TRACING THE CULPRIT Part six: More gram-negative cocci and bacilli Institute for microbiology presents L [USEMAP] Summary Clinical characteristics – G– cocci Clinical characteristics – „other G– bacilli Diagnostics of G– cocci Diagnostics of „other G– bacilli“ Clinical characteristics – G- cocci Story One •Johny 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… Certainly, you know, what is the culprit •Of course, it was Neisseria gonorrhoeae (gonococcus) causing gonorrhoea. •Gonorrhoea is urethritis, in females also cervicitis; asymptomatically or symptomatically gonococci are found in pharynx and rectum, too. •In females, it is not a colpitis (vaginal inflammation), so it is not recommended to perform vaginal swabs in gonorrhoea. Story two •Lucy studied for four weeks for the examination of physiology. She did not leave the house at all and only sat and read. At the exam she had intention, that she is not able to say a word, but finally she passed with „E“. •In the evening, she visited a dancing party with friends. The party was full of smoke and they danced all night. Next day, Lucy was not well, she started to have fever and rash. (continuing) •In this moment, Lucy was hospitalized at infection clinic. In ambulance she failed unconscious and doctors said that it is a metabolic failure. After ten hours of attempts to keep Lucy‘s vital functions, that had no effect, Lucy died. •Such a course of infection may be caused by a dangerous culprit. Some of his strains are present in throat of healthy persons… 10 NEME skin_rash_wellcometrust 10 http://www.infektionsbiologie.ch And the culprit is… •… Neisseria meningitidis or meningococcus •Meningococcus causes meningitis, but • also sepsis and other serious problems; • all this is product of clonal strains. •Other strains are completely innocent and studies say that about ten percent of population are throat carriers of meningococcus. •Virulence is related mostly with protein antigens •Polysaccharide antigens determine preventability by vaccination • • Why the infection comes sometimes, and sometimes does not •The 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 •Meningococcus is transmitted by a narrow contact. Invasive infection is more likely when mucous membranes are damaged, e. g. by smoking or previous viral infection. •Infection is often present after too big physical activity after long inactivity period •Meningococcal infection is serious, but quite rare in Europe. In some other parts of the world, the situation is different – see next slide. Meningococcal meningitis is worldwide very important infection •„Meningitis belt“, area of extended presence of meningococcal meningitis 800px-Meningococcal_Meningitis_Range.svg.png By Ninjatacoshell - Own work, derived from File:BlankMap-Africa.svg, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=17051365 Treatment •It is necessary to ensure patient‘s survival (to follow haemorrhagia and acidobasic equilibrium) •In the same time, antibiotics are administered •Drug of choice in meningococcal diseases, according to guidelines, is still classical penicillin. Nevertheless, we would rather use it for less serious infections. For meningitis, we would use 3rd generation cephalosporins (ceftriaxon – good access to the CSF), or other antibiotics (macrolids, tetracyclins, quinolones) Prevention by vaccination •As the incidence is not high in Europe (although the lethality is), usually not the whole population, but just risk groups are vaccinated (soldiers, people in contact with a risky strain) •The problem exists with serogroup B, as its antigenic determinant is weak. Long time there was no vaccination against this group. Recently, a new vaccine exists, but its effectiveness is only 74 % (preliminary data) Vaccines •There are differences between them. Old polysaccharide vaccines give less protection than new conjugated vaccines •There exist also difference in serogroups (C only, A + C or tetravaccine A + C + W135 + Y) •B and C are the most common types in Czechia, but e. g. Mecca hajj (حج مكة‎) pilgrims need get vaccinated against W135 Let‘s compare Neisserias: 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“ Neisserias The least delicate, air transport possible The least delicate, grows even on poor blood agar Story three •Annie was crying and touching her ear. Her mother measured her temperature, and it was elevated. •At general practitioner's, Annie was examined and diagnosis of otitis media was set •As her tympanon was already broken, the pus was taken for examination •AMOCLEN (amoxicilin) was used for treatment immediately. 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 P1010003a [USEMAP] Moraxella (Branhamella) catarrhalis Foto Mikrobiologický ústav Clinical characteristics – „other G– bacilli“ Story four •There 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 •After an 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 finally stopped. Legionaire‘s disease •It is a disease caused by Legionella pneumophila •Legionaire‘s disease is a variant of disease that is quite severe; another variant, Pontiac fever, is rather mild •The bacterium have reservoir in water installation, air condition, etc. •During building new hospital departments (but also senior houses, hotels, spa…) legionelosis prevention should be taken, mostly at planning water pipe system (namely it is necessary to avoid blind branches, which cannot be run through by hot water or a