Diagnosis of Infectious Diseases by Laboratory Methods Flashcards
Diagnosis of Infectious Disease reasoning
- clinical syndromes are rarely specific for single pathogens
- having an understanding of the organisms most frequently associated with a clinical presentation allows for empiric therapy until definitive laboratory results are available
Five approaches to detect cause of infections
- microscopy
- culture
- detection of bacterial antigens
- demonstration of specific nucleic acids (molecular techniques)
- detection of antibodies directed against the organism (serology)
Typical turn around times using conventional clinical microbiology techniques
- Day 1: Specimen processing, plating and staining
- Day 2: Culture examination; identification and sensitivity tests set up
- Day 3: Identification and Sensitivity Tests Read
- Day 4: Physician Review of Culture Results
Certain cultures can take longer to grow and therefore culture results take longer: fastidious bacteria: 2-4weeks, Fungi: up to 4 to 6 weeks, Mycobacteria (TB): up to 8 weeks
Specimen collection
- proper collection and transport of specimens is critical to the quality of results in the lab
- poor samples result in failure to isolate the causative organism which leads to improper treatment
- collect speciment in acute phase of infection
- use proper technique
Microscopic Examination of Infected Materials
- various stains used to
- confirm submitted material is representative
- identify cellular components and debris of inflammation to estimate the probability of infection
- identify specific infectious agents
- guide physicians to early treatment with antibiotics
- support or refute initial physician initial diagnosis
Gram staining
- demonstrates cellular material and inflammatory response as well as shape and arrangmenet of bacteria
- Gram positive boxy rods with spores - Bacillus
- Gram positive pleomorphic rods- Corynebacterium
- Gram positive cocci clusters- Staphylococcus aureus
- Gram positive cocci chains- Streptococcus
- Gram negative boxy rods- E. coli
- Gram negative pleomorphic rods- Haemophilus influenza
Common Things Happen Commonly
-knowledge of the most frequent agents associated with specific infections- and their gram reaction and morphology- can allow for presumptive ID before culture or other test results are available
Newborns
Gram neg rods- E. coil
Gram pos cocci chain- Group B strep
Gram pos rods- Listeria monocytogenes
Children
Gram pos cocci pairs- Strep pneumoniae
Gram neg diplococci- Neisseria meningitidis
pleomorphic gram neg rods- Haemophilis influenzae
Detection of Bacterial Antigens from Clinical Material
- a variety of immunoassays are available for detectial antigens directly from clinical material
- assays are generally 100% specific- however vary in their sensitivity
- decreased sensitivity may be due to improper specimen collection and transport, inappropriate timing of collection or assay format
- in some cases, antigen detection is the method of choice in detecting an infectious agent (detecting Legionella in urine is more sensitive than in culture or DFA staining)
Antigen Detection Formations
- Latax agglutination- an antibody is coated onto a latax particle- when a sample contains the antigen it causes visible agglutination of the particles
- Coagglutination- antibodies are bound to bacteria- when a sample contains the antigen it causes visible agglutination of the bacteria
- Direct fluorescent antibody (DFA)- antibodies are tagged with a fluorescent dye
ELIZA
- antigen detection format
- a microplate is precoated with capture antibody
- labeled antibody is added and binds to captured antigen
- TMB substrate solution is added to the wells and color develops in proportion to the amount of analyte present in the sample
Immunochromatographic assays
- the sample mobilizes gold particles coated with monoclonal antibody
- if antigen is present in the sample, a complex is formed between the capture antibdoy and the monoclonal antibody gold conjugate which can be seen visually
Rapid Diagnosis of Group A Strep
- a number of assays are available that allow for the detection of S pyogenes directly from a swab of the tonsillar area
- although not 100% sensitive (mostly due to specimen collection) the assays approach 100% specificity
Serological Diagnosis of Infectious Diseases
- not all infectious agents have available antigen assays or culture techniques making the detection of specific antibodies diagnostically useful
- uses of serology infectious disease:
- determine disease susceptibility or immunity
- diagnose a current (acute) or previous infection
Advantage:
- specimen (usually serum) is easy to obtain
- serologic tests are widely available
- ease of specimen transport
Disadvantage:
- IgG tests require acute and convalescent sera in some disease states
- IgM tests can have false positives and false negative results
- 2-3 week delay in diagnosis in infections with short incubation period
Determining Current or Recent Infections
- IgM after infection:
- appears in serum in 1-2 weeks
- persists for 2-3 months, consistent with current or recent infection
IgG after infectionL
- 2-3 weeks after infection
- may persist for life
- may represent somewhat recent infection or immunity
Antibody titers:
- amount of antibody at a particular dilution of patient serum
- results expressed as titer: 1:2, 1:4, 1:8
- higher dilution is consistent with higher level of antibody in patient serum
Interpreting antibody test results
- acute and convalescent antibody titers
- used to assess IgG levels at 2 phases
Acute phase-collected after exposure or symptom onset
Convalescent phase: sample collected 2-4 weeks later
-a 4-fold rise in titer of paired sera collected 2-4 weeks apart verifies recent infection and is considered diagnostically significant
Nucleic acid based tests
- increasing used for detection and identification of microorganisms and viruses directly from clinical speciments
- identification from culture material
Allows for detection and ID of causative agents that:
- are difficult to culture
- fastidious (difficult to grow)
- slow growing
- highly infectious agents that are dangerous to culture
- increased sensitivity over current methods
- detect carrier/colonization
- detection of antimicrobial resistance genes
Herpes Simplex Encephalitis (HSE)
- HSV is most common cause of sporatic, fatal encephalitis
- infection of brain parenchyma, especially the temperol and frontal lobes- hemorrhage and necrossis
- complication of
- primary infection of neonate- HIV-2
- reactivation of latent disease in older children and adults- HSV- 1
- you can do a CSF PCR- even though not FDA approved you can do RT-PCR home brew assay
- use melt curve analysis to distinguish between HSV-1 and HSV-2
Limitations of NAAT
- relatively expensive- reagents, equipment
- availiblilty-relatively few FDA-cleared assays
- still need for organism isolation/ AST
- dectect both live and dead organisms
- risk of contamination and false positives
- not 100% sensitive OR specific
Culture and Recovery of Microorganisms
- traditional method of determining causative agent
- microorganisms have specific nutritional and growth requirements
- clinical labs use a variety of media to enhance the recovery of microorganisms: labs do not culture for all organisms all the time, need to know what is routine
Initial Differentiation of Microorganisms Based on Growth Characteristics
-initial colony observation allows for preliminary identification based on size, topography, opacity, and differential reactions based on agar
lactose fermenting gram neg rod: Escherichia, Klebsiella, Citobacter, Enterobacter
Non-lactose: Protreus, Morganella, Salmonella, Shigella, Pseudomonas
Film Array
- multiplex PCR
- have multiarray at the very end
- about 1 hour turn around
- 20 respiratory viruses and bacteria in one assay
Potential Future
-use smartphone app to STD test partners
