Laboratory investigations

1. Laboratory Investigations
Dr Nneka U. Igboeli, PhD, FPCPharm
Department Of Clinical Pharmacy &
Pharmacy Management
University Of Nigeria, Nsukka

2. Learning Outcomes
•Appreciate manifestation of infectious diseases
•Understand the role of clinical microbiology in infectious disease diagnosis
•Know the basic methods used in microbiologic examinations
•Know the organisms that cause infectious diseases
•Understand diagnosis of common infectious diseases and the role of point-of-care
testing in disease management

3. Learning Objectives
•Understand the rationale for microbial testing
•Know the clinical conditions that can warrant microbial testing
•Apply the knowledge of microbial examination in achieving
expected patient outcomes

4. OUTLINE
•Introduction
•Manifestations of Infections
•Clinical Microbiology
•Microbiologic Examinations
•Common Infectious Organisms
•Diagnosis of some common infectious diseases
•Point-of-care testing in Microbiology

5. Introduction
• Microbial Causes of Infection
• Infections may be caused by bacteria, viruses, fungi, and parasites.
• The pathogen may be
• Exogenous (acquired from environmental or animal sources or from other
persons) or
• Endogenous (from the normal flora).
• The diagnosis of infectious disease is best achieved by:
• Applying in-depth knowledge of both medical and laboratory science
• Principles of epidemiology and pharmacokinetics of antibiotics
• Integrating a strategic view of host–parasite interactions

6. Introduction
• Quality of the specimens received for analysis is invaluable
• The clinician needs confidence that the results provided by the
microbiology laboratory are accurate, significant, and clinically
relevant
• Laboratory requires that all microbiology specimens be properly
selected, collected, and transported to optimize analysis and
interpretation

7. Manifestations of Infection
• The clinical presentation of an infectious disease reflects the interaction
between the host and the microorganism.
• This interaction is affected by the host immune status and microbial virulence
factors
• Signs and symptoms vary according to the site and severity of infection
• Other factors include sex, age, underlying diseases/conditions, presence of
implanted prosthetic devices or materials
• Signs and symptoms may be localized or may be systemic, with fever, chills,
and hypotension
• Acute infection generally associated with an increased white blood cell count,
fever, and localized signs
• These manifestations may be absent in less severe disease

8. Manifestations of Infection
• More severe infection, including sepsis, may be associated with hypotension,
disseminated intravascular coagulation, and end-organ damage
• Other workup for sepsis or other severe infections apart from microbial tests
includes –
• Chest radiography and CT scanning
• Abdominopelvic Ultrasonography, CT scanning and MRI
• Site-specific soft tissue imaging using ultrasonography, CT scanning, or MRI
to assess possible abscess, fluid collection, or necrotizing skin infection
• Complete blood count (leukopenia = WBC count < 4000/µL,
thrombocytopenia = platelet count < 100,000/µL, and anaemia may be
observed in sepsis), Leukocytosis = WBC count > 12,000/µL, Neutrophils –
40% - 70% of WBC, Lymphocytes – 20% - 40% of WBC, Monocytes – 0% -
11% of WBC, Eosinophils – 0% - 8% of WBC, Basophils – 0% - 3% of WBC

9. Manifestations of Infection
•Plasma C-reactive protein (CRP) (elevated)*
•Serum Lactate (for sepsis, > 2 mmol/L)
•Plasma procalcitonin (elevated)
•Plasma glucose level (for sepsis, > 140mg/dL or
7.7mmol/L in the absence of diabetes)
• Some cases may be sufficiently characteristic to suggest the
diagnosis; however, they are often nonspecific
•Diagnosis requires a composite of information, including
history, physical examination, radiographic findings, and
laboratory data

10. Clinical Microbiology
• Clinical microbiology is a science of interpretive judgment
• Is my patient’s illness caused by a microbe?
• If so, what is it?
• What is the susceptibility profile of the organism so therapy can be targeted?
• Sensitivity – probability that a test says a person has a disease when in fact
they do have the disease
• Specificity – probability that the test says a person does not have a disease
when in fact they are disease free

