Vol. 30, No. 8JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2104-21070095-1137/92/082104-04$02.00/0Copyright © 1992, American Society for Microbiology

Typing of Histoplasma capsulatum by Restriction FragmentLength Polymorphisms in a Nuclear Gene

ELIZABETH J. KEATH,1* GEORGE S. KOBAYASHI,2-5 AND GERALD MEDOFF2'5

Department ofBiology, Saint Louis University, St. Louis, Missouri, 63103,1 and Division of InfectiousDisease, Department of Medicine,2 Division of Laboratory Medicine, Departments ofPathology andMedicine, Division ofDermatology, Department ofMedicine,4 and Department ofMicrobiology

and Immunology,5 Washington University School ofMedicine, St. Louis, Missouri 63110

Received 24 January 1992/Accepted 18 May 1992

We previously described yps-3, a Histoplasma-specific nuclear gene probe useful in the identification ofHistoplasma capsulatum. By using restriction fragment length polymorphisms (RFLPs) ofDNA detected by theyps-3 gene and mitochondrial DNA, 76 clinical and soil isolates of H. capsulatum were classified. The majorityof North American isolates obtained from endemic regions of the midwestern United States were members ofthe previously characterized class 2, although four clinical isolates from different patients with AIDS from thatregion were grouped in class 1 with the temperature-sensitive Downs strain. A Florida soil isolate (FLS1) wasplaced in class 4 on the basis ofRFLP with both probes. Two American Type Culture Collection strains (G184Band G186B) from Panama were grouped into class 3 by this analysis. A group of five H. capsulatum isolatesobtained from patients with AIDS in New York City were typed into a new class 5 on the basis of yps-3polymorphisms; those organisms fell into two broad mitochondrial DNA patterns, designated 5b and 5c. Twonew isolates from Panama were also members of this broad yps-3 class 5 group, but they exhibited a distinctmitochondrial DNA profile (class 5a). A sixth class was detected in DNA obtained from a patient with AIDSfrom Panama; that DNA had unique RFLP profiles with respect to both probes. These observations suggestthat the Histoplasma-specificyps-3 gene probe is a sensitive tool for typing H. capsulatum in clinical specimens.Additionally, these studies provide molecular support for the hypothesis that AIDS-associated histoplasmosisin nonendemic areas is due to reactivation of a previously acquired infection.

Although Histoplasma capsulatum, the etiologic agent ofhistoplasmosis, has been recognized as an important fungalpathogen in immunocompromised hosts, particularly thegroup with AIDS (6, 7), little is known about its epidemiol-ogy and pathogenesis in immunodeficient individuals. Forexample, systemic histoplasmosis has been reported in pa-tients with AIDS who do not reside in regions wherehistoplasmosis is endemic (13), leading to the suggestion thatthe disease resulted from reactivation of previously acquiredH. capsulatum infections. However, there have been nodirect molecular tests of this hypothesis.

Molecular techniques have provided powerful tools for thedirect analysis of the genomes of many pathogens (3, 10).Our previous studies have exploited differences in restrictionfragment length polymorphisms (RFLPs) of mitochondrialDNA (mtDNA) or ribosomal DNA to divide clinical and soilisolates of H. capsulatum into four classes (16). Althoughthis approach has proved successful for H. capsulatum (17,19), Candida albicans (12, 14), and Blastomyces dermatiditis(5), classification of strains is most simply achieved in thediagnostic laboratory by (i) using specific probes whichhybridize exclusively with the infectious agent and (ii) ap-plying molecular clones from nuclear segments of the ge-nome which are constant over many generations. A recentlycloned nuclear sequence, yps-3, addresses both criteriabecause it is a Histoplasma-specific sequence which distin-guishes among classes of the organism (8).

In the study described here, we applied RFLP analysiswith both yps-3 and mtDNA probes to H. capsulatumisolates obtained from additional sources, including patients

* Corresponding author.

with AIDS, in an effort to discover previously undetectedclasses of the organism. On the basis of our results, wepropose a new system for typing H. capsulatum based onRFLP at the nuclear yps-3 locus supplemented by mtDNAsubtyping; the system identified two new classes and fournew subclasses of this pathogen on the basis of differenceswithin mtDNA. This system also provided a convenientmethod of testing the reactivation theory of H. capsulatuminfection in patients with AIDS.

