STUDY GUIDE - UMF - Iuliu Haţieganu

STUDY GUIDE 2009-2010
MICROBIOLOGY DEPARTMENT
III-rd YEAR LABORATORIES
Content: Clinical Microbiology:
Laboratories/Practical activities program:
Hours/week: Practical activities (laboratory): 2
Hours/semester: Practical activities (laboratory): 28
Teaching staff:
Assistant professor Dr. Carmen Costache
Teaching assistant professor Dr. Ioana Colosi
Consultation schedule:
Assistant professor: Dr. Carmen Costache: Thursday 12-14
Teaching assistant professor: Dr. Ioana Colosi: Friday 12-13
Laboratories
L 1
Laboratory diagnosis in
bacterial respiratory tract
infections: angina,
pneumonia,
L 2
Laboratory diagnosis in
atypical pneumonia
L 3
Laboratory diagnosis of
CNS infections: Bacterial
meningitis
L 4
Laboratory diagnosis of
bacterial digestive tract
infections, blood and
sexually transmitted
diseases
L 5 Laboratory diagnosis of
fungal respiratory tract
infections (pneumonia) and
fungal CNS and skin
infections,
Laboratory diagnosis in
infections with blood
protozoa, sexually
Laboratory diagnosis in infections with Corynebacterium
spp.
Laboratory diagnosis in infections with Streptococcus spp.
Laboratory diagnosis in infections with Staphylococcus spp.
Laboratory diagnosis in infections laboratory diagnosis in
tuberculosis Mycobacterium spp.
Laboratory diagnosis in infections with Bordetella spp.
Laboratory diagnosis in infections with Chlamydia spp.
Laboratory diagnosis in infections with Mycoplasma spp.
Laboratory diagnosis in infections with Legionella
pneumophila
Laboratory diagnosis in infections with Haemophilus spp.
Laboratory diagnosis in infections with Neisseria spp.
Laboratory diagnosis in infections with Clostridium spp.
Laboratory diagnosis in infections with Bacillus spp.
Laboratory diagnosis in infections with Enterobacteria
Laboratory diagnosis in infections with Escherichia,
Salmonella, Shigella, Vibrio
Laboratory diagnosis in infections with Pseudomonas,
Laboratory diagnosis in infections with Treponema spp.
Laboratory diagnosis in infections with Ricketsia spp.
Brucella spp, Leptospira spp
Laboratory diagnosis in infections with Pneumocystis
Aspergillus, Cryptococcus
Laboratory diagnosis in infections with Candida,
Dermatophytes
Laboratory diagnosis in infections with Trichomonas,
Laboratory diagnosis in infections with Plasmodium,
Laboratory diagnosis in infections with Toxoplasma,
1

transmitted protozoa, of
CNS and of visceral and
congenital infections
L 6 Laboratory diagnosis of
parasitic digestive tract
infections
L 7 Laboratory diagnosis of
parasitic visceral infections
Laboratory diagnosis in infections with intestinal protozoa:
Giardia, Entamoeba, Criptosporidium
Laboratory diagnosis in infections with intestinal
Nematodes: Ascaris lumbricoides, Trichuris trichiura,
Enterobius vermicularis ,Trichinella spiralis, Ancylostoma
duodenalis, Strongyloides stercoralis
Laboratory diagnosis in infections with intestinal Flatworms:
Fasciola, Tenia, Diphylobotrium, Echinococcus,
Laboratory diagnosis in Cisticercosis, Hydatic cyst, Alveolar
echinococcosis
TABLE OF CONTENTS
LABORATORY DIAGNOSIS IN BACTERIAL INFECTIONS...................................... 4
RESPIRATORY TRACT INFECTIONS........................................................................... 4
LABORATORY DIAGNOSIS IN DIPHTERIA ........................................................... 4
GENUS CORYNEBACTERIUM ................................................................................. 4
LABORATORY DIAGNOSIS IN STREPTOCOCCAL INFECTION: ANGINA,
SCARLET FEVER, BACTERIAL PNEUMONIA ....................................................... 5
GENUS STREPTOCOCCUS ...................................................................................... 5
LABORATORY DIAGNOSIS OF WHOOPING COUGH ......................................... 8
BORDETELLA PERTUSIS....................................................................................... 8
LABORATORY DIAGNOSIS OF ATYPICAL PNEUMONIA.................................. 9
GENUS MYCOPLASMA............................................................................................. 9
GENUS CHLAMYDIA............................................................................................ 10
LEGIONELLA PNEUMOPHILA............................................................................ 11
LABORATORY DIAGNOSIS IN TUBERCULOSIS:GENUS MYCOBACTERIUM12
LABORATORY DIAGNOSIS IN PYOGENIC INFECTION ........................................ 13
GENUS STAPHYLOCOCCUS ................................................................................ 13
LABORATORY DIAGNOSIS IN CENTRAL NERVOUS SYSTEM INFECTION –
BACTERIAL MENINGITIS............................................................................................ 15
GENUS HAEMOPHILUS........................................................................................ 15
GENUS NEISSERIA............................................................................................... 17
GENUS CLOSTRIDIUM- Laboratory diagnosis in tetanus, botulism ........... 19
GENUS BACILLUS. ................................................................................................ 21
LABORATORY DIAGNOSIS IN DIGESTIVE TRACT INFECTIONS ....................... 23
LABORATORY DIAGNOSIS IN INFECTIONS WITH ENTEROBACTERIA
ENTEROBACTERIACEAE FAMILY ........................................................................ 23
GENUS SALMONELLA.......................................................................................... 24
GENUS SHIGELLA ................................................................................................. 24
OPPORTUNISTIC ENTERICS................................................................................. 25
2

