JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/97/$04.0010

Jan. 1997, p. 313–316 Vol. 35, No. 1

Copyright q 1997, American Society for Microbiology

Candida and Torulopsis: a Blinded Evaluation of Use of PseudohyphaFormation as Basis for Identification of Medically Important YeastsF. C. ODDS,1* M. G. RINALDI,2 C. R. COOPER, JR.,3 A. FOTHERGILL,2 L. PASARELL,3 AND M. R. MCGINNIS3

Department of Bacteriology and Mycology, Janssen Research Foundation, 2340-Beerse, Belgium1; Fungus Testing Laboratory,University of Texas Health Science Center, San Antonio, Texas 782842; and Medical Mycology Research Center, Center for

Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston, Texas 775553

Received 26 January 1996/Returned for modification 12 April 1996/Accepted 16 October 1996

Seventy yeast isolates representing species in the genera Candida and Torulopsis but excluding Candidaalbicans were examined in three laboratories for production of pseudohyphae in Dalmau cultures. The micro-scopic morphology of the isolates was scrutinized by four individuals experienced in yeast identification andthree inexperienced persons, all of whom were blinded as to the putative identification of the yeasts. For 49(70%) of the 70 isolates, the seven observers recorded comparable scores for morphology, but 5 (7%) of theisolates showed extreme variation in recorded morphologies, from true hyphae formed to no pseudohyphaeformed. Isolates of Candida parapsilosis and Torulopsis glabrata consistently did and did not form pseudohy-phae, respectively: however, other Candida and Torulopsis spp. did not always express their expected morphol-ogies. In 48 (19%) of 252 readings (seven observers), 36 isolates of Candida spp. were scored as forming nopseudohyphae, and in 22 (9.2%) of 238 readings, 34 isolates of Torulopsis spp. were recorded as forming truehyphae or pseudohyphae. These results show that pseudohypha formation is not a reliable characteristic foridentification of yeasts at the genus level; we suggest that the merger of Torulopsis spp. into the genus Candidashould be finally accepted.

The genera Candida and Torulopsis have traditionally con-tained asexual yeasts that have no distinguishing morphologicor other characteristics to facilitate a more precise classifica-tion. They are essentially heterogeneous-form genera whosemembers are defined by negative rather than positive proper-ties (1, 9, 10). Classification within the genus Candida or Toru-lopsis does not necessarily imply close taxonomic relationshipsamong member species.The taxonomic status of the genera Candida and Torulopsis

has been a source of controversy for many years. The twogenera have been distinguished by their ability (Candida) orlack of ability (Torulopsis) to form pseudohyphae when cul-tured under microaerophilic conditions or on an appropriateagar medium by the so-called Dalmau technique, which in-volves scoring the inoculum into the agar (4). In an influentialmonograph on yeast taxonomy published in 1970, Van Udenand Buckley considered that the distinction between Candidaand Torulopsis spp. was “arbitrary and artificial” but retainedthe separation to avoid the “confusion and justified irritation”that abolition of the generic distinction would create amongthose who “use or encounter” species in these genera (9).Nevertheless, in 1978 Yarrow and Meyer (12) formally pro-posed deletion of the genus Torulopsis and the transfer ofTorulopsis spp. into the genus Candida. This proposal wasrejected in two later publications (3, 5). Because 18 years havepassed since the proposal to merge Torulopsis spp. into thegenus Candida was made, we felt that a scientific reappraisal ofthe validity of separate identifications of Candida and Torulop-sis spp. was appropriate. The intention of the study was toassess the consistency of recording observations of pseudohy-pha formation by personnel experienced and inexperienced in

evaluating growth on Dalmau cultures and who were blindedas to the likely identification of the isolates.The three participating laboratories followed similar proto-

cols. Each laboratory prepared triplicate subcultures from sin-gle colonies of 24 yeasts selected to provide 11 to 12 isolatesrepresentative of species in the genus Torulopsis and 11 to 12representatives of Candida spp. Inclusion of one yeast isolatefrom a genus other than Candida or Torulopsis was permittedin each panel as a general control for identifications to thegenus level. Isolates of Candida albicans were specifically ex-cluded, since this species is particularly distinctive by micro-scopic examination. The yeast cultures were randomly codedand distributed to all participants. Dalmau plate cultures wereset up according to the methods routinely used in that labora-tory, and the plates were examined microscopically after 48 hof incubation by at least two individuals, one of whom wasexperienced and the other of whom was inexperienced in read-ing yeast and pseudohyphal morphologies.Techniques used for Dalmau cultures were as follows. In