MATERIALS AND METHODS

Organisms and culture conditions. Stock cultures of H.capsulatum G222B (ATCC 26034), G186B (ATCC 26030),and G184B (ATCC 26028) were obtained from the AmericanType Culture Collection. The Downs strain (ATCC 38904)and three isolates which resemble this strain with respect toRFLP, temperature sensitivity, and virulence (16) wereobtained from separate patients with AIDS and disseminatedhistoplasmosis; these strains are permanent members of ourlaboratory collection. FLS1, a soil isolate from Florida, hasbeen described previously (16). All other strains evaluated inthis study were from clinical isolates. Isolate RW wascultured from a patient presenting with acute pulmonaryhistoplasmosis at the Washington University School of Med-icine after recent travel in Central America. Isolates fromtwo other areas within the endemic Midwest region werealso evaluated. H. capsulatum cultures from 16 patients withAIDS from Kansas City, Mo., were provided by M. Gupta,Trinity Lutheran Hospital. Three groups of isolates fromIndianapolis, Ind., were obtained from L. J. Wheat andincluded 9 isolates from non-AIDS patients with pulmonaryhistoplasmosis, eight cultures from AIDS patients, and 10

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CLASSIFICATION OF H. CAPSULATUM 2105

isolates from non-AIDS patients. Five H. capsulatum iso-lates (039498 [039], 130757 [130], 139524 [139], 140907 [140],182181 [182]) from patients with AIDS in New York Citywere obtained from Memorial Sloan Kettering Cancer Insti-tute. Three isolates (589, 862, 1718) from Panama (M.Martin, Universidad de Panama, Facultad de Medicinas),were also subjected to RFLP analysis. All cultures weremaintained in 2% glucose-1% yeast extract medium in themycelial phase at 22°C.

Isolation of DNA, gel electrophoresis, and Southern blots.High-molecular-weight DNA was prepared from various H.capsulatum isolates by breaking the mycelia under liquidnitrogen with a mortar and pestle (1) and processing asdescribed previously (17) by treatment with RNase A andproteinase K. Restriction enzymes were from PromegaBiotec.Genomic DNA was digested with BglII, electrophoresed

in 0.7% agarose gels in a borate buffer system (2), andtransferred to a nylon membrane (Nytran; Schleicher &Schuell, Inc., Keene, N.H.) by the method of Southern (15).Blots were hybridized initially with a pooled probe repre-senting mtDNA from H. capsulatum G217B (17). Followingautoradiography, the blot was stripped as recommended bythe manufacturer and rehybridized with the 1.85-kb HindIIIfragment containing the yps-3 sequence (8). All hybridiza-tions were performed at 65°C in an aqueous hybridizationmixture containing 7% sodium dodecyl sulfate (SDS), 1%bovine serum albumin, 0.5 M sodium phosphate (pH 7.2), 1mM EDTA, and 106 cpm of nick-translated probe per ml, asdescribed previously (9). Blots were washed in 2x SSC (lxSSC is 0.15 M sodium chloride plus 0.015 M sodium citrate)-0.1% SDS at room temperature to remove excess probe andwere then processed by washing them three times at 65°C in0.lx SSC-0.1% SDS.

RESULTS

RFLPs at theyps-3 and mitochondrial loci in H. capsulatum.A total of 45 different H. capsulatum isolates obtained frompatients in the midwestern United States were evaluated forRFLPs. Isolates were obtained from immunocompetent pa-tients and from patients with AIDS, and no patient hadrecently traveled to other regions endemic for histoplasmo-sis. Forty-one of these isolates, which were from the St.Louis, Kansas City, and Indianapolis areas, exhibited ayps-3 and mtDNA RFLP profile consistent with that of theprototype North American strain G222B (data not shown)(Fig. 1) and were therefore designated class 2 (17, 18). Nonew mitochondrial oryps-3 classes emerged from a compar-ison of 19 H. capsulatum isolates obtained from immuno-competent patients or 8 isolates from patients with AIDSfrom Indianapolis (data not shown). Three previously de-scribed isolates from patients with AIDS who were lifelongresidents of the St. Louis metropolitan area and an isolate(88-175) obtained from a patient with AIDS from Texas havebeen typed with the Downs strain (class 1) by a similaranalysis (16). For example, BglII fragments of 2.0, 1.8, and0.9 kb were observed in hybridizations with the yps-3 probein both Downs and 88-175 DNAs (Fig. 1B), and identicalmitochondrial RFLP profiles were observed (Fig. 1A).