11. • Specimens are selected on the basis of signs and symptoms,
• Should be representative of the disease process, and
• Should be collected before administration of antimicrobial agents.
• The specimen amount and the rapidity of transport to the laboratory
influence the test results
• Efforts must be made to minimize specimen contamination, especially
for skin and mucous membranes that have large and diverse indigenous
flora e.g. transtracheal puncture, suprapubic bladder puncture
• If impossible to collect uncontaminated specimen, decontamination
procedures, cultures on selective media, or quantitative cultures
must be used
Specimen Selection, Collection, and Processing

12. Microbiologic Examination
• i) Direct Examination and Techniques:
• Direct examination of specimens reveals gross pathology. Microscopy may
identify microorganisms
• Immunofluorescence, immuno-peroxidase staining, and other immunoassays may
detect specific microbial antigens
• Genetic probes identify genus- or species-specific DNA or RNA sequences
• ii) Culture:
• Isolation of infectious agents frequently requires specialized media. Nonselective
(non-inhibitory) media permit the growth of many microorganisms e.g. more
sensitive liquid (broth) media for isolation of small numbers of microbes, less
sensitive solid (agar) media provide isolated colonies that can be quantified
• Differential cultures utilizes differential carbohydrate fermentation capabilities
of microbes by incorporating one or more carbohydrates along with a suitable PH
indicator e.g. eosin methylene blue or MacConkey agar to isolate enteric bacilli

13. Microbiologic Examination
• Culture (contd):
• Selective media contain inhibitory substances that permit the isolation of
specific types of microorganisms e.g. Thayer-Martin medium to isolate
Neisseria gonorrhoeae
• Number of bacteria in specimens may be used to define presence of
infection e.g. UTIs accompanied by bacteriuria of about ≥ 104 CFU/ml –
quantitative culture
• Chlamydiae and viruses can be cultured, but isolation requires inoculation
into animals, thus, rickettsial infection is diagnosed serologically

14. Microbiologic Examination
• Determination of Isolate Pathogenicity:
• A positive culture may represent colonization, contamination or infection
• Colonization indicates that bacteria are present at the site, however, they are not
actively causing infection
• Poor sampling techniques or inappropriate handling of specimens (aseptic
handling) may cause this
• Contamination differs from colonization in that these isolates are not truly at the
site in question
• Microbial Identification in cultures:
• Turbidity, gas formation, colony and cellular morphology may permit
preliminary identification.
• Growth characteristics under various conditions, utilization of carbohydrates
and other substrates, enzymatic activity, immunoassays, and genetic probes
are also used

15. Microbiologic Examination
• iii) Serodiagnosis:
• A high or rising titre of specific IgG antibodies or the presence
of specific IgM antibodies may suggest or confirm a diagnosis
e.g. Hepatitis virus, HIV-1
• Disadvantages – lag time between onset of infection and
development of antibodies, may reflect a past infection,
immunosuppressed patients may be unable to mount an
antibody response
• iv)Antimicrobial Susceptibility:
• Microorganisms, particularly bacteria, are tested in vitro to
determine whether they are susceptible to antimicrobial agents
• Susceptibility tests are performed by either disk diffusion or a
dilution method

16. Microbiologic Examination
• Antimicrobial Susceptibility (contd):
• Well standardized for gram negative and gram positive organisms, but not as
established for anaerobes and fungi
• In contrast, susceptibility testing is available for Candida species to azoles in
the treatment of candidiasis
• The MIC is the minimum concentration at which an antimicrobial inhibits the
growth of organisms – does not indicate whether the organism is actually killed
• The MIC is determined through broth dilution method using serial dilutions of
antimicrobials in different wells/tubes
• In some disease states (e.g., endocarditis), bactericidal therapy is necessary
• Minimum bactericidal concentration (MBC) is used to determine the killing
activity associated with an antimicrobial
• The MBC is determined by taking an aliquot from each clear MIC tube for
subculture onto agar plates
• The concentration at which no significant bacterial growth (99.9% of the
original inoculum) is observed; this plate is considered the MBC

