p10-laboratory-diagnosis bacterial _0.pptx

Lab diagnosis of bacterial infections

1. Laboratory tests

2. Objectives Identify the main causes of outbreak in the work zone Know the different types of samples and diagnostic tests that can be conducted, based on the infection dynamics Review the role of the laboratory coordinated with surveillance to characterize outbreaks

3. Most common etiologies in India Febrile syndrome: – Dengue, Chikungunya, Zika, yellow fever, influenza, leptospirosis, malaria Icteric febrile syndrome: – Yellow fever, leptospirosis, malaria, hepatitis Hemorrhagic icteric febrile syndrome: – Yellow fever, leptospirosis Hemorrhagic febrile syndrome: – Dengue, yellow fever, leptospirosis, hantavirus Neurological syndromes: – Bacterial, viral (Zika and viral encephalitis), polio, botulism Respiratory syndromes: – Influenza, hantavirus, diphtheria and other respiratory viruses Acute diarrheic disease: – Cholera, rotavirus, norovirus, enterobacteria Exanthematous disease: – Zika, measles, rubella

4. Most used tests for diagnosing etiologic agents Serological: – ELISA (IgM and IgG) – Direct and indirect immunofluorescence Molecular: – Polymerase chain reaction (PCR) – Real time PCR – Sequencing Microscopic: – Optical microscopy -Fluorescent M - Electron M Specimen type, collection, and conservation – Examples of type of sample and appropriate conservation Culture: – isolation Identification Antibiotic sensitivity testing

5. 1- Microscopy Microorganisms can be examined microscopically for: a- Bacterial motility: Hanging drop method: A drop of bacterial suspension is placed between a cover slip and glass slid b- Morphology and staining reactions of bacteria: Simple stain: methylene blue stain Gram stain: differentiation between Gm+ve and Gm– ve bacteria . Primary stain (Crystal violet) . Mordant (Grams Iodine mixture) . Decolorization (ethyl alcohol) . Secondary stain ( Saffranin ) Ziehl- Neelsen stain: staining acid fast bacilli . Apply strong carbol fuchsin with heat . Decolorization (H 2 SO 4 20% and ethyl alcohol . Counter stain ( methylen blue)

6. 2- Culture Techniques * Culture media are used for: - Isolation and identification of pathogenic organisms - Antimicrobial sensitivity tests * Types of culture media: a- Liquid media: - Nutrient broth: meat extract and peptone - Peptone water for preparation sugar media - Growth of bacteria detected by turbidity b- Solid media: - Colonial appearance - Hemolytic activity - Pigment production

7. 1- simple media: Nutrient agar 2- Enriched media: media of high nutritive value . Blood agar . Chocolate agar . Loffler’s serum 3- Selective media: allow needed bacteria to grow . Lowenstein–Jensen medium . MacConkeys agar . Mannitol Salt Agar 4- Indicator media: to different. between lact. and non lact. ferment . MacConkeys medium . Eosine Methlyne blue Agar 5- Anaerobic media: for anaerobic cultivation . Deep agar, Robertson’s Cooked Meat Medium Types of solid media

8. Colonial appearance on culture media * Colony morphology: . Shape . Size . Edge of colony . Color * Growth pattern in broth: . Uniform turbidity . Sediment or surface pellicle * Pigment production: . Endopigment production ( Staph. aureus ) . Exopigment production ( Ps. aeruginosa ) * Haemolysis on blood agar: . Complete haemolysis ( Strept. Pyogenes ) . Partial haemolysis ( Strept. Viridans ) * Growth on MacConkey’s medium: . Rose pink colonies (Lactose fermenters) . Pale yellow colonies (Non lactose fermenters )

9. 3- Biochemical Reaction Use of substrates and sugars to identify pathogens: a- Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar b- Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface e- H 2 S production: Depends on production H 2 S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate

10. 3- Biochemical Reaction (cont.) c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator d- Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Color of medium turns pink (positive) e- Action on milk: Fermentation of lactose with acid production Red color if litmus indicator is added