LABORATORY DIAGNOSIS IN INFECTIONS WITH OTHER GRAM
NEGATIVE BACILLI 27
GENUS PSEUDOMONAS....................................................................................... 27
GENUS VIBRIO....................................................................................................... 28
HELICOBACTER PYLORI..................................................................................... 30
LABORATORY DIAGNOSIS OF INFECTIONS WITH BRUCELLA SPP. .......... 31
LABORATORY DIAGNOSIS IN SEXUAL TRANSMITTED DISEASES.................. 32
LABORATORY DIAGNOSIS OF INFECTIONS WITH TREPONEMA PALLIDUM
LABORATORY DIAGNOSIS OF INFECTIONS WITH LEPTOSPIRA SPP............... 34
LABORATORY DIAGNOSIS OF INFECTIONS WITH RICKETTSIA........................ 35
LABORATORY DIAGNOSIS OF INFECTIONS WITH PARASITES .................. 37
LABORATORY DIAGNOSIS OF INFECTIONS WITH PROTOZOA.................. 38
DIGESTIVE TRACT INFECTIONS: Giardia, Entamoeba, Criptosporidium................. 38
CONGENITAL INFECTIONS: Toxoplasma.................................................................. 40
BLOOD INFECTIONS:Plasmodium……………………………………………………49
SEXUALLY TRANSMITTED DISEASES: Trichomonas …………………………… 51
LABORATORY DIAGNOSIS OF INFECTIONS WITH NEMATODES............... 44
Ascaris lumbricoides......................................................................................................... 45
Trichuris trichiura (whipworm), trichocephalus .............................................................. 45
Enterobius vermicularis (pinworm).................................................................................. 46
Trichinella spiralis............................................................................................................ 47
Ancylostoma duodenalis (hookworm) .............................................................................. 48
Strongyloides stercoralis .................................................................................................. 48
Enterobius vermicularis …………………………………………………… …………. 58
LABORATORY DIAGNOSIS OF INFECTIONS WITH PLATYHELMINTS ... 50
TREMATODES Fasciola .................................................................................................. 51
CESTODES (TAPEWORMS) Tenia, Diphylobotrium, Echinococcus.............................. 52
DISEASES PRODUCED BY NEMATODES LARVAE: Cisticercosis, Hydatic cyst,
Alveolar echinococcosis.................................................................................................... 55
LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS .................................... 58
Laboratory diagnosis in infections with Pneumocystis jirovecii...................................... 59
Laboratory diagnosis in infections with Aspergillus........................................................ 59
Laboratory diagnosis in infections with Candida ............................................................. 60
Laboratory diagnosis in infections with Cryptococcus .................................................... 61
Laboratory diagnosis in infections with Dermatophytes.................................................. 61
3

LABORATORY DIAGNOSIS IN BACTERIAL INFECTIONS
RESPIRATORY TRACT INFECTIONS
LABORATORY DIAGNOSIS IN DIPHTERIA
GENUS CORYNEBACTERIUM
Summary
General properties
Species
Laboratory diagnosis in diphteria
Practical activities:
- Gram, Neisser staining, observation and interpretation
- Inoculation and observation of Corynebacterium spp. colonies on culture media
Educational objectives
Essential
Corynebacterium
- species members of the normal flora of skin, naso- and oropharynx, urogenital and
gastrointestinal tract = diphteroids (C. hoffmani, C. xerosis)
- Corynebacterium diphteriae (C.diphteriae) = pathogen => diphtheria.
General properties
- Pleomorphic Gram (+) rods, often with clubbed ends, arranged in pairs and trios
- may contain intracellular metachromatic granules.
- Noncapsulated, nonmotile, aerobes, catalase +
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: 2 swabs from carriers (1 nasal and 1 pharyngeal swab)
3 pharyngeal swabs and 1 nasal swab from patients.
2. Microscopic examination
- Gram smear
- Neisser smear: metachromatic corpuscles (Babes-Ernst) are visible (bluish green)
3. Inoculation on media
- Loffler medium,
- blood agar plate,
- OCST medium (enrichment medium) and after 18-24 hours on Tinsdale medium
4. Identification based on:
- morphologic characteristics: Gram stained smear from colonies developed on Loffler
media shows Gram (+) rods, with clubbed ends, arranged in pairs or trios (frequent like
Chinese characters).
- culture properties:
- Tinsdale medium: small black colonies develop, with a brownish aura
- biochemical properties: differentiate between C.diphteriae and diphteromorfs
- pathogenic properties: production of toxin
- in vitro: Elek’s test
- in vivo: exotoxin production - dermonecrotic lesion in rabbit
B. Indirect diagnosis
- epidemiologic value
4

- biologic method: intradermal reaction (Schick test)
Important
Cultural properties: 3 biotypes (epidemiologic purposes.)
- C.diphteriae gravis (R colonies),
- C.diphteriae mitis (S colonies),
- C.diphteriae intermedius (S colonies, but with irregular edges)
Interpretation of IDR Shick
(+) reaction means an inflammatory reaction greater than 10mm, which is observed in
persons who do not have or have insufficient antibodies ( < 0,03 UIA/ml; in USA they
consider 0.15 UIA/ml) to neutralize the injected toxin.
(-) reaction means an inflammatory reaction smaller than 10 mm, which indicates that the
individual is able to neutralize the injected toxin.
Useful
Schick test technique: involves intradermic injection of a very small amount of toxin
(1/50DLM, contained in 0.2 ml)
- results are read after 48-96 hours
Optional
Elek’s test is a double diffusion test performed directly on the surface of an agar plate. A
filter paper strip is impregnated with antiserum to the toxin. If the strain is a toxin
producing strain, precipitation of the toxin with the antitoxin antibodies will form a
precipitation line.
Questions and reviewing
Corynebacterium dyphterie has the following characteristics
a. grow always as smooth type colonies
b. produce endotoxin
c. may produce exotoxin
d. may form rough type colonies
e. belong to normal flora of the skin
LABORATORY DIAGNOSIS IN STREPTOCOCCAL INFECTION: ANGINA,
SCARLET FEVER, BACTERIAL PNEUMONIA
GENUS STREPTOCOCCUS
Summary
General properties
Classification
Laboratory diagnosis in streptococcal angina, scarlet fever
Laboratory diagnosis in bacterial pneumonia
Practical activities:
- Gram staining, observation & interpretation
- Inoculation and observation of Streptococcus spp. colonies on culture media
- Latex agglutination reaction
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- Susceptibility testing to antibiotics
Educational objectives
Essential
General properties
- Gram positive cocci that form linear chains of varying length.
- Catalase negative.
- Mostly facultative aerobes; some strict anaerobes
Classification
A. By hemolysis on blood agar
β - hemolytic streptococci:: complete lysis of erythrocytes on blood agar (β hemolysis):
strepotoccoci group A (Streptococcus pyogenes), group B, C, G
α – hemolytic streptoccoci: incomplete, green hemolysis on blood agar (α hemolysis):
S.pneumoniae, S.viridans
γ - non hemolytic streptococci: S.lactis
B. By determinants of antigenicity classified by Rebecca Lancefield
- Polysacharide “C” (in the cell wall) allows grouping of streptococci in Lancefield
groups, from A to T
Laboratory diagnosis in streptococcal infection (angina, scarlet fever)
Beta-hemolytic streptococci
A. Direct diagnosis
1. Sample collection: pharyngeal exudates
2. Microscopic examination: irrelevant (saprobic streptococci)
3. Isolation on blood agar
4. Identification based on:
- morphologic properties: gram positive, round or ovalar cocci, arranged in chains
- cultural properties: small translucent colonies (< 1 mm) with -hemolysis
- biochemical properties
- antigenic properties - classification into groups: latex agglutination.
B. Indirect diagnosis
a) ASLO test, (Antibodies against Streptolysine O)
- Interpretation
b) IDR Dick: based on neutralization of the erythrogenic toxin by the antitoxin
antibodies
- Purpose
- Interpretation
Laboratory diagnosis in bacterial pneumonia
Alfa- hemolytic strepcococci - Streptococcus pneumoniae (pneumococcus)
General properties: diplococci, ovoid or lancet-shaped, capsulated
A. Direct diagnosis
1. Sample collection: sputum and 2-3 venous blood samples
2. Microscopic examination:
- Gram smear in which we can see lancet-shaped, G (+), diplococci,
- Quellung test (enhance the appearance of capsule)
6