Figure 1A shows the results of genomic Southern blotanalysis with the mtDNA probe. Consistent with previousfindings, the Central American isolate G186B and Floridaisolate FLS1 gave unique mitochondrial and yps-3 finger-prints and were grouped into classes 3 and 4, respectively(Fig. 1). However, five previously undetected mitochondrial

mtDNAco

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W_WEm,^

B3 yps3com azN ss _ rv co

cN.o6 -- _o N X~ S O N Ct 'O

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FIG. 1. Southern blot analysis of representative H. capsulatumisolates. (A) BglII-restricted whole-cell DNAs from 10 isolates weretransferred by Southern blotting and were hybridized with a pooledmtDNA probe from strain G217B. The sizes of HindIll-generatedbacteriophage lambda DNA fragments are indicated on the left ofthe panels. The relative positions of unique fragments with themtDNA RFLP profile are designated by arrows. Isolates 88-175(Texas) and Downs exhibited a common pattern in this analysis,although slight differences were observed with HaeIII digests (datanot shown). (B) The blot shown in panel A was stripped andrehybridized with the 1.85-kb HindllIyps-3 nuclear gene probe. Thenine apparent mtDNA RFLP classes seen in panel A resolved intosix classes based on the nuclear gene probe. The sizes of thecharacteristic BglII fragments are given in kilobases.

profiles were observed. These were seen in two isolates(isolates 140 and 130) from patients of Puerto Rican extrac-tion who lived in the New York City area, two Panamanianisolates (isolates 1718 and 589), and the St. Louis isolate(isolate RW) obtained from a patient who had recentlytraveled to Central America. Differences among these iso-lates and prototypic organisms such as Downs, G222B,G186B, and FLS1 are highlighted by arrows in Fig. 1. Slightchanges in the BglII restriction pattern were responsible forthe observed differences among these isolates. For example,the 4.8- and 1.1-kb BglII fragments seen in isolate RWpresumably result from an additional restriction site presentin that strain; that site was absent from the 5.9-kb fragmentobserved in isolate 130. Similar comparisons for isolates 140and 589 argue for slight alterations within the mitochondrialgenome which contribute to the observed polymorphism.To evaluate the relationships among these organisms at a

different, nuclear locus, the blot was stripped and rehybrid-ized with the 1.85-kb HindIII fragment from yps-3. As seenin Fig. 1B, this Histoplasma-specific probe detected poly-morphic BglII fragments in isolates Downs (class 1), G222B(class 2), G186B (class 3), and FLS1 (class 4), generatingunique patterns consistent with the previous system ofclassification based on mitochondrial DNA polymorphisms(17, 19). Moreover, this Southern blot analysis groupedisolates 140, 589, 130, and RW within the same nuclear probeclass, since the yps-3 gene detected identical 12- and 2.4-kbBglII fragments in all four organisms. On the basis of thisanalysis, these isolates were grouped as members of a broadyps-3 class 5 (Table 1), encompassing four mitochondrialsubclasses (a to d). By extension of this typing system,isolate 1718 resolved into a unique class (class 6) on the basisof the results obtained with both yps-3 and mtDNA probes.

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2106 KEATH ET AL.

