Lab Investigation Bacterial Infections.pptx

1. Dr Niraj Gupta

2.  Understand Importance of Test
 Clinical Diagnosis Remains Gold Standard
 Avoid Antibiotic Abuse
 Understanding Errors

3.  The challenge of the clinical diagnostic
laboratory is to provide accurate, clinically
relevant information in a timely manner
 Laboratory diagnosis of bacterial infections
begins at the bedside, not in the laboratory,
with the appropriate collection and transport of
specimens

5.  Culture Based Tests
 Non Culture Based Tests

7.  Whereas technologic advancements have been made in
the area of direct pathogen identification the isolation
of live organisms is still critical, in particular because of
the need to determine antimicrobial susceptibility
results.
 Bacterial culture depends on the growth of organisms
on various different types of culture media, including
selective, differential, and enriched.

8.  Bloodstream Infection
 Respiratory Tract Infections
 Central Nervous System Infections
 Urinary Tract Infections

9.  Given the significant impact of sepsis, the
detection of microorganisms circulating in the
bloodstream of a patient is among the most
critical roles of the clinical microbiology
Laboratory
 A positive blood culture can confirm a cause of
the septic event as well as allow appropriate
antimicrobial susceptibility testing and thus
optimal antibiotic therapy

10.  The automated blood culture systems routinely used in
microbiology laboratories are described as
continuously monitored because the system takes
periodic readings to evaluate possiblegrowth of
microorganisms.
 Several systems are currently available with differences
in detection methods, but all perform comparably
 This constant monitoring is accomplished without
entering the bottle, and thus the contamination rate for
these systems is lower than that of the lysis-
centrifugation system

11.  The single variable with the greatest impact on
the sensitivity of detection of bacterial
pathogens in blood is the volume of blood
 The most common approach to guiding
appropriate volume of blood cultured from
pediatric patients is the implementation of a
weight-based guideline

12.  Several studies have shown that delays in entry of blood
culture bottles to the blood culture system can result in
falsely negative cultures and increases in time to detection
 Optimally, the blood culture bottle should be loaded onto
the blood culture system within 4 hours of collection
 If delay in entry to the blood culture system is required,
during the delay time, the sample should not be incubated
at 37°C (98.6°F) because preincubation may significantly
compromise results.
 Optimal temperature for transport of blood culture bottles is
room temperature

13.  With use of the automated systems, both routine and
fastidious organisms will be detected within a 5-day
incubation time.
 This 5-day incubation time is adequate for detection of
fastidious organisms such as Brucella,
Francisella,Haemophilus, Actinobacillus, Cardiobacterium,
Eikenella, Kingella,Abiotrophia, and Granulicatella.
 Importantly, there are limitations to the routine blood
culture; for example, Streptobacillus moniliformis is
sensitive to even low levels of the anticoagulant SPS,
which is added tosome aerobic blood culture bottles

14.  In cases with suspected rat-bite fever, the
recommendation would be to include an
anaerobic blood culture bottle because the
anaerobic bottles contain a lower level of SPS.
 Certain organisms are so slow growing that
they will likely be missed with the routine 5-
day incubation time of a standard blood
culture; examples of these pathogens are
filamentous fungi, Nocardia spp., and slow-
growingmycobacterial species.

15.  It is best to send separate specimens for
specialized fungal ormycobacterial testing if
these organisms are suspected

16.  After the blood culture is flagged as positive, organism identification
 and susceptibility testing can be performed. Although traditional
 identification and antimicrobial susceptibility results can take up to 48
 hours to obtain, the introduction of mass spectrometry and molecular
 technologies to the clinical microbiology laboratory has dramatically
 decreased the time to definitive organism identification.31 Several rapid
 molecular tests are available that can identify organisms to species or
 genus level and for a limited number of drugs can provide information
 on antimicrobial resistance.11 Two examples of gram-positive organisms
 with known resistance phenotypes that can be identified with rapid
 molecular tests are Enterococcus faecium resistant to vancomycin and
 Staphylococcus aureus resistant to methicillin. For gram-negative organisms,
 although information regarding species-level identification can
 be obtained, there are no molecular assays that can predict susceptibility.
 Importantly, the addition of rapid identification by molecular tests or
 mass spectrometry does not replace the need to grow the organism in
 order to perform traditional susceptibility testing

17.  The upper respiratory tract
 Lower respiratory tract includes the structures
beyond the larynx.

18.  Samples collected from the upper respiratory
tract include swabs and washes and are not
considered sterile
 Whereas samples collected from the lower
respiratory tract typically require more
invasive collection techniques, such as
bronchoalveolar lavage or lung biopsy, and
may be considered sterile

19.  Upper respiratory tract infections account for
more physicians’ office visits than any other
type of infection and although most of these
infections are the result of viral infections,
bacterial etiologies are also important.
 Examples of infections that fall into the “upper
respiratory” category include pharyngitis, otitis
media, and otitis externa.