3. Isolation on chocolate agar
- inoculation in white mouse => septicemia and death after 24 to 48 hours (pure culture
of pneumococcus from spleen and heart blood)
4. Identification based on:
- morphologic properties: gram positive, ovoid or lancet-shaped, capsulated diplococci
- cultural properties: on chocolate agar => yellowish hemolysis;
On blood agar => -hemolysis
On liquid media => opaque.
Colonies: smooth, mucoid aspect
- biochemical properties: used for confirmation of pneumococcal etiology in bacterial
pneumonia: they are lysed by bile
- serologic testing => determination of the isolate’s serological type.
Important
ASLO test : principle and technique
IDR Dick: principle and technique
blood should be cultured in liquid media (hemoculture) and multiple blind passages must
be made.
- optochin sensitivity testing (pneumococci are inhibited by optochin-a quinine
derivative);
Useful
Serology: Quellung reaction: the interaction of capsular polysaccharide and the
corresponding antigen changes the optical properties of the capsule, causing it to appear
swollen under the microscope.
Laboratory diagnosis in infections with Enterococci
Essential
General properties
Diseases
- nosocomial infections, urinary tract infections, endocarditis, meningitis, abdominal and
pelvic infections
Laboratory diagnosis
Direct diagnosis
1. Sample collection: sputum, blood, urine
2. Microscopic examination: Gram staining
3. Isolation on culture media
- chocolate agar, blood agar
- selective media with bile
4. Identification based on:
- morphologic properties
- cultural properties
- biochemical properties: bile-esculine test, growth in 6.5% NaCl medium
5. Antibiotic susceptibility testing
Important
Species: E.fecalis, E.faecium
Questions and reviewing
7

The ASLO reaction is used in diagnostic of :
a) rheumatic fever
b) staphylococcal infections
c) acute glomerulonephritis
d) infections produced by Streptococcus viridans
e) infections produced by Streptococcus pyogenes.
β-haemolytic streptococcus group A:
a. is also known as Streptococcus pneumoniae
b. is also known as Streptococcus pyogenes
c. produces rheumatic fever
d. produces acute glomerulonephritis
e. produces diphteria.
LABORATORY DIAGNOSIS OF WHOOPING COUGH
BORDETELLA PERTUSIS
Summary
General properties
Infections
Laboratory diagnosis of whooping cough
Practical activities:
- stained smears, observation & interpretation
- performing antigen-antibodies reactions and interpretation
Educational objectives
Essential
General characteristics
- small Gram-negative coccobacillus
- aerobic
- nutritionally fastidious
- colonizes the cilia of the mammalian respiratory epithelium
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: particularities
2. Microscopic examination
3. Isolation on Bordet-Gengou media (potato, glycerine, defibrinated blood, agar)
Important
4. Identification based on:
- morphologic properties: encapsulated Gram negative coccobacilli
- cultural properties: haemolytic, fastidious microorganisms (they grow slow), look like a
half of a pearl.
B. Indirect diagnosis
a) Serologic
-agglutination in tubes
8

Useful:
- antigenic characteristics of Bordetella pertussis: 14 different K antigens, which
characterized all the three species.
- pathogenic properties of Bordetella pertussis:
-intranasal inoculation in mouse will result in bronchopneumonia;
-intra-dermal inoculation in rabbit will produce dermal necrosis
- Biologic diagnostic: IDR (intradermal reaction)
Optional:
Composition of Bordet-Gengou media (potato, glycerine, defibrinated blood, agar)
LABORATORY DIAGNOSIS OF ATYPICAL PNEUMONIA
GENUS MYCOPLASMA
Summary
General properties
Species
Infections
Laboratory diagnosis
Practical activities:
- stained smears, observation & interpretation
- performing antigen-antibodies reactions and interpretation
Essential
General properties
- smallest free-living bacteria
- lack cell wall => do not stain with conventional bacteriologic stains and naturally
resistant to β-lactamins.
Mycoplasma pneumoniae "atypical" pneumonia.
Laboratory diagnosis - difficult during acute phase
A. Direct diagnosis
1. Sample collection
2. Microscopic examination: irrelevant, do not stain.
3. Inoculation on media: complex nutritional requirements (lipids)
4. Identification:
- colony - "fried-egg" shape, with a raised centre and a thinner outer edge.
- PCR
Important
Other mycoplasmas
Mycoplasma hominis pelvic inflammatory disease.
Ureoplasma urealyticum nongonococcal urethritis.
- produces urease
Useful
B. Indirect (serological) diagnosis
- cold agglutinins: IgM autoantibody against type O red blood cells
9

- complement fixation
Optional
Technique of antigen-antibodies reactions.
GENUS CHLAMYDIA
Summary
General properties
Species
Infections
Laboratory diagnosis
Practical activities:
- stained smears, observation & interpretation
- performing antigen-antibodies reactions and interpretation
Educational objectives
Essential
General properties
- obligate intracellular organisms
Representatives
–Chlamydia psittaci
–Chlamydia pneumoniae
–Chlamydia trachomatis
1. Chlamydia psittaci
- primary atypical pneumonia
2. Chlamydia pneumoniae
- upper respiratory infections
- antigenically distinct
- inclusion bodies “pear-shaped”
- later evolves into mild bronchopneumonia
3. Chlamydia trachomatis
- trachoma (chronic follicular keratitis) - serotypes A, B, B4 and C
- Sexually transmitted disease (serotypes D through K)
- adults: nongonococcal urethritis, mucopurulent cervicitis
- neonates: inclusion conjunctivitis, pneumonia
- LGV (serotypes L1, L2, and L3) = lymphogranuloma venereum ( systemic
involvement) – sexually transmitted diseases.
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection (according to the symptoms)
2. Microscopic examination: Giemsa staining: small, intracellular inclusions in host’s
cells.
3. Inoculation - cellular media with cycloheximide, tissue cultures (McCoy), embrionated
egg, mice
4. Identification :
- microscopic properties: Giemsa stained smears – ”bull’s eye”
- antigenic properties:
10