TABLE 1. Classification of H. capsulatum isolates byyps-3 polymorphisms

yps-3 frag- mtDNA Prototype Geographic No. ofClass ment size(kb) subgroup isolate site isolates

1 2.0, 1.8, 0.9 Downs Missouri 5ab2 3.2, 2.0 G222B North America 45b3 2.4, 1.8 G186B Panama 2b,c4 1.8, 0.9 FLS1 Florida lb,d5 12, 2.4 a 589 Panama 2b5 12, 2.4 b 130 New York 4b

(Puerto Rico)5 12, 2.4 c 140 New York 2b

(Puerto Rico)5 12, 2.4 d RW Missouri (Central lb

America)6 4.2 1718 Panama lb

a From Spitzer et al. (16).b This study.c From Vincent et al. (19) and unpublished data.d From Spitzer et al. (17).

Classification ofH. capsulatum isolates by polymorphisms ina nuclear gene. By Southern blot analysis with theyps-3 geneprobe, more than 50 clinical isolates of H. capsulatum fromNorth and Central America were grouped into the classifi-cation scheme detailed in Table 1. Based on that strategy,organisms were placed into classes 1 to 6 b on the basis ofyps-3-defined RFLPs and were then subtyped by usingmtDNA polymorphisms. The previously described classes 1to 4 (17, 19) are consistent with this scheme, which empha-sizes relationships among the isolates at the nuclear genelevel. As seen in Table 1, two newyps-3 classes, designatedclasses 5 and 6, emerged from this analysis. Class 5 exhibitedthe greatest degree of heterogeneity at the mitochondrialDNA level, with four subgroups assigned to that class. Thisdual classification system eliminated the need for scrutiniz-ing ethidium bromide-stained gel profiles of variable qualityby providing a highly sensitive screen of two different DNAsequences derived from two genomes for each isolate tested.

Reactivation of H. capsulatum infection in patients withAIDS. The typing strategy described in this report was usedto evaluate H. capsulatum isolates in patients with AIDSpresenting for treatment in New York City. All patients hada history of intravenous drug abuse and were immigrantsfrom Puerto Rico, a region endemic for Histoplasma infec-tion. Although the possibility of travel to other areaseudemic for Histoplasma infection was not excluded forthese patients, socioeconomic considerations made wide-spread travel unlikely. As seen in Fig. 2A, three differentmitochondrial subclasses were observed in the five isolatesfrom patients with AIDS (isolates 182, 140, 130, 139, and130) on the basis of BglII polymorphisms. Two separateisolates from the right lung (isolate 140) and bone marrow(isolate 139) from a single patient gave identical profiles withBglII (Fig. 2A) and PvuII (data not shown), irrespective ofthe culture site.To assess the relationship among these isolates at the

nuclear gene level, the blot was stripped and rehybridizedwith the 1.85-kb HindIII fragment representing the yps-3sequence (Fig. 2B). BglII digests of prototype DNAs from allknown classes obtained from North and Central Americawere electrophoresed in parallel. All of the isolates frompatients with AIDS were members ofyps-3 class 5, exhibit-ing BglII fragments of 12 and 2.4 kb. Moreover, this yps-3

A:L)mtDNA

N S t. ,., ;,

:,x.- -*

-,l;f,yps3,;

i

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FIG. 2. RFLP analysis ofH. capsulatum isolates from immigrantpatients with AIDS in the New York City area. Isolate DNAs (182,130, 039, 140/139) were obtained from four Puerto Rican patientswith AIDS and disseminated histoplasmosis; isolates 140 and 139were cultured from different sites from the same individual. DNAswere digested with BglII in parallel with DNAs from representativeclass 1 to 3 organisms (Downs, G186B, G222B) and from threePanamanian isolates (1718, 589, 862). (A) Hybridization with apooled mtDNA probe delineated two mtDNA subclasses within thePuerto Rican isolates and two additional subclasses in Panamanianstrains 1718, 589, and 862. (B) Rehybridization of the Southern blotshown in panel A with the 1.85-kb HindIII yps-3 gene probedemonstrates common profiles of 12 and 2.4 kb for the nuclear genein the all Puerto Rican and two of the Panamanian isolates (isolates589 and 862). Isolate 1718 (Panama) gave a unique RFLP profile,with a single band at 4.2 kb, with the yps-3 gene probe.

