822
A.J.C.P.—Vol.
LETTERS T O T H E EDITOR
Table 1. Laboratory Results in Patients with Antithrombin V Loeliger and Hers
Harbaugh et at.
64
Hemorrhagic tendency
Yes
Yes
Bleeding time
Normal
Normal
Clotting time
Prolonged
Prolonged
Activated partial thromboplastin time
Not done
Prolonged
Recalcification time
Prolonged
Not done
Prothrombin time
Prolonged
Prolonged
Basic prothrombin consumption
Normal
Normal
Clot retraction
Normal
Normal
Factor V
Slightly decreased
Slightly decreased
Other factor assays
Normal
Normal
R E X B. C O N N ,
Antithrombin II
Decreased
Decreased
Department of Laboratory Medicine The Johns Hopkins Hospital Baltimore, Maryland 21205
Antithrombin III
Decreased
Decreased
Thrombin time
Prolonged
Prolonged
Antithrombin: Corrected by protamine sulfate Absorbed by BaS0 4 Heat stable at 56 C.
No No Yes
No No Yes
Reptilase time
Normal*
Normal
Antithromboplastin activity
No
Yes
M. E U L A L I A H A R B A U G H , E L E A N O R M. H I L L ,
M.T.
M.D.
M.D.
References
This result was not published in original paper.
1. Harbaugh ME, Hill EM, Conn RB: Antithrombin and antithromboplastin activity accompanying IgG myeloma. Am J Clin Pathol 63:57-67, 1975 2. Loeliger EA, Hers JF: Chronic antithrombinaemia (antithrombin V) with haemorrhagic diathesis in a case of rheumatoid arthritis with hypergammaglobulinaemia. T h r o m b Diath Haemorrh 1:499-526, 1975
Morphologic Identification of Pathogenic Yeasts To the Editor:—In many medical laboratories, pathogenic yeasts are identified using the scheme developed by Dolan. 1 This scheme has been criticized,2 Received May 1, 1975; received revised manuscript J u n e 18, 1975;acceptedforpublicationJune 18,1975. Work done at the Department of Pathology, University of Auckland School of Medicine, Auckland, New Zealand. Presented at the annual meeting of North Island Pathologists of New Zealand, Roturoa, November 1974. Supported by the government of New Zealand under the Commonwealth Medical Aid Programme. Key words: Pathogenic yeasts; Candida; Cryptococcus; Saccharomyces; Trichosporon; Geotrichum; Germ tube; Capsule; Chlamydospore.
but it is more practicable for reasonably e x p e r i e n c e d mycologists at medical laboratories where rapid identification of pathogenic organisms is a requisite. We have recently shown 3 that the germ tubes of Candida albicans, as well as the characteristic mycelial morphology of Trichosporon and most species of Candida can be demonstrated by the direct microscopic examination of a single preparation under cover of a coverglass on 0.1% glucose agar that has been incubated for three hours at 37 C. and then for 4 8 - 7 2 hours at 22 C. Subsequent studies with clinical isolates
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
classified as having antithrombin V. Loeliger and Hers were the first investigators to identify the acquired direct inhibitor of thrombin, which they named "antithrombin V." Subsequent authors used the same term to designate an inhibitor of fibrinogen polymerization having physical properties similar to the thrombin inhibitor. Despite these similarities, there appear to be sufficient dissimilarities between these two inhibitors to warrant a change in nomenclature that can indicate the mode of action of each. Perhaps, as suggested in Drs. Hensen and Loeliger's letter, "polymerization inhibitor" would be appropriate.
December 1975
LETTERS TO THE EDITOR
823
YEAST O-V/. GLUCOSE AGAR 3 h, 570C EXAMINE
FOR GERM TUBES FURTHER INCUBATION 4 8 - 7 2 h, i r C EXAMINE FOB HyPHftE EXAMINE FOR
WGROSIN fr COVERGLASS EXAMINE FOR
CAPSULE
+
ABTHROSPOP.ES
+ CBYPTOCOCCUS*SP. RH00OTORULA SP. SMALL BLASTO" SPORES WITHOUT ASCOSPORES
SACCHAROTORULOPSIS SP* MVCES LIKE 5P§
TYPICAL1 MORPHOLOGY IDENTIFIES
RECTANGULAR ARTHROSPORES
C.ALBICANS C.5TELLAT0IDEA C.TROPICALIS C.PSEUDOTROPICALI5 C.KRUSEI C. PARAPSILOSIS C-GUILLIERGEOTRICHUM* 5 P. MONDII
OVAL ARTHROSPORES WITH YEASTS
TRICHO 5P0R0N SP.