- complement fixation
- ELISA
- detect LPS in urethral and cervical specimens
- PCR (polymerase chain reaction)
B. Indirect diagnosis
Serologic: complement fixation, immunofluorescence
Important life cycle:
- elementary body = infectious, extracellular form
- reticulate (initial) body = replicative, intracellular
Chlamydia psittaci and Chlamydia pneumonie - multiple small inclusion bodies scattered
around the nucleus,
Chlamydia trachomatis - single large inclusion body.
- intermediate/inclusion body
–condensation of the reticulate bodies
–looks like a “bull’s eye” under MO
Useful : Symptoms of diseases.
Optional
- C. trachomatis - inclusions containing glycogen
- C. psittaci and C. pneumoniae - inclusions that do not contain glycogen.
- glycogen-filled inclusions are visualized by staining with iodin
Biologic diagnosis: LGV- Freni IDR
LEGIONELLA PNEUMOPHILA
Summary
General properties
Pathogenesis and disease
Laboratory diagnosis
Educational objectives
Essential atypical pneumonia
General properties
- it is the most important representant.
- it causes pneumonia
- Gram-negative rods that stain very poor with standard Gram stain
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection
2. Microscopic examination: Gram stained smear reveal neutrophils but no bacteria
3. Inoculation on media
4. Identification: Fluorescent-antibody staining: Legionella pneumophila antigens in the
urine or lung tissue.
B. Indirect (serologic) diagnosis - increase antibody titer by immunofluorescence assay,
complement fixation.
Important: Cold-agglutinins titer does not rise in Legionella pneumonia, in contrast to
pneumonia caused by Mycoplasma.
11

Useful: Legionaires' disease.
Optional
Special staining: silver impregnation stain (Dieterle)
Required special media to grow- media enriched in iron and cystein.
LABORATORY DIAGNOSIS IN TUBERCULOSIS
GENUS MYCOBACTERIUM
Summary
General properties
Classification
Laboratory diagnosis in tuberculosis
Practical activities:
- Ziehl-Neelsen staining, observation and interpretation
- Observation and drawing of M.tuberculosis colonies on culture media
(Lowenstein –Jensen)
Educational objectives
Essential
Cell wall composition => acid-alcohol resistance (AAR) or acid-fast => Ziehl-Neelsen
staining.
Species (classification)
Diseases
Laboratory diagnosis in tuberculosis
A. Direct diagnosis
Specimen collection
Microscopic examination (acid fast staining): red slender rods placed in capitals or
isolated against a blue background formed by destroyed cells, other bacteria, mucus, and
fibrin.
Inoculation on culture media- Lowenstein-Jensen
Identification
- morphology and staining
- culture properties - M.tuberculosis: R, dry, cauliflower-like
- biochemical characteristics
B. Indirect diagnosis
- intradermal reaction (IDR) to M.tuberculosis antigen = tuberculin or its protein-purified
derivative, PPD) due to the delayed-type of hypersensitivity. It is done by Mantoux test.
- Interpretation:
- 48-72 hours later by measuring the indurate erythematous reaction:
0-9 mm = (-) IDR: the person does not have cellular immunity against tubercle
bacilli (is not infected)
> 10 mm = (+) IDR: does not necessarily mean the patient has tuberculosis (if it
is so, the reaction is very big and ugly, with necrosis). It only means it has been
previously exposed to the bacterium or has been vaccinated.
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Immunoprophylaxis: Administration of bacille Calmette-Guerin (BCG vaccine)
Important:
- M.tuberculosis complex
o M.tuberculosis, M. africanum, M.microti, M.bovis
- MOTT ( Mycobacteria Other Than Tuberculosis)
- False IDR reaction (+):
o cross-reactions with other mycobacterium;
o (-): anergy after influenza, chicken-pox, IS
- BCG = an attenuated strain derived from M.bovis
Useful:
- Primary infection
- Active tuberculosis
Tuberculostatics: Isoniazide and Rifampin for 6 months, together with Ethambutol and
Pyrazinamide for the first two months.
Other antituberculous drugs: Streptomycin, Cycloserine, Ethionamide, Fluoroquinolones,
Kanamycine, and PAS
Optional:
- counting of bacilli on stained smear
- Other culture media : Broth, Middlebrook
- pathogenic properties in laboratory animals => Conclusion: CMI (cellular
mediated immunity) develops tuberculin
Questions and reviewing:
1. For human infection with M.tuberculosis the following statements are true
a. immunity is humoral
b. immunity is cellular
c. immunity is tested in vivo by IDR to tuberculin
d. immunity usually demonstrated by a 4 fold rise in the titer of antibodies
e. the prophylaxis is done by vaccination with DTP
2. Proof of the presence of active disease caused by Mycobacterium tuberculosis is
provided by which one of the following diagnostic measures?
a. the tuberculin test (IDR)
b. clinical findings ( weight loss, night sweats, cough, low-grade fever)
c. finding acid-fast organism in sputum
d. isolation of M.tuberculosis in culture
e. positive O&P examination.
PYOGENIC INFECTION
GENUS STAPHYLOCOCCUS
Summary
General properties
Laboratory diagnosis in infections with Staphylococcus spp.
Practical activities:
- Gram staining, observation & interpretation
13

- Inoculation and observation of Streptococcus spp. colonies on culture media
- Latex agglutination reaction
- Susceptibility testing to antibiotics
Essential
Staphylococcus aureus
General properties
- colonizes the skin and mucous membranes (30% of normal humans, 70% in healthcare
personal)
- catalase positive
- aerobic conditions
- grow in presence of 7.5% sodium chloride (halotolerants)
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: pus, blood, nasal and pharyngeal exudates, urine, etc.
2. Microscopic examination:
- Gram smear: clusters of Gram positive cocci on a background formed by fibrin, necrotic
desquamated cells.
3. Inoculation on media
- grow well under condition that is usually inhibitory to other bacteria
(e.g. 7.5% sodium chloride or 40% bile)
- blood agar
- manitol salt agar (Chapman media) => selective and differential
4. Identification based on:
- morphology and staining: G (+) cocci arranged in clusters
- cultural properties: smooth, shiny colonies (S) with different pigmentation depending on
the species: white (S.epidermidis), white or yellow (S.aureus) and white or gray
(S.saprophyticus).
S.aureus produce hemolysis (incomplete = ), which can transform into complete
hemolysis () at 4 ºC.
- biochemical properties: catalase +, coagulase + (S.aureus), manitol fermentation and
oxidation + (S.aureus)
5. Sensitivity to antibiotics and treatment
- must be performed because the resistance is very frequent and easily acquired from one
strain to another
- oxacillin, vancomicin, cephalosporins, erythromycin or clindamycin
Important:
Representants (more than 30 species):
- Staphylococcus aureus responsible for most staphilococcal infections in humans
- Staphylococcus epidermidis causes opportunistic infections in immunosuppressed
patients.
- Staphylococcus saprophyticus is opportunistic, cause urinary tract infections in women.
Useful: Differentiating characteristics of staphylococci
Optional:
Staphylococcus grows best at 37 C but become pigmented best at 20-25 C
14