20.  Otitis externa and otitis media are usually
diagnosed clinically and managed empirically;
however, in cases of spontaneous perforation of
the tympanic membrane or when
myringotomy/tympanocentesis is performed,
fluid can be sent for culture and microbiologic
evaluation.
 Fluid is the optimal specimen for bacterial culture.
 Nasopharyngealspecimens are not representative
of the etiologic agents of otitis media

21.  Laboratory diagnosis of lower respiratory tract
infections, primarily pneumonia, is complicated by the
difficulty of collecting appropriate lower respiratory
tract samples.
 Delays in transport of specimens to the laboratory may
result in overgrowth of significant pathogens by
nonpathogenic colonizing flora or the loss of
significant Pathogens.
 Whereas organisms isolated from specimens such as
pleural fluid or tissue from lung biopsy must always be
consideredsignificant, these highly invasive procedures
are relatively rarely performed.

22.  The most common specimens collected to aid
in diagnosis of pneumonia include
 sputa and
 tracheal washes or
 aspirates.
 Results from cultures of these specimen sources
in any scenario are complicated by the presence
of normal upper airway flora, and the
determination of significance for any given
isolated organism is difficult

23.  Specimen quality is evaluated on the basis of
 the presence and quantity of organisms,
 squamous epithelial cells,
 and polymorphonuclear leukocytes
 In the case of tracheal secretions or aspirates
from ventilated, low-birth-weight infants, large
numbers of polymorphonuclear leukocytes
were associated with the length of intubation
and not the development of respiratory
symptoms

24.  BAL is not as commonly performed because of the
more invasive nature of the procedure
 Bronchoalveolar lavage specimens provide quality
lower respiratory tract samples.
 The initial aliquot removed will be contaminated with
upper airway secretions and should be discarded
before bacterial culture.
 In an effort to aid in establishing significance of various
pathogens isolated from bronchoalveolar lavage
cultures, some laboratories perform quantitative
cultures and establish cutoffs for significance

25.  Cerebrospinal fluid (CSF) specimens are
collected by lumbar puncture and must be
transported to the laboratory at room
temperature and processed without delay

26.  If sequential tubes of CSF are collected
 the first tube is not ideal for microbiologic studies because of
the possibility of contamination with skin flora or blood from
the initial lumbar puncture, which can alterculture results.
 The second tube should be sent for microbiologic
studieswhenever possible.
 Although bacterial stain and culture can be performed
on small amounts of fluid, it is recommended to send
larger amounts of CSF because of the typically low
number of microorganisms present in the specimen
 For isolation of mycobacteria from CSF, a minimum of
5 mL of specimen is recommended

27.  Positive Gram stain results can offer useful information
about organism morphology and Gram stain phenotype; but
large numbers of organisms are needed for visualization,
and anegative Gram stain result does not indicate a lack of
pathogenic organisms in the CSF.
 Staining with acridine orange (a fluorescent stain) increases
the sensitivity of detection of microorganisms, but this stain
does not provide information about the Gram-stain
phenotype(e.g., gram positive or gram negative).
 CSF specimens should be inoculated onto both chocolate
agar and sheep blood agar and incubatedfor 72 hours.
Routine inoculation of broth-based media is not
necessaryand may result in isolation of contaminants

28.  The Film Array Meningitis/Encephalitis (ME)
Panel (BioFire Diagnostics)
 is the only US Food and Drug Administration
(FDA)-cleared PCR-based panel for detecting
pathogens including bacteria, viruses, and
yeast from CSF specimens
 Importantly, because of the need to perform
antimicrobial susceptibility testing, nucleic acid
testing should only be considered as an adjunct
to routine bacterial culture

29.  Pediatric urinary tract infections are most
commonly caused by
 gram negative
 enteric bacteria
 Escherichia coli causes 80% of infections.
 Othercommon pathogens include but are not limited
to Klebsiella spp., Proteus spp., Enterobacter spp., group
B streptococcus and Enterococcus spp.,
 and occasionally Pseudomonas spp

30.  Urinary tract pathogens must be isolated and
identified in and among a background of other
normal bacterial flora.
 To distinguish organisms that colonize the
distal urethra from true urinary tract
pathogens, quantitative bacterial culture is
performed on urine specimens

31.  Catheterized specimens are an acceptable choice for
patients in whom the clean-catch method is not
possible.
 Soiled diapers and urine collected by affixing a bag to
the perineum are not acceptable specimens.
 To prevent misleading results, it is important that urine
specimens be kept refrigerated if they are not
inoculated in a timely manner (<2 hours);
 in cases of prolonged storageof urine at ambient
temperature, false-positive culture results can
beobtained

32.  Current recommendations based on previous
studies state that
 50,000 CFU/mL of a single uropathogenic
organism in a properly collected urine
specimen in the presence of pyuria is indicative
of a urinary tract infection
 Any quantity of bacterial growth from urine
collected through a suprapubicaspiration is
considered significant.

