VEQ PARASSITI 2014 FIRENZE, 14 DICEMBRE 2015 DOTT.SSA CHIARA VETTORI LABORATORIO ANALISI OSPEDALE VERSILIA

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1 VEQ PARASSITI 2014 FIRENZE, 14 DICEMBRE 2015 DOTT.SSA CHIARA VETTORI LABORATORIO ANALISI OSPEDALE VERSILIA 1

2 Parassiti ematici Plasmodium malariae Plasmodium vivax Plasmodium falciparum (parassitemia) Negativo 2

3 Phylum Apicomplexa 34

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7 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 3 Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Plasmodium malariae - Tutti gli stadi /. 100% Plasmodio 85% P.malariae 3.5% P.falciparum Risultato Numero % Score Plasmodium malariae Plasmodium malariae Tutte le forme di sviluppo Plasmodium malariae Trofozoiti e Gametociti Plasmodium malariae Gametociti Plasmodium malariae Trofozoiti Plasmodium malariae Schizonti e Gametociti Plasmodium malariae Trofozoiti e Schizonti Plasmodium malariae Schizonti Plasmodium species Plasmodium species Trofozoiti Plasmodium species Schizonti Plasmodium vivax Plasmodium falciparum Schizonti Plasmodium ovale Plasmodium falciparum Plasmodium vivax Trofozoiti Plasmodium ovale Trofozoiti e Schizonti Plasmodium falciparum Trofozoiti e Schizonti Plasmodium ovale Trofozoiti Plasmodium ovale Schizonti NON ESEGUITO 8 n.a. = non assegnato 7

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10 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 9 Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Plasmodium vivax - Tutti gli stadi /. 100% Plasmodio 83.1% P.vivax 6.9% P.vivax + altro 7.9% P.falciparum Risultato Numero % Score Plasmodium vivax Trofozoiti e Gametociti Plasmodium vivax Tutte le forme di sviluppo Plasmodium vivax Plasmodium vivax Trofozoiti Plasmodium vivax Trofozoiti e Schizonti Plasmodium vivax Gametociti Plasmodium species Plasmodium vivax Tutte le forme di sviluppo/plasmodium falciparum Trofozoiti Plasmodium species Trofozoiti Plasmodium vivax Trofozoiti e Gametociti/Plasmodium falciparum Trofozoiti Plasmodium vivax Trofozoiti/Plasmodium falciparum Trofozoiti Plasmodium species Tutte le forme di sviluppo Plasmodium vivax/plasmodium ovale Plasmodium vivax Tutte le forme di sviluppo/plasmodium falciparum Trofozoiti e S Plasmodium falciparum Trofozoiti Plasmodium ovale Trofozoiti e Gametociti/Plasmodium falciparum Trofozoiti e Gam Plasmodium falciparum Trofozoiti e Gametociti Plasmodium ovale Trofozoiti e Gametociti Plasmodium falciparum Trofozoiti e Schizonti Plasmodium malariae Plasmodium ovale Tutte le forme di sviluppo NON ESEGUITO 7 n.a. = non assegnato 10

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13 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 6 Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Plasmodium falciparum - trofozoiti /. /. 99.5% Plasmodio 79% P.falciparum 11% P.vivax Risultato Numero % Score Plasmodium falciparum Trofozoiti Plasmodium falciparum Plasmodium falciparum Trofozoiti e Schizonti Plasmodium falciparum Schizonti Plasmodium falciparum Merozoiti Plasmodium falciparum Trofozoiti e Gametociti Plasmodium falciparum Schizonti e Gametociti Plasmodium species Plasmodium species Trofozoiti Plasmodium species Trofozoiti e Schizonti Plasmodium vivax Trofozoiti Plasmodium vivax Plasmodium ovale Trofozoiti Plasmodium malariae Trofozoiti Plasmodium vivax Trofozoiti e Gametociti Plasmodium vivax Schizonti/Merozoiti Plasmodium vivax Schizonti Plasmodium vivax Tutte le forme di sviluppo Plasmodium vivax Trofozoiti e Schizonti Plasmodium malariae Microfilarie non identificate NON ESEGUITO 8 n.a. = non assegnato 13