Questions and reviewing
Selective medium for staphylococci is:
a. blood agar
b. Muller-Hinton media
c. Lowenstein Jensen media
d. Chapman media
e. Drigalski media
Staphylococcus aureus have the following characteristics:
a. produce hemolysis on blood agar
b. ferment manitol on Chapman media
c. produce a goldish pigment
d. produce hydrogen sulfide
e. produce a grey pigment.
CENTRAL NERVOUS SYSTEM INFECTION – BACTERIAL
MENINGITIS
GENUS HAEMOPHILUS
Summary
General properties
Species
Infections
Laboratory diagnosis
Practical activities:
- Gram staining, observation and interpretation
- Colonies of Haemophilus, Neisseria: observation and interpretation
- stained smears, observation and interpretation
- performing antigen-antibodies reactions and interpretation
Essential
General properties
- Gram negative cocobacilli (short rods)
- grow only on media containing blood (growth required 2 factors):
- factor X (hematin, a precursor of hemin)
- factor V (nicotinamide-adenine dinucleotide, NAD)
Genus Haemophilus - Species
- Haemophilus influenzae
- Haemophilus parainfluenzae
- Haemophilus aegypticus
- Haemophilus ducreyi
- Haemophilus haemolyticus
- Haemophilus parahaemolyticus
15

Haemophilus influenzae
- meningitis
- acute bacterial epiglottitis (children between 2 and 5 years of age; can cause respiratory
obstruction and arrest)
- otitis media and sinusitis, in young children
- bacteremia - always caused by capsulated strains
- bronchitis and pneumonia
A. Direct diagnosis
1. Sample collection: CSF, sputum, pharyngeal exudates, blood (for type b), and pus
2. Microscopic examination (important in meningitis): Gram negative coccobacilli
- Quellung reaction is the classic approach to serotyping and diagnosis
- immunofluorescence
- microscopic examination - important for H.aegipticus and H.ducrey (difficult to culture)
3. Inoculation on media
- blood agar, chocolate agar, Levinthal media with lysed erythrocytes.
- Satellitism:
- H.influenzae may grow on blood agar plates if Staphylococcus aureus is
simultaneously seeded.
4. Identification based on:
- morphological characteristics
- cultural characteristics
- antigenic characteristics
- six major serotypes (a to f), defined by antisera to capsular polysaccharide.
- the most important one is the serotype b.
Important
Haemophilus ducreyi
- causes chancroid, a venereal disease
- painful genital ulcers, edema, and regional lymphadenopathy.
- treated with sulfonamides or aminoglycosides.
Useful
Some of the Haemophilus species are found in oral cavity and pharynx.
Haemophilus
- does not require factor X for growth
- causes pneumonia and endocarditis.
Quellung reaction is the classic approach to serotyping and diagnosis
- a small drop of CSF + specific antiserum on a slide => if specificity is met than the
capsule will appear larger in the smear.
Optional
Factor X (a heme compound) is present in blood agar plates, and, with the addition of the
feeder bacteria, there are two sources of factor V (NAD): produced by S.aureus itself and
NAD released from red cells lysed by the α-hemolysin of S.aureus.
Questions and reviewing:
Which one of the following microorganisms cause respiratory infections ?
A.
a. Streptococcus pyogenes
16

. Corynebacterium diphteriae
c. Bordetella pertusis
d. Haemophilus influenzae
e. Staphylococcus aureus
B.
Mention the disease they are producing and methods for diagnosis.
Which one of the microorganisms are Gram (+) and which are Gram (+) bacteria ?
GENUS NEISSERIA
Summary
Species
Infections
Laboratory diagnosis of meningococcal meningitis
Laboratory diagnosis of infections with gonococcus
Practical activities:
- Gram staining, observation & interpretation
- Colonies of Neisseria: observation & interpretation
- stained smears, observation & interpretation
- performing antigen-antibodies reactions and interpretation
Educational objectives
Essential
General characteristics
- Gram negative cocci, kidney or bean-shaped, often seen as diplococci
- grow best on chocolate agar
Species
a) Extracellular, saprobic neisseria (e.g. Neisseria catarhalis, Neisseria flava, Neisseria
perflava, Neisseria sicca) found in respiratory and genito-urinary tract
b) Intracellular, pathogenic species - Neisseria meningitidis (meningococcus)
- Neisseria gonorrhoeae (gonococcus)
Neisseria meningitidis (meningococcus)
Essential
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: cerebrospinal fluid (CSF), blood
2. Microscopic examination
- Gram stain: Gram negative intracellular diplococci, kidney shaped
- Ziehl-Neelsen stain will eventually exclude the tuberculosis etiology.
3. Inoculation on culture media
- chocolate agar
- Műller-Hinton media with antibiotics (Vancomicine, Colistine, Nistatin)
Important
4. Identification based on:
17

- morphologic properties
- cultural properties: S grey colonies
- biochemical properties: catalase (+), oxidase (+), produces acid from glucose and
maltose
- antigenic properties: latex agglutination.
Useful:
All neisseria are sensitive to drying and must be plated immediately in fresh, moist, warm
media
Incubation at 37ºC, 48 hours at CO 2 10-20%
Culture and isolation is necessary to determine susceptibility to antimicrobial agents
Optional:
The Quellung reaction
Vincent Bellot precipitation reaction: CSF in a test tube will contain polysaccharidic
antigen (originated from lysis of the meningococci ) + antimeningococcal serum will
form an opaque ring at the interface.
Gonococcus (Neisseria gonorrhoeae)
Laboratory diagnosis
Essential
A. Direct diagnosis
1. Sample collection
2. Microscopic examination:
- Gram staining - intracellular Gram negative diplococci
- staining with metilen blue may be also considered
3. Inoculation on media:
- chocolate agar
- Thayer-Martin
- Műller-Hinton media with antibiotics (Vancomicine, Colistine, Nistatin)
4. Identification based on:
- morphologic properties
- cultural properties: S, grey colonies
- biochemical properties: catalase (+), oxidase (+), produces acid from glucose only.
-antigenic properties
Important:
All Neisseria are sensitive to drying and must be plated immediately in fresh, moist,
warm media. Incubation at 37ºC, 48 hours at CO 2 10-20%
Useful: Gonorrhea is the most commonly diagnosed infectious disease in the U.S and a
lot of other countries.
Questions and reviewing:
1.Which from following bacteria are Gram-negative diplococci :
a. Corynebacterium diphteriae
b. Clostridium tetani
c. Streptococcus pneumoniae
d. Neisseria meningitidis
e. Neisseria gonorrhoeae
18