11. f- Oxidase test: Some bacteria produce Oxidase enzyme Detection by adding few drops of colorless oxidase reagent Colonies turn deep purple in color (positive) g- Catalase test: Some bacteria produce catalase enzyme Addition of H 2 O 2 lead to production of gas bubbles (O 2 production) h- Coagulase test: Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method i- Urease test: Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH 3 Alklinity of media and change color of indicator from yellow to pink

12. Serological tests (Serology) Two types: direct and indirect tests – Direct tests: based on the antigen antibody reaction to investigate the presence of antigen. That is, these tests investigate the presence of the etiologic agent or one of its components. Example: direct immunofluorescence. – Indirect tests: based on detecting specific antibodies against the agent or one of its components, which indirectly allow us to assume that the pathogen being investigated was present in the host at some point Detect the presence of antibodies Cheaper than molecular tests Based on the antigen antibody reaction and constitute a valuable tool in diagnosing infectious disease

13. Serological tests (Serology) Important concepts for interpreting serological results: Positive IgM is related to a recent infection Increased antibody titer in paired samples indicates a recent infection – First sample: negative – Second sample: antibodies present – Antibody titer in the second sample > 4x the antibody titer in the first sample Positive IgG indicates (generally) a past infection

14. Types of samples and appropriate collection Primary infection Secondary infection

15. Serological tests (Serology) Immunofluorescence – Immunofluorescence techniques may be applied as techniques to investigate the presence of viral, parasitic, bacterial, or mycotic antigens in clinical samples or antibodies produced against them in the patient’s serum. ELISA (enzyme-linked immunosorbent assay) – Immunoenzymatic techniques are tests based on antibody antigen reaction, making it possible to detect the presence of antibodies produced against viral, parasitic, bacterial, or mycotic antigens in clinical specimens in the patient’s serum. – The ELISA technique may be used to detect IgM or IgG.

16. Molecular tests Are based on detecting the etiologic agent’s genome. Make it possible to detect the etiologic agent’s RNA or DNA with high sensitivity and specificity. Are more costly than serology. Make it possible to confirm the presence of the etiologic agent in clinical samples. Make it possible to detect RNA or DNA even in very small sample volumes.

17. Molecular tests Polymerase chain reaction (end point PCR) – The end point PCR is based on amplifying and visualizing the complementary DNA or ADNA (cDNA). This technique consists of amplifying and visualizing a specific DNA fragment from the etiologic agent. Real time PCR – Also known as quantitative PCR, this test is a variant of the polymerase chain reaction (PCR). – Real time PCR makes it possible to amplify and at the same time absolutely quantify specific DNA or cDNA molecules

18. Microscopic Consists of using special instruments such as magnifying glasses and microscopes to magnify the size of structures that are invisible to the naked human eye, such as viruses, bacteria, and parasites. Microscopic observation is necessary in the field of parasitology. Modes of observation vary based on strategies employed and range from micrometric to nanometric observation. Therefore, various types of microscopes are required, such as photonic and electronic (scanning, transmission, and atomic energy).

19. Microscopic Optical microscopy technique allows us to visualize bacteria and parasites simply by examining the sample with a microscope. For bacteria, a Gram stain (a purple stain) is often performed first. Bacteria is classified as follows: – Gram positive (appear blue because they retain the Gram stain) – Gram negative (appear red because they do not capture the stain) A blood swab is performed to detect parasites found in the blood, such as filariasis, malaria, or babesiosis. For this test, a drop of blood is placed on a microscope slide, stained, and examined under the microscope to visualize the parasite. Optical microscopy

20. Sample type, collection, and conservation Successful laboratory diagnosis depends on two factors prior to the laboratory phase: Selecting the type of sample: It is important to select the correct type of sample, based on clinical material and days since the onset of symptoms, whenever it is possible to obtain a positive result (serologically or molecularly) Sample quality: There are three factors that have a direct influence on the quality of the sample to be received by the laboratory: – Correct collection – Conservation of the cold chain during transport – Conservation at the appropriate temperature based on the processing time

21. Examples of sample type and appropriate conservation Dengue, Chikungunya, yellow fever, hantavirus, leptospirosis Zika, measles, and rubella Hepatitis Influenza and other respiratory viruses Bacterial and viral encephalitis Rotavirus, norovirus, enterobacteria Malaria Meningeal disease