14 Azienda Ospedaliero - Universitaria Careggi CENTRO REGIONALE DI RIFERIMENTO S.O.D. Sicurezza e Qualità in Laboratorio Struttura Certificata UNI EN ISO 9001:2008 Direttore dr. Massimo Quercioli tel fax Data 08/10/2015 Oggetto: Programma di V.E.Q. Parassitologia 2015 Calcolo della parassitemia: Striscio sottile Si conta il numero di emazie parassitate su un totale di emazie che varia da almeno a o più a seconda dei casi e si esprime la parassitemia in percentuale (%). (Nr Emazie Parassitate/Nr Emazie Contate) x 100 = Parassitemia % Se la parassitemia è alta (>1%) è possibile esaminare solo emazie; se è bassa (<1%) si dovranno esaminare emazie o più. Più elevato è il numero di emazie contate, più preciso è il calcolo della parassitemia. Le emazie poliparassitate vengono contate come una sola emazia infetta, mentre i gametociti non devono essere contati. 14

15 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 6 Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Plasmodium falciparum - trofozoiti /. /. Risultato Numero % Score da 1.0% a 2.0% n.a. da 2.1% a 3.0% n.a. da 3.1% a 4.0% n.a. da 0.5% a 0.99% n.a. da 4.1% a 5.0% n.a. da 0.1% a 0.49% n.a. da 5.1% a 6.0% n.a. da 9.1% a 10.0% n.a. da 0.05% a 0.09% n.a. < 0.05% n.a. da 6.1% a 7.0% n.a. da 8.1% a 9.0% n.a. >10% n.a. 25% Indice Parassitemico (solo per P. falciparum) Valore Atteso : 2.2 Totale partecipanti: % NON ESEGUITO 49 n.a. = non assegnato 15

16 Azienda Ospedaliero - Universitaria Careggi CENTRO REGIONALE DI RIFERIMENTO S.O.D. Sicurezza e Qualità in Laboratorio Struttura Certificata UNI EN ISO 9001:2008 Direttore dr. Massimo Quercioli tel fax Data 08/10/2015 Oggetto: Programma di V.E.Q. Parassitologia 2015 Si comunica che è stata modificata la modalità di inserimento dei risultati della parassitemia. Dal prossimo invio di un campione per parassitologia ematica sarà possibile inserire il valore esatto della parassitemia riscontrata e non più il range in cui il valore riscontrato era compreso. Sarà possibile inserire due cifre intere e due cifre decimali, quindi valori compresi tra 0,01 e 99,99. La valutazione della parassitemia, ovvero del numero di parassiti presenti nel sangue, è utile in tutte le infezioni da Plasmodi e indispensabile nelle infezioni da Plasmodium falciparum per due motivi: la gravità dell infezione è correlata al numero di emazie parassitate. Ad esempio, una parassitemia del 2% ( parassiti/μl di sangue) è già considerata indice di infezione severa, valori superiori al 5% ( /μL) possono avere un significato diagnostico sfavorevole; sorveglianza del trattamento per verificarne l efficacia e soprattutto per evidenziare le possibili resistenze al trattamento. La conta dei parassiti deve essere effettuata quotidianamente fino alla sparizione dei parassiti. Saranno elaborati unicamente i risultati di parassitemia riguardanti le infezioni da P. falciparum. Per la valutazione della parassitemia viene calcolata la media e la deviazione standard dei risultati inviati dai laboratori. Sono considerati corretti i risultati all interno di ± 1DS, parzialmente corretti i risultati compresi tra ± 1 e 2 DS ed errati i risultati > di ± 2DS. 16

17 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 6 40,0 67.4% VALORE ATTESO: 2,2% Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Plasmodium falciparum - trofozoiti /. /. 36,2 30,0 31,2 20,0 10,0 0,0 12,8 8,5 5 2,1 0,7 0 0,7 2,1 0,7 < >10 17

18 Manser et al. Malaria Journal 2013, 12:428 RESEARCH Open Access Estimating the parasitaemia of Plasmodium falciparum: experience from a national EQA scheme Monika Manser 1, Catherine Olufsen 1, Nick Andrews 2 and Peter L Chiodini 1,3* Abstract Background: To examine performance of the identification and estimation of percentage parasitaemia of Plasmodium falciparum in stained blood films distributed in the UK National External Quality Assessment Scheme (UKNEQAS) Blood Parasitology Scheme. Methods: Analysis of performance for the diagnosis and estimation of the percentage parasitaemia of P. falciparum in Giemsa-stained thin blood films was made over a 15-year period to look for trends in performance. Results: An average of 25% of participants failed to estimate the percentage parasitaemia, 17% overestimated and 8% underestimated, whilst 5% misidentified the malaria species present. Conclusions: Although the results achieved by participants for other blood parasites have shown an overall improvement, the level of performance for estimation of the parasitaemia of P. falciparum remains unchanged over 15 years. Possible reasons include incorrect calculation, not examining the correct part of the film and not examining an adequate number of microscope fields. 18