* -FOR IDENTIFICATION OF THEIR SPECIES ASSIMILATION STUDIES WOULD BE REQUIRED. ®-OCCASIONALLY CAN PRODUCE SHORT HYPHAE. Note:WHEN UNCERTAIN CONFIRM IDENTIFICATION BY FERMENTATION AND ASSIMILATION TESTS. CONFIRM DIFFERENTIATION OF GEOTRICHUM FROM TRICHOSPORON BY UREASE TEST.
FIG. 1. A scheme for the identification of pathogenic yeasts.
of other yeasts (Cryptococcus neoformans, 6; Cryptococcus albidus, 6; Torulopsis glabrata, 33; Saccharomyces cerevisiae, 4) have shown that all isolates of Cryptococcus form capsules on this medium. This can be easily demonstrated in those organisms that have failed to develop hyphae after 72 hours of growth at 22 C. by placing a drop of nigrosin, then a coverglass, over the lines of inoculation. Direct microscopic examination of such preparations also helps to differentiate the noncapsulated organisms, since those of S. cerevisiae are larger than those of T. glabrata. After 72 hours of growth on this medium, all isolates of S. cerevisiae formed ascospores that could be demonstrated in Ziehl Neelsen-stained smears.
identification of pathogenic yeasts. T o evaluate the practicability of the scheme, a total of 265 clinical isolates comprising the yeasts mentioned above, plus isolates of Candida and Trichosporon (C. albicans, 140; C. tropicalis, 22; C. pseudotropicalis, 3; C. krusei, 11; C. parapsilosis, 33; C. guillermondii, 33; Trichosporon cutaneum, 2) reported earlier 3 and two isolates of Geotrichum were identified independently on the basis of their individual morphology, carbohydrate fermentation and assimilation patterns together with their ability to produce urease, as well as following the scheme. Using the scheme, except for the two isolates of C. parapsilosis, all isolates of Candida could be placed in their respective species as determined by other tests, On the basis of these observations, we and the remaining isolates could be placed developed a scheme (Fig. 1) for the similarly in their respective genera.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
LARGE BLASTOSPORES 6ASCOSPORES
C . ALBICANS C. STELLATOIDEA
824
LETTERS TO THE EDITOR
While the present scheme has all the merits of Dolan's scheme in that the majority of pathogenic yeasts can be identified mainly on morphologic examination with minimal use of the more laborious time-consuming procedures of fermentation and assimilation tests, it has added advantages in that only a single preparation on a simple, inexpensive, easily pre-
64
pared medium is used, and is examined directly at various stages. It may, however, be noted that laboratory personnel unfamiliar with the morphology of these organisms will have some initial difficulties in using the scheme; initially, they should confirm identifications by biochemical tests. However, in due time they will develop reasonable confidence in the identification of most pathogenic yeasts on the morphologic basis suggested. K. R. J O S H I , M.D.,
PH.D.
Reader in Pathology and Microbiology S. P. Medical College Bikaner (Raj.), India References 1. Dolan CT: A practical approach to identification of yeast-like organisms. Am J Clin Pathol 55: 580-590, 1971 2. English MP: Identifying yeasts. Med Lab Technol 31:327-333, 1974 3. Joshi KR, Bremner DA, Parr DN, et al: The morphological identification of pathogenic yeasts using carbohydrate media. J Clin Pathol 28:18-24, 1975
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Though isolates of Candida stellatoidea and Rhodotorula were not studied in this series, these have been included in the scheme for the sake of completion. C. stellatoidea, which forms germ tubes and chlamydospores, must be differentiated from C. albicans by its inability to assimilate sucrose. However, this species is becoming an infrequent clinical isolate, possibly because of availability of better technics for assimilation studies. Organisms of Rhodotorula species, being capsulated, should be differentiated from those of Cryptococcus by their colored (pink or coral) colonies. 2
AJ.C.P.—Vol.
