TRACING THE CRIMINAL Part six: More gram-negative cocci and bacilli Institute for microbiology shows L [USEMAP] Survey of topics Clinical characteristics – G– cocci Clinical characteristics – „other G– bacilli Diagnostics of G– cocci Diagnostics of „other G– bacilli“ Clinical characteristics – G– cocci Story One nJohny was very childish, he had no experience with women even in 20 years of age. His friends made fun of him. Once they made a plan: they gave him lots of spirits and paid a prostitute for him. Johny had a feeling, that he is finally a man… only before pus started to drop from his urethra… 41 kapavka 56 pár Certainly, you know, what is the criminal 11 Neisseria gonorrhoeae nOf course, it is Neiseria gonorrhoeae, (gonococcus) causing gonorrhoea. nGonorrhoea is urethritis, in females also cervicitis; asymptomatically or symptomatically gonococci are found in pharynx and rectum, too. nIn females, it is not a colpitis (vaginal inflamation), so it is not recommended to perform vaginal swabs in gonorrea. Partner of Johny of that night… 42 gonorrhea-4 žena nova.medicina.cz 55 chlamgon www.lcusd.net Story two nLucy learned four weeks for the examination of physiology. She did not leave the house at all and only sat at textbooks. At the exam she had intention, that she is not able to say a word, but finally she passed with „E“. nIn the evening, she visited a dancing party with friends. The party was full of smoke and they danced during all night. Next day, Lucy was not well, she started to have fever and rash. (continuing) nIn this moment, Lucy was hospitalized at infection clinic. In ambulance she failed unconcious and doctors constated metabolic failure. After ten hours of attempts to keep Lucy‘s vital functions, that had no effect, Lucy died. nSuch a course of infection may be caused by a dangerous criminal. Some of his strains are present in throat of healthy persons… 10 NEME skin_rash_wellcometrust 10 http://www.infektionsbiologie.ch And the criminal is… n… Neisseria meningitidis or meningococcus nMeningococcus causes meningitis, but also sepsis and other serious problems; all this is product of clonal strains. nOther strains are completelly inocents and studies say that some ten percent of population are throat carriers of meningococcus. nVirulence is related mostly with protein antigens nPolysacharidic antigens determine preventability by vaccination 12 Neisseria meningitidis http://www.waterscan.co.yu Why the infecion comes sometimes, and sometimes does not nThe invasive infection is only present, when the strain is highly virulent (specific clones of the microorganism) and the host organism is ready to get infected nMeningococcus is transmitted by a narow contact. Invasive infection is more likely when mucous membranes are dammaged, e. g. by smoking or previous viral infection. nInfection is often present after too big physical activity after long inactivity period How they come into the tissue 08 interaktion 07 odběr likvoru CSF sampling http://www.infektionsbiologie.ch http://www.infektionsbiologie.ch Meningococcal meningitis is worldwide very important infection 06 meningitis pás 09 dítě nemocné meningitidou 05 NEME „Meningitis belt“, area of extended presence of meningococcal meningitis all pictures: http://www.infektionsbiologie.ch Let‘s compare neisseriae: In vivo In vitro Gonococcus The most delicate, sexual transmission only The most delicate, grows on chocolate agar only Meningococcus Less delicate, short distance air transport Less delicate, if blood agar is enriched, growth is enabled So called „oral“ neisseriae The least delicate, air transport possible The least delicate, grows even on poor blood agar 18 meningokokken_250 www.meningitis.de Story three nAnnie was crying and touching her ear. Her mother measured her temperature, and it was elevated. nAt general practicioner‘s, Annie was examined and diagnosis of otitis media was set nAs her tympanon was already broken, the pus was taken for examination nAmoxicilin was used for threatment imediatelly. Later, a susceptible pathogen was found. And this pathogen was •Moraxella (sub-genus Branhamella) catarrhalis – another Gram-negative coccus •This organism is often present in small amounts even in healthy persons. •In pure culture causes sinusitis, otitis media, pharyngitis etc. •Her sub-genus (originally genus) name Branhamella was