19 Figure 5 The range of parasitemia reported by participants on a blood film with an intended result of 1.5%. 19

20 Many participants fail to specify the percentage parasitaemia perhaps because it is not part of their routine practice. Counting of red blood cells infected with parasites of P. falciparum is essential and the percentage parasitaemia should always be reported as this has implications for prognosis and the pattern of treatment employed [6]. Participants are penalized for failing to do so. 20

21 OVERESTIMATION Overestimation of parasitaemia could to be due to their counting the number of trophozoites per 100 red blood cells and not the number of parasitized red blood cells. A red blood cell infected with multiple parasites counts as one parasitized red cell. Another reason could be including gametocytes when calculating the parasitaemia and counting all the malaria parasites present in a mixed infection could also contribute to overestimating the percentage parasitaemia. This is demonstrated in specimen 14 which contained trophozoites of P. falciparum with a reference parasitaemia of 16% but also contained trophozoites of Plasmodium vivax. 35% of participants overestimated the parasitaemia compared with 33% and 25% who overestimated the parasitaemia in specimens 15 and 16, which had a reference parasitaemia of 20%, but contained only trophozoites of P. falciparum. Similarly for specimen 8 which contained trophozoites of P. falciparum with a reference parasitaemia of 2.5%, but also contained trophozoites of Plasmodium ovale. Nineteen percent of participants overestimated the parasitaemia compared to 11% and 14% in specimens 6 and 7 respectively both of which had a similar parasitaemia, but contained trophozoites of P. falciparum alone. When calculating the parasitaemia of Plasmodium falciparum, only the trophozoite stages are counted and the gametocytes and other malaria parasite species are excluded from the result [6]. NUMERO DI EMAZIE PARASSITATE EMAZIE POLIPARASSITATE INFEZIONI MISTE NON CONTARE I GAMETOCITI 21

22 Those participants who underestimate the parasitaemia may not be counting an adequate number of fields. It is recommended that 40 fields of a thin film are counted, especially when the parasitaemia is low due to the possible uneven distribution of parasites, Examining the wrong part of the film can contribute to an inaccurate calculation, i.e. examining fields that are too thick or too thin. The area where the red cells are touching and not overlapping or too far apart should be examined Calculation error is another possible source of wrong results. The recommended procedure for estimating the percentage parasitaemia in a thin blood film is by expressing the number of parasitised red blood cells per 100 cells. A minimum of 1,000 cells should be counted. The quantification can be facilitated by the use of a Miller square [6]. CONTARE ALMENO 40 CAMPI ESAMINARE LE AREE CORRETTE CONTARE ALMENO 1000 EMAZIE 22

23 Laboratory diagnosis of malaria Plasmodium spp. Determination of Parasitemia Determination of parasitemia can be done using both thick and thin smears. Thick smears: The number of parasites/µl of blood is determined by enumerating the number of parasites in relation to the standard number of WBCs/µl (8000). No. Parasites (8000 No. WBCs counted) = No. parasites per µl of blood 23

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26 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 12 Striscio di sangue color. con Giemsa a ph 7.2 Microrganismo Presente: Assenza di Parssiti /. 14.8% Plasmodio Risultato Numero % Score Assenza di parassiti Plasmodium species Leishmania species Amastigoti Plasmodium malariae Plasmodium vivax Trofozoiti Plasmodium falciparum Trofozoiti Plasmodium malariae Gametociti Plasmodium vivax Microfilarie non identificate Microfilarie Plasmodium vivax Merozoiti Microfilarie non identificate Loa loa Microfilarie Plasmodium vivax Schizonti e Gametociti Plasmodium species Trofozoiti e Gametociti Wuchereria bancrofti Babesia species Merozoiti Plasmodium vivax Gametociti Plasmodium vivax Tutte le forme di sviluppo Plasmodium species Gametociti Plasmodium falciparum Tutte le forme di sviluppo Plasmodium species Trofozoiti Plasmodium malariae Schizonti Parassiti non identificati Plasmodium malariae Trofozoiti NON ESEGUITO 7 n.a. = non assegnato 26

27 100,0 negativo Assenza di parassiti plasmodium 80,0 85,2 90,8 84,5 92,2 87,8 84,1 91,4 81,6 86,4 78,3 60,0 40,0 20,0 3,9 7,0 8,1 3,7 14,0 13,2 8,8 7, ,3 14,8 27

