In the name of God

1. In the name of God Diagnostic microbiology

2. Background • Microbiology is the study of cellular and acellular cells and agents. • Cellular micro-organisms are consisted of prokaryotics bacteria and archea- and eukaryotics such as fungi (yeat, moulds) , protoza, and algae. • In regard to their size, smallest bacteria such as mycoplasma are 0.1-0.3 and the largest eukaryotic such as helminth have diameter of >1000

3. Acellular infectious agents are nonliving structures. Some have nucleic acids either DNA and/or RNA • Some have no any nuclear elements and are just protein in nature prions • Some of these acellular agents are toxins produced by bacteria and fungi. • Some bacterial products or fungal metabolites are beneficial and are antimicrobial agents. • Microbial toxin are metabolite which can cause intoxication rather than infection.

4. Acellular infectious agents: • Perions : are structurally deformed proteins, when they change in tertiary configuration they can invade neural cells. Prions are the causative agents of scrapies in sheep, mad cow disease in cows and cattle and spongy form encephalitis in human. • Nanobacteria : are newly diagnosed and very small bacterial form of life. these are assumptions about their role in renal stone formation and some form of atherosclerosis. • Toxins : are microbial substances that are able to induce damage to host cells, an immunogenic or allergenic response and/or fever such as • Endotoxins of G(-) bacteria • Neurotoxins of Cl-Tetani • Entrotoxins of E-coli vibriocholera • Aflatoxins of asepergillus rubratoxin • Algal toxin such as saxtoxin and hepatotoxin

5. Microbes can be found in nature ubiquitously in water, soil, decade organic materials, and also they can colonies, the external surface of the human body (skin) and exposed internal mucosal surface all the time colonization or infection doesn’t mean disease production.

6. Microbes that can cause infectious disease are called pathogens. Some of them are true pathogens meaning that when they colonies to special site or receptors they cause infectious disease, like Niesseria gonorhea and shigella species etc,… and some of them produce disease under certain conditions, opportunistic like pseudomonas spp and many others.

7. In order to initiate an infectious disease microbes needs to: • Attachment to surface epithelium • Ability to multiply at site of entry or attachment • Evasion of host defense • Production of toxins, enzyme, that can destroy cells and tissues.

8. What are virulence factors: • Virulence factor are: • cell structure component : LPS of the G(- )bacteria • cell structure appendages : pili, resistance genes on plasmids etc,… • Capsule : which inhibit phagocutosis, capsule in strep pneumonia, bacillus antheracis • Slime layer production : exopolysaccharide production by a mixture of organisms biofilm production • Microbial toxins : diphtheriatoxin, cholera toxin

9. Some of these virulence factors are inherited, capsule production, cell destructive enzymes. (Leokocidin, Hemolysins, DNAes, RNAes, Coagulase, phospholipase, Lipase, etc,…) and some of these virulence factors may be acquired by genetic phenomena like transformation, genetic transduction (Scarlet fever toxin, diphtheria toxin and genetic conjugation, transfer of resistant factors.)

10. Some microbes are able to live in extracellular environment and cause an acute pus forming infectious disease such as staph, aureus, strep pyogens coryrebacterium diphtheria, Neissena meningitidies Neissena gonorrlea etc,… • Some bacteria should live intracellularly in order to cause disease such as chlamydia causative agents of trachoma, preumonia, and genital infections. • Ehrlichia specis which produce disease when they multiply within macrophages causing disease known as human monocythic Ehrliosis. • Anaplasma phagocytophilnm live within granulocytes plasmodium Spp(malaria) and baesia which produce intra erythrocytic disease.

11. Some microbes are facultative in respect to disease production such as leishmania specis, M.TB, listeria monocytogenes. • Some microbes such as Brucella species, Salmunella species, Yersinia pestis, fancisella can alive intracellulary. • They produce some enzyme, which combat with intracellular killing mechanisms.

12. Some algae also can cause infection such as genus prototheca. Prototheca live in soil and decaying matter. They can enter wound especially in foot. they can also invade lymphatic and produce systemic and fatal infection. Prototheca can be grown in the laboratory, they produce yeastlike, colonies.