Which from following bacteria
1. Neisseria; 2. Haemophilus; 3. Brucella; 4. Treponema.
Have the following morphological properties:
a) are cocci in clusters
b) are Gram negative diplococci
c) form long chains of cocci
d) are spirochetes.
e) are capsulated diplococci
f) are coccobacilli.
GENUS CLOSTRIDIUM
Laboratory diagnosis in infections with Clostridium spp.
Summary
Laboratory diagnosis in botulism
Laboratory diagnosis in tetanus
Laboratory diagnosis in gas gangrene
Practical activities:
- Analyzing smears from wounds (tetanus, gas-gangrene) Gram staining, observation and
interpretation
Essential
General characteristics:
- Gram positive spore forming rods, obligate anaerobes
Representants
- Clostridium botulinum→ botulism.
- Clostridium tetani
- Clostridium perfringens, C.histoliticum, C.aedematiens, etc. - the germs of the gas
gangrene
- Clostridium difficile
1. Clostridium botulinum
Laboratory diagnosis
Essential
A. Direct diagnosis
1. Sample collection
2. Microscopic examination and staining properties: non capsulated Gram (+) rods,
containing subterminal spores, with a diameter greater than the diameter of the bacilli,
motile.
3. Inoculation on media (media for anaerobes)
4. Identification based on:
- morphological features
- cultural properties
- antigenic properties: 8 antigenic types of toxin - demonstrated on mice.
19

Important: Media for anaerobes (Veillon, thioglicolic broth, etc.), nutrient or blood agar
in anaerobe technique (Ott).
Optional: Biochemical properties
2. Clostridium tetani
Essential: Diagnosis is primarily by clinical presentation because culture and
identification take a few days.
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection
2. Microscopical examination: long Gram positive bacilli, terminal spherical and bulging
spores (”drum stick”).
Important
3. Inoculation on media (media for anaerobes)
4. Identification based on:
- morphological properties
- cultural properties: on liquid media - smell of burned ivory.
- pathogenic properties: inoculated in mouse at the base of the tail →convulsion and
paralysis.
Useful
In vivo neutralization test: guinea pigs protected with 500 IU of antitetanic serum do not
develop the disease.
Optional
Cultural properties: on solid media and semisolid media.
Biochemical properties
3. Clostridium perfringens and gas gangrene
Essential
Species:
- Anaerobes: Clostridium septicum, Clostridium histolyticum Clostridium sporogenes
Clostridium aedematiens
Important
Laboratory diagnosis
1. Sample collection
2. Microscopical examination - compulsory: short, thick, capsulated, Gram positive rods,
(Moller staining for spores).
3. Inoculation on specific media for anaerobes
Useful
4. Identification - difficult
- morphological properties
- cultural properties
- biochemical properties (H 2 S, fermentation of sugar with gas production)
- antigenic properties: 5 types of exotoxins (A – E) - slide agglutination.
Symptoms of gas gangrene
Optional: pathogenic properties: inoculation in mouse, guinea pig and rabbit.
20

4. Clostridium difficile
Laboratory diagnosis
Important: detection of toxins in faeces by EIA.
Useful: Culture, isolation and identification
Optional: identification of the secreted exotoxins
Questions and reviewing:
Which of the microorganisms listed is usually acquired through contaminated food or
water ?
a. Salmonella typhi
b. Shigella dysenteriae
c.Vibrio cholerae
d. Clostridium tetani
e. Clostridium perfringens
f. Clostridium botulinum
Mention the methods for diagnosis.
Which one of the microorganisms are Gram (+) and which are Gram (+) bacilli ?
GENUS BACILLUS
Laboratory diagnosis in infections with Bacillus spp.
Practical activities:
- Analyzing smears in Gram staining with Bacillus spp., observation and
interpretation
Essential
General properties
- aerobic, spore-forming bacterium
- Gram positive rods
- oval, central spores.
Representants
- Bacillus anthracis
- Bacillus cereus
- Bacillus subtilis
Bacillus anthracis
- the most significant human pathogen of this genus, the cause of anthrax.
Anthrax = an antropozoonosis (mainly occurs in animals: goat, sheep herds, and only
accidentally can infect humans).
Pathogenesis
- the exotoxin: 3 proteins associated in a complex → cell death and edema
- the capsule: a polypeptide of D-glutamic acid
Diseases
1. Cutaneous anthrax (most usual)
2. Pulmonary anthrax (wool sorter’s disease)
21

3. Gastrointestinal anthrax
Laboratory diagnosis
A. Direct or bacteriological diagnosis
1. Sample collection (depending on the location)
- serosity from the papula
- sputum, feces, CSF
2. Microscopic examination of a Gram stained smear: large capsulated Gram-positive
bacilli displayed in diplo or isolated (like bamboo sticks);
3. Inoculation on media: simple agar or blood agar.
4. Identification is based on:
- morphologic and staining properties: large Gram-positive rods in short-to-long chains;
spores are ellipsoidal, central and do not swell the sporangium. B. anthracis is nonmotile.
- culture properties: R (rough), nonhemolytic colonies
- biochemical properties: degrades carbohydrates with no gas formation, liquify gelatine.
Treatment
Penicillin
Important
Antigenic properties: capsular antigen and a polysacharidic antigen in the cell wall → the
ring precipitation reaction (Ascoli reaction) for a retrospective diagnosis in dead animals.
Pathogenesis in laboratory animals: intraperitoneal inoculation in white mouse → lethal
septicemia. B.anthracis may be seen in the spleen smear.
Pasteur’s living spore vaccine from a noncapsulated strain is used for the vaccination of
herds.
Nonliving vaccines are used for the protection of humans at risk of occupational
exposure.
Useful
Bacteremia and secondary infections (meningitis), are complications of all three forms.
Optional
Biochemical properties of Bacillus anthracis:
B. anthracis - penicilline susceptibility: on a penicillin containing medium the rods will
become round = spheroplasts and will resemble a string of pearls.
Bacillus cereus and Bacillus subtilis
Important
General properties
- widely distributed in environment
- they cause diseases mainly in immunocompromised patients.
- aerobic spore-forming bacterium
Food poisoning caused by B. cereus – when foods are prepared and held without proper
refrigeration for several hours before being served.
B.cereus is resistant to penicillin so the drug of choice is clindamycin (aminoglycosides,
tetracycline, and erythromycin are also effective).
B.subtilis - infect immunocompromised patients (intravenous drug users) →septicemia.
Penicillin is the drug of choice.
Useful
Habitat
22

B. cereus
- in soil, on vegetables, and in many raw and processed foods.
- a common food contaminant: cooked meat and vegetables, boiled or fried rice, vanilla
sauce, custards, soups, and raw vegetable sprouts.
Prophylaxis
- depend on destruction by a heat process and temperature control to prevent spore
germination and multiplication of vegetative cells in cooked, ready-to-eat foods.
Questions and reviewing:
Which of the following are Gram positive capsulated diplobacilli:
a. E.coli
b. Neisseria meningitidis
c. Corynebacterium diphteriae
d. Bacillus anthracis
e. Tuberculosis bacilli
In the fall of 2001, a series of letters containing spores of Bacillus anthracis were mailed
to members of media and to US Senate offices. The result was 22 cases of anthrax with 5
deaths. The heat resistance of bacterial spores, such as those of B.anthracis is due to
a. the presence of large amounts of diaminopimelic acid in their structure
b. their dehydrate state
c. large amounts of calcium dipicolinate
d. large amounts of D-glutamic acid
e. the presence of lipid A in their structure.
DIGESTIVE TRACT INFECTIONS
ENTEROBACTERIACEAE FAMILY
Summary
General properties
Classification
Laboratory diagnosis in infections with enterics
Practical activities:
- Gram staining, observation & interpretation
- Inoculation and observation of E.coli, Samonella, Shigella, Klebsiella, Proteus,
Pseudomonas colonies on culture media
- Agglutination reaction on the slide
- Susceptibility testing to antibiotics
Essential
General properties of Enterobacteriaceae family
- Genuses and species
- Habitat
- staining properties
23