28 Parassitologia fecale Protozoi (cisti, trofozoiti) Giardia duodenalis (lamblia) Entamoeba histolytica/dispar Cystoisospora belli (oocisti) Elminti (uova, larve) Nematodi Ascaris lumbricoides Strongyloides stercoralis Trematodi (digenei) Paragonimus Cestodi Diphyllobotrium latum Hymenolepis nana 28

29 10-14 mcm assostile corpi parabasi 2-4 nuclei Cisti 29

30 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze REGIONE TOSCANA V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 5 Codice Lab. 00 Sospensione formolata di Feci Microrganismo Presente: Giardia intestinalis - cisti /Rara ed incostante presenza di B. hominis /. /. Giardia 96.6% Risultato Numero % Score Giardia duodenalis (lamblia) Cisti Giardia duodenalis (lamblia) Giardia duodenalis (lamblia) Cisti/Blastocystis hominis Cisti Giardia duodenalis (lamblia) Cisti e Trofozoiti/Blastocystis hominis Giardia duodenalis (lamblia) Cisti/Blastocystis hominis Giardia duodenalis (lamblia) Uova Giardia duodenalis (lamblia) Cisti/Endolimax nana Giardia duodenalis (lamblia) Cisti/Strongyloides stercoralis Larve rabditoidi Giardia duodenalis (lamblia) Cisti/Entamoeba species Cisti Giardia duodenalis (lamblia) Cisti/Taenia species Uova Giardia duodenalis (lamblia) Cisti/Endolimax nana Cisti Assenza di parassiti Enterobius vermicularis Uova Strongyloides stercoralis Larve Paragonimus species Uova Ancylostoma/Necator species Larve NON ESEGUITO 6 n.a. = non assegnato 30

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32 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze REGIONE TOSCANA V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 7 Codice Lab. 0 Sospensione formolata di Feci Microrganismo Presente: E.histolytica/dispar - Cisti / Incostante presenza di: E.coli - E.nana - B.hominis /. Risultato Numero % Score Entamoeba histolytica/dispar Cisti Entamoeba histolytica/dispar Entamoeba histolytica/dispar Cisti e Trofozoiti Entamoeba histolytica/dispar Cisti/Endolimax nana Cisti Entamoeba histolytica/dispar Cisti/Blastocystis hominis Cisti Entamoeba histolytica/dispar Cisti/Blastocystis hominis Entamoeba histolytica/dispar Tutte le forme di sviluppo Entamoeba histolytica/dispar/endolimax nana Entamoeba histolytica/dispar Cisti e Trofozoiti/Blastocystis hominis Entamoeba histolytica/dispar Trofozoiti Entamoeba histolytica/dispar Cisti/Entamoeba coli Cisti Entamoeba histolytica/dispar Cisti e Trofozoiti/Endolimax nana Cisti Entamoeba histolytica/dispar Cisti/Chilomastix mesnili Trofozoiti Entamoeba species Cisti Entamoeba coli Cisti Entamoeba coli Entamoeba species Assenza di parassiti Entamoeba coli Cisti e Trofozoiti Entamoeba species Cisti/Blastocystis hominis Cisti Blastocystis hominis Tutte le forme di sviluppo Endolimax nana Cisti Blastocystis hominis Entamoeba histolytica/dispar/ascaris lumbricoides Endolimax nana Entamoeba coli Cisti/Ascaris lumbricoides Entamoeba hartmanni Cisti Hymenolepis nana Uova Ascaris lumbricoides Uova Ascaris lumbricoides Dicrocoelium species Uova Entamoeba hartmanni Oocisti Schistosoma japonicum Uova Entamoeba coli Cisti e Trofozoiti/Entamoeba hartmanni Cisti e Trofozoiti Ascaris lumbricoides Uova/Entamoeba hartmanni Cisti Hymenolepis diminuta NON ESEGUITO 4 n.a. = non assegnato 32 E.histolytica/dispar 71% Colorazione (tricromica) Immunocromatografia Biologia molecolare Sierologia

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34 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 1 Sospensione formolata di Feci Microrganismo Presente: Ascaris lumbricoides - Uova /. 98% Risultato Numero % Score Ascaris lumbricoides Uova Ascaris lumbricoides Ascaris lumbricoides Uova non fertili Ascaris lumbricoides Larve Ascaris lumbricoides Uova/Uova non fertili Ascaris lumbricoides Uova/Ancylostoma/Necator species Tutte le forme di sviluppo Giardia duodenalis (lamblia) Cisti Cryptosporidium parvum Oocisti Giardia duodenalis (lamblia) Enterobius vermicularis Uova NON ESEGUITO 3 n.a. = non assegnato 34