17. COMMON INFECTIOUS ORGANISMS IN
SYSTEMS
• Bloodstream infections and infections of the cardiovascular system
• HACEK (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and
Kingella) bacteria
• Brucella species, Candida spp, Staphylococcus spp, Streptococcus spp,
Enterococcus spp
• Listeria monocytogenes, Enterobacteriaceae, Pseudomonas spp, Acinetobacter spp
• Central nervous system infections
• Viruses (Coxsackie B virus, Coxsackie A virus, Echovirus, Polio virus,
Adenovirus, HIV, Mumps virus, Cytomegalovirus
• Parasites (Trypanosoma cruzi, Trichinella spiralis, Toxoplasma gondii)
• Mycobacterium tuberculosis, fungi (Cryptococcus neoformans) and bacteria
(Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes,
Streptococcus agalactiae, Haemophilus influenzae, Escherichia coli)

18. COMMON INFECTIOUS ORGANISMS IN SYSTEMS
• Ocular infections
• Bacteria (Aerobes: Streptococcus, Enterococcus, Staphylococcus,
Enterobacteriaceae, Haemophilus, Pseudomonas spp; Anaerobes:
Peptostreptococcus, Veillonella, Bacteroides, Fusobacterium, Prevotella spp,
Cutibacterium (Propionibacterium) acnes)
• Nocardia spp; Mycobacterium spp; Fungi
• Soft tissue infections of the head and neck
• Haemophilus influenzae, Streptococcus pneumoniae, β-hemolytic streptococci,
Staphylococcus aureus, Neisseria meningitidis
• Acute infection: Streptococcus pyogenes, Staphylococcus aureus,
Streptococcus anginosus (milleri) group, Mixed aerobic and anaerobic bacterial
flora of the oral cavity
• Chronic infection: Mycobacterium avium complex, Mycobacterium tuberculosis,
Other mycobacteria
• Peritonsillar abscess: Streptococcus pyogenes, Staphylococcus aureus,
Streptococcus anginosus (milleri) group, Arcanobacterium haemolyticum, Mixed

19. Other Commonly Infected Systems
1. Upper Respiratory Tract Bacterial And
Fungal Infections
2. Lower Respiratory Tract Infections
3. Infections Of The Gastrointestinal Tract
4. Intra-abdominal Infections
5. Bone And Joint Infections
6. Urinary Tract Infections
7. Genital Infections
8. Skin And Soft Tissue Infections
9. Arthropod-borne Infections
10.Viral Syndromes
11.Blood And Tissue Parasite Infections

20. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES
• Central Nervous System
• i) Meningitis – symptom triad of fever, stiff neck, and altered mental status in
adults; in neonates and infants → irritability and poor feeding reported along
with fever; elderly → sign may be absent or subtle
• Diagnosis – CSF gram stain and culture, as well as cultures obtained from
potential sites of infection e.g. blood, sputum, urine
• CSF gram stain positive in > 50% of acute bacterial meningitis and directs
initial empiric therapy
• CSF culture nearly always positive except in antecedent receipt of antibiotic
therapy

21. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES

22. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES
• CARDIOVASCULAR
• i) Infective endocarditis – Nonspecific manifestation include fatigue, weight
loss, low grade and remittent fever, night sweats; Musculoskeletal
complaints (e.g., arthralgias, myalgias, and back pain) are common and
may mimic rheumatic disease
• Fever may be absent in 30% to 40% of patients older than 60 years of
age
• Other symptoms can include lethargy, anorexia, malaise, nausea, and
vomiting
• Cardiac murmurs are present in more than 85% of patients with
endocarditis

23. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES
• RESPIRATORY TRACT
• i)Pneumonia - manifest acutely with high fever, chills, tachypnoea,
tachycardia, and productive cough, abnormal WBC count, physical
examination findings include crackles, rhonchi, bronchial breath sounds
• Diagnosis - chest radiograph or other imaging technique usually reveals an
infiltrate
• Pre-treatment blood cultures and a respiratory sample (expectorated or induced
sputum or endotracheal aspirate in intubated patients) should be obtained
• Although these cultures are often negative, when they are positive, they allow
fine-tuning of the empirical antibiotic selection

24. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES
• ii) Tuberculosis - Active disease is characterized by fever, chills, night sweats,
weight loss, and changes on chest radiography
• Diagnosis - Diagnosis of active disease includes tuberculin skin testing, chest
radiography, and sputum collection for acid-fast bacilli stain and culture
• Nucleic acid amplification tests and interferon-γ release assays may aid in the
diagnosis of tuberculosis
• The gold standard for laboratory confirmation of TB is culture
• Moderate elevation of the white blood cell count, with an increase in
monocytes and eosinophils, and anaemia are the most common hematologic
manifestations of TB

25. DIAGNOSIS OF COMMON INFECTIOUS
DISEASES
• GASTROINTESTINAL
• i) Infectious Diarrhoea - three or more episodes of loose stools, or any loose
stool with blood during a 24-hour period, which may be accompanied by nausea,
vomiting, or abdominal cramping
• Inflammatory diarrheal illnesses are characterized by the presence of bloody or
mucoid stools
• Diagnosis - stool specimens with red blood cells (RBCs) or occult blood, or
those that contain large numbers of white blood cells (WBCs) or markers of
faecal leukocytes such as lactoferrin, suggest infection attributable to invasive
pathogens
• Identification of bacterial toxins in stool specimens is a useful diagnostic tool,
because only the toxigenic strains of C. difficile are pathogenic

26. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• Routine microbiologic analysis is made using selective media favouring the
growth of the pathogen(s) of interest
• Stool cultures are recommended for patients with one of the following: severe
diarrhoea; bloody stools; or stools containing leukocytes, lactoferrin, or occult
blood; or an oral temperature of at least 101.3◦F; and for patients with persistent
diarrhoea who have not been given empiric antimicrobials
• ii) Typhoid Fever - High fever, abdominal pain, headache, dry cough
• Diagnosis – culture – isolation of organism in blood, stool or bone marrow;
•Widal test (serodiagnosis)– somatic (O) and flagellar (H) antigen titres, O (6-8
days, often active) and H (10 -12 days, often carriers) antigens, results in fractions
– denominator main parameter – normal range 1/20 – 1/80, Moderate sensitivity
and specificity
• Antimicrobial susceptibility test (MCS)
• Rapid diagnostic test

27. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• URINARY TRACT INFECTIONS – symptoms of lower UTI (e.g., cystitis) include
dysuria, frequent urination, suprapubic pain, haematuria, and back pain
• Patients with upper tract infection (e.g., acute pyelonephritis) often present with similar
findings as well as loin pain, costovertebral angle tenderness, fever, chills, nausea, and
vomiting
• Diagnosis – Urinalysis - macroscopic analysis describing the colour of the urine;
measuring its specific gravity; and estimating the pH and glucose, protein, ketone,
blood, and bilirubin contents using a rapid “dipstick” method
• Microscopic examination of urine sediment from centrifugation for presence and
quantity of leukocytes, erythrocytes, epithelial cells, crystals, casts, and bacteria
• Pyuria (i.e., ≥8 WBC per mL) of unspun urine or 2–5 WBC/HPF of centrifuged urine)
• A rapid diagnostic dipstick test for the detection of bacteriuria, the nitrite test, detects
nitrite formation from the reduction of nitrates by bacteria
• Gold-standard criterion for the diagnosis of UTI is the urine culture with a positive

28. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• STIs
• i) Gonorrhoea - purulent discharge associated with dysuria in men,
vaginal discharge in females
• Diagnosis - intracellular gram-negative diplococci in the gram-stained
exudate confirms the diagnosis in symptomatic men, endocervical
culture in females and urethral cultures in males
• ii) Chlamydia trachomatis - clinical syndromes caused by C.
trachomatis, includes cervicitis, urethritis, endometritis, and salpingitis
in women, and urethritis, prostatitis, proctitis, and Reiter syndrome in
men
• Diagnosis - culture, direct immunofluorescence assay (DFA), enzyme
immunoassay (EIA), or nucleic acid amplification tests (NAAT) of
endocervical or male urethral swabs

29. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• iii) Syphilis – from painless papule called chancre in primary syphilis to skin
lesions in secondary syphilis. The tertiary syphilis comes with pathologic
processes involving the skin, bones, central nervous system, and cardiovascular
system
• Diagnosis – Dark-field microscopic examination of exudates from chancre,
nontreponemal serology tests e.g. venereal disease research laboratory (VDRL)
and rapig plasma regain (RPR) Card Test, treponemal test requiring fluorescence
microscopy - FTA-Abs test
• iv) Vulvovaginal Candidiasis (VVC)- vulvar and vaginal pruritus, vaginal
soreness, vulvar burning, dyspareunia, and a thick, white vaginal discharge that
appears to be curd like
• Diagnosis – microscopic presence of Candida using a wet mount preparation with
10% KOH or a Gram stain of the vaginal discharge, vaginal discharge culture

30. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• Viral Hepatitis - can present as either an acute with jaundice or chronic
illness with inflammatory liver condition with ongoing hepatocellular
necrosis
• Diagnosis - acute hepatitis comes with increased serum
aminotransferase concentrations greater than 2.5 times the upper limit
of normal (Liver function test), Serologies are useful in diagnosing viral
hepatitis
• Antibodies against hepatitis A virus (anti-HAV), hepatitis B surface
antigen (HBsAg), and hepatitis C virus (anti-HCV) are useful diagnostic
tests
• A diagnosis of acute HAV infection includes the presence of
immunoglobulin M (IgM) anti-HAV
• If HBsAg is present, further testing for hepatitis B envelope antigen
(HBeAg) and HBV DNA confirms the presence of active viral replication

31. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• HIV/AIDS - Mononucleosis-like, cold or flu-like symptoms may occur 6 to 12
weeks after infection, lymphadenopathy, fever, rash, headache, Fatigue,
diarrhea, sore throat, neurologic manifestations with a mirage of opportunistic
infections like persistent herpes-zoster infection (shingles), oral candidiasis
(thrush), oral hairy leukoplakia, Kaposi’s sarcoma (KS)
• Diagnosis – Nucleic acid test (NAT) for actual virus – detect HIV in 10-33
days after exposure; Antigen/antibody tests for HIV antigens and antibodies –
detects 18 – 90 days after exposure; Antibody tests for HIV antibodies – detects
HIV in 23 – 90 days

32. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• Covid-19 – at least two clinical findings of the following – fever,
radiographic features, normal or lower WBC, or reduced lymphocyte
count
• Diagnosis – reverse transcription polymerase chain reaction (RT-PCR)
using lower respiratory tract (LRT)specimen: sputum and/or
endotracheal aspirate or bronchoalveolar; OR if impossible or in
asymptomatic contacts, upper respiratory tract(URT) specimen:
nasopharyngeal and oropharyngeal swab, or wash in ambulatory
patients with preference for combined swab collection
• High viral load detected 5-6 days of onset of symptoms with higher
sensitivity with nasopharyngeal swab; highest yield in LRT specimens
• Serological testing detects antigens and antibodies directed against the
virus and the average time for seroconversion is 12 days

33. DIAGNOSIS OF COMMON INFECTIOUS DISEASES
• PARASITIC INFECTIONS
• i) Malaria – Fever, chills, headache, malaise, nausea, and vomiting
• Diagnosis – standard microscopy, Rapid diagnostic test, PCR
• ii) Amoebiasis - Persistent mucoid / bloody diarrhoea, Abdominal pain,
fever/chills
• Diagnosis - Stool: microscopy for cysts and motile organisms (amoebic
dysentery), Full Blood Count, Chest radiograph (in amoebic liver
abscess), Abdominal Ultrasound Scan
• iii) Giardiasis - profuse watery stools, abdominal distension, and
cramping for several weeks
• Diagnosis - Full blood count, stool microscopy and faecal fat
assessment, Jejunal biopsy

