MED201_Principles_of_diagnosis_of_bacterial_Infectious_Diseases (2).pdf

Principles of Diagnosis of bacterial Diseases 1 & 2 2023-24 Dr. Raj Department of Microbiology College of Medicine, IAU [email protected] Learning Objectives • After attending this lecture, you are expected to know ✓ ✓ ✓ ✓ ✓ ✓ ✓ Importance of specimen (sample) collection and transport Principl...

Principles of Diagnosis of bacterial Diseases 1 & 2 2023-24 Dr. Raj Department of Microbiology College of Medicine, IAU [email protected] Learning Objectives • After attending this lecture, you are expected to know ✓ ✓ ✓ ✓ ✓ ✓ ✓ Importance of specimen (sample) collection and transport Principles of different methods used to identify bacteria Working principles of different microscopes Various culture media & culture methods used to grow bacteria Biochemical tests, Antigen detection tests used to identify Principles of serological and molecular tests Rapid diagnostic tests – principles Identification of bacteria depends on • Quality of specimen - Good quality specimens are needed for reliable microbiological diagnosis • Type or nature of specimen • Collection of specimen – follow proper procedure • Transport of specimen- as early as possible • Method of diagnosis (type of diagnostic test) – right method Type of specimen • Depends on the site of infection • Examples: Infection Specimen 1 Upper respiratory Tract infection (URTI) 2 Lower respiratory tract infection (LRTI) 3 Urinary tract infection (UTI) 4 Gastrointestinal tract infection (GTI) Stool sample 5 Wound infection Swab or biopsy 6 1Swab 2Biopsy Ear infection Earswab 7 Tissue infection STopyTraping 1Nasopharyngeal swab,2nasal swab, 3 thraot swab ad 1 Sputum,2 tracheal aspirate, Ibi 3broncheoalveolar lavage (BAL) inBronchi Urine sample sample of feces Ear swab Swab, biopsy, or scraping Specimen Collection Where are you collecting from and in what container? Fluids Swabs Tissues Bottles & tubes Eyes, ears, nose, throat Wounds, ulcers, genital Pus, urine, sputum, Bile, CSF, blood Biopsy or scraping Avoid contamination with normal flora What are the specimens that could be contaminated with normal flora? pm Transport of specimen To avoid loss of viability of the microorganism: • Time is very precious • Time: Transport as quickly as possible • Temperature : correct temperature • Preservation: transport media prevent dryness and maintain viability • Remember anaerobes – Thioglycolate broth Tools - Specimen Collection and Transport Good communication with the microbiologist is essential for proper diagnosis AND treatment of infectious diseases Which Methods for Identification • Depends on ✓ Type of specimen ✓ Expected organism ✓ Availability of culture Laboratory diagnostic methods ✓ Direct Microscopy Antigen detection Culture (growth) Genome detection ✓ Indirect serology (Abs) m Laboratory diagnostic methods Infectious agents are identified by various methods Microscopy Light microscope Dark field microscope Fluorescent microscope Cultural method Aerobic Anaerobic Non-cultural methods Antigen detection Antibody detection PCR test Rapid diagnostic test (POCT, ICT) Dark field microscopy • To test for spirochetes such as Treponema pallidum • The micrscope excludes the unscattered beam from the image. So the background appears black • It only collects scattered beam from the objects, so the bacteria appears illuminating Treponema pallidum (Spirochetes) Microscopy with staining • Differential stains: WP ✓ Gram stain: Gram positive and Gram-negative bacteria ✓ Acid-fast stain: e.g. Ziehl Neelsen and Kinyoun [Mycobacteria] ✓ Giemsa stain: some intracellular bacteria in tissues • Negative staining such as India ink: detect the capsule e.g., in Cryptococcus neoformans (yeast) • Fluorescent stain: such as Auramine-rhodamine stain used to detect Mycobacterium tuberculosis Microscopy-Differential Stains Gram stain • • • • • • • The most commonly used stain in the microbiology laboratory Provides presumptive diagnosis Can be rapid identification method – very imp. CSF meningitis Gram reaction - +ve or -ve Identify the morphology and arrangement of bacteria The direction of the treatment can be decided But does not tell us about the exact species of bacteria, its not complete identification method Gram stain • 4 steps with 4 different chemicals or stains ✓ ✓ ✓ ✓ Primary stain - crystal violet Fixative or mordant - iodine Decolorizer - acetone or alcohol Secondary stain or counter stain - safranin • Most crucial step in Gram staining is decolorization ✓ Underdecolorization → G-ve can be seen as G+ve ✓ Overdecolorization → G+ve can be seen as G-ve • Decolorizer should not be left too long or too short Primary stain Mordant or fixative Decolorization secondary stain or counter stain Microscopy with staining • Gram Stain Gram positive cocci Gram positive rods Gram positive diplococci Staphylococcus aureus Bacillus anthracis Streptococcus pneumoniae 15 Microscopy with staining • Gram negative cocci Neisseria gonorrhoeae Gram negative diplococci Neisseria meningitidis Pus cells Gram negative diplococci Microscopy with staining Gram Negative Rods Gram negative rods in urine specimen Proteus species Gram negative rods in pus specimen Vibrio cholerae in faeces E. coli (the disease cholera) Gram