35 ELMINTI: UOVA E DIMENSIONI Opisthorchis Clonorchis Capillaria Taenia Hymenolepis nana Trichuris Enterobius Ascaris Anchylostoma Diphyllobotrium Hymenolepis diminuta Trycostrongilus Nanophyetus 35 Paragonimus Ascaris sterile

36 Larva rabditoide esofago 36

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38 G.bianchi Eosinofili:62.7% 38

39 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 4 Sospensione formolata di Feci Microrganismo Presente: Strongyloides stercoralis / Larve rabditoidi /. /. 95.3% Risultato Numero % Score Strongyloides stercoralis Larve rabditoidi Strongyloides stercoralis Larve Strongyloides stercoralis Strongyloides stercoralis Larve strongiloidi Strongyloides stercoralis Larve rabditoidi/larve strongiloidi Assenza di parassiti Ancylostoma/Necator species Larve Blastocystis hominis Cisti Ancylostoma/Necator species Ancylostoma/Necator species Larve rabditoidi NON ESEGUITO 3 n.a. = non assegnato 39

40 opercolo giallo-bruno non embrionate protuberanza 40

41 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 2 Sospensione formolata di Feci Microrganismo Presente: Diphyllobothrium latum - Uova /. 88.9% Risultato Numero % Score Diphyllobothrium latum Uova Diphyllobothrium latum Diphyllobothrium species Uova Diphyllobothrium latum Larve Assenza di parassiti Uova non identificate % Fasciola hepatica Uova Fasciola hepatica Fasciola hepatica/fasciolopsis buski Uova Strongyloides stercoralis Larve rabditoidi Paragonimus species Giardia duodenalis (lamblia) Taenia species Uova Strongyloides stercoralis Larve Chilomastix mesnili Cisti Cryptosporidium species Cisti Ancylostoma/Necator species Uova Entamoeba coli Trofozoiti Ascaris lumbricoides Schistosoma japonicum Uova Dicrocoelium species Uova Endolimax nana Cisti Strongyloides stercoralis/dicrocoelium species NON ESEGUITO 3 n.a. = non assegnato 41

42 ELMINTI: UOVA E DIMENSIONI Opisthorchis Clonorchis Capillaria Taenia Hymenolepis nana Trichuris Enterobius Ascaris Anchylostoma Diphyllobotrium Hymenolepis diminuta Trycostrongilus Nanophyetus 42 Paragonimus Ascaris sterile

43 guscio sottile filamenti polari uncini 43

44 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 10 Sospensione formolata di Feci Microrganismo Presente: Hymenolepis nana - uova /. Incostante presenza di Blastocystis RE Co 94.1% Risultato Numero % Score Hymenolepis nana Uova Hymenolepis nana Embriofori Hymenolepis nana Hymenolepis nana Uova/Blastocystis hominis Cisti Hymenolepis nana Uova/Blastocystis hominis Hymenolepis nana Embriofori/Blastocystis hominis Cisti Hymenolepis nana Embriofori/Blastocystis hominis Tutte le forme di sviluppo Hymenolepis nana/blastocystis hominis Hymenolepis nana Trofozoiti % Hymenolepis diminuta Uova Hymenolepis diminuta Hymenolepis nana Uova/Endolimax nana Cisti Hymenolepis nana Uova/Entamoeba coli Cisti Hymenolepis nana Uova/Entamoeba histolytica/dispar Cisti Hymenolepis diminuta Embriofori Hymenolepis diminuta Cisti Diphyllobothrium latum Uova Strongyloides stercoralis Larve Entamoeba hartmanni Cisti NON ESEGUITO 3 n.a. = non assegnato 44

45 ELMINTI: UOVA E DIMENSIONI Opisthorchis Clonorchis Capillaria Taenia Hymenolepis nana Trichuris Enterobius Ascaris Anchylostoma Diphyllobotrium Hymenolepis diminuta Trycostrongilus Nanophyetus 45 Paragonimus Ascaris sterile

46 Life Cycle: un verme adulto vive 4-6 settimane Autoinfestazione per anni 75 milioni portatori uova embrionate infettanti cisticercoide nell insetto o nel villo intestinale 46