13. Clinically important fungi and moulds dermatophytes: which produce infection in hair, nail and skin such as epidermophyton trichophyton, candida yeast. Fungi which can cause mucosal infection such as mucromycosis, asperiogillus Fungi which can cause systemic infection such as yeast, histoplasma capsulatum etc,…

14. Clinically important protozoa Malaria: plasmodium species Leishmania species (kala-azar, mucocutonous infection) Amoeba: E-histolytica Ciliate: Balantidium coli Flagellate: Trichomonas, Giardia Helminths crypthosporidium etc,…

15. Clinically important viruses • Hepatits viruses, such as HBV, HAV, HCV, HDV, HEV, HGV • HIV and related immunodefeciency viruses HIV1 and HIV2 • Herpes simplex and related viruses such as varicellazoster virus, VZV, cytomegalovious CMV • Epsteinbar virus EBV • Crimian congo hemorhagic fever virus CCHFV • Ebola viruses • Influenzae viruses, Entero viruses and many others.

16. Acellular proteinous structure causing infecious disease Prions: prions are protein in naturre. When their tertiary structure is change due to mutation, it becomes infectious. Prions are assumed to be the cause degerative disease of nerve cells. prions are the causative agents of certzfeid Jacob syndrome in human. Madcow disease in cow and cattles. Scrapies in sheep and goats.

17. Optimal times for specimen collection In the suspected case of salmonellosis typhoid and paratyphoid-take a blood culture in the first week of infection. Serology test in the end of the second week and urine and stool culture during the second and third week may show positive result.

18. In case of bacteremia and sepsis blood is taken for culture in order to isolate the suspected organism. In every case try to obtain blood for culture before antibiotic therapy. If this is not unavoidable try to use culture media which contains resins to bind to the suspended antibiotics.

19. Specimen collection for microbiology examination Once an infectious disease is suspected and after physician request for specimen collection all quality criteria should carried out. Although culturing and isolation of the infecting microbe is the gold standard there are situations in which the pathogen can’t be isolated by routine culturing methods and media.

20. This may be due to the bacterium or microbe in state of VBNC meaning that although the bacteria are alive (viable) but nonculturable. The change in the PH of infected site may cause decrease in viability and although presence of many enzymes produced by the inflammatory cells (peroxidases, phospholipases, RNAes, DNAes) may result in destruction of microbes.

21. Prior antibiotic therapy and incomplete dose may be another cause in which although the disease is yet present but the infecting agent is inactivated. In the case of wound infectious first cleans the superficial pus and dirt by alcohol and then if the wound is open draw the specimen from edge of the infection sites without touching the adjacent skin.

22. In the case of cervical and endometrial culture don’t contaminate the specimen with vaginal secretions. In the case of UTI suspected patient, the best way is to obtain a clean cached early morning mid stream urine or any urine remained in the bladder at least for 4 hours.

23. Taking urine specimen by catheters is not advised unless other wise there is no any simpler way. Suprapubic urinecollection should be remained for very small children in which collection of CVU is not possible to them. In case of streptococcal pharynsitisthe peritonsillar fossae and postpheraryngeal wall is swabed

24. In case of lower respiratory tract infection try to obtain the sputum by a deep cough. It is better that patient have had a mouth wash and after that a deeply coughed sputum be taken in wide mouth sterile container. In the case of any deeply seated abcess or pus loculation, the best way of specimen collection is by aspiration.

25. In the case of sinusitis the best specimen is obtained through suctioning of sinus discharge, otherwise with a flexible sterile swab via the nostriles enter the sinus. In the case of otitis media, carefully enter a sterile swab toward the site of infection. In the case of meningitis- after LP done by physician transport the tube, as soon possible and centrifuge the content of the second tube for culturing and smear .

26. Improper specimen collection This is a physician or an oriented HCW to take specimen from the actual site of infection As it is possible do not mix the specimen with micriflora present in the site. As it is possible try to take specimen before any chemotheraputic (antibiotic) are used.

27. As it is possible try to take sufficient amount of specimen quality and quantity of specimen is of importance. As soon as possible transfer the specimen to the lab, in a safe manner

28. Rejection criteria in CML specimens • Reject specimen taken in broken or leaky container • Reject voided urine, vomitous for an aerobic culture. • Reject saliva and mouth wash as sputum specimen.