33.  The most common manifestation of
gastrointestinal (GI) infection is viral
 Feces should be collected in a sterile container and
transported to the laboratory in a timely fashion to
avoid over growthwith normal flora and the
potential loss of pathogenic organisms
 If rapid transport to the laboratory is not possible,
transport media such as Cary-Blair can be used to
stabilize the organisms during transportto the
laboratory.

34.  Because of the presence of high quantities of
commensal
 flora in feces, stool cultures require a
combination of both selective
 and differential media to identify potential
pathogens

35.  Owing to the variability in the stool culture
process,
 the clinician must inquire as to what pathogens are
routinely screened for and which pathogens may
require a request for specialized culture methods
in the testing laboratory
 Several recently developed PCR-based, multiplex
GI panels offer a reduction in time to result and
detect a broad range of organisms including
bacterial, viral, and protozoal pathogens

37.  Several non–culture-based approaches are used
to detect bacterial
 infections, including microscopy, detection of
bacterial antigens or
 antibodies against the organism, and detection
of nucleic acids by
 molecular methods

38.  Simple yet reliable technique for demonstrating
the presence of microorganisms as well as
inflammatory cells in the specimen
 Differential (Gram, Kinyoun, Giemsa) and
fluorescent (acridine orange, auramine-
rhodamine) stains aremost commonly used

39.  The Gram stain is a rapid, inexpensive method for
assessing the presence of an etiologic agent in the
specimen
 Other differential stains routinely used for
bacterial identification are
 the Kinyoun stain for Mycobacterium spp.,
 the modified Kinyoun stain for Nocardia spp. and
Streptomyces spp.,
 and the Wright-Giemsa stain for bacteria (Borrelia) or
bacterial inclusions (Anaplasma, Ehrlichia) in clinical
specimens.

40.  Provides rapid identification of a variety of pathogens isolated
from clinical specimens within hours.
 This technology uses unique protein spectral profiles of the
microorganisms to identify organisms based on known
catalogueddatabases of these profiles.
 In contrast to traditional biochemical based methods of
identification of microorganisms, MALDI-TOF-MS allows the
identification of organism with minimal culture growth and as
such shortens the time to identification significantly.
 Two commercially available MALDI-TOF-MS platforms are
cleared by the FDA for organism identification on culture isolates,
but no system has yet been cleared for organism identification of
direct specimens

41.  Nucleic acid amplification tests (NAATs) detect
bacterial nucleic acid from either direct specimen
or cultured bacterial isolates.
 NAATs have become increasingly important tools
for the detection and identificationof infectious
diseases.
 The introduction of nucleic acid–based testing has
provided increased sensitivity of detection and
decreased time to detection for many bacterial
pathogens

42.  Molecular techniques can be particularly useful for the detection
and identification of fastidious organisms (those that have
specialized growth conditions or are slow or noncultivable under
laboratory conditions)
 There are numerous molecular tests to aid in the identification of
poorly cultivable or atypical organisms,
 including Chlamydia trachomatis,
 Neisseria gonorrhoeae,
 Mycobacterium tuberculosis,
 Bordetella pertussis,
 Bartonella henselae,
 Mycoplasma pneumoniae, and many others.
 Thenumber of pathogens that can be detected by NAATs is
rapidly increasing, as are the specimen types from which
molecular testing can beperformed

43.  Bordetella pertussis
 Tuberculosis
 Clostridium difficile
 Neisseria gonorrhoeae and Chlamydia
trachomatis

44.  Sequence-based technologies have been used to
improve the speed and accuracy of identification
of organisms that are difficult to identify by
conventional methods or to detect and identify
uncultivable organisms.
 New high-throughput sequencing technologies,
also known as nextgeneration sequencing, have
replaced the commonly used Sanger sequencing
for clinical microbiology application
 For bacterial identification, 16S ribosomal genes
are common targets of interest.

45.  There are several open-access ribosomal sequence
databases that can be used to produce alignments of
the 16S ribosomal sequences andsubsequent species
assignment.
 By comparing the organism’s 16S rRNA gene sequence
with sequences found in databases of known
organisms, the identity of etiologic agents from clinical
specimens can be successfully determined.
 Other technologies,
 such as pyrosequencing and
 single-cell and whole-genome sequencing methods, are also
promising and powerful tools in clinical microbiology

46.  Thank you!