22. Dengue, Chikungunya, yellow fever, hantavirus, leptospirosis Type of sample: serum Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than 1 week) Laboratory diagnosis: – 1 to 5 days following onset of symptoms: PCR – 5 to 10 days following the onset of symptoms: PCR + ELISA IgM

23. Zika, measles, and rubella Type of sample: serum Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than one week) Laboratory diagnosis: – 1 to 5 following the onset of symptoms: PCR – 5 to 10 days following the onset of symptoms: PCR + ELISA IgM

24. Zika, measles, and rubella Type of sample: urine Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than 1 week) Laboratory diagnosis: – 1 to 15 days following the onset of symptoms: PCR

25. Zika – congenital syndrome or fatal cases Sample Quantity Transport medium Transport conditions Conservation >1 week Laboratory test Mother’s serum 5–7 mL No additives 4 / 8 °C - 20 / - 70 °C PCR, ELISA IgM, PRNT, others Umbilical cord blood 5–7 mL No additives 4 / 8 °C - 20 / - 70 °C PCR, ELISA IgM, PRNT, others Placenta 0,5–1 mL Buffered formalin 4 °C – TA* 4 °C – TA* Immunohistochemical Placenta 5–7 mL Saline solution 4 / 8 °C - 20 / - 70 °C PCR Umbilical cord (tissue) Buffered formalin 4 °C – TA* 4 °C – TA* Immunohistochemical Umbilical cord (tissue) Saline solution 4 / 8 °C - 20 / - 70 °C PCR Newborn’s blood 0,5–1 mL No additives 4 / 8 °C - 20 / - 70 °C PCR, ELISA IgM, PRNT, others Newborn’s CSF** 0,5 mL No additives 4 / 8 °C - 20 / - 70 °C PCR, ELISA IgM, PRNT, others Mother’s total blood 5–7 mL EDTA, others 4 / 8 °C 4 °C Biochemical, others Newborn’s total blood 2–5 mL EDTA, others 4 / 8 °C 4 °C Biochemical, others Tissue** 3x3 cm (approx) Buffered formalin 4 °C – TA* 4 °C Immunohistochemical Tissue** 3x3 cm (approx) Saline solution 4 / 8 °C - 20 / - 70 °C PCR * Ambient temperature ** Under medical indication for suspected neurological syndrome *** Fatal cases: brain, liver, kidney, others

26. Hepatitis Type of sample: serum Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than 1 week) Laboratory diagnosis: – 1 to 5 days after onset of symptoms: PCR + Elisa HBsAg – 5 to 10 after onset of symptoms: PCR + ELISA IgM

27. Influenza and other respiratory viruses Type of sample: nasopharyngeal swab Quantity: 2 nylon swabs Transport medium: viral transport medium or saline solution (3 mL) Transport conditions: 2 to 8°C Conservation: 4°C until aliquots are prepared; aliquots to a -20°C (up to 48 hours) and -70°C (period greater than 48 hours) Laboratory diagnosis: – PCR or IF (only typification) followed by PCR

28. Influenza and other respiratory viruses Type of sample: nasopharyngeal aspiration; nasopharyngeal wash Material: suction device Transport medium: saline solution Transport conditions: 2 to 8°C Conservation: 4°C until aliquots are prepared; aliquots to a -20°C (up to 48 hours) and -70°C (period greater than 48 hours) Laboratory diagnosis: – PCR or IF (only typification) followed by PCR

29. Bacterial and viral encephalitis Type of sample: serum Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than 1 week) Laboratory diagnosis: – 1 to 5 following onset of symptoms: PCR – 5 to 10 following onset of symptoms: PCR + ELISA IgM

30. Bacterial and viral encephalitis Type of sample: blood Quantity: 3 to 7 mL Transport medium: no additives Transport conditions: 2 to 8°C Conservation: -20°C (up to 1 week) / -70°C (period greater than 1 week) Laboratory diagnosis: – 1 to 5 following onset of symptoms: PCR – 5 to 10 following onset of symptoms: PCR + ELISA IgM

31. Rotavirus, norovirus, enterobacteria Type of sample: fecal material Conservation: 2 to 8°C Laboratory diagnosis: – Generally, molecular methods are used based on PCR and ELISA kits for antigen detection

32. Malaria Type of sample: total blood Conservation: 2 to 4°C Laboratory diagnosis: – Large drop (extended) for microscopy

33. Meningeal disease Bacteria culture Hemoculture PCR Viral isolation in cell culture

34. Specimen Selection, Collection, and Processing The quantity material must be adequate Specimens are selected on the basis of signs and symptoms, should be representative of the disease process Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful. Meaningful specimens to diagnose bacterial infections must be secured before antimicrobial drugs are administered.

