Transcript laboratory diagnosis of infectious diseases

LABORATORY DIAGNOSIS OF
INFECTIOUS DISEASES
OBJECTIVES
Know available diagnostic technologies for ID
Understand specific specimen for specific
diagnostic test
Understand procedure for specimen collection
DIAGNOSTIC TECHNOLOGIES
Gram stain/Microscopy
Cultures.
Three major culture media
Enrichment: chocolate and sheep blood
Selective: Thayer-Martin
Differential: MacConkey-ability to ferment lactose
DIAGNOSTIC TECHNOLOGIES
Antigen Detection Assays
Most has poor sensitivity and specificity
57% sensitivity and 98% specificity for pneumococcal pneumonia
Conc. Urine EIA for Legionella pneumophila serogroup 1 has 89%
sensitivity and 100% specificity
Immunochromatographic assay has better
sensitivity and are faster
DIAGNOSTIC TECHNOLOGIES
Immunoserology
Hemagglutination
EIA
Latex agglutination
Compliment fixation
Immunoflorecent
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
MOLECULAR DIAGNOSTICS
Most widely used is PCR
High sensitivity
High specificity
Diversity
Nucleic acid probes
Do not amplify DNA
MOLECULAR DIAGNOSTICS
Polymerase Chain Reaction
Specific PCR: Uses primers to known DNA targets. So
far 31 clinical bacterial gene sequence are known and
38 in progress
• 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
MOLECULAR DIAGNOSTICS
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
Leading uses for nucleic acid
based tests
Nonculturable agents
Human papilloma virus
Hepatitis B virus
Fastidious, slow-growing agents
Mycobacterium tuberculosis
Legionella pneumophilia
Highly infectious agents that are dangerous to
culture
Francisella tularensis
Brucella species
Coccidioidis immitis
Leading uses for nucleic acid
based tests
In situ detection of infectious agents
Helicobacter pylori
Toxoplasma gondii
Agents present in low numbers
HIV in antibody negative patients
CMV in transplanted organs
Organisms present in small volume specimens
Intra-ocular fluid
Forensic samples
Leading uses for nucleic acid
based tests
Differentiation of antigenically similar agents
May be important for detecting specific virus genotypes
associated with human cancers (Papilloma viruses)
Antiviral drug susceptibility testing
May be important in helping to decide anti-viral
therapy to use in HIV infections
Non-viable organisms
Organisms tied up in immune complexes
Leading uses for nucleic acid
based tests
Molecular epidemiology
To identify point sources for hospital and
community-based outbreaks
To predict virulence
Culture confirmation
OTHER USES OF MOLECULAR
DIAGNOSTICS
Viral load monitoring
Viral genotyping
Bacterial resistance detection
Bacterial genotyping
LIMITATION OF PCR TECHNOLOGIES
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
LIMITATION OF PCR TECHNOLOGIES
Specimen should be frozen until
amplification
No antimicrobial sensitivity is available
Needs the clinician to name the suspect
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
Pathogens
Generic name of
test
Mechanism
Sensitivity
(%)
Specificity
(%)
Positive
Predictive
Value (%)
Negative
Predictive
Value (%)
Time to perform
test
Respiratory
Syncytial
Virus
RSV
Qualitative
detection of
RSV
antigen by
immunoassa
y
89-93
93 - 99
96-97
80-89
15 minutes
Influenza A Virus
Rapid Flu A
Detection
of
influenza
A
nucleoprot
ein antigen
78-82
92 - 94
80-91
90-97
10-15 minutes
Influenzas
Virus
Rapid Flu B
Detection
of
influenza B
nucleoprotei
n antigen
58-71
96 - 97
Not available
Not available
10-15 minutes
Rapid Flu A & B
Non-differential
detection of
both
influenza A
& B by
neuraminida
se enzyme
assay
52-73
92-99
95-98
74-80
22 minutes
Immunoas
say
B
Influenza A & B
Viruses
Mechanism
Sensitivity
(%)
Detects group A
staphylococcal
carbohydrate antigen
by immunoassay
89-94
(Compared
culture)
Helicobacter pylori
Detects
immunoglobulin G
antibodies specific
to H. pylori
85-90
(Compared to
biopsy)
Borrelia burgdorferi
Detects antibodies to
B. burgdorferi using
recombinant antigen
72
(Compared
ELISA)
Pathogens
Group
Streptococcus
A
Specificity
(%)
Positive
Predictive
Value (%)
Negative
Predictive
Value (%)
Time to perform test
95-99
55-89
90-97
5 minutes
80-89
85
79
5-10 minutes
97
Not available
Not available
20 minutes
to
to
Collection and Processing of Clinical Specimen
Diagnostic Technologies
Culture
Antigen detection
Serology
Molecular diagnostics
Rapid Diagnostic Test
CLIA-Waived tests
Other rapid non-CLIA waived tests
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Most important aspect of laboratory
medicine
Insufficient quantity
Contamination
Improper transport media
Delay in transportation
Inappropriate storage
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collecting Blood
Clean with 70% ethyl alcohol
Disinfect with 10% povidone-iodine
Allow to dry for at least 1 minute
No wiping!