47 Original Article: Brief Report Malignant Transformation of Hymenolepis nana in a Human Host Atis Muehlenbachs, M.D., Ph.D., Julu Bhatnagar, Ph.D., Carlos A. Agudelo, M.D., Alicia Hidron, M.D., Mark L. Eberhard, Ph.D., Blaine A. Mathison, B.S.M.(A.S.C.P.), Michael A. Frace, Ph.D., Akira Ito, Ph.D., Maureen G. Metcalfe, M.S., Dominique C. Rollin, M.D., Govinda S. Visvesvara, Ph.D., Cau D. Pham, Ph.D., Tara L. Jones, Ph.D., Patricia W. Greer, M.T., Alejandro Vélez Hoyos, M.D., Peter D. Olson, Ph.D., Lucy R. Diazgranados, M.D., and Sherif R. Zaki, M.D., Ph.D. N Engl J Med Volume 373(19): November 5, 2015

48 In this case report, malignant transformation and metastatic spread of Hymenolepis nana, the dwarf tapeworm, was identified in a patient with advanced HIV infection. Summary Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer. 48

49 Radiographic and Pathological Features of Malignant Hymenolepis nana. Muehlenbachs A et al. N Engl J Med 2015;373:

50 This case posed a diagnostic conundrum. The proliferative cells had overt features of a malignant process they invaded adjacent tissue, had a crowded and disordered growth pattern, and were monomorphic, with morphologic features that are characteristic of stem cells (a high nucleus-to-cytoplasm ratio) but the small cell size (<10 µm in diameter) suggested infection with an unfamiliar, possibly unicellular, eukaryotic organism. Infection with a plasmodial slime mold (phylum, Amoebozoa; class, Myxogastria) was considered because of the prominent syncytia formation. Although many cestode tissues are syncytial notably, their tegument a tapeworm infection was initially considered less likely because of the primitive appearance of the atypical cells, the complete absence of architecture that was identifiable as tapeworm tissue, and the rarity of previously reported cases of invasive cestodiasis. 2,3 During our laboratory investigations, the le- 50

51 Confirmation of H. nana Infection. Muehlenbachs A et al. N Engl J Med 2015;373:

52 Opercolo Guscio spesso Non embrionale 52

53 polmone, cervello Asia e Africa Ingestione crostacei crudi 53

54 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 8 Sospensione formolata di Feci Microrganismo Presente: Paragonimus spp - Uova 91.1% Risultato Numero % Score Paragonimus species Uova Paragonimus species Paragonimus species Uova non fertili Paragonimus species Cisti Paragonimus species Uova/Chilomastix mesnili Trofozoiti Paragonimus species Larve Paragonimus species/opistorchis/clonorchis species Fasciola hepatica/fasciolopsis buski Uova Diphyllobothrium latum Uova Fasciola hepatica Fasciolopsis buski Uova Fasciola hepatica Uova Diphyllobothrium latum Clonorchis sinensis Uova Opistorchis/Clonorchis species Clonorchis sinensis Fasciolopsis buski Opistorchis/Clonorchis species Uova NON ESEGUITO 3 n.a. = non assegnato 54

55 ELMINTI: UOVA E DIMENSIONI Opisthorchis Clonorchis Capillaria Taenia Hymenolepis nana Trichuris Enterobius Ascaris Anchylostoma Diphyllobotrium Hymenolepis diminuta Trycostrongilus Nanophyetus 55 Paragonimus Ascaris sterile

56 Oocisti 56

57 S.O.D. Sicurezza e Qualità A.O.U.Careggi-Firenze V.E.Q. in PARASSITOLOGIA Ciclo 2014 Campione N 11 Sospensione formolata di Feci Microrganismo Presente: Oocisti di Cystoisospora belli. Presenti, non in tutti i campioni, r Entamoeba coli Risultato inviato: 65.4% Risultato Numero % Score Cystoisospora (Isospora) belli Oocisti Cystoisospora (Isospora) belli Cystoisospora (Isospora) belli Cisti Cystoisospora (Isospora) belli Oocisti/Entamoeba coli Cisti Cystoisospora (Isospora) belli Oocisti/Entamoeba species Cisti Cystoisospora (Isospora) belli Uova Assenza di parassiti Entamoeba coli Strongyloides stercoralis Larve Heterophyes/Metagonimus species Uova Balantidium coli Entamoeba histolytica/dispar Cisti Schistosoma mansoni Uova Amebe non identificate/larve non identificate Entamoeba histolytica/dispar Cisti/Entamoeba coli Cisti Giardia duodenalis (lamblia) Cisti Trichuris trichiura Larve Entamoeba hartmanni NON ESEGUITO 3 n.a. = non assegnato 57