laboratory A, yeasts from an overnight culture at 308C ondiluted casein hydrolysate-yeast extract-glucose medium (6)were cross-streaked with an inoculating loop on plates of 1%cream of rice–1% Tween 80 agar with firmness sufficient toscrape the agar surface lightly during inoculation. Sterile glasscoverslips were pressed over an inoculated area. The plateswere incubated at 258C. In laboratories B and C, yeasts from24- to 48-h cultures incubated at 308C on Sabouraud glucoseagar were lightly streaked onto the surface of cornmeal agarcontaining Tween 80. Sterile glass coverslips were placed overthe streak marks, and the plates were incubated at 308C for48 h.In two of the participating laboratories, the isolates were

fully identified to the species level, when possible, on the basisof physiological profiles and according to the methods rou-tinely used in each laboratory. Laboratory A used the APIID32C identification galleries (Biomerieux, Marcy-l’Etoile,France). When the measured API profile led to a result de-

* Corresponding author. Mailing address: Department of Bacteriol-ogy and Mycology, Janssen Research Foundation, B-2340 Beerse, Bel-gium. Phone: 32 14-603004. Fax: 32 14-605403.

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scribed as excellent identification in the reference chart andthe morphology of the yeast conformed to the ID32C results,that identification was recorded; when the API ID32C-basedidentification was less than excellent, further physiological pro-files on API 20C yeast galleries (Biomerieux) and classicalassimilation broth tests were utilized to establish the identifi-cation of the isolate. Laboratory B used the API20C identifi-cation system (BioMerieux Vitek, Hazelwood, Mo.) as well asadditional tests as necessary according to the manufacturer’sinstructions. Pseudohypha formation as a criterion for identi-fication of yeasts was set up and read independently of theblinded evaluation. When laboratories A and B disagreed overan identification, the isolates were subjected to traditional fer-mentation and assimilation tests to establish the correct iden-tity, in consultation with a yeast reference laboratory in oneinstance.Table 1 lists the 72 yeast isolates examined, the species

identifications as determined by laboratories A and B, and themorphology scores for each isolate according to each of sevenobservers. A total of four experienced (A to D) and threeinexperienced (E to G) observers recorded the morphology ofeach isolate with a score of from 1 to 4, as follows: 1, unequiv-ocal true hyphae seen; 2, unequivocal pseudohyphae (but nottrue hyphae) seen; 3, pseudohyphae possibly present but equiv-ocal; 4, definitely no formation of pseudohyphae (Table 1).One of the isolates identified as Saccharomyces cerevisiae andanother as a yeast-like variant of Wangiella dermatitidis wereexcluded from further study.On the basis of the data in Table 1, it was calculated that

experienced observers of yeast microscopic morphology re-corded a score of 3 (equivocal pseudohypha formation) 31times, 11.1% of all 280 scores they recorded, compared withinexperienced observers, who recorded 37 equivocal scores(17.6% of 210 total scores). These differences were not statis-tically significant. Agreement between morphology scores re-corded for individual isolates was perfect (identical scores) ordiffered by only 1 point for 49 (70%) of the isolates whenexamined by experienced observers and for 51 (73%) of theisolates when examined by inexperienced observers. The num-bers (percentages) of isolates for which morphology scoresover the entire range (1 to 4) were recorded by different ob-servers were five (7%) and four (6%) for experienced andinexperienced individuals, respectively.Table 2 summarizes the score data according to the species

of yeast examined. For both experienced and inexperiencedobservers, most isolates of Candida parapsilosis and Torulopsisglabrata were given morphology scores indicating consistentpseudohypha formation and no pseudohypha formation, re-spectively, by these two species. T. glabrata isolates were scoredas forming unequivocal pseudohyphae, i.e., a score of 2, in 4 ofa total of 189 readings, and all 4 of these aberrant readingswere from inexperienced observers. Similarly, for C. parapsi-losis isolates, nothing was given a score of 1 (true hyphae) fromthe 52 total readings from experienced observers, comparedwith 2 of 36 readings by inexperienced observers.For several of the less frequently represented species in the