negative comma shaped rods Microscopy with staining Acid fast stain for Mycobacterium spp Positive acid fast bacilli (AFB) smear Ziehl Neelsen stain Mycobacterium tuberculosis Microscopy with staining • India ink (negative stain): used for rapid detection of capsule in clinical specimens ✓ Ink darkens background- not the cell ( Negative Staining) Streptococcus pneumoniae Cryptococcus neoformans ➢ Polysaccharide capsule around the cell prevents staining Microscopy with staining • Fluorescent microscopy ✓ Fluorescent dyes AY o auramine-rhodamine stain ✓ Direct fluorescent antibody stain Acid Fast Bacilli (AFB) in clinical specimens Mycobacterium tuberculosis Treponema pallidum Chlamydia trachomatis Chlamydia trachomatis Immunofluorescence staining using monoclonal Abs conjugated to a fluorescent stain Microscopy – limitations • Experience and knowledge to interpret • May be time consuming for screening of smears • Evaluation of specimen where normal flora are present ✓ Urine ✓ Sputum • Many pathogens are indistinguishable from normal flora • Technical difficulties : ✓ Smears too thick, too thin, nothing? ✓ Only Mucoid or granular material ..no organism Laboratory diagnostic methods Microscopy examination Direct- examination Microscopy-staining Culture Non-culture methods Culture • The process of growing microorganisms in artificial culture media • Specimens inoculated on to culture media that contains the required nutrient for growth and then • incubated at suitable temperature to allow the growth of Bacteria • Grow & multiply, form colonies (a large number of bacteria) How is this (colony) useful? • Colonial Morphology – bacteria can be identified • To study bacterial morphology (gram stain) • Biochemical tests and biotyping : to demonstrate biochemical features and for typing • Antibiotic sensitivity pattern (to decide antibiotics) • All this helps in identification and management Colonial Morphology Proteus spp Staph aureus value Types of Culture Media • Based on appearance or consistency, culture media are grouped into: ✓ Liquid (or broth) media – broths ✓ Semisolid media - nutrient agar ✓ solid media – Blood agar, Mac conkey, Chocolate agar Based on the growth detection, culture media are classified as: Conventional culture media: Prepared from nutrients - aqueous extract of meat, peptone. ➢ Simple/Basal media - Nutrient agar ➢ Enriched media – Blood agar ➢ Enrichment media - alkaline peptone water, Selenite F broth, tetrathionate broth ➢ Selective media - TCBS, XLD, ➢ Differential media - MacConkey ➢ Transport media - Stuart, alkaline peptone H20 ➢ Anaerobic media- Robertson cooked meat broth Automated culture media: Mainly available for blood and sterile body fluid culture. BACTEC BACT –D Difference between enriched and enrichment media is that enriched media supports the growth of nutritionally demanding bacteria, whereas enrichment media prevents the growth of undesired or contaminated microorganisms. Enriched- Blood agar, chocolate agar, Loeffler's serum, MacConkey agar, blood culture media Enriched Media • Basal medium added with additional nutrients - blood, serum or egg • In addition to non-fastidious organisms, support the growth of fastidious nutritionally exacting bacteria. • Prepared by adding 5-10% of sheep blood to the molten nutrient agar at 450C. Blood agar • Tests the hemolytic property of the bacteria (Staph aureus and Strep pyogenes) which may be either✓ i) partial or α (green) hemolysis and ✓ ii) complete or β hemolysis Enriched Media- Chocolate agar: • Heated blood agar, prepared by adding 5 -10% of sheep blood to the molten nutrient agar at 70°C. • More nutritious than blood agar - supports certain highly fastidious bacteria - Haemophilus influenzae that does not grow on blood agar. • Neisseria species (Gonococci, Meningococci) • Thayer martin media (modified chocolate agar with antibiotics to inhibit gram positive bacteria) – selective medium for Gonococci Selective Media • Solid media containing inhibitory substances - inhibit the normal flora present in the specimen and allow the pathogens to grow. • Encourages growth selectively, no unwanted organism grows Selective Media Lowenstein–Jensen medium for Mycobacterium tuberculosis TCBS agar for Vibrio cholerae Potassium tellurite media With corynebacterium Note black colonies Selective Media DCA (Deoxycholate Citrate Agar) for salmonella and shigella Salmonella on DCA XLD (Xylose Lysine Deoxycholate) agar for salmonella and shigella Transport Media • Used for the transport of the clinical specimens suspected to contain delicate organism or when delay is expected while transporting the specimens from the site of collection to the laboratory • Bacteria do not multiply in the transport media - they only remain viable. Differential Media • Differentiate between two groups of bacteria - by using an indicator. Differential Media (Cont..) MacConkey agar for E coli CLED agar for urinary pathogens LF NLF Used mainly to grow Gram negative rods Blood Culture and Media used for Blood Culture • Recovery of bacteria from blood is difficult - they are usually present in lesser quantity and many of the blood pathogens are fastidious (highly selective, need special nutrients) • Enriched media (Blood agar) - used for isolating