RFLP pattern was identical to that observed in isolates 589and 862, which were H. capsulatum isolates obtained frompatients with AIDS and disseminated histoplasmosis fromPanama. A common 2.4-kb BglII fragment was also ob-served in another Panamanian isolate, G186B (class 3). Incontrast, no common yps-3 fragments were observed whenthese isolates from patients with AIDS were compared withthe Downs or G222B isolates prevalent in North America.These findings demonstrate that the patients with AIDS inthe New York City area examined in the study describedhere did not acquire H. capsulatum infection by the preva-lent class 1 or 2 organisms while they were living in NorthAmerica and are most consistent with reactivation of a latentHistoplasma infection because of prior infection in CentralAmerica.

DISCUSSION

The strategy for molecular classification of the H. capsu-latum isolates described here makes it possible to rapidlyclassify organisms into broad yps-3 classes on the basis ofpolymorphisms within this nuclear gene. Environmental aswell as clinical specimens can be evaluated by the initialyps-3 hybridization profile, and polymorphisms withinmtDNA can be used to further subdivide the isolates. Thisapproach holds significant advantages over previously re-ported classifications systems (17, 19). Those systems havegenerally relied on patterns on ethidium bromide-stainedgels that are subject to variations in the amount of loadedDNA, labor-intensive DNA extractions, and a lack of sen-sitivity. In contrast, the initial class assignment describedabove is based on hybridization with a Histoplasma-specific

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CLASSIFICATION OF H. CAPSULATUM 2107

nuclear sequence which can detect as little as 120 to 250 ngof H. capsulatum DNA and which eliminates diagnosticuncertainties in differentiating Histoplasma from other fungisuch as B. dermatiditis, Candida species, Chrysosporiumkeratinophilum, and Sepedonium chrysospermum (9).

Subtyping with mtDNA probes permits discrimination ofstrains within a single yps-3 class and makes it possible toaddress questions of regional or local epidemiology. Al-though other restriction digests such as HaeIII, HhaI, andPstI have been used to demonstrate mtDNA polymorphismsand may be useful in some detailed applications, a conve-nient single digest-single blot can be used for bothyps-3 andmtDNA analyses.

This study demonstrates that, among the isolates frompatients with AIDS in the New York City area whichpresumably originated from Puerto Rico, significant diver-sity within mtDNA may exist within a single H. capsulatumyps-3 class. Two mitochondrial subclasses (subclasses 5band 5c) were observed in these isolates and were distinctfrom the profiles (subclasses Sa and Sd) obtained with otherorganisms from the Central American region that wereanalyzed). This diversity at the mitochondrial level wasresolved by analysis at the yps-3 locus, by which all isolatesexhibited the same hybridization pattern. Since the obtainedyps-3 pattern was clearly distinct from that observed in thepredominant North American isolates (classes 1 and 2), itappears likely that the patients with AIDS in New York Citywere not infected by H. capsulatum strains that wereprevalent in that region but, rather, manifested histoplasmo-sis through reactivation of previously acquired class 5 iso-lates. A more extensive survey of H. capsulatum isolatesfrom soil or clinical specimens from Puerto Rico wouldprovide a more direct test of this hypothesis. Anotherpossibility is that the New York City area has a completelyunique strain of H. capsulatum compared with other NorthAmerican strains and appears to infect only patients withAIDS. Additional H. capsulatum isolates from non-AIDSpatients in the New York metropolitan area have not beenavailable for direct testing of this hypothesis.The reason for the heterogeneity in the class 5 isolates of

H. capsulatum is unclear. It may be that the temperateCentral American region, as well as the spatial separation ofthe Puerto Rican island and the Panamanian isthmus habi-tats, provided a unique environment appropriate for thedivergence of the mitochondrial genomes. Interestingly,extensive polymorphism in the mtDNA restriction patternshas been observed among serotype C isolates of Cryptococ-cus neoformans var. gattii (18), a dimorphic fungal pathogenwith a recently characterized ecological niche (4) whichcauses frequent infections in tropical regions (11).

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant AI 28950

(to E.J.K.) from the National Institutes of Allergy and InfectiousDisease and grant AI 07015 to the Medical Mycology Center fromthe National Institutes of Health.We gratefully acknowledge the technical assistance of Sharon

Travis and Audrey Painter.

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