study, the scores given for pseudohypha formation were moreoften wide-ranging or inappropriate (on the basis of nopseudohypha formation by Torulopsis spp.), regardless of theexperience of the observer in judging yeast cell morphology.All four isolates of Candida lusitaniae were scored principallyas morphology level 3 or 4, i.e., equivocal or no pseudohyphaformation, while the expected score of 2 (unequivocal pseudo-hyphae) was recorded for only 4 of 28 observations. Isolates ofCandida krusei showed a similar although less pronounced

tendency to elicit unexpected scores of 3 and 4, regardless ofobserver experience.The definition of a Candida species as one that forms

pseudohyphae and of a Torulopsis species as one that forms nopseudohyphae predicts that isolates of Candida spp. shouldnever receive a score of 4, while scores of 1 and 2 are compat-ible with the genus definition, and a score of 3 indicates ob-server indecision. Similarly, isolates of Torulopsis spp. shouldnever be scored as 1 or 2, since unequivocal formation of trueor pseudohyphae is incompatible with the genus definition. Inthe present study, scores of 4 were assigned to yeasts identifiedas Candida spp. on 46 occasions, representing 18.8% of 245total readings (35 isolates and seven observers), and scores of1 or 2 were assigned to yeasts identified as Torulopsis spp. on23 occasions, representing 9.8% of 245 total readings.Reidentifications of yeasts by laboratories A and B were

concordant for 62 (89%) of the 70 Candida and Torulopsisisolates. Discrepancies were noted as follows: two C. kruseiisolates identified as Candida norvegensis, two C. krusei isolatesidentified as Torulopsis inconspicua, one T. inconspicua isolateidentified as C. krusei, one T. candida isolate identified asCandida guilliermondii, one C. guilliermondii isolate identifiedas T. candida, and one Torulopsis pintolopesii (not identified inlaboratory B). Identifications of Candida pelliculosa in onelaboratory and Hansenula anomala in the other were not re-garded as discrepant, since the former species is the anamorph ofthe other. The species C. parapsilosis (13 isolates) and T. glabrata(26 isolates) were the two most frequently represented amongthose tested, and agreement for identification of these specieswas 100% between laboratories A and B. Single isolates ofCandida lambica, Candida lipolytica, Candida tropicalis, andCandida valida and four isolates of C. lusitaniae were alsoidentified concordantly by both laboratories. These resultsshow that identifications of yeasts based predominantly oncommercially supplied kits can lead to confusion, particularlybetween the triad C. krusei, C. norvegensis, and T. inconspicua.Confusion between identifications of C. guilliermondii and T.candida is often difficult to avoid, since these biologically dif-ferent species share indistinguishable physiological properties(1); only careful and exhaustive morphological examination forpseudohypha formation can successfully discriminate betweenthese species at the phenotypic level.Two of the three laboratories submitted only isolates of the

species C. glabrata as representatives of the genus Torulopsis.Neither laboratory routinely isolates yeasts from samples suchas skin and nails or from veterinary sources, where other Toru-lopsis species are often found. Although T. glabrata is the mostcommonly encountered representative of the genus Torulopsisamong clinically isolated yeasts, it is not representative of thewhole genus, as indicated by the difficulties and inconsistenciesencountered with the isolates of T. candida, T. inconspicua,and T. pintolopesii in the study. The last species, which is thepredominant commensal yeast found in rodents, could noteven be identified with the commercial kits used for clinicalyeast isolates because it was not included in their databases.(The identity of the isolate of C. pintolopesii used in this studywas in fact determined by the Centraalbureau voor Schimmel-cultures in Delft, The Netherlands.)This study was designed to assess whether pseudohypha for-

mation is a reliable criterion for distinguishing among yeasts.McGinnis defined a pseudohypha as “a series of blastoconidiathat have remained attached to each other, forming a hy-phalike filament” (4). Van der Walt’s description of “forma-tion of a filamentous structure consisting of cells which ariseexclusively by budding” (8) is essentially identical. However,scrutiny of the illustrations of various Candida spp. in the