microorganisms from blood. • Available either as Conventional(traditional) or Automated media • Automated Blood Culture Techniques ✓ Principle: When bacteria multiply, they produce CO2 by metabolism, decreases the pH of the medium, triggers the sensor present at the bottom of the bottle. Metabolites are detected in system through a sensor system ✓ Continuous automated monitoring - monitored for the microbial growth - every 10 minutes ✓ Composition: Tryptic soy broth and/or brain heart infusion broth + polymeric Resin beads - adsorb and neutralize the antimicrobials present in blood specimen. ✓ 1. BacT/ALERT 2. BACTEC (BD Diagnostics) • Disadvantages of Automated systems of Blood Culture ✓ High cost of the instrument and culture bottles ✓ Inability to observe the colony morphology as liquid medium is used Anaerobic Culture Media • Contain reducing substances which take up oxygen and create lower redox potential permit the growth of obligate anaerobes, such as Clostridium. • Robertson’s cooked meat (RCM) broth • Thioglycolate broth • Anaerobic blood agar CULTURE METHODS • Aerobic & Anaerobic methods • Inoculate the specimen on to appropriate culture media, followed by incubate the culture plates in appropriate conditions (Temp, Ph, O2, CO2, nutrients, light, etc). o • Most of the pathogenic bacteria (medically important) are aerobes or facultative anaerobes; grow best at 37°C. • Therefore, the inoculated culture plates are incubated at 37°C aerobically overnight in an incubator • If anaerobic organisms suspected – anaerobic culture methods CULTURE METHODS Bacterial Incubator Biosafety Cabinet (BSC) Incubatory Conditions • The incubatory conditions may vary depending upon the bacteria to be isolated. ✓ For capnophilic bacteria (Campylobacter) : Candle jar is used. Here, inoculated media are placed inside a jar, along with a lighted candle and then jar is sealed ✓ For microaerophilic bacteria, such as Campylobacter and Helicobacter require 5% oxygen for optimum growth ✓ For obligate anaerobes, anaerobic culture methods are used Anaerobic culture methods Anaerobic culture methods includes: ➢ Evacuation and Replacement of oxygen -McIntosh and Filde’s anaerobic jar ➢ Reducing Agents - Robertson cooked meat broth ➢ Absorption of Oxygen by Chemical Methods- GasPak ➢ Anaerobic Glove box and Anaerobic WorkStation ➢ Pre-reduced Anaerobically Sterilized (PRAS) Indicator of anaerobiosis ✓ Chemical indicator: Reduced methylene blue remains colorless in anaerobic conditions, but turns blue on exposure to oxygen ✓ Biological indicator using obligate aerobe such as Pseudomonas: The absence of its growth indicates that complete anaerobiosis has been achieved. Anaerobic culture methods GasPak system Anoxomat Anaerobic Glove box Anaerobic workstation Culture – limitations • Require appropriate collection and transport to maintain viability. • Require specific growth factor to grow : Fastidious organisms such as Haemophilus influenzae • Safety concerns with pathogenic organisms: such as Mycobacterium tuberculosis (only cultivated in BSL-3 lab) • Some organisms cannot be cultured: such Treponema pallidum • Risk of contamination • Requires long time- Hours - days (bacteria) Laboratory diagnostic methods Microscopy examination Culture (colony) Direct- examination Wet mount Non-culture methods Antigen detection Antibody detection Genome detection Microscopy-staining Colonial morphology Antibiotic susceptibility testing Biochemical assays Biochemical tests * Used to identify bacteria by demonstrating biochemical activities (enzymes, breakdown products, metabolites). Test Examples of Organisms giving Positive results Catalase test Staphylococci, Enterobacteriaceae Indole test Escherichia coli Urease test Proteus, H pylori, Klebsiella Citrate test Klebsiella Oxidase test Vibrio, Pseudomonas Coagulase test Staph aureus • Students are instructed to refer practical guide for more information about these Biochemical tests(principles, procedures etc etc) • Questions can be asked based on these principles, procedures Activity: • On what culture media would you cultivate these bacteria Bacteria Streptococcus pyogenes Haemophilus influenzae Salmonella typhi Neisseria meningitidis Vibrio cholerae Staphylococcus aureus Escherichia coli Culture media Blood Agar dilate Again Type (enriched, selective,…) β hemolysis choclate IBS Blood Agar Melon by Lactose Fermentose Activity: think, pair, and share • What type of sample would you collect for infection Patient suffering from gastrointeritis Sore throat Central nervous system infection Urtherral discharge Genital ulcer Eye infection Skin infection specimen Container (sterile/nonsterile) SAQs for practice • Name any 2 rapid diagnostic tests that can be used to identify bacteria • • • • • • • Name different microscopies used to identify bacteria Name any 4 biochemical test used to identify bacteria Name one selective culture media used in bacteriology lab Name one enriched culture media used in bacteriology lab Name one differential culture media used in bacteriology lab Anaerobic culture methods What are the advantages of MALDI-TOF References • Goering et al- Mims Medical Microbiology- 5th edition, Chapter 31. • Jawetz Melnick & Adelbergs- Medical Microbiology 26th edition, Chapter 47. 44