58 Classe Apicomplexa At time of excretion, the immature oocyst contains usually one sporoblast (more rarely two). In further maturation after excretion, the sporoblast divides in two (the oocyst now contains two sporoblasts); the sporoblasts secrete a cyst wall, thus becoming sporocysts; and the sporocysts divide twice to produce four sporozoites each. Infection occurs by ingestion of sporocystscontaining oocysts: the sporocysts excyst in the small intestine and release their sporozoites, which invade the epithelial cells and initiate schizogony. Upon rupture of the schizonts, the merozoites are released, invade new epithelial cells, and continue the cycle of asexual multiplication. Trophozoites develop into schizonts which contain multiple merozoites. After a minimum of one week, the sexual stage begins with the development of male and female gametocytes. Fertilization results in the development of oocysts that are excreted in the stool. 58

59 A B C Cystoisospora belli oocysts were detected in an unstained wet mount prepared after concentration of the stool specimen by the formalin ethyl acetate technique (Panel A, arrow) and in a fecal smear stained by Kinyoun s modified acid-fast staining method (Panel B). Cystoisospora oocysts are thin-walled and ellipsoidal and range from 20 to 33 µm in length and from 10 to 19 µm in width. 19 Oocysts are immature (unsporulated) when found in freshly passed feces and may contain a single, spherical sporoblast (arrowhead) or may lack a distinct sporoblast (arrows). 19 After a few days of room-temperature incubation, the oocysts mature to sporulated oocysts containing two sporocysts (Panel C, arrowheads). Oocysts will show autofluorescence when wet mounts are examined by fluorescence microscopy, making detection easier. Geographic Distribution Worldwide, especially in tropical and subtropical areas. Infection occurs in immunodepressed individuals, and outbreaks have been reported in institutionalized groups in the United States. Clinical Presentation Infection causes acute, nonbloody diarrhea with crampy abdominal pain, which can last for weeks and result in malabsorption and weight loss. In immunodepressed patients, and in infants and children, the diarrhea can be severe. Eosinophilia may be present (differently from other protozoan infections). 59

60 Key points for laboratory identification of Cyclospora, Cryptosporidium, and Cystoisosporabelli A B C Oocysts in stool smears stained with modified acid-fast stain: A Cryptosporidium sp. B Cyclospora cayetanensis C Cystoisospora belli Oocysts in stool smears stained with safranin stain: D Cryptosporidium sp. E Cyclospora cayetanensis F Cystoisospora belli D E F G H I Wet mount preparations Cyclospora cayetanensis sequence showing sporulation event from unsporulated oocyst (far left) G to sporulated oocyst containing 2 sporocysts (far right) under differential interference contrast (DIC) H I Cystoisospora belli (UV fluorescence microscopy) Cyclospora cayetanensis (UV fluorescence microscopy) 60

61 Sarcocystis 61

62 The Nobel Assembly at Karolinska Institutet has today decided to award the 2015 Nobel Prize in Physiology or Medicine with one half jointly to William C. Campbell and Satoshi Ōmura for their discoveries concerning a novel therapy against infections caused by roundworm parasites and the other half to Youyou Tu for her discoveries concerning a novel therapy against Malaria Diseases caused by parasites have plagued humankind for millennia and constitute a major global health problem. In particular, parasitic diseases affect the world s poorest populations and represent a huge barrier to improving human health and wellbeing. This year s Nobel Laureates have developed therapies that have revolutionized the treatment of some of the most devastating parasitic diseases. 62

63 William C. Campbell and Satoshi Ōmura discovered a new drug, Avermectin, the derivatives of which have radically lowered the incidence of River Blindness and Lymphatic Filariasis, as well as showing efficacy against an expanding number of other parasitic diseases. Youyou Tu discovered Artemisinin, a drug that has significantly reduced the mortality rates for patients suffering from Malaria. These two discoveries have provided humankind with powerful new means to combat these debilitating diseases that affect hundreds of millions of people annually. The consequences in terms of improved human health and reduced suffering are immeasurable. Figure 1: The 2015 Nobel Prize in Physiology or Medicine awards discoveries regarding novel therapies for some of the most devastating parasitic diseases: River Blindness, Lymphatic Filariasis (Elephantiasis) and Malaria. The distribution of these diseases is quite similar and is collectively shown in blue on the world map. 63