derived from Sarah Branham, one of first women scientists. She was one of brave women to show man that not only men can be good in science Moraxella catarrhalis 49 fig2moraxella 50 moraxella 48 MoraxellaBranhamellaCatarrhalis http://www.medicine.uiowa.edu http://www.uni-ulm.de http://www.microbelibrary.org [USEMAP] Clinical characteristics – „other G– bacilli“ Story four nThere was a big movement in the hospital that day: three more patients, all of them seniors, became ill, and again it was the same – breathing problems and fever nAfter a thoroughful examination, the laboratory found the pathogen not only in patient secretions, but also in hot water pipes of the hospital. The pipes had to be rebuilt and only after that the infections were stopped finally. Legionaire‘s disease nIt is a disease caused by Legionella pneumophila nLegionaire‘s disease is a variant of disease that is quite severe; another variant, Pontiac fever, is rather mild nThe bacterium ofther have reservoir in water instalation, air condition, etc. nWhen building new hospital departments (but also senior houses, hotels, spa…) measures to prevent legionelosis should be taken, mostly at planing water pipe system (no blind branches). [USEMAP] 21 legionella 22 legionella 23 legionella-glow Legionella www.rivm.nl/infectieziektenbulletin www.chemistryquestion.com mcb.berkeley.edu 66 legionella http://www.eldersllp.com Legionaire‘s disease 64 LEPN rtg plic http://aapredbook.aappublications.org Legionella and temperature 67 teploty LEPN http://www.engr.psu.edu Some ways of water disinfection 68 legionela-01 desinfekce 69 legionela-02 UV desinfekce 70 legionela-03 chorová df 71 legionela-04 filtrace 72 legionela-05 filtrace 2 1 hot temperature 2 UV rays 3 filtration 4 chloration 1 2 3 4 http://www.koncept-ekotech.com 65 Legionella%20Certificate „Other Gram-negative bacteria“ nIt is not a real „group“ or „familly“. Nevertheless, these are quite rare bacteria, usually not growing on Endo agar, some of them growing on blood agar, and causing various diseases nBesides Legionella, we should mention at least three genera: Bordetella, Brucella and Francisella Genus Bordetella nB. pertussis and B. parapertussis cause whooping cough nB. bronchiseptica causes various pathologies in humans and animals. nWhooping cough is very rare due to vaccination nPernasal swab is used when necessary (swab from nasopharynx, taken throung nasal cavity) 32 Bordetella npswab http://www.dsf.health.state.pa.us Bordetella 2 BOPE 33 BOPE 35 DTP-HB_4 www2.mf.uni-lj.si www.gpo.or.th http://medinfo.ufl.edu 74 bordetella a teploty Bordetella and temperature http://www.rivm.nl/infectieziektenbulletin Bordetella was isolated in 1906 by Jules Bordet et Octave Gengou 73 Bordet a Gengou www.pasteur.fr 34 Jak se dělá pertusová vakcína Pertussis vaccine production www.stedim.com 31 Bordetella cat_symptoms Bordetella bronchiseptica: cat infection www.nobivacbb.com Genus Brucella nIt is a causative agent of zoonoses nBrucella abortus is a bovine pathogen. It infects commonly bovine placenta, causing abortions in cattle. In human it causes so named Bang disease (fever, problems with various organs etc.) nOther brucellae are Brucella suis from hogs, Brucella melitensis from sheep and goats and Brucella canis from dogs 76 Brucella abortus abortované tele 77 240px-BrucellaMelitensis 30 Brucella melitensis Brucella http://pathport.vbi.vt.edu http://upload.wikimedia.org 26 Brucella fig28_1 gsbs.utmb.edu 27 Brucella fig28_2 gsbs.utmb.edu 29 fig28_4 gsbs.utmb.edu 28 fig28_3 Remembering Brucella gsbs.utmb.edu/microbook melitensis = derived from island of Malta (Melita in Latin) 75 Brucella http://www.nlm.nih.gov Genus Francisella nMost important species – F. tularensis nCauses tularemia – „hare plague“ nGamekeepers, but even more cooks preparing game are in risk of infections nThe organism may infect wounds, but also it is possible to inhalate it, thus leading to pneumoniae 34 francisella Francisella 38 francisella1 04a tulara66 www.infektionsnetz.at www.wnysmart.org/tularemia www.stedim.com 39 tularaemie_r 37 francisella2 microbes.historique.net www.infektionsnetz.at 59 tular2002 61 PULTUL 62 images%20med 63 tularemie http://www.antropozoonosi.it (4×) From report about epidemiological situation (IX/2008) – I (abbreviated) nTularemia – A21: Woman, born 1970 from Valtice, gardener. 