34. Point-of-care testing in Microbiology
• Point-of-care (POC) test are test designed for use at or near the site where the
patient is located that do not require permanent dedicated space and where the
testing is performed outside the physical facilities of the clinical laboratories
• POC testing provides access to rapid diagnosis and predictive value key to
realization of patient outcomes
• Necessary in resource-limited settings where lack of access to medical testing
is a major reason for failure of healthcare services
• Applied in non-traditional environments, such as clinics, pharmacies, or homes
• General WHO framework for POCT development is based on the
“ASSURED” criteria: Affordable, Sensitive, Specific, User friendly, Rapid and
robust, Equipment-free, and Deliverable to end users

35. Point-of-care testing in Microbiology
• Two types of POCTs: small bench-top analyzers and hand-held single-use devices
• Lateral flow enzyme immunoassays (EIAs) for bacterial, viral, fungal, or parasite
antigens has a long history as POCTs
• Lateral flow tests or strips rely on the binding of a microbial antigen present in the
clinical sample to a primary antibody conjugated to signal, typically a gold
impregnated molecule or a fluorescent marker
• Bound antibody-antigen complex(es) then migrates either under the effect of a lysis
buffer or by capillarity in a solid substrate to generate detectable signals
• Lateral flow assays are generally inexpensive and easy to use, but suffer from
limited sensitivity, particularly when compared with nucleic acid amplification
assays (NAATs)
• These immunoassays include agglutination, immunochromatographic and
immunofiltration tests
• Non-immunological POC tests based on nucleic acid detection are also available for
few organisms

36. Point-of-care testing in Microbiology
• Advantages – decreased analytical time, cost-effective, antibiotic stewardship by limiting
overuse of antibiotics
• Clerc O and Gerub G. Routine use of point-of-care tests: usefulness and application in clinical microbiology. The Authors journal Compilation
European Society of Clinical Microbiology and Infectious Diseases 2010:16 -1054-1061

37. Point-of-care testing in Microbiology

38. Case Study – Typhoid fever
• AB is a 35 year-old man who works in UBA bank. He is the bank
manager and has been stressed at work lately. Three days ago, he
was feeling so week after coming back from work by 10.00pm and
by the next day he felt his temperature was mildly high (in his own
words). He suspects that he has malaria and typhoid because,
according to him he always have these ailments every other month.
He then decided to do a lab test two days ago and he collected the
result yesterday evening. The result is pasted in the next page and he
wants an interpretation for the result.
• Should AB take an antibiotics for typhoid fever?

39. Case Study – Typhoid fever contd

40. Case Study – Pneumonia
• AL is 45-year old woman is hospitalized with a sustained high fever of 39.4C, and
shortness of breath. Her cough is productive and she appears to be in acute distress.
CBC and MP tests were ordered and the result are as follows:
• Total WBC count – 18,000/µL
• Neutrophils – 76%
• Bands – 13%
• Lymphocytes – 10%
• Monocytes – 0%
• Eosinophils – 1%
• Basophils – 0%
• MP - Negative
• A diagnosis of pneumonia is suspected.
• How is lab result consistent with bacterial infection?
• Which other lab test(s) would you suggest for AL?

41. References
• Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical
Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at
Galveston; 1996. Chapter 10
• Fournier PE, Drancourt M, Colson P, et al. Modern clinical microbiology: new
challenges and solutions. Nat Rev Microbiol 2013;11: 574-585.
https://doi.org/10.1038/nrmicro3068
• Zayed RA, Omran D, Zayed AA. Covid-19 clinical and laboratory diagnosis
overview. Journal of Egyptian Public Health Association 2021;96:25.
• Clerc O and Gerub G. Routine use of point-of-care tests: usefulness and
application in clinical microbiology. The Authors journal Compilation European
Society of Clinical Microbiology and Infectious Diseases 2010:16 -1054-1061

42. Thanks for listening