64 From bacteria and plants to novel anti-parasite therapies After decades of limited progress in developing durable therapies for parasitic diseases, the discoveries by this year s Laureates radically changed the situation. Satoshi Ōmura, a Japanese microbiologist and expert in isolating natural products, focused on a group of bacteria, Streptomyces, which lives in the soil and was known to produce a plethora of agents with antibacterial activities (including Streptomycin discovered by Selman Waksman, Nobel Prize 1952). Equipped with extraordinary skills in developing unique methods for large-scale culturing and characterization of these bacteria, Ōmura isolated new strains of Streptomyces from soil samples and successfully cultured them in the laboratory. From many thousand different cultures, he selected about 50 of the most promising, with the intent that they would be further analyzed for their activity against harmful microorganisms (Figure 2). Figure 2: Satoshi Ōmura searched for novel strains of Streptomyces bacteria as a source for new bioactive compounds. He isolated microbes from soil samples in Japan, cultured them in the laboratory (inset to left) and characterized many thousands of Streptomyces cultures. From those, he selected around 50 cultures that appeared most promising, and one of these cultures later turned out to be Streptomyces avermitilis (inset to right), the source of Avermectin. 64

65 William C. Campbell, an expert in parasite biology working in the USA, acquired Ōmura s Streptomyces cultures and explored their efficacy. Campbell showed that a component from one of the cultures was remarkably efficient against parasites in domestic and farm animals. The bioactive agent was purified and named Avermectin, which was subsequently chemically modified to a more effective compound called Ivermectin. Ivermectin was later tested in humans with parasitic infections and effectively killed parasite larvae (microfilaria) (Figure 3). Collectively, Ōmura and Campbell s contributions led to the discovery of a new class of drugs with extraordinary efficacy against parasitic diseases. Figure 3: William C. Campbell discovered that one of Ōmura s Streptomyces cultures was very effective in killing off parasites and the active compound, Avermectin, was purified. Avermectin was further modified to Ivermectin, which turned out to be highly effective in both animals and humans against a variety of parasites, including those that cause River Blindness and Lymphatic Filariasis. 65

66 Malaria was traditionally treated by chloroquine or quinine, but with declining success. By the late 1960s, efforts to eradicate Malaria had failed and the disease was on the rise. At that time, Youyou Tu in China turned to traditional herbal medicine to tackle the challenge of developing novel Malaria therapies. From a large-scale screen of herbal remedies in Malaria-infected animals, an extract from the plant Artemisia annua emerged as an interesting candidate. However, the results were inconsistent, so Tu revisited the ancient literature and discovered clues that guided her in her quest to successfully extract the active component from Artemisia annua. Tu was the first to show that this component, later called Artemisinin, was highly effective against the Malaria parasite, both in infected animals and in humans (Figure 4). Artemisinin represents a new class of antimalarial agents that rapidly kill the Malaria parasites at an early stage of their development, which explains its unprecedented potency in the treatment of severe Malaria. Figure 4: Youyou Tu searched ancient literature on herbal medicine in her quest to develop novel malaria therapies. The plant Artemisia annua turned out to be an interesting candidate, and Tu developed a purification procedure, which rendered the active agent, Artemisinin, a drug that is remarkably effective against Malaria. 66

67 Avermectin, Artemisinin and global health The discoveries of Avermectin and Artemisinin have fundamentally changed the treatment of parasitic diseases. Today the Avermectin-derivative Ivermectin is used in all parts of the world that are plagued by parasitic diseases. Ivermectin is highly effective against a range of parasites, has limited side effects and is freely available across the globe. The importance of Ivermectin for improving the health and wellbeing of millions of individuals with River Blindness and Lymphatic Filariasis, primarily in the poorest regions of the world, is immeasurable. Treatment is so successful that these diseases are on the verge of eradication, which would be a major feat in the medical history of humankind. Malaria infects close to 200 million individuals yearly. Artemisinin is used in all Malaria-ridden parts of the world. When used in combination therapy, it is estimated to reduce mortality from Malaria by more than 20% overall and by more than 30% in children. For Africa alone, this means that more than lives are saved each year. The discoveries of Avermectin and Artemisinin have revolutionized therapy for patients suffering from devastating parasitic diseases. Campbell, Ōmura and Tu have transformed the treatment of parasitic diseases. The global impact of their discoveries and the resulting benefit to mankind are immeasurable. 67