TRACING THE CRIMINAL Part nine: Criminals in spiral form Institute for microbiology shows L From anthem of medical students „Diabetes mellitus, icterus et vomitus“ n(Second part) nTreponema pallidum nGonococcus ruber nUlcus molle, ulcus durum nMolle est reparaturum nNos curabit … n(name of a suitable urologist of dermatovenerologist) Causes syfilis Old name of causative agent of gonorrhoea (N. gonorrhoeae) Chancroid – caused by Haemophilus ducreyi Chancre – one of typical symptomas of syphilis (Melody of Gaudeamus igitur, iuvenes dum sumus) 23 syph1 www.med.sc.edu [USEMAP] Survey of topics Clinical characteristics of spiral bacteria Microbiological characteristics & dg. of spirochets Clinical characteristics of spiral bacteria Story one nRoseanne Pinkspot started to have pink spots on her body. She thought, that probably… Oh yes, several weeks ago whe participated on a girl scout camp and several times during the camp she had a tick. nHer GP sent her to children infection clinic, and experienced infectionist confirmed, that most likelly it is the disease that Roseanne supposed. For sure, she took serum for antibody detection… 37 lymerash 15 borri9 www.borrelia.de www.med.sc.edu Erythema migrans nThis is a picture of Erythema migrans of student M. M., who kindly agreed to let it for use in education C:\Uživatel\Ondra\Obrázky a fotky\Odborné\ECM Michal Makuša 2u.jpg The causative agent was nBorrelia afzelii, one of borreliae, causing Lyme disease and belonging to the group Borrelia burgdorgeri sensu lato (= „broad sense of meaning“) nThis species „in broad sense“ is divided into several species „in narrower sense“. The most important are B. garinii, B. afzelii and B. burgdorferi sensu stricto nWhile in the USA mostlly the third of them is common and joint symptomatology is common, in Europe two first borreliae are more common, and the typical disease is neuroborreliosis nBesides lyme diseases there exist other species causing recurrent fever (B. duttoni, B. recurrentis) 19 ISlarva http://www.pasteur.fr 21 Borrelia Borrelia burgdorferi 02a borrelia http://www.presse.uni-wuppertal.de gemi.mpl.ird.fr 16 cyklus bore Lyme disease – a tick borne zoonosis 38 lymecycle 20 zecke http://www.presse.uni-wuppertal.de www.niaid.nih.gov www.med.sc.edu 17 Borr recurentis Borrelia recurrentis http://medinfo.ufl.edu Story two (virtual, but basis is from a real story) nWhen Phyllis found, that she really needs pervitin, and more and more, she decided to earn money by her own body. nWhen the client paid more, she went with him without a preservative, she used anticonception and she felt more OK nThen she fell in love and decided to have a child. She stopped the anticonception and was happy. Helmut will be a good father… Story two – continuing nSo Phyllis was pregnant. But she found heerself a genital ulcus and her gynecologist took blood for serological examination. It was positive. Phyllis did not want interruption, it was too late and she wanted her child. nPhyllis was treated, but the antibiotic was not chosen properly. The child was born ill and after two weeks it died because of a secondary Klebsiella septicaemia 11 treponema The criminal was nTreponema pallidum, causing syphilis (lues) nSyphilis is a classic sexual disease. It is transmitted sexually only. But it is a systemic disease – in developped stages the whole body is affected (gummata, aortal dissection, neurolues, psychical symptomas) nSome subspecies of T. pallidum and some other treponemas cause other, differently transmitted diseases (framboesia – yaws, T. pertenue) nSome treponemas are non-pathogenous www.primer.ru 07 Treponema%20pallidum 13 TreponemaPallidum uhavax.hartford.edu http://nl.wikipedia.org Treponema pallidum C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Syfilis dodatky\53 Syph stádia.jpg www.geocities.com Course of syphilis Course of syphilis 08 chancre1 primary syphilis („chancre“) secondary syphilis 09 syph%20sec%20tongue uhavax.hartford.edu (2×) C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Syfilis dodatky\51 terciární syfilis.jpg C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Syfilis dodatky\52 syfilitické aneurysma aortálního oblouku.jpg C:\Documents and Settings\u Svate Anny\Dokumenty\Obrázky\Syfilis dodatky\54 last stage syphilis.jpg www.common-place.org www.geocities.com www.nlm.nih.gov Tertiary syphilis Syphilis 394px-Syphilis-poster-wpa-cure 25 syph3 www.med.sc.edu en.wikipedia.org Yaws (framboesia) 26 yaws www.med.sc.edu Story three nMr. Ratter was an employee of NWPS Ltd. (Nowhere Water Pipes and Sewage) nHis job was sewage cleaning. He knew all sewage corridors. He also knew rat habits, he liked rats and he understood them. nNevertheless, once there was some mis-understanding between him and the leader of rat group and Mr. Ratter was bitten to his leg. nSome time after this, Mr. Ratter was hospitalized with icterus and bleeding… 40 lepto-kidney Kidney with the corresponding disease www.med.sc.edu This is not Mr. Ratter, but his Venezuelan colleague with a similar fate… 43 paciente_icterico http://www.ceniap.gov.ve The disease is caused by… nLeptospira interrogans ser. Icterohemorrhagiae nFormerly individual serovars of Leptospira were considered to be individual species, now all pathogenic ones are taken as a part of species Leptospira interrogans (second species Leptospira biflexa is non-pathogenic) nSymptomatology varies, from „flu-typhoid“ symptomas of serovar Grippotyphosa (field fever, canefield fever) to jaundice and bleeding (Weil disease, as in mr. Ratter) in serovar