- cultural properties
- biochemical properties
Classification:
- opportunistic pathogens (E.coli, Klebsiella, Proteus, Enterobacter, Serratia,
Yersinia enterocolitica)
- true pathogens: Salmonella, Shigella, Yersinia pestis and some pathogenic strains
of E.coli ( EAEC- enteroadherent E.coli, ETEC-enterotoxigenic E.coli, EHEC –
enterohemorrhagic E.coli)
Diseases produced
GENUS SALMONELLA
General properties
Classification
S.typi, S.paratyphi A and B, S.enteritidis
Diseases
Major salmonelosis: typhoid and paratyphoid fever (S.typi, S.paratyphi)
Minor salmonelosis: gastroenteritis, food poisoning (S.typhimurium, S.anatum,
S.enteritidis)
Laboratory diagnosis
A. Direct (bacteriological diagnosis)
Specimen collection
Microscopic examination: irrelevant
Inoculation on culture media: media for enterics, with lactose
Identification
- morphology and staining: in a Gram smear- red rods
- culture properties: round, smooth (S), lactose negative colonies
- biochemical characteristics
o production of H 2 S (black colonies)
- antigenic characteristics (agglutination reaction on the slide)
antigen O, antigen H, antigen Vi
Interpretations
B. Indirect diagnosis (serologic diagnosis)
Widal reaction: antibodies against S.typhi, S.paratyphi A and B.
Interpretation
Important
Habitat
Transmission
Biochemical properties of Salmonella
Useful: Symptoms of typhoid fever and food poisoning.
Optional: Technique for Widal reaction.
Essential
General properties
GENUS SHIGELLA
24

Classification: the most important species: S.dysenteriae Shiga, S.flexneri, S.boydii,
S.sonnei
Diseases: bacterial dysentery (shigellosis)
Laboratory diagnosis
A. Direct (bacteriological diagnosis)
Specimen collection
Microscopic examination: only orientative
Inoculation on culture media: media for enterics, with lactose
Identification
- morphology and staining: in a Gram smear- red rods
- culture properties: round, smooth (S), lactose negative colonies
- biochemical characteristic
- indole and metal red positive
- do not produce H 2 S
- antigenic characteristics (agglutination reaction on the slide)
Important
Biochemical properties of Shigella (lactose negative colonies on MacConkey's or
Drigalsky agar, do not produce gas by fermentation, do not grow on citrate or sodium
acetate).
Useful: Symptoms of dysentery
Optional: Sereny test
OPPORTUNISTIC ENTERICS
Educational objectives
Essential
General properties of E.coli, Klebsiella, Proteus, Pseudomonas, Vibrio, Helicobacter
- species
- Habitat
Diseases produced
Laboratory diagnosis
- staining properties
- cultural properties and biochemical properties
- antigenic properties
- antibiotic susceptibility
GENUS ESCHERICHIA
E.coli
General properties
- the most abundant facultative anaerobe in the colon and feces
- ferments lactose
Diseases
1. Community -acquired urinary tract infections
2. Nosocomial (hospital-acquired) urinary tract infections
25

3. Neonatal meningitis
4. Diarrhea caused by enterotoxigenic (ETEC) E.coli.
5. Infection with enteropathogenic E.coli (EPEC) results in a dysenteria-like syndrome.
Laboratory diagnostic in infections with opportunistic enterics
It is only a direct diagnosis.
1. Sample collection: according to the patient’s symptoms
2. Microscopic examination: Gram negative bacilli
- capsulated (in CSF, sputum, otic secretion) => may be Klebsiella
- Gram negative rods in urine => urinary infection
- no relevance in feces
3. Inoculation on media for enterics
4. Identification based on:
- morphological features: Gram negative rods = red; if polymorphs may be Proteus sp.; if
cocobacillar may be Yersinia sp.
- cultural features:
-Klebsiella colonies: mucous
-Proteus colonies: “cat eyes” on Istrate media (green with a black middle);
concentric waves on Drigalski media ( invasion);
- biochemical analysis
- antigenic properties (serotyping): agglutination on the slide.
Antimicrobial susceptibility test
Important: Important biochemical test for the final identification of the species:
- Indole, metil red, H 2 S negative, and Voges-Proskauer, citrate positive for Klebsiella
- Only Proteus sp. produces phenylalanin-dezaminaze.
Biochemical characteristics of E.coli
- produces indole from tryptophan (indole +)
- it decarboxylates lysine (+)
- it utilizes acetate as its only source of carbon
- it is motile
Useful: Specific serotypes are indicated by the initial designation of the major antigen,
and then (for E.coli) by a number. Ex. O111:K55:H3
Questions and reviewing
Which of the microorganisms listed is usually acquired through contaminated food or
water?
a. Salmonella typhi
b. Shigella dysenteriae
c. Streptococcus pneumoniae
d. Clostridium tetani
Mention the disease they are producing (in the right side of each of the listed agents)
What kind of samples is collected and which are their characteristics?
Which of the following agents may be responsible for bacterial dysenteria:
a. Streptococcus pyogenes
b. Shigella spp.
c. Corynebacterium diphteriae
26

d. Mycobacterium tuberculosis
e. Salmonella spp.
LABORATORY DIAGNOSIS IN INFECTIONS WITH OTHER GRAM
NEGATIVE BACILLI
GENUS PSEUDOMONAS
Summary
General properties
Species
Infections
Laboratory diagnosis
Practical activities:
- Gram staining, observation & interpretation
- Inoculation and observation of Pseudomonas colonies on culture media
- Susceptibility testing to antibiotics
Educational objectives
Essential
P.aeruginosa - the most important human pathogen of the genus;
General properties
- Gram negative rod, capsulated, aerobic
- oxidase (+)
- pigment producers - pyoverdin => green coloration of the agar plates
Diseases
- opportunistic and nosocomial infections (extensive burn, trauma to the skin, urinary
tract manipulations, cystic fibrosis):
1. Purulent infections of burn or surgical lesions
2. Urinary tract infections
3. Respiratory tract infection
4. Enteritis
5. Meningitis
6. Septicemia
Laboratory diagnosis: requires culture and isolation
1. Sample collection: pus, urine, CSF, sputum, feces, light antiseptic solutions, soap.
2. Microscopic examination: mobile, Gram negative bacilli
3. Isolation on media with lactose => Lac (-) colonies
4. Identification based on:
- morphologic properties
- cultural propertie: pigment producers, S colonies with a sweet smell of grapes (or lime)
- biochemical properties: oxidase (+), Lac (-)
- antigenic properties: agglutination with serum containing antibodies against O and H
Antibiotic susceptibility must be determined in all cases (Pseudomonas spp. are
naturally resistant to a variety of antibiotics and in addition acquired resistance is very
common).
27

