Diagnosis of Infectious Diseases by Laboratory Methods Flashcards Preview

Microbiology/Immunology > Diagnosis of Infectious Diseases by Laboratory Methods > Flashcards

Flashcards in Diagnosis of Infectious Diseases by Laboratory Methods Deck (22)
1

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

2

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)

3

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

4

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

5

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

6

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

7

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

8

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)

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

Film Array

-multiplex PCR
-have multiarray at the very end
-about 1 hour turn around
-20 respiratory viruses and bacteria in one assay

22

Potential Future

-use smartphone app to STD test partners