ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 6, No. 1 Copyright © 1976, Institute for Clinical Science

Laboratory Diagnosis of Parasitic and Fungal Diseases of the Central Nervous System THOMAS M. SODEMAN, M.D. and NANCY DOCK (ASCP-MT) Department of Pathology, The University of Michigan Medical Center, Ann Arbor, MI 48104

ABSTRACT This review is presented to bring attention to those fungal and parasitic organisms that have been associated with central nervous system (CNS) infection and to offer an approach for handling their laboratory diagnosis. Treatment of the cerebrospinal fluid (CSF) to yield best results on direct smear examination and cul­ ture are discussed. Culture procedures and methods to be done are given in chart form. Those immunologic tests useful in supporting the diagnosis of fungal or parasitic CNS infections are also included. Introduction recognition of the offending organism will Central nervous system (CNS) infections result from the clinical history and exami­ of parasitic organisms in the United States nation of a variety of specimen types. In the represent a minor problem. When present, case of fungal infections, cultural isolation is however, early recognition is essential if an usually more likely than with parasites. adequate therapeutic response is expected. For this reason, clinical microbiology labora­ Approach to Diagnosis of Parasitic tories should develop an approach for han­ Meningoencephalitis dling this less common diagnostic problem. In table I are listed the more common Involvement of the CNS with fungal parasites involving the CNS for which the organisms is an increasing diagnostic prob­ following approach is adapted. This list is lem as more immune disrupted patients are grouped into protozoan, platyhelminthes seen secondary to advancing malignancy or and with appropriate references. complex chemotherapeutic regimens. Most Those organisms likely to be acquired in the microbiology laboratories have within their United States are noted. The remaining armament diagnostic procedures for the organisms would present as imported cases more common opportunistic organisms. with a parasitic etiology suggested by their This review is presented to bring attention social history. With the exception of amoe­ to those fungal and parasitic organisms that bic meningoencephalitis, diagnosis of the have been associated with CNS infection primary infection would most probably de­ and to offer an approach for handling their velop from examination of blood, feces or laboratory diagnosis. Discussion will be other body fluids. limited primarily to examination of the In figure 1 is outlined a schematic ap­ cerebrospinal fluid (CSF) realizing, however, proach to handling CSF specimens when that in most parasitic and fungal infections, parasitic infections are considered. It is 47 4 8 SODEMAN AND DOCK

TABLE I concentration. Commonly, centrifugation Parasites Capable of Producing CNS Infections has been used at 2500 rpm for 10 minutes. The sediment and a small amount of fluid Protozoans should be kept for direct examination and ♦Primary Amebic Hartmannella2 ® culture. The supernate is retained for bio­ Naegleria1 3 *Entamoeba chemical and immunologic procedures. An *Toxoplasma gondii71* alternate approach is the adaption of the cy­ Plasmodium falciparum23 Trypanosoma cruzi18 tology filtration concentration procedure48 Trypanosoma gambiense77 Trypanosoma rhodesiense35 where CSF is aseptically passed through a 2 Platyhelminthes to 5 n pore size Nucleopore filter. If culture sp.12'®3 Fasciola hepatica1*^ is indicated, one half of the membrane may Paragonimus westermani1*5 be applied to media and the other half Heterophyes heterophyes2^ Metagonimus yokagawai2 examined unstained or stained by the Pa­ *Taenia solium ()9'58 Multiceps multiceps7 panicolaou procedure. The Nucleopore filter *Echinococcus granulosus65 provides a transparent medium. If pores Nematoda *Trichinella spiralis34 prove distracting, procedures are available *Strongyloides stercoralis Angiostrongylus cantonensis3'4 to eliminate their outline. *Ascaris 1umbricoides5 2 Since few parasites may be cultured in the * (visceral migrans)®® ^ routine laboratory, examination of the well Dipetalonema perstans1 prepared smear is most likely to yield results. Loa loa73 Adult forms, eggs or larva could potentially be found in CSF from all of the organisms ♦Infection encountered in the United States listed in table 1. Only in two infections, essential that as large a volume as possible of however, should organisms be routinely CSF be obtained. Quantities of 5 to 10 ml found; amoebic meningoencephalitis and are not unreasonable, if clinically possible, African trypanosomiasis. because of the variety of procedures to be Primary isolation from the sediment on conducted and the scarcity of organisms. culture media may be successful with proto­ The initial step in handling CSF is one of zoan organisms. Cultural procedures for helminths are complex and outside the