Clean the rubber stopper of the bottle
Use alcohol for Bactec bottle to prevent cracking
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collect enough blood
1-2ml in neonate
2-3ml in infants
3-5ml in children
10-20ml in adolescent
Rapid inoculation
A 3 hour delay result in 25% reduction in recovery of S.
pneumoniae
Paisley JW, Lauer BA. Pediatric blood cultures. Clin Lab Med 1994; 14: 17
Roback MG, Tsai AK, Hanson KL. Delayed incubation of blood culture bottles: Effect
on recovery rate of S. pneumoniae. Pediatr Emerg Care 1994; 10: 268
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collecting urine
Clean-voided midstream urine
Use of urine bag
Catheterized specimen/ Suprapubic aspiration
Collecting CSF
CSF is hypotonic
Refrigeration can render fastidious bacteria non-viable
Cell count decreases by 32% after 1 hour and 50% after 2 hours
Steele RW, Mormer DJ, O’Brien MD, et al. Leukocyte survival in
cerebrospinal fluid. J Clin Microbiol 1986; 23: 965
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Insufficient quantity/quality
Small quantity for optimal analysis
Poor specimen e.g. eye cultures for chlamydiae should
have enough cellular element
Contamination
During collection
During transport
Contamination in the lab
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Improper transport media
Prevent drying
Maintain optimal physiochemical environment
Prevent oxidation and destruction of enzymes
Provide adequate nutrients
Three major culture media
Enrichment: chocolate and sheep blood
Selective: Thayer-Martin
Differential: MacConkey-ability to ferment lactose
COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Delay in transportation
Holding conditions are specimen or pathogen
specific
Urine: 2˚ C to 8˚C
Inoculated blood: 35˚ C to 37˚C
SPECIFIC EXAMPLES
Specimen for isolation of N. gonorrhoeae should be
inoculated into a specific media, transported within 30
minutes of collection, incubated at 35˚-37˚C in 5-10% co2
Stool for ova & parasite should be placed in preservatives
CSF is held in room temperature and never refrigerated
Stool for C. difficle must be refrigerated or frozen
SUMMARY
If you are not sure of how to collect a specimen
call microbiology before collection
If you are unsure what to send, call ID before
collection
If you are not sure of specific clinical syndrome,
save a sample
You are asked to attend the delivery of a term infant because the baby is small
for gestational age, and prenatal ultrasonography revealed periventricular
cerebral calcifications. The infant’s birth weight is 2,000 g. On physical
examination, you note hepatosplenomegaly and a petechial rash on the face and
trunk.
Of the following, the BEST laboratory test for diagnosing the cause of these findings is
A. Nasopharyngeal culture for herpes simplex
B. Rapid plasma reagin for syphilis
C. Serum immunoglobulin (Ig) G titer for rubella
D. Serum IgM titer for toxoplasmosis
E. Urine culture for cytomegalovirus
A 12-year-old girl comes to your office complaining of headache, malaise, fever to
101 F (38.3  C), rhinorrhea, and a sore throat for the past 3 to 4 days. Most recently,
she developed hoarseness and cough. On physical examination, you note crackles and
wheezes throughout the lung fields. Chest radiography demonstrates interstitial
infiltrates in the lower lung fields bilaterally.
Of the following, the BEST test to order to confirm the patient’s diagnosis
is:
A. Direct fluorescent antibody for Bordetella pertussis
B. Enzyme immunoassay for respiratory syncytial virus
C. Serology for Mycoplasma pneumoniae
D. Throat culture for group A Streptococcus
E. Viral culture for parainfluenza
A 16-year-old boy presents with a 5-day history of low-grade fever, headache,
mild nasal congestion, and a persistent cough associated with posttussive
syncope, he reports that it is hard for him to catch his breath after one of his
coughing episodes. His immunizations are up to date. Several of his classmates
are ill with similar symptoms. Chest radiography results are normal.