Important
Determinants of pathogenicity
- enterotoxin (LPS)
- exotoxins and others
Tested AB: Ticarcillin, Piperacillin, Mezlocillin, Aztreonam, Imipenem, III and IV
generation of cephalosporins (Ceftazidime, Cefoperazone), aminoglicosides (gentamicin,
tobramycin, amikacin), quinolones (Ciprofloxacin).
Useful: Transmission: food, dirty hands, respiratory equipment, intravenous fluids (for
nosocomial infections).
P.aeruginosa
- it behaves opportunistically in debilitated and immune compromised patients.
- natural habitat: soil and water
Optional: Cultural properties:
- pigment producers - pyocyanin
- fluorescein (under ultraviolet light)
- on blood-agar => total hemolysis
Pathogenic characteristics (animals):
- for the enterotoxin the ligaturated intestine test
- for the exotoxin the dermonecrosis test.
Questions and reviewing:
A very resistant to antibiotics, Gram negative, blue-green pigment producer bacillus was
isolated from the lesions of a burned patient. It is most probably:
a. Klebsiella pneumoniae
b. Staphylococus aureus
c. Beta-hemolytic streptococci
d. Pseudomonas aeruginosa
e. Shigella flexneri
GENUS VIBRIO
Summary
General properties
Species
Infections
Laboratory diagnosis
Practical activities:
- Gram staining, observation and interpretation
Essential
General properties
- Gram negative, comma-shaped rod
- oxidase positive
- motile with single polar flagellum
Representants
28

- Vibrio cholerae
- Vibrio parahemoliticus
- is a halophilic marine organism
- is acquired by ingestion of raw or improperly cooked seafood
Diseases
Vibrio cholerae - the major pathogen is the cause of cholera
Vibrio parahemoliticus causes food poisoning characterized by diarrhea.
Laboratory diagnostic in cholera
A. Direct diagnosis
1. Sample collection
2. Microscopic examination: comma-shaped, mobile, Gram negative rods
3. Inoculation on media: selective media (alkaline pH).
4. Identification is based on:
- morphologic properties
- cultural properties: smooth, colorless colonies
- biochemical properties:
- oxidase positive (differentiation of enterics)
- antigenic properties: agglutination with O1 => V.cholerae
B. Indirect diagnosis
Serologic: a rise in antibody titer in acute and convalescent-phase sera.
Important
Cholera: watery diarrhea in large volumes, "rice-water" stool, dehydration (adequate
replacement of water and electrolytes), loss of electrolytes leads to cardiac and renal
failure, acidosis and hypokalaemia.
Useful:
Transmission, reservoir, epidemic.
Antibiotic susceptibility test: Tetracycline, Chloramfenicol, Sulfamides
Optional
- antigenic properties: agglutination => classified into 3 serotypes (AB, AC, ABC);
- biochemical properties => biotypes: classic or El Tor
-non-agglutinant => NAG
-atypical, non-toxigenic
- vibriono-lysis with complement: agglutination, granulation, lysis
-pathogenic characteristics: enterotoxin production => is demonstrated by ligaturated
intestine test.
El Tor characteristics:
- produces hemolysis on blood agar
- acetoinic fermentation of glucose: Voges-Proskauer test = (+)
- is resistant to Polimixin B
Questions and reviewing:
Cholera toxin has the following characteristics:
a. is produced by V.cholerae
b. is an exotoxin which enhances adenilat cyclase activity ,
29

c. results in loss of water (watery diarrhea)
d. is more likely to occur in persons with very high gastric acidity
e. is frequent neutralized by alkaline pH of the bile
HELICOBACTER PYLORI
Summary
General properties
Infections
Laboratory diagnosis
Educational objectives
Essential
- infection is associated (not determined) with chronic gastritis, gastric peptic ulcers, and
gastric carcinoma
General properties:
- Gram negative, spiral shaped (helical) rod
- urease producing
Gold standard for diagnosis: gastric endoscopies and biopsy.
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: biopsy specimen of the gastric mucosa
2. Microscopic examination: Gram negative helical rods.
3. Biochemical properties:
- rapid test: detects H.pylori based on its production of urease on the biopsied tissue
- noninvasive test of urease activity: principle of breath test and urine test
B. Indirect (serologic) diagnosis
ELISA – antibodies (IgG) anti H.pylori
Interpretation
Important: Pathogenesis
Useful
Transmission: person-to-person
Symptoms
Antibiotics: claritromycin, doxycycline, and metronidazole
Optional
Helicobacter pylori
General properties:
- capnophilic (grow best in 10% CO 2 )
- microaerophilic
Questions and reviewing
Watery diarrhea can be produced by:
a) E. coli enterotoxigen
b) influenza virus
c) Helicobacter pylori
d) Streptococcus pneumoniae
e) Vibrio cholerae.
30

GENUS BRUCELLA
Laboratory diagnosis of infections with Brucella spp.
Summary
Species
Infections
Laboratory diagnosis
Educational objectives
Essential
General properties
- small Gram negative cocobacilli,
=> brucelosis - antropozoonosis
Disease
- brucellosis ="undulant fever" ("wave-like" nature of the fever which rises and
falls over weeks in untreated patients) = "Mediterranean fever" and "Malta fever"
- cattle affected with Brucella abortus have high incidences of abortions
Species
Brucella melitensis – goats and sheep
Brucella suis - pigs
Brucella abortus - cattle
Brucella ovis –sheep
Brucella canis – dogs
Transmission
- contaminated or untreated milk (and its derivates)
- direct contact with infected animals (also includes contact with their carcasses)
- have the unique property of being able to penetrate through intact human skin
- brucellosis - usually associated with the consumption of unpasteurized milk and soft
cheeses made from the milk of infected animals and with occupational exposure of
veterinarians and slaughterhouse workers.
Important:
A. Direct diagnosis
1. Sample collection: blood (when the fever is high), CSF, urine, bone marrow aspirate,
liver biopsy
3. Isolation on culture media:
- on media with veal liver (Castañeda method)
- grow slow (48h) with transparent colonies, which may become brown
- BACTEC System
4. Identification based on:
- cultural properties
- biochemical characteristics: growth may be impaired by different dies: fuxin, malachit
green, tionin => species may be differentiated