EXAMINATION FOR PARASITES realm of routine laboratories. In table II are listed the organisms in which culture tech­ niques may prove useful. The medias CEREBROSPINAL FLUID available and appropriate references are also listed. Techniques for protozoan and concentration helminth cultures have been reviewed by a number of authors.29'69,71,72 Cultural tech­ niques have proven most useful for primary

TABLE II

Readily Culturable Parasites

Ameba Entamoeba Axenic broth2® Monophasic® Diphasic11 ( Table 2 ) Hartmannela-Naegleria Tryptocase Sov20 Plain agarllf'*7 Figure 1. A schematic approach to handling cere­ Trypanosomes NNN^ brospinal fluid specimens when parasitic infections are Diphasic72 considered. LABORATORY DIAGNOSIS PARASITIC & FUNGAL MENINGOENCEPHALITIDES 4 9 amoebic meningoencephalitis resulting from testing, where useful, is also indicated. A either Naegleria or Hartmannella. Excellent review of the sérodiagnostic procedure is growth may be obtained in trypticase soy available in Faust et al29 and Kagan.41 Wall75 broth or agar and on plain agar with Entero- reported that experience with the use of bacter aerogenes or Escherichia coli as nu­ sérodiagnostic procedures with CSF suggests trient sources. In addition, intranasal or ce­ that the titers run ten fold lower than in sera. rebral injection of CSF into mice may be Parasitic CNS infections where immu­ used to isolate amoeba. Naegleria has a flag­ nologic procedures on CSF could prove ellated stage (which may be induced to form most useful are those caused by cestodes, by moisture) as well as an amoeboid form. Toxocara canis () and Trypanosomes are the only other group that amoebas. can be readily cultured. However, most The preceding approach should provide a laboratories do not maintain the necessary mechanism for screening CSF for parasitic media listed in table II. Diagnosis may be infections. With the exception of primary made by animal inoculation or examination amoebic meningoencephalitis, the diagnosis of blood, bone marrow or material as of CNS involvement will most likely be well as by serodiagnosis. made on clinical history and findings of a Immunologic procedures have been used primary infection. The CSF biochemical and primarily for serodiagnosis of parasitic in­ cellular response will appear non-specific in fection with only casual reference to their most cases with a normal or elevated use with CSF. In table III are listed pro­ opening pressure, elevated protein and cedures that are available for immuno­ depressed sugar. Elevated IgM levels may be logic diagnosis of the more common CNS in­ found in trypanosomiasis.51 The cellular fections involving parasites. These proce­ response will be predominantly mononu­ dures were developed for blood. Skin clear though polymorphonuclear leukocytes may predominate. Two hints that a para­ TABLE III site is involved may be the presence of a Immunologic Procedures meningitis from which no microorganism is cultured and in which eosinophils are found. Organism Tests CNS infections in which parasites may be seen in CSF are those in which multiple pro­ Entamoeba CF FA IHA PPT Hartmannella CF liferating forms or eggs are produced. Such a Naegleria None Toxoplasma CF FA I HA SK Dye Test state would exist in amoebiasis, trypano­ Toxocara IHA+ BF+ PVC somiasis, filiariasis, , tox­ Trypanosoma CF FAIHA SK cruzi oplasmosis, and infections African CF FA with Angiostrongylus. Cerebrospinal fluid Trypanosomes Schistosomes CF FA IHA SK BF localization of organisms is less likely in Taenia solium CF IHA PPT (Cysticercosis) cystic lesions caused by cestodes or in adult Echinococcus CF+ FA IHA+ SK+ BF+ or larval ectopic infections from nematodes Fasciola CF FA IHA hepatica and platyhelminthes. Paragonimus CF SK In the United States only three parasites Trichinella CF+ PPT SK+ BF+ Plasmodium FA IHA pose immediate problems; agents of primary (malaria) Filaria CF IHA PPTSK BF amoebic meningoencephalitis, visceral larva Strongyloides CF PPT SK migrans and toxoplasmosis. Primary amoe­ bic meningoencephalitis results from the CF = complement fixation; FA = fluorescent ; IHA = indirect hemagglutination; SK = skin test; free living soil amebas, Hartmannella PPT = precipitation; BF = bentonite flocculation; PVC = passive cutaneous anaphylaxis. and Naegleria. These infections have been +Available in the United States predominantly associated with swimming in 5 0 SODEMAN AND DOCK fresh water lakes or pools and are felt to be TABLE IV acquired via the nasal passage. Up to 1973, Higher Bacteria and Fungi 62 cases have been reported in the United Capable of Producing CNS Infection