Ictero-hemorragiae. (At least these two serovars are quite simple for remembering, try to remeber at least them J) [USEMAP] Microbiologic characteristics and diagnostics of spirochets Spirochets nBorreliae (but also treponemas and leptospiras) are spirochets, i. e. spiral bacteria. nTheir cell wall is close to a gram-negative one, but they do not Gram stain mostly because they are very thin. So we microscopy them only using dark-field or fluorescence microscopy, or imunofluorescence (¹ fluorescence) nSpirochets commonly are not culturable (some of them can be cultured experimentally, but it has no practical meaning) 14 hechemy2 www.wadsworth.org Treponema pallidum 12 Ro0304F8Treponema http://www.revoptom.com Treponema: direct methods nDirect diagnostics is rare, also because often there is hardly something to take. Only patients with chancre are available for scrapping. nMicroscopy: It is possible to use wet mount – dark field. It is strange, that although is is a wet mount, immersion is used (treponemas are very subtle). Besides that, fluorescence staining can be used nNeither culture nor biochemical methods are used nAntigen detection can be performed by direct IMF nAnimal experiment: There exist so named RIT – Rabbit infectivity test nPCR diagnostics is more and more important. This is an exception – besides chancre scrapping, it is also possible to send full blood for examination. 10 treponema0 www.primer.ru Direct syphilis diagnostics – survey nRIT – Rabbit infectivity test. For ethical reasons, but also as it is too much work, the RIT is minimized today. nDark field – shining Treponema pallidum is observed againts the dark field nDirect IMF – another direct, but difficult method nPCR – also from blood New Zealand Rabbit used for RIT newzealand7 www.rockinjawrabbits.com Treponema Dark field microscopy Zástin www.medmicro.info rays Preparation Only rays flexed at the preparation come to the obeserver‘s eye. Therefore, the observer‘s eye can see dark field with shining object(s) FTA_ABS Immunofluorescence www.medmicro.info Treponema: indirect methods nWe use non-treponema tests, where cardiolipin from bovine hearths acts as an antigen, and treponema tests, where we have a real antigen from Treponema pallidum nDiagnostics is composed of screening and confirmation. We confirm everything that was positive or at least borderline at screening, in reasonable cases even negative results. nScreening usually consists of a non-treponema and a treponema test nConfirmation is performed by highly specific treponema tests The most important indirect tests for lues Historical BWR – Bordet Wassermann Nontr. Screening RRR – Rapid Reagin Test or RPR or VDRL test MHA-TP (TPHA) Treponema Confirmatory ELISA FTA-ABS (indir. imunofluor.) Western Blotting Historical, or superconfirmation TPIT (Treponema Pallidum Imobilisation Test) = Nelson MHA-TP – test for passive haemaglutination; now RBC use replaced by polycelluslose RRR and TPHA nIn RRR, the well with turbidity is positive (it looks like the positive control). It is necessary shake the panel, otherwise the reaction would not be visible. nTPHA is an agglutination on carrier (RBC). A „potato shaped formation“ is positive, a dense dot is negative MHA-TP – to remember Aglutinace na nosičích nPositive – agglutinate formed, viewed from up as clot of irregular shape nNegative – RBC (polycelulose particles in newer variant) fall to bottom forming a regular dense dot viewed from up TPHA – reading: TPHA_hodnoceni RRR – reading: turbidity = positive, no turbidity = negative www.medmicro.info Indications for confirmation nScreening reactions are performed always, when somebody is to be tested for syphilis (including e. g. pregnant women that are not at all supposed to be positive). Screening reactions are performed only qualitatively or semiquantitatively nIndication for confirmation is: nany positive or at least borderline result in RRR and/or MHA-TP reaction, OR npresence of suspicious lesions on body, or anamnesis of risky sexual intercourse – here even in case of negativity of both reactions ELISA, Western blotting and PCR in spirochetal diagnostics nELISA, Western blotting and PCR – all of them are used in spirochets simillarly as in other microbes – see J09 and J10 topics in spring term. nPositive are patients with values of absorbance higher than a given value (CAL – calibration well, cut off etc.) nExamination of IgG and IgM antibodies is important, mere IgG positivity is just a proof of a previous infection. nPCR is used in diagnostics of syphilis and Lyme disease. It is usually positive sooner than methods detecting antibodies. 53 kansen_02 Borrelia and leptospira – course of investigation nBorrelia: Mostly serology, event. PCR. In serology, IgM (typical fot an early infection) and IgG antibodies are detected using ELISA method, positive finding is confirmed by Western blotting. Western blotting is more specific. nLeptospira: Dark field microscopy and culture in special medium are used idsc.nih.go.jp C:\Uživatel\Ondra\Obrázky a fotky\Odborné\Z internetu\Medici\Spirochety\41 leptospira.jpg Leptospira interrogans www.ucmp.berkeley.edu 39 leptospira Leptospira in the electronoptic microscope www.med.sc.edu Leptospiral diagnostics nMicroscopy of leptospira leptospiry www.medmicro.info More diagnostic opportunities in leptospira (latex agglutination) 47 leptospirosis4 46 leptospirosis1 49 leptospirosis3 52 leptospirosis2 4× www.thailabonline.com The End Leptos-Rasch [USEMAP] www.asci.org/artikel754.html Jody Rasch: Leptospira 60" x 70" – acrylic on canvas