Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts

International Journal for Parasitology 17 "0887# 0770Ð0772 Research note Fuchsin ~uorescence and auto~uorescence in Cryptosporidium\ Isospora and Cy...

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International Journal for Parasitology 17 "0887# 0770Ð0772

Research note

Fuchsin ~uorescence and auto~uorescence in Cryptosporidium\ Isospora and Cyclospora oocysts M[ Varea\ A[ Clavel\ O[ Doiz\ F[J[ Castillo\ M[C[ Rubio\ R[ Gomez!Lus Servicio de Microbiolog(a\ Hospital Cl(nico Universitario\ 49998 Zaragoza\ Spain Received 07 May 0887^ received in revised form 09 July 0887^ accepted 09 July 0887

Abstract Cryptosporidium parvum and Isospora belli oocysts stained with carbolÐfuchsin\ as in a modi_ed ZiehlÐNeelsen technique\ ~uoresce bright red under green light "435 nm#[ Cryptosporidium oocysts tend to ~uoresce more brightly the less intensely stained they appear under transmitted light^ this is not the case with Isospora[ Fuchsin!stained Cyclospora cayetanensis oocysts ~uoresce rather dimly\ but those not taking the dye retain their typical auto~uorescence[ Cryp! tosporidium and Isospora oocysts are also auto~uorescent\ appearing violet under u[v[ light "254 nm#\ and green under violet "394 nm# and blueÐviolet light "325 nm#[ Their auto~uorescence does not survive the staining procedure[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ Keywords] Cryptosporidium^ Isospora^ Cyclospora^ Fuchsin^ Fluorescence^ Auto~uorescence

Staining of cryptosporidial oocysts by a modi_ed ZiehlÐNeelsen procedure was _rst described in 0870 ð0Ł[ It has since been noticed that this technique does not stain all Cryptosporidium parvum oocysts evenly ð1\ 2Ł[ A modi_ed ZiehlÐNeelsen stain "meth! anol _xation\ carbolÐfuchsin for 19 min\ 6) sul! phuric acid for 29 s\ malachite green for 29 s# is used routinely at our laboratory for the diagnosis of cryptosporidiosis[ In our experience\ weakly and non!stained oocysts are frequently seen "or rather not seen# along with well!stained ones on the same

 Corresponding author[ Fax] ¦23!865 650 553^ e!mail] aclavelÝposta[unizar[es[[

slide when it is observed under transmitted light "Fig[ 0A#[ However\ things are di}erent when such a modi! _ed ZiehlÐNeelsen!stained slide is examined in an epi~uorescence microscope[ Under u[v[ light "exci! tation wavelength 254 nm\ long pass _lter 319 nm] _lter set F!0# oocysts are not noticeable[ Under violet "394Ð384 nm] F!1# and blueÐviolet light "325Ð 419 nm] F!2# they show a faint orangeÐred ~u! orescence\ but under green light "435Ð489 nm] F!3#\ most of these apparently poorly stained oocysts ~uoresce bright red "Fig[ 0B#[ The ~uorescence intensity appears to be approximately inversely pro! portional to fuchsin uptake as visible in transmitted light] oocysts displaying the darkest red typically yield the dimmest ~uorescence\ while those most

9919!6408:87:, ! see front matter Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ PII] S 9 9 1 9 ! 6 4 0 8 " 8 7 # 9 9 0 3 5 ! 4

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M[ Varea et al[:International Journal for Parasitology 17 "0887# 0770Ð0772

Fig[ 0[ "A# Cryptosporidium parvum oocysts in human stool concentrated by formalinÐethyl acetate sedimentation[ Modi_ed ZiehlÐ Neelsen stain[ "B# Same _eld as in "A#\ epi~uorescence microscopy "excitation 435 nm\ long pass 489 nm#[ "C# Methanol!_xed\ unstained Cryptosporidium parvum oocysts[ Epi~uorescence microscopy "excitation 394 nm\ long pass 384 nm#[ "D# Methanol!_xed\ unstained Isospora belli oocyst[ Epi~uorescence microscopy "excitation 394 nm\ long pass 384 nm#[ "E# Isospora sp[ oocysts in human stool[ Modi_ed ZiehlÐNeelsen stain[ "F# Same _eld as in "E#\ epi~uorescence microscopy "excitation 435 nm\ long pass 489 nm#[ Scale bars] "A#Ð"C#\ 09 mm^ "D#Ð"F#\ 29 mm[

vividly ~uorescent frequently show but a very light pinkish tinge on transmitted light "Fig[ 0A\ B] arrowed oocysts#[ In order to test the possibility that this phenom! enon was actually due to oocyst auto~uorescence\ concentrated oocysts were methanol!_xed and mounted in a suitable mounting medium "Eukittþ\ O[ Kindler GmbH and Co[\ Freiburg#[ Flu! orescence was indeed observed\ but with a di}erent pattern] violet with the F!0 set\ green with F!1 and