States, all but one fatal. Symptoms resemble 1. Organisms with a Predilection for the CNS a fulminant bacterial meningitis. Cerebro­ Cryptococcus neoforiaans Cladosporium trichoides (bantianum)10'51f'6 3 spinal fluid findings are of a bloody, purulent Nocardia asteroides2^'2^ nature and amoebas may be seen. Iron he­ Phycomycetes (mucor, shizopus, absidia)® 2. Organisms Found in the CNS as a Secondary Focus matoxylin stains, however, may be of Disseminated Disease Coccidioides inmitis®® necessary. Cultures and animal inoculation Candida albicans will confirm the diagnosis.13,21,48 Blastomyces dermatitidis17 Histoplasma capsulatum67 It is not uncommon to have CNS involve­ 3. Organisms Very Rarely Found in the CNS Sporothrix schenckii^8 ment in infections of visceral larva migrans Aspergillus fumigatus1*® owing to Toxocara canis. This infection is Actinomyces israelii usually associated with a heavy and hypergammaglobulinemia in a child suffering from pica. Serologic procedures are common and well described CNS infection of questionable value.42 of this group. The second set of organisms Toxoplasmosis both congenital and re­ shown in table IV causes deep mycoses lapsing may present symptomatic cerebral which may disseminate through the blood to involvement. It is estimated that at least one several organs—one of which may be the third of the adults have been infected with brain. The remaining mycotic agents have toxoplasmosis, though most asymptomat­ been only rarely reported to have CNS in­ ically. With the increase in immunosup- volvement. pressed patients, the relapsing form is being Patients suspected of having fungal men­ seen in which CSN involvement is the ingitis should have a thorough CSF exami­ primary problem.30,74 Cerebrospinal fluid nation and culture. An approach to handling examination has shown a varying cellular CSF is noted in figure 2. Clinical information response. The opening pressure may be normal or elevated. The protein content is EXAMINATION FOR FUNGUS increased with no change in the glucose. Sabin Feldman dye titer may be expected SERUM CEREBROSPINAL FLUID from the CSF. Diagnosis can require intra- " T " concentration--1------peritoneal inoculation of mice or hamsters. r supernate sediment Approach to Diagnosis of Fungal Meningoencephalitis In table IV are listed fungi (and two Actinomycetes) that have been reported in CNS infections. The first group of organisms India Ink Sabouraud's

preferentially attacks the CNS when in­ Unstained ^

troduced into the body. Cryptococcus neo- Gram stain Brain - Heart formans establishes a progressive meningitis, I nfusion Periodic acid ^ Cladosporium trichoides causes brain ab­ Schiff 24 c scesses, Nocardia disseminates to the CNS 37 C ( Table 5 I in a high proportion of infections and I Table 6 I various Phycomycetes cause a rhinocerebral Figure 2. A schematic approach to handling cere­ invasion in severely compromised hosts. brospinal fluid specimens when fungal infections are Cryptococcal meningitis is by far the most considered. LABORATORY DIAGNOSIS PARASITIC & FUNGAL MENINGOENCEPHALITIDES 51 is essential and must be communicated cimens suspected of containing fungi. In 50 to the microbiology laboratory, especially percent of cases of Cryptococcal meningitis, when the CSF is in small quantity, so that the organisms can be demonstrated by this the best use of the fluid is made. Unless the method.37 Extreme caution must be taken specimen is grossly bloody or if the quantity not to mistake red blood cells, mononuclear is less than 1.0 ml, it should be concentrated leukocytes or non-encapsulated yeast cells either by centrifugation or Millipore fil­ for Cryptococcus neojormans. Use of an ad­ tration.32 Use of the sediment from centri­ ditional direct smear technique may assist in fuged CSF enables inoculation of several finding other fungal agents. A modified pe­ media types that may be incubated at riodic acid-Schiff stain33 is especially good different temperatures, allowing a variety of for demonstration of Candida and other growth conditions. Concentration by Milli­ yeasts and will serve as an additional micro­ pore filtration limits the number of media scopic scan of the CSF. A Gram stain is that can be used for culture. Ease of han­ useful when Nocardia is suspected. dling and availability and expense of ma­ Unidentifiable forms, cellular debris and terials used in centrifugation make it a questionable yeast forms seen on an India preferable technique for routine processing ink preparation may be reexamined by of CSF. Direct smears should be made from looking at unstained CSF for structural de­ the sediment material to increase the chance tail. of finding fungal forms in CSF. Because of the low numbers of organisms In table V are outlined fungal structures present, demonstration of the fungal forms that may be seen in CSF. An India ink on direct smear is usually not successful. preparation to screen for Cryptococcus neo- Culture is therefore imperative. Readily formans should be done on all CSF spe­ available culture media, simple techniques

