the EIA on testing of serum. The LFA has also been shown to have high sensitivity in detection of all serotypes (A–D). The test has been approved by the Food and Drug Administration for use on serum and CSF.
The introduction of the lateral flow assay has the potential to revolutionize cryptococcal diagnosis in resource limited settings. It is as simple to perform as a urine pregnancy test, and therefore does not require trained laboratory personnel. It can be performed in the field and does not require sophisticated laboratory equipment, refrigeration and/or centrifugation.
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