A.J.C.P.—Vol.
LETTERS T O T H E EDITOR
Table 1. Laboratory Results in Patients with Antithrombin V Loeliger and Hers
Harbaugh et at.
64
Hemorrhagic tendency
Yes
Yes
Bleeding time
Normal
Normal
Clotting time
Prolonged
Prolonged
Activated partial thromboplastin time
Not done
Prolonged
Recalcification time
Prolonged
Not done
Prothrombin time
Prolonged
Prolonged
Basic prothrombin consumption
Normal
Normal
Clot retraction
Normal
Normal
Factor V
Slightly decreased
Slightly decreased
Other factor assays
Normal
Normal
R E X B. C O N N ,
Antithrombin II
Decreased
Decreased
Department of Laboratory Medicine The Johns Hopkins Hospital Baltimore, Maryland 21205
Antithrombin III
Decreased
Decreased
Thrombin time
Prolonged
Prolonged
Antithrombin: Corrected by protamine sulfate Absorbed by BaS0 4 Heat stable at 56 C.
No No Yes
No No Yes
Reptilase time
Normal*
Normal
Antithromboplastin activity
No
Yes
M. E U L A L I A H A R B A U G H , E L E A N O R M. H I L L ,
M.T.
M.D.
M.D.
References
This result was not published in original paper.
1. Harbaugh ME, Hill EM, Conn RB: Antithrombin and antithromboplastin activity accompanying IgG myeloma. Am J Clin Pathol 63:57-67, 1975 2. Loeliger EA, Hers JF: Chronic antithrombinaemia (antithrombin V) with haemorrhagic diathesis in a case of rheumatoid arthritis with hypergammaglobulinaemia. T h r o m b Diath Haemorrh 1:499-526, 1975
Morphologic Identification of Pathogenic Yeasts To the Editor:—In many medical laboratories, pathogenic yeasts are identified using the scheme developed by Dolan. 1 This scheme has been criticized,2 Received May 1, 1975; received revised manuscript J u n e 18, 1975;acceptedforpublicationJune 18,1975. Work done at the Department of Pathology, University of Auckland School of Medicine, Auckland, New Zealand. Presented at the annual meeting of North Island Pathologists of New Zealand, Roturoa, November 1974. Supported by the government of New Zealand under the Commonwealth Medical Aid Programme. Key words: Pathogenic yeasts; Candida; Cryptococcus; Saccharomyces; Trichosporon; Geotrichum; Germ tube; Capsule; Chlamydospore.
but it is more practicable for reasonably e x p e r i e n c e d mycologists at medical laboratories where rapid identification of pathogenic organisms is a requisite. We have recently shown 3 that the germ tubes of Candida albicans, as well as the characteristic mycelial morphology of Trichosporon and most species of Candida can be demonstrated by the direct microscopic examination of a single preparation under cover of a coverglass on 0.1% glucose agar that has been incubated for three hours at 37 C. and then for 4 8 - 7 2 hours at 22 C. Subsequent studies with clinical isolates
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
classified as having antithrombin V. Loeliger and Hers were the first investigators to identify the acquired direct inhibitor of thrombin, which they named "antithrombin V." Subsequent authors used the same term to designate an inhibitor of fibrinogen polymerization having physical properties similar to the thrombin inhibitor. Despite these similarities, there appear to be sufficient dissimilarities between these two inhibitors to warrant a change in nomenclature that can indicate the mode of action of each. Perhaps, as suggested in Drs. Hensen and Loeliger's letter, "polymerization inhibitor" would be appropriate.
December 1975
LETTERS TO THE EDITOR
823
YEAST O-V/. GLUCOSE AGAR 3 h, 570C EXAMINE
FOR GERM TUBES FURTHER INCUBATION 4 8 - 7 2 h, i r C EXAMINE FOB HyPHftE EXAMINE FOR
WGROSIN fr COVERGLASS EXAMINE FOR
CAPSULE
+
ABTHROSPOP.ES
+ CBYPTOCOCCUS*SP. RH00OTORULA SP. SMALL BLASTO" SPORES WITHOUT ASCOSPORES
SACCHAROTORULOPSIS SP* MVCES LIKE 5P§
TYPICAL1 MORPHOLOGY IDENTIFIES
RECTANGULAR ARTHROSPORES
C.ALBICANS C.5TELLAT0IDEA C.TROPICALIS C.PSEUDOTROPICALI5 C.KRUSEI C. PARAPSILOSIS C-GUILLIERGEOTRICHUM* 5 P. MONDII
OVAL ARTHROSPORES WITH YEASTS
TRICHO 5P0R0N SP.