TABLE V

Direct Smears of Cerebrospinal Fluid

Stain Explanation of Findings Organism

India ink Encapsulated yeast cell, with or without single bud; Cryptococcus capsular material displaces India ink up to three neoformans times the diameter of the cell yeast - 2 to 8 \i capsule - 8 to 0 u Unstained Oval yeasts, with or without buds, with or without Possible exam pseudohyphae Candida yeasts - 2 to 8 p Thick-walled, broad-based bud on single yeast cell; Possible no capsule Blastomyces yeast - 5 to 20 U hyphal fragments - 5 to 30 v Possible coenocytic Phycomycete .pigmented Cladosporium septate ^ ^non-pigmented Aspergillus Gram stain Gram positive, filamentous, branching but fragmented, Possible thin bacilli Nocardia bacilli - 1 Ovoid yeast cells, with or without buds, with or Possible pseudohyphae Candida Periodic-Acid Yeast cells, with or without buds and pseudohyphae Yeast Schiff Thin, filamentous bacilli Bacteria hyphal particles do not stain well 5 2 SODEMAN AND DOCK

TABLE VI APPROXIMATE IDENTIFICATION TIME

Growth Times for Primary Isolation COLONIAL MORPHOLOGY MICROSCOPIC EXAMINATION

Organism Days Organism Days Germ tube small“’ large Candida albicans I I Candida 1 - 2 Coccidioides 6-12 ( presumptive ) ACTINOMYCETE MOLD Cryptococcus 1-4 Sporothrix 8-12 4 hours Phycomycetes 2-4 Cladosporiurn 10 - 18 3-5 Blastomyces 10 - 18 biochemical Aspergillus differentiation Nocardia 5-10 Histoplasma 14 - 20 biochemical differentiation I Nocardia 2 - 10 days