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F!2\ and not noticeable with F!3[ The ~uorescence faded quickly\ more so the more intense the illumi! nation[ The best contrast was achieved under violet light "F!1# "Fig[ 0C#[ In order to _nd out to which reagent the observed ~uorescence was due\ oocysts stained only with either carbolÐfuchsin or malachite green were then examined^ ~uorescence was observed exclusively in the former\ with the same pattern as in fully modi! _ed ZiehlÐNeelsen!stained oocysts[

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M[ Varea et al[:International Journal for Parasitology 17 "0887# 0770Ð0772

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Table 0 Fuchsin!associated ~uorescence and auto~uorescence in coccidian oocysts] intensity and colour depending on excitation wavelength Excitation wavelength Fuchsin ~uorescence] 254 nm 394 nm 325 nm 435 nm Auto~uorescence] 254 nm 394 nm 325 nm 435 nm

Cryptosporidium parvum

Isospora belli

Cyclospora cayetanensis

* 2OrangeÐred 2OrangeÐred ¦:¦¦¦Red a

* 2OrangeÐred 2OrangeÐred ¦:¦¦¦Red

* 2OrangeÐred 2OrangeÐred 2Red

¦Violet ¦¦Green ¦Green *

¦Violet ¦¦Green ¦Green *

¦¦Blue ¦¦Green ¦Green *

b

a

Fluorescence seems to be roughly inversely proportional to the intensity of dying as visible in transmitted light[ Cyclospora oocysts not stained after being submitted to the modi_ed ZiehlÐNeelsen technique retain auto~uorescence[

b

Fuchsin!~uorescence and auto~uorescence were observed in all of the 19 Cryptosporidium isolates examined for them\ both fresh and aged\ unpre! served and preserved either in 09) formalin or 1[4) potassium dichromate[ Isospora belli oocysts are also acid!fast ð3Ł[ Two available I[ belli isolates "one 7!months!old\ one fresh# were examined[ They gave the same results as Cryptosporidium "Fig[ 0CÐE#\ except that no relationship between transmitted light appearance and ~uorescence intensity was evident[ This auto~uorescence pattern is similar to that reported for Cyclospora cayetanensis oocysts ð4\ 5Ł[ One available\ aged Cyclospora isolate was there! fore methanol!_xed and examined with the same results\ except that ~uorescence under F!0 was more intense and more blue than violet in shade[ This sample was also submitted to the modi_ed ZiehlÐ Neelsen technique[ Stained Cyclospora oocysts ~uoresced poorly with the fuchsin pattern\ while unstained ones "which were the vast majority# retained\ at least partially\ their auto~uorescence pattern[ Cryptosporidium and Isospora oocysts are usually thought not to be ~uorescent\ and this has even been noted as a di}erential feature between them

and Cyclospora ð6Ł[ However\ the results reported here "summarised in Table 0# suggest that this is not always the case\ and di}erentiation based on this feature may be inaccurate[ References ð0Ł Henriksen SA\ Pohlenz JFL[ Staining of cryptosporidia by a modi_ed ZiehlÐNeelsen technique[ Acta Vet Scand 0870^11]483Ð485[ ð1Ł Baxby D\ Blundell N[ Sensitive\ rapid\ simple methods for detecting Cryptosporidium in faeces[ Lancet 0872^1]0038[ ð2Ł Clavel A\ Arnal AC\ Sanchez EC\ et al[ Morphological and staining alterations of Cryptosporidium oocysts contained in 09) formalin!preserved faeces[ Res Rev Parasitol 0883^ 43"3#]162Ð164[ ð3Ł Ng E\ Markell EK\ Fleming RL\ Fried M[ Demonstration of Isospora belli by acid!fast stain in a patient with acquired immune de_ciency syndrome[ J Clin Microbiol 0873^19"2#] 273Ð275[ ð4Ł Long EG\ White EH\ Carmichael WW\ et al[ Morphologic and staining characteristics of a cyanobacterium!like organ! ism associated with diarrhea[ J Infect Dis 0880^053] 088Ð191[ ð5Ł Ortega YR\ Sterling CR[ Cyclospora cayetanensis] epi! demiology and diagnosis[ Clin Microbiol Newsl 0885^ 07"11#]058Ð065[ ð6Ł Soave R[ Cyclospora] an overview[ Clin Infect Dis 0885^12]318Ð324[