35. 4- Animal pathogenicity * Animal pathogenicity test: Animals commonly used are guinea pigs, rabbits, mice * Importance of pathogenicity test: - Differentiate pathogenic and non pathogenic - Isolation organism in pure form - To test ability of toxin production - Evaluation of vaccines and antibiotics

36. Serological identification A- Direct serological tests: - Identification of unknown organism - Detection of microbial antigens by using specific known antibodies - Serogrouping and serotyping of isolated organism B- Indirect serological tests: - Detection of specific and non specific antibodies ( IgM & IgG ) by using antigens or organisms

37. DIAGNOSTIC TECHNOLOGIES Immunoserology Hemagglutination EIA Latex agglutination Complement fixation Immunoflorecent

38. RAPID DIAGNOSTIC TESTS High sensitivity and specificity High negative and positive predictive values High accuracy compared to gold standard Simple to perform Rapid turn around time Cost effective

39. LIMITATIONS OF CONVENTIONAL CLINICAL MICROBIOLOGY Culture Labor intensive Need for special media Prolonged period of time to culture Some organisms are uncultivable on artificial media Potential health hazards Antigen Detection Negative tests require confirmation Effected by poor specimen collection Low microbe burden Serology Unhelpful during early stage of infection Not quite useful in immunocompromised patients

40. Molecular Biology Techniques A- Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labeled probe Probe: consists of specific short sequence of labeled single- stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA Then It is detected by a labeled probe C- Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis

41. MOLECULAR DIAGNOSTICS Most widely used is PCR High sensitivity High specificity Diversity Nucleic acid probes Do not amplify DNA

42. Polymerase Chain Reaction *Specific PCR: Uses primers to known DNA targets. Use when conventional diagnostics are inadequate, time consuming, difficult and hazardous *Broad range PCR : uses complementary primers to conserved regions shared by a given taxonomic group Used in cases of B. henselae and Mycobacterium spp *Multiplex PCR Uses single clinical specimen to investigate several potential pathogens simultaneously Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6, EBV, Enteroviruses *Real-time PCR Utilizes a fluorescent labeled probe Requires small volumes thus takes 30-60 minutes to complete

43. Real time PCR for Diagnosis of Infectious Disease Detect PCR product during synthesis Requires fluorescence-based detection and specialized detection instrumentation Advantages Less time for results Improved analytical sensitivity Broad applicability (target characterization, load determination etc)

44. OTHER USES OF MOLECULAR DIAGNOSTICS Viral load monitoring Viral genotyping Bacterial resistance detection Bacterial genotyping

45. LIMITATION OF PCR TECHNOLOGIES Specimen should be frozen until amplification No antimicrobial sensitivity is available Needs the clinician to name the suspect Cost False positives caused by amplification of contaminants Only sample from normally sterile sites should be considered for broad-range PCR Specimen is required to be refrigerated or stored in alcohol before processing

46. Antimicrobial Susceptibility testing Introduction: Identification of a bacterial isolate from a patient provides guidance in the choice of an appropriate antibiotic for treatment Many bacterial species are not uniformly susceptible to a particular anti-bacterial compound This is particularly evident among the Enterobacteriaceae , Staphylococcus spp., and Pseudomonas spp. The wide variation in susceptibility and high frequencies of drug resistance among strains in many bacterial species necessitates the determination of levels of resistance or susceptibility as a basis for the selection of the proper antibiotic for chemotherapy

47. Antimicrobial Susceptibility testing can be down by three ways: Minimum Inhibitory Concentration (MIC) Disk Diffusion Method Minimum Bactericidal Concentration (MBC)

48. Most important aspect of laboratory medicine Insufficient quantity Contamination Improper transport media Delay in transportation Inappropriate storage