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monograph by Van Uden and Buckley (9) shows that thedefinition embraces weakly organized chains of nearly spheri-cal yeast cells as well as highly organized branched filamentsmade up of greatly elongated component cells. In much olderyeast taxonomies, different styles and appearances of pseudo-hyphae were used to distinguish yeast taxa (11); however, thesecharacteristics were later abandoned, largely because of theirvariability.In a numerical taxonomic analysis of Candida and Torulopsis

spp. (7), no natural separation between the two genera wasevident on the basis of the combined phenotypic characteristicsof the yeasts. Moreover, pseudohypha formation did not ap-pear as a significant taxonomic marker for any of the clustersof species that emerged from that analysis. The terms “prim-itive pseudomycelium,” “rudimentary pseudomycelium,” and“sparse pseudomycelium” can be found in standard descrip-tions for several species in the genus Candida (9) and the genusTorulopsis (10). Our study showed that inexperienced observ-ers recorded equivocal morphologies (score of 3) more oftenthan experienced observers (17 versus 11%) and that theformer were responsible for almost all of the inappropriatemorphology scores recorded for the otherwise well-recognizedspecies C. parapsilosis and T. glabrata. Nevertheless, the differ-ences in observer equivocation were not statistically significant,and a generally high level of variability was seen for pseudohy-pha scores regardless of the experience of the observer; 19 to21 of 70 isolates showed ranges of two or three score differ-ences, and inappropriate morphology scores (pseudohyphaefor a Torulopsis sp. and no pseudohyphae for a Candida sp.)were recorded overall for 69 (14%) of the 490 readings made.Therefore, we conclude that pseudohypha formation is toounreliable a characteristic to allow for dependable identifica-tion of medically important yeasts in the separate genera Can-dida and Torulopsis.For differentiation of some species, e.g., C. guilliermondii

and Torulopsis famata, pseudohypha formation remains theonly reliable characteristic. However, it is clear from the pres-ent study that correct observation of pseudohypha formation isvulnerable to both observer variation and technical artifactsthat emphasize the need for care beyond the norms of rapidthroughput in clinical microbiology laboratories in the identi-fication of such difficult species.We suggest that the proposal of Yarrow and Meyer (12) to

merge isolates previously classified as Torulopsis spp. into the

TABLE 1. Details of the 72 yeast isolates studied, the results oftheir reidentifications in two laboratories, and the morphology

scores reported by each of seven observersa

Yeastrefer-enceno.

Yeast identification Morphology scored by thefollowing observers:

Laboratory A Laboratory B A B C D E F G

1 T. glabrata T. glabrata 4 4 4 4 4 4 42 C. krusei C. krusei 3 3 2 4 4 4 23 C. norvegensis C. kruseib 2 2 1 1 1 1 14 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 25 T. glabrata T. glabrata 4 4 4 4 4 3 46 C. lusitaniae C. lusitaniae 4 4 4 4 4 4 47 T. glabrata T. glabrata 4 4 4 3 4 4 48 No identification W. dermatitidisc 2 2 4 4 1 4 49 C. lipolytica C. lipolytica 2 1 1 2 1 2 110 C. lusitaniae C. lusitaniae 4 4 2 4 3 4 311 C. krusei C. krusei 3 3 2 3 4 4 212 T. glabrata T. glabrata 4 4 4 4 3 4 413 T. glabrata T. glabrata 4 4 4 4 3 4 414 T. glabrata T. glabrata 4 4 4 4 4 4 415 C. krusei C. krusei 2 3 2 4 2 4 216 T. glabrata T. glabrata 4 3 3 3 2 3 317 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 218 T. glabrata T. glabrata 4 4 4 4 4 4 419 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 220 C. krusei C. krusei 2 2 1 4 3 4 221 T. glabrata T. glabrata 4 4 4 4 4 4 422 T. glabrata T. glabrata 4 4 4 4 4 4 423 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 224 T. glabrata T. glabrata 4 4 4 4 3 4 425 C. parapsilosis C. parapsilosis 2 2 2 2 2 1 226 C. krusei C. krusei 2 2 2 1 3 1 227 T. glabrata T. glabrata 4 4 4 4 2 3 428 C. guilliermondii C. guilliermondii 2 2 3 2 2 1 329 T. candida T. candida 3 4 2 4 4 4 430 C. valida C. krusei 4 2 2 1 3 1 131 C. lambica C. lambica 2 2 2 1 2 1 232 T. inconspicua C. kruseib 4 2 2 1 4 1 133 S. cerevisiae S. cerevisiae 2 4 2 4 2 3 234 C. pelliculosa H. anomala 4 3 2 3 2 2 335 T. candida T. candida 3 4 2 4 2 4 336 C. norvegensis C. kruseib 4 2 4 2 2 2 437 T. pintolopesiid No identification 2 4 2 4 3 3 438 T. inconspicuab C. krusei 4 4 2 4 4 2 339 T. glabrata T. glabrata 4 4 4 4 4 2 440 C. guilliermondii C. guilliermondii 4 2 2 1 3 4 341 T. candidab C. guilliermondii 3 3 2 4 4 3 342 T. candida T. candida 4 4 3 4 2 4 443 C. guilliermondii C. guilliermondii 2 2 2 1 2 1 244 C. guilliermondiib T. candida 3 4 2 4 4 4 345 T. inconspicua C. kruseib 4 2 2 1 4 1 146 C. pelliculosa H. anomala 4 3 2 3 2 4 347 C. pelliculosa H. anomala 4 3 2 3 2 3 348 T. glabrata T. glabrata 4 4 4 4 4 3 449 T. glabrata T. glabrata 4 4 4 4 3 4 450 T. glabrata T. glabrata 4 4 4 4 4 4 451 C. parapsilosis C. parapsilosis 2 2 2 2 1 4 252 C. krusei C. krusei 4 2 2 3 4 4 153 T. glabrata T. glabrata 4 4 4 4 4 3 454 T. glabrata T. glabrata 4 4 4 4 4 4 455 T. glabrata T. glabrata 3 3 3 4 4 3 456 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 257 T. glabrata T. glabrata 4 3 4 4 4 4 458 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 259 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 260 T. glabrata T. glabrata 4 4 3 3 2 3 361 T. glabrata T. glabrata 4 4 4 4 4 3 362 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 263 T. glabrata T. glabrata 4 4 4 4 4 4 464 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 2

Continued

TABLE 1—Continued

Yeastrefer-enceno.

Yeast identification Morphology scored by thefollowing observers:

Laboratory A Laboratory B A B C D E F G

65 C. parapsilosis C. parapsilosis 2 2 2 2 2 2 266 T. glabrata T. glabrata 4 4 4 4 4 3 467 C. lusitaniae C. lusitaniae 3 4 2 4 3 4 468 T. glabrata T. glabrata 4 4 4 4 4 4 469 C. lusitaniae C. lusitaniae 2 4 4 4 2 3 470 C. tropicalis C. tropicalis 2 1 2 1 1 1 171 T. glabrata T. glabrata 4 4 4 4 4 4 472 T. glabrata T. glabrata 4 4 4 4 4 4 4

a Scores are as follows: 1, true hypha formation; 2, unequivocal pseudohyphaformation; 3, equivocal pseudohypha formation; 4, no pseudohyphae formed.b Correct identification based on traditional tests.c An albino, mutant strain (Mel3 [ATCC44504]) of this normally darkly pig-

mented yeast was employed in this study.d This identification was confirmed by the Centraalbureau voor Schimmelcul-

tures at Delft, The Netherlands.

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genus Candida for the reasons outlined by those authors andby Davis (2) be accepted. We recognize that many medicalmycologists will be reluctant to abandon the name Torulopsisand that much of the reluctance is based on the conspicuouslydifferent morphology of T. glabrata from that of clinically en-countered Candida spp. Our study has shown that, even withinthe small subset of species represented by clinical yeast iso-lates, pseudohypha formation is not a dependable distinguish-ing character for routine yeast identification at the genericlevel. Because of this, and because the endlessly continuedcontroversy over classification and nomenclature of medical

yeasts is unhelpful to medical practitioners unspecialized inmycology, we encourage taxonomists to redress present uncer-tainties over the status of precedence of the name Torulopsis byformally proposing the conservation of Candida over Torulop-sis.