20th June GP visit for T 38°C, sore throat, enlarged lymphonodes next to right clavicula. The lymphonode was extirpated. First serological examination negative, second (2nd July) positive nBrucellosis – A23: Man, born 1972, Brno. 10th May intestinal problems, febrilia, hepatopatia, dg. proven serologically. Brucella abortus positive, CFT 1:8. In anamnesis a travel to Borneo (Kalimantan) in april, trek in a tropical forest. Delayed report. n(Reported by Public Health Office for South Moravia) From report about epidemiological situation (IX/2008) – II nPertussis (A37.0): 4 cases reported (Brno-environment, Hodonín), people aged 14 to 17, all vaccinated, one with missing re-vaccintion nParapertussis (A37.1): 3 cases of disease, coinfection, Brno environment, Hodonín [USEMAP] Diagnostics of G– cocci Neisseriae and Moraxellae – characteristics I nMicroscopy: G – diplococci, coffee bean shaped, often intraleucocytar (see next slide) 20 10 http://medicine.plosjournals.org Some neisseriae a moraxellae may be prolonged and so they might be coccobacilli or bacilli (e. g. Neisseria elongata) Gonococci 43 Gonorrhoea2 negomik2 www.medmicro.info, photo O. Z. http://www.warwickshire.gov.uk 05 NEME Meningococcus http://www.infektionsbiologie.ch 53 NEGO a NEME www.mfi.ku.dk Observation of gonorrhoea smear nGonococci (but also meningococci) are Gram-negative diplococci, coffee-bean shaped, mostly intracelullar. Presence of cocci inside leucocytes is their typical property. NEGO_MIK_4 In females, the microscopy is slightly different from males. Baba1 € � www.medmicro.info NEGO_MIK_4 NEGO_MIK_4 WBC with gonococci www.medmicro.info Neisseriae and Moraxellae – characteristics II nCulture: tiny, colourless or yellowish (according to the species) colony, growing (species specific) on blood or chocolate agar nBlood agar or chocolat agar necessary also for diffusion disc test 16 Colonies http://www.ratsteachmicro.com Gonococcus à ß Meningococcus NeEc4 Culture of Neisseria and Moraxella nThere exist differences between G– cocci in culture properties: nOral neisseriae and moraxellae (O+M) grow on blood agar (BA). nMeningococci (ME) grow only on nutrient-rich variants of BA. nGonococci (GO) do not grow on BA at all, they require chocolat agar. NeEc4 Foto O. Z. BA BA+ ChA GO NO NO YES ME NO YES YES O+M YES YES YES negoca www.medmicro.info Gonococcus atb testing Neisseriae and Moraxellae – characteristics III nBiochemical diagnostic: all of them are catalase positive, oxidase positive; Moraxella catarrhalis also positive in a specific test, called INAC (indoxyl-acetate test) nAntigen analysis: performed usually by means of by latex agglutination, very important in meningococci for differentiation between serogroup B (not preventable by vaccination) and other serogroups (preventable) Basic biochemical tests nQuick tests with diagnostic strips simplify the diagnostics nNeisseriae are oxidase positive, moraxellae too, but their reaction might be late. nMoraxella is typically positive in INAC test nINAC test is simillar to oxidase test, but the strip should be moistened and one has to wait 5 minutes. The colour is blue-green. Species determination of neisseriae and moraxellae nFor detailed identification of neisseriae and moraxellae, biochemical tests are used, in Czechia mostly NEISSERIAtest, in other countries other tests (at those below or on the next slide) nBoth pathogenous neisseriae have little biochemical activity: Gonococcus splits glucose only, Meningococcus glucose and maltose. 17 gcmc_sugars 14 Neisseria_meningitidis_galerie http://medecinepharmacie.univ-fcomte.fr Neisseriae and Moraxellae – differential diagnostics 1 nGram staining: G – (diplo)cocci nOxidase differenciates some other G– cocci (e. g. Acinetobacter – a G– non-fermenter – is also often coccoid) nGrowth on various media differenciates ngonococcus (growth on chocolate agar only), nmeningococcus (growth on rich blood agar and chocolate agar) noral neisseriae (growth on both poor and rich blood agar and chocolate agar) nM. catarrhalis (growth like oral neisseriae) Neisseriae and Moraxellae – differential diagnostics 2 nINAC test (a strip test similar to oxidase test) – positive in Moraxella catarrhalis nComplex biochemical test (NEISSERIAtest), is used especially for mutual differenciation of oral neisseriae nAntigen analysis (determination of meningococcal serogroup in invasive