Of the following, the test that is MOST likely to aid in the diagnosis of
this patient is:
A. Cold agglutinin test
B. Mantoux skin test with purified protein derivative
C. Monospot
D. Pertussis direct fluorescent antibody
E. Sputum Gram stain
A 5-year-old girl develops fever, swelling of the parotid
gland, and headache.
Of the following, the BEST diagnostic test for this child is:
A. Bacterial culture of parotid duct secretions
B. Epstein-Barr virus serology
C. Mumps serology
D. Serum amylase
E. Viral culture of respiratory secretions
A 3-year-old boy presents with a large, non-tender, rubbery anterior
cervical lymph node. You prescribe a course of dicloxacillin, but there is
no change in the node. Results of Mantoux purified protein derivative
skin test reveal 8 mm in duration.
Of the following, the BEST diagnostic procedure to undertake in this
patient now is:
A. Biopsy of the node
B. Chest radiography
C. Excision of the node
D. Gastric aspirates for culture
E. Needle aspiration of the node
A newborn has evidence of symmetric intrauterine growth restriction.
Evaluation reveals microcephaly with intracranial calcifications, “salt and
pepper” retinopathy, hearing deficit, enlarged liver and spleen, and
purpura. Laboratory evaluation documents thrombocytopenia.
Of the following, the test MOST likely to confirm the diagnosis in this
infant is:
A. Cytology of a conjunctival swab
B. Rapid plasma reagin test
C. Serology of blood
D. Urine assay for interferon
E. Viral culture of urine
Of the following, the BEST direct stain to detect
Mycobacterium tuberculosis is the
A. Calcofluor white stain
B. Giemsa stain
C. Gram stain
D. Kinyoun stain
E. Periodic acid-Schiff stain
A 9-year-old boy has had a nonproductive cough for the past 3 weeks. He has
been afebrile and otherwise feeling well. On physical examination, you note
widespread rales. Chest radiography reveals bilateral, diffuse infiltrates. You
diagnose pneumonia, most likely due to Mycoplasma pneumoniae.
Of the following, the test that would BEST confirm the diagnosis is:
A. Bacterial culture of sputum
B. Blood culture
C. Gram stain of sputum
D. Mycoplasma-specific immunoglobulin M
E. Serum cold agglutinins
A 5-year-old boy is hospitalized in January with fever and seizures.
Lumbar puncture reveals clear cerebrospinal fluid that has a white
blood cell count of 47/ cu mm, all of which are lymphocytes. On
physical examination, he appears obtunded but arouses with painful
stimuli. Neurologic examination reveals no focal findings.
Of the following, the diagnostic test that is MOST likely to reveal the
etiology of this child’s illness is:
A. Bacterial culture of cerebrospinal fluid of herpes simplex
B. Polymerase chain reaction test of cerebrospinal fluid for
herpes simplex
C. Streptococcus pneumoniae bacterial antigen test of
cerebrospinal fluid
D. Viral culture of cerebrospinal fluid
E. Viral culture of nasopharyngeal and rectal swabs
A newborn has hepatosplenomegaly, purpuric rash, jaundice,
thrombocytopenia, and microcephaly. Computed tomography
of the head demonstrates cerebral calcifications
Of the following, the MOST appropriate diagnostic testing for this infant
includes:
A. Maternal human immunodeficiency virus serology
B. Serologic testing of mother and infant for
cytomegalovirus
C. Serologic testing of mother and infant for Toxoplasma
D. VDRL on infant and maternal sera
E. Viral culture of swabs of infant’s throat and
conjunctivae
A 12-year-old girl is brought to your office of evaluation
following 4 weeks of diarrhea, abdominal pain, and weight
loss. You suspect giardiasis.
Of the following, the MOST reliable next step to establish the diagnosis
is to examine a single stool sample for:
A. Giardia antigen
B. Leukocytes
C. Ova and parasites
D. pH
E. Reducing substances
A 4-month-old infant develops severe paroxysmal coughing
10 days after the onset of nasal congestion and rhinorrhea.
His mother reports that often 15 to 20 coughs occur in rapid
succession.
Of the following, the BEST test to establish the diagnosis is:
A. Bronchoscopy that demonstrates the presence of a
foreign body
B. Culture of a nasal swab that grows a small gramnegative coccobacillus
C. Culture of a nasal swab that shows viral growth
D. pH probe that demonstrates gastroesophageal reflux
E. Skin testing with demonstration of allergies