We acknowledge the technical assistance of Leanna Jackson andMarc Van Der Flaes.

REFERENCES

1. Barnett, J. A., R. W. Payne, and D. Yarrow. 1990. Yeasts: characteristics andidentification, 2nd ed. Cambridge University Press, Cambridge, UnitedKingdom.

2. Davis, C. 1986. Torulopsis glabrata. Mycopathologia 96:191–193.3. Kane, J., S. Krajden, and R. C. Summerbell. 1986. Torulopsis: still legitimatename for medically important yeast. Can. Med. Assoc. J. 135:274–275.

4. McGinnis, M. R. 1980. Laboratory handbook of medical mycology. Aca-demic Press, New York.

5. McGinnis, M. R., L. Ajello, E. S. Beneke, E. Drouhet, N. L. Goodman, C. J.Halde, L. D. Haley, J. Kane, G. A. Land, A. A. Padhye, D. H. Pincus, M. G.Rinaldi, A. L. Rogers, I. F. Slakin, W. A. Schell, and I. Weitzman. 1984.Taxonomic and nomenclatural evaluation of the genera Candida and Toru-lopsis. J. Clin. Microbiol. 20:813–814.

6. Odds, F. C. 1993. Effects of temperature on anti-Candida activities of anti-fungal antibiotics. Antimicrob. Agents Chemother. 37:685–691.

7. Odds, F. C., M. J. Sackin, and D. Jones. 1990. Numerical taxonomic analysisof imperfect yeast species in Candida and Torulopsis shows no basis forgeneric separation. J. Gen. Microbiol. 136:761–765.

8. Van der Walt, J. P. 1970. Criteria and methods used in classification, p.34–113. In J. Lodder (ed.), The yeasts: a taxonomic study, 2nd ed. North-Holland Publishing Company, Amsterdam.

9. Van Uden, N., and H. Buckley. 1970. Candida Berkhout, p. 898–1087. In J.Lodder (ed.), The yeasts: a taxonomic study, 2nd ed. North-Holland Pub-lishing Company, Amsterdam.

10. Van Uden, N., and M. Vidal-Leiria. 1970. Torulopsis Berlese, p. 1235–1308.In J. Lodder (ed.), The yeasts: a taxonomic study, 2nd ed. North-HollandPublishing Company, Amsterdam.

11. Wickerham, L. J. 1951. Technical bulletin no. 1029. U.S. Department ofAgriculture, Washington, D.C.

12. Yarrow, D., and S. A. Meyer. 1978. Proposal for the amendment of thediagnosis of the genus Candida Berkhout nom cons. Int. J. System. Bacteriol.28:611–615.

TABLE 2. Summary of morphology scores for 70 yeast isolatesin the genus Candida or Torulopsisa

Yeast species No. ofisolates

With experiencedobservers, no. oftimes morphologyreceived the

following scores:

With inexperiencedobservers, no. oftimes morphologyreceived the

following scores:

1 2 3 4 1 2 3 4

C. guilliermondii 4 2 9 2 3 2 3 4 3C. krusei 10 6 19 7 8 9 8 2 11C. lambica 1 1 3 0 0 1 2 0 0C. lipolytica 1 2 2 0 0 2 1 0 0C. lusitaniae 4 0 3 1 12 0 1 4 7C. parapsilosis 12 0 48 0 0 2 33 0 1C. pelliculosa 3 0 3 6 3 0 4 4 1C. tropicalis 1 2 2 0 0 3 0 0 0C. valida 1 1 2 0 1 2 0 1 0T. candida 4 0 3 5 8 0 2 3 7T. glabrata 27 0 0 10 98 0 4 16 61T. inconspicua 1 0 1 0 3 0 1 2 0T. pintolopesii 1 0 2 0 2 0 0 2 1

a The numbers of times that isolates within each species were scored as 1, 2, 3,or 4 by four experienced and three inexperienced observers are tabulated.

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