infections) Neisseria gonorrhoeae – sampling nIn gonorrhoea suspicion it is very important to perform sampling properly. Despite all care it is likely that the pathogen would not be able to survive. That is why it is recomended to send also smear on a slide from cervix and urethra (but not rectum and pharynx) nSo „complete gonorrhoea examination“ consists of following parts: nurethral swab in Amies + smear ncervical swab in Amies + smear (�) nrectal swab in Amies (no smear) nphagyngeal swab in Amies (no smear) Antibiotic susceptibility testing in neisseriae and related bacteria nAntibiotic susceptibility in pathogenous neisseriae is determined on media, on which they are able to grow, i. e. not on MH agar nFirst election drug for meningococcus is even today classical penicillin. It is used also for Gonococcus. Other drugs are macrolids, quinolones or cefriaxone. In neisseriae, zones are often large and confluent. negoca When the zones are so large that is is not possible to measure them, we do not measure them and we write down simply „susceptible“. Hypothetical margins of zones are in green: mention, that they are mostly either confluent, or behind the margin of the Petri dish! www.medmicro.info, photo O. Z. A set of antibiotics againts pathogenic neisseriae Antibiotic Abbr. Reference zone Penicillin (basic penicillin) AMP 28 mm Chloramphenicol C 18 mm Azithromycin (macrolide) AZM 12 mm Doxycycline (tetracykline) DO 19 mm Ampicillin (aminopenicillin) AMP 29 mm Co-trimoxazol (mixture) SXT 16 mm Somewhere, E-test is used 52 meningocoque_3_Pharo www.actu-pharo.com Antigen detection / antigen analysis nAgglutination set for CSF agglutination is used for identification of pathogens. In case of a may be identified. nIn Meningococcus also the serogroups may be assessed. Therefore we can use it also for antigen analysis of an already cultured strain. 47 vakcína meningo NeEc3 www.baxter-ecommerce.com Photo O. Z. Antigens detected at CSF antigen analysis nNeisseria meningitidis A nNeisseria meningitidis B teens, children nNeisseria meningitidis C nN. meningitidis Y/W135 nHaemophilus influenzae b children nStreptococcus pneumoniae seniors nStreptococcus agalactiae newborns nIn green colour there is the age group, where the infection is the most typical [USEMAP] Diagnostics of „other G– bacilli“ „Other gram negative“ characteristic nMicroscopy: G – bacilli, often short nCulture: we use mostly special media (BG for Bordetella, BCYE for Legionella etc.) nBiochemical diagnostic: some characteristics might be used nAntigen analysis: sometimes usefull nIndirect methods used, mostly for tularemia nDifferential diagnostics is not algoritmic here. Usually specimens are sent to the laboratory with suspicion for legionelosis, whooping cough, Bang disease etc. This strange way of inoculation is used, as the experience showed, that it increases successfull diagnostics. n1) inoculation of central field (to a drop of penicillin) n2) Spiral to margins n3) Radial rays Bordetella: Inoculation on BG agar 73 Bordet a Gengou Bordet Gengou 2 25 Legionella_pneumophila_isolement_BCYE BCYE medium for Legionella http://medecinepharmacie.univ-fcomte.fr Buffered Charcoal Yeast Extract Francisella diagnostics: Reading the agglutination set nTitre is counted, i. e. the highest dilution giving still a positive reaction Aglutinace Tularemie detail www.medmicro.info 1:10 1:20 etc. Any titer (i. e. everything except negative results) is interpreted as suspicious! The End 40 bunny [USEMAP] www.siumed.edu How to build the buildings for health care to prevent legionellosis nto ensure properties of the building for the healthcare (enouth space for personel, hygiene, storage etc.) nProtection of hot and cool water nProtection of sewage and solid waste nProtection of heating, air conditioning etc. nAlready when searching for an architect it is recommended to ensure that the architect has basic knowledge of healthcare management [USEMAP] Especially for legionellosis nThe infection highly related with the status of the building is legionelisus. nIn many cases an outrbreak of legionellosis is a result of bad project of water pipes, air conditioning etc. nIn case of water pipes, especially blind stream branches, that cannot be washed through and so they might as a reservoir of legionellosis nCorrection is only possible by rebuilding all the pipe system [USEMAP]