Mucor and several relatively uncomplicated iden­ Candida Cryptococcus Rhizopus neoformans tification schema can be found in excellent albicans Absidia mycology manuals.8,47 Plain Sabouraud’s 3 days 6 days Aspergillus dextrose and brain-heart infusion agars at Coccidioides Sporothrix 24° and 37° can be used to culture all of the Cladosporium fungi (plus Nocardia) from CSF. Antimi­ Blastomyces crobial agents such as chloramphenicol and Histoplasma gentamycin should not be added to the * Immediate identification upon mature colonial formation of characteristic media owing to the inhibitory effects on microscopic structures Nocardia. Cyclohexamide will inhibit several Figure 3. The approximate time needed for iden­ yeast forms and should also be avoided. The tification of the organisms. limiting factor in identification is the time needed for the fungi to grow. In table VI are a combination of both antigen and antibody shown an approximation of expected growth tests should be done.61 Indirect fluorescent times for primary isolation. The additional and tube agglutination tests for antibody time needed for identification, once the plus latex agglutination tests for antigen colony has developed, is shown in figure 3. together give 95 percent reliability in diag­ Suggestions for tests, techniques and mor­ nosis of progressive cryptococcosis. The phological descriptions used in identification latex agglutination test may be done directly of these fungi have been published.8,47,79 In table VII15,18 are listed those immuno­ TABLE VII logic tests that can be effective in support of Immunologic Tests the diagnosis of a systemic mycotic in­ fection. This area is difficult to evalutate be­ Useful Diagnosis Tests cause it is rapidly expanding. In addition, Predilection for central nervous system several of the fungi are ubiquitous op­ Cryptococcus neoformans LA AG IFA portunists, and tests for their antibody are Cladosporium trichoides None Phycomycetes None often inadequate in differentiating antibody Nocardia asteroides None Secondary focus of dissemination owing to prior exposure from antibody of Coccidioides immitis PPT CF AGDD LA active disease. The immunologic tests can be Candida albicans PPT AG Blastomyces dermatitidis AGDD IFA most effectively used for diagnosis of cryp- Histoplasma capsulatum CF AGDD Very rarely found tococcal and coccidioidal meningitis. Sporothrix schenckii None formed in the early stages of Aspergillus fumigatus AGDD cryptococcal dissemination are neutralized Actinomyces israelii *AGDD IFA by the strongly antigenic capsule of the LA - latex agglutination; AG - agglutinin titer; yeast. Therefore, antibody may not be de­ IFA - indirect fluorescent antibody; PPT - precipitin titer; CF - complement fixation; tectable although large amounts of anti­ AGDD - agar gel double diffusion. genic substance are present. Because of this, ♦Experimental only. LABORATORY DIAGNOSIS PARASITIC & FUNGAL MENINCOENCEPHALITIDES 5 3 on CSF with approximately 70 percent reli­ This syndrome is rare. The organism s mode ability.43 of entry from the soil to the brain is not For coccidioidomycosis, the older com­ known, nor is the reason for its apparent plement fixation and tube precipitin tests neurotrophic disease. If pus is taken from the done together give significant titers in 85 brain site, the darkly pigmented, percent of culture proven cases. The newer large organisms could most likely be dem­ agar gel double diffusion and latex aggluti­ onstrated on smear and could be easily cul­ nation tests done together are 93 percent tured. comparable. In some instances the meninges Phycomycetes (Mucor, Rhizophus, Ab- may be the first site of dissemination from sidia) cause CNS infections in debilitated pulmonary coccidioidomycosis. Tests for an­ hosts. Seventy-five percent of the cases of tibody in the CSF should be done if CNS phycomycosis involving the brain are found symptoms occur. Usually complement fixing in patients with uncontrolled diabetes antibodies from the blood do not diffuse into mellitus.38 The organisms extend to the the CSF. Therefore, detection of antibody in brain directly from the nasal sinuses. Clinical the CSF would indicate active meningitis. manifestations clearly show increasing facial However, finding antibody in the CSF in low swellings. titers when CSF findings are normal should When Cryptococcus neoformans causes be carefully evaluated.76 CNS infection, it can usually be diagnosed This approach to direct smear exami­ rapidly by India ink preparation and cultural nation, cultural attempt and immunologic methods. The organism grows and can be testing should provide procedures for diag­ identified within a week. Well established nosis of all likely fungal causes of CNS in­ immunological tests can be used to confirm fections. The CSF findings in the various the diagnosis. fungal meningitides with exception of Can­ It can not be emphasized enough that dida meningitis are the same: increase in when either a fungal or parasitic etiology is leukocytes with a predominance of lym­ being considered in the differential diagnosis phocytes, moderately decreased glucose and of meningoencephalitis, consultation be­ elevated protein content. This picture may tween the attending physician and the labo­ resemble that occurring in tuberculous men­ ratory is absolutely necessary. The added ad­ ingitis. Therefore, culture of the spec­ vantage that such contact gives to the imen is of utmost importance to differentiate evaluating laboratory through a knowledge the cause. In Candida meningitis, CSF of the patient’s social and physical history leukocyte counts are much higher with pre­ will allow the development of a combined dominance of polymorphonuclear leuko­ analysis of laboratory findings, and skin cytes. testing. This can direct processing of the In cases when meningitis is a mani­ CSF to yield the maximum information. festation of a disseminated fungal disease (as Without such contact, considerable delay or in histoplasmosis, blastomycosis, nocardi­ even a failure to diagnose the organism will osis, candidiasis, coccidioidomycosis), the occur. causative agents will most likely be recognized from sources other than CSF. References From respiratory specimens in particular, 1. Adolph, P. E., Kagan, I. G., and McQuay, R. M.: the causative organisms will probably be Diagnosis and treatment of Acanthocheilonema culturable as well as evident on direct smear perstans . Amer. J. Trop. Med. Hyg. 11:76- 80, 1962. prior to CNS involvement. 2. Africa, C. M.: Ova in the spinal cord of man. Cladosporium in the CNS is usually found Phillippine J. Sci. 62:393-399, 1937. in the brain tissue at autopsy, not in the CSF. 3. Alicata, S. E.: Angtostrongylus cantonensis (Nem- 5 4 SODEMAN AND DOCK

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G.: Serologic diagnosis. Visceral larva mi- W. M.: The isolation of additional strains of patho­ grans. Clin. Pediat. 7:508-509, 1968. genic Hartmanella Sp (Acanthamoeba). Amer. J. 43. Kaufmen, L. and Blumer, S.: Value and in­ Clin. Path. 45:383-387, 1965. terpretation of serological tests for diagnosis of 21. Culbertson, C. G., Smith, J. W., Cohen, H. K., and Cryptococcosis. Appl. Microbiol. 16:1907-1912, Minner, J. R.: Experimental infection of mice and 1968. LABORATORY DIAGNOSIS PARASITIC & FUNGAL MENINGOENCEPHALITIDES 5 5

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