* -FOR IDENTIFICATION OF THEIR SPECIES ASSIMILATION STUDIES WOULD BE REQUIRED. ®-OCCASIONALLY CAN PRODUCE SHORT HYPHAE. Note:WHEN UNCERTAIN CONFIRM IDENTIFICATION BY FERMENTATION AND ASSIMILATION TESTS. CONFIRM DIFFERENTIATION OF GEOTRICHUM FROM TRICHOSPORON BY UREASE TEST.
FIG. 1. A scheme for the identification of pathogenic yeasts.
of other yeasts (Cryptococcus neoformans, 6; Cryptococcus albidus, 6; Torulopsis glabrata, 33; Saccharomyces cerevisiae, 4) have shown that all isolates of Cryptococcus form capsules on this medium. This can be easily demonstrated in those organisms that have failed to develop hyphae after 72 hours of growth at 22 C. by placing a drop of nigrosin, then a coverglass, over the lines of inoculation. Direct microscopic examination of such preparations also helps to differentiate the noncapsulated organisms, since those of S. cerevisiae are larger than those of T. glabrata. After 72 hours of growth on this medium, all isolates of S. cerevisiae formed ascospores that could be demonstrated in Ziehl Neelsen-stained smears.
identification of pathogenic yeasts. T o evaluate the practicability of the scheme, a total of 265 clinical isolates comprising the yeasts mentioned above, plus isolates of Candida and Trichosporon (C. albicans, 140; C. tropicalis, 22; C. pseudotropicalis, 3; C. krusei, 11; C. parapsilosis, 33; C. guillermondii, 33; Trichosporon cutaneum, 2) reported earlier 3 and two isolates of Geotrichum were identified independently on the basis of their individual morphology, carbohydrate fermentation and assimilation patterns together with their ability to produce urease, as well as following the scheme. Using the scheme, except for the two isolates of C. parapsilosis, all isolates of Candida could be placed in their respective species as determined by other tests, On the basis of these observations, we and the remaining isolates could be placed developed a scheme (Fig. 1) for the similarly in their respective genera.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
LARGE BLASTOSPORES 6ASCOSPORES
C . ALBICANS C. STELLATOIDEA
824
LETTERS TO THE EDITOR
While the present scheme has all the merits of Dolan's scheme in that the majority of pathogenic yeasts can be identified mainly on morphologic examination with minimal use of the more laborious time-consuming procedures of fermentation and assimilation tests, it has added advantages in that only a single preparation on a simple, inexpensive, easily pre-
64
pared medium is used, and is examined directly at various stages. It may, however, be noted that laboratory personnel unfamiliar with the morphology of these organisms will have some initial difficulties in using the scheme; initially, they should confirm identifications by biochemical tests. However, in due time they will develop reasonable confidence in the identification of most pathogenic yeasts on the morphologic basis suggested. K. R. J O S H I , M.D.,
PH.D.
Reader in Pathology and Microbiology S. P. Medical College Bikaner (Raj.), India References 1. Dolan CT: A practical approach to identification of yeast-like organisms. Am J Clin Pathol 55: 580-590, 1971 2. English MP: Identifying yeasts. Med Lab Technol 31:327-333, 1974 3. Joshi KR, Bremner DA, Parr DN, et al: The morphological identification of pathogenic yeasts using carbohydrate media. J Clin Pathol 28:18-24, 1975
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Though isolates of Candida stellatoidea and Rhodotorula were not studied in this series, these have been included in the scheme for the sake of completion. C. stellatoidea, which forms germ tubes and chlamydospores, must be differentiated from C. albicans by its inability to assimilate sucrose. However, this species is becoming an infrequent clinical isolate, possibly because of availability of better technics for assimilation studies. Organisms of Rhodotorula species, being capsulated, should be differentiated from those of Cryptococcus by their colored (pink or coral) colonies. 2
AJ.C.P.—Vol.