disinfectant when necessary). [USEMAP] „Other Gram-negative bacteria“ •It is not a real „group“ or „family“. Nevertheless, these are quite rare bacteria, usually not growing on Endo agar, some of them growing on blood agar, and causing various diseases •Besides Legionella, we should mention at least three genera: Bordetella, Brucella and Francisella Genus Bordetella •B. pertussis and B. parapertussis cause whooping cough •B. bronchiseptica causes various pathologies in humans and animals. •Whooping cough is very rare due to vaccination •Pernasal swab is used when necessary (swab from nasopharynx, taken through nasal cavity) Genus Brucella •It is a causative agent of zoonoses •Brucella abortus is a bovine pathogen. It commonly infects bovine placenta, causing abortions in cattle. In human it causes so named Bang disease (fever, problems with various organs etc.) •Other brucellae are Brucella suis from hogs, Brucella melitensis from sheep and goats and Brucella canis from dogs Genus Francisella •Most important species – F. tularensis •Causes tularemia – „hare plague“ •Gamekeepers, but even more cooks preparing game are in risk of infections •The organism may infect wounds, but also it is possible to inhale it, thus leading to pneumoniae From report about epidemiological situation (IX/2008) – I (abbreviated) •Tularemia – A21: Woman, born 1970 from Valtice, gardener. 20th June GP visit for T 38°C, sore throat, enlarged lymph nodes next to right clavicle. The lymph node was extirpated. First serological examination negative, second (2nd July) positive •Brucellosis – 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. •(Reported by Public Health Office for South Moravia) [USEMAP] From report about epidemiological situation (IX/2008) – II •Pertussis (A37.0): 4 cases reported (Brno-environment, Hodonín), people aged 14 to 17, all vaccinated, one with missing re-vaccination •Parapertussis (A37.1): 3 cases of disease, coinfection, Brno environment, Hodonín Diagnostics of G– cocci Neisseria gonorrhoeae – sampling •In 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 recommended to send also smear on a slide from cervix and urethra (but not rectum and pharynx) •So „complete gonorrhoea examination“ consists of following parts: –urethral swab in Amies + smear –cervical swab in Amies + smear (�) –rectal swab in Amies (no smear) –pharyngeal swab in Amies (no smear) Sampling and acute diagnostics in purulent meningitis •In case of suspicion for purulent meningitis, usually cerebrospinal fluid is taken, eventually also blood for blood culture. CSF can be examinated biochemically, cytologically and microbiologically. •The person taking CSF can mention, that CSF is turbid and flows out under the pressure •In laboratory, two quick methods are available •microscopy •direct antigen detection •Not regarding the diagnostics and its results, the most important is instant start of patient treatment! Neisserias and Moraxellas – characteristics 1 •Microscopy: G – diplococci, coffee bean shaped, often intraleucocytar (see next slide) •Some Neisserias a Moraxellas may be prolonged and so they might be coccobacilli or bacilli (e. g. Neisseria elongata) Observation of gonorrhoea smear •Gonococci (but also meningococci) are Gram-negative diplococci, coffee-bean shaped, mostly intracellular. Presence of cocci inside leucocytes is their typical property. NEGO_MIK_4 Baba1 In females, the microscopy is slightly different from males. € � Institute for microbiology NEGO_MIK_4 NEGO_MIK_4 WBC with gonococci Institute for microbiology Neisserias and Moraxellas – characteristics 2 •Culture: tiny, colourless or yellowish (according to the species) colony, growing (species specific) on blood or chocolate agar •Blood agar or chocolate agar necessary also for diffusion disc test Culture of Neisseria and Moraxella • There exist differences between G– cocci in culture properties: –Oral Neisserias and Moraxellas (O+M) grow on blood agar (BA). –Meningococci (ME) grow only on nutrient-rich variants of BA. –Gonococci (GO) do not grow on BA at all, they require chocolate agar. NeEc4 Foto O. Z. BA BA+ ChA GO NO NO YES ME NO YES YES O+M YES YES YES negoca Institute for microbiology Gonococcus atb testing Neisserias and Moraxellas – characteristics 3 •Biochemical diagnostics: all of them are catalase positive, oxidase positive; Moraxella catarrhalis also positive in a specific test called INAC (indoxyl-acetate test) •Antigen analysis: performed usually by means of latex agglutination, very important in meningococci for differentiation between serogroups (for finding a proper vaccine) Basic biochemical tests •Quick tests with diagnostic strips simplify the diagnostics •Neisserias are oxidase positive, Moraxellas too, but their reaction might be late. •Moraxella is typically positive in INAC test •INAC test is similar to oxidase test, but the strip should be moistened and one has to wait 5 minutes. The colour is blue-green. Species determination of Neisserias and Moraxellas •For detailed identification of Neisserias and Moraxellas, biochemical tests are used, in Czechia mostly NEISSERIAtest, in other countries other tests (below or on the next slide) •Both pathogenic Neisserias have little biochemical activity: Gonococcus splits glucose only, meningococcus glucose and maltose. Neisserias and Moraxellas – differential diagnostics 1 •Gram staining: G – (diplo)cocci •Oxidase differentiates some other G– cocci (e. g. Acinetobacter – a G– non-fermenter – is also often coccoid) •Growth on various media differentiates –gonococcus (growth on chocolate agar only), –meningococcus (growth on rich blood agar and chocolate agar) –oral Neisserias (growth on both poor and rich blood agar and chocolate agar) –M. catarrhalis (growth like oral Neisserias) Neisserias and Moraxellas – differential diagnostics 2 •INAC test (a strip test similar to oxidase test) – positive in Moraxella catarrhalis •Complex biochemical test (NEISSERIAtest), is used especially for mutual differentiation of oral Neisserias •Antigen analysis (determination of meningococcal serogroup in invasive infections) Antibiotic susceptibility testing in Neisserias and related bacteria •Antibiotic susceptibility in pathogenic Neisserias is determined on media, on which they are able to grow, i. e. not on MH agar •First drug of choice for meningococcus is classical penicillin. It is used also for Gonococcus. Other drugs are macrolids, quinolones or cefriaxone. In Neisserias, zones are often large and confluent. •If the zones are so large that it is not possible to measure the diameter, we measure the radius and multiply by two. •Hypothetical margins of zones are in green: mention, that they are mostly either confluent, or behind the margin of the Petri dish! negoca Institute for microbiology, photo O. Z. A set of antibiotics against Neisseria gonorrhoeae Antibiotic Abbr. Reference zone / concentration* Penicillin (penicillin) – E-test P S ≤ 0,06 R > 0,25 Cefotaxime (3G cephalosporin) CTX S ≥ 34 R < 34 Meropenem (karbapenem) MEM S ≥ 30 R < 30 Azithromycin (azalide) AZM S ≥ 20 R < 20 Ciprofloxacin (fluoroquinolone) CIP S ≥ 35 R < 33 *In diffusion disc tests reference zone in mm, in e-tests breakpoint in mg/l A set of antibiotics against Neisseria gonorrhoeae Antibiotic Abbr. Reference zone / concentration* Penicillin (penicillin) – E-test P S ≤ 0,06 R > 1 Cefuroxime (2G cephalosporin) CXM S ≥ 31 R < 26 Cefotaxim (3G CS) – E-test CTX S ≤ 0,12 R > 0,12 Azithromycin (azalide) AZM S ≥ 25 R < 25 Tetracycline (tetracycline) TE S ≥ 38 R < 30 Ciprofloxacin (fluoroquinolone) CIP S ≥ 41 R < 28 *In diffusion disc tests reference zone in mm, in e-tests breakpoint in mg/l Antigen detection / antigen analysis n NeEc3 Photo O. Z. •Agglutination set for CSF agglutination is used for identification of pathogens. •In Meningococcus also the serogroups may be assessed. Therefore we can use it also for antigen analysis of an already cultured strain. • Antigens detected at CSF antigen analysis •Neisseria meningitidis A •Neisseria meningitidis B teens, young adults, children •Neisseria meningitidis C •N. meningitidis Y/W135 •Haemophilus influenzae b toddlers (prior to vaccination) •Streptococcus pneumoniae seniors •Streptococcus agalactiae newborns •In green colour there is the age group, where the infection is the most typical [USEMAP] Diagnostics of „other G– bacilli“ „Other gram negative“ characteristic •Microscopy: G – bacilli, often short •Culture: we use mostly special media (BG for Bordetella, BCYE for Legionella etc.) •Biochemical diagnostic: some characteristics might be used •Antigen analysis: sometimes useful •Indirect methods used, mostly for tularaemia •Differential diagnostics is not algorithmic here. Usually specimens are sent to the laboratory with suspicion for legionellosis, whooping cough, Bang disease etc. Bordet Gengou 2 Bordetella: Inoculation on Bordet-Gengou (BG) agar •This strange way of inoculation is used, as the experience showed, that it increases successful diagnostics. •1) inoculation of central field (to a drop of penicillin) •2) Spiral to margins •3) Radial rays 25 Legionella_pneumophila_isolement_BCYE BCYE medium for Legionella http://medecinepharmacie.univ-fcomte.fr Buffered Charcoal Yeast Extract Francisella diagnostics: Reading the agglutination set •Titre is counted, i. e. the highest dilution giving still a positive reaction Aglutinace Tularemie detail Institute for microbiology 1:10 1:20 etc. Any titre (i. e. everything except negative results) is interpreted as suspicious! Brucella diagnostics •Serology reactions, e. g. ELISA, are used, but the diagnostics is only performed by specialized laboratories • •Major-general Sir David Bruce (1855-1931) Davidbruce.JPG By see above - Dietmar Steverding. The history of African trypanosomiasis. Parasites & Vectors 2008, 1:3doi:10.1186/1756-3305-1-3, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=4306063 [USEMAP] The End H:\Monika-práce\atlas\fotky na výměnu 16-12-2016\Segreganty 2.jpg Legionella pneumophila – photo by Dr. V. Drašar How to build the buildings for health care to prevent legionellosis •to ensure properties of the building for the healthcare (enough space for personnel, hygiene, storage etc.) •Protection of hot and cool water •Protection of sewage and solid waste •Protection of heating, air conditioning etc. •Already when searching for an architect it is recommended to ensure that the architect has basic knowledge of healthcare management [USEMAP] Especially for legionellosis •The infection highly related with the status of the building is legionellosis. •In many cases an outbreak of legionellosis is a result of bad project of water pipes, air conditioning etc. •In case of water pipes, especially blind stream branches, that cannot be washed through and so they might as a reservoir of legionellosis •Correction is only possible by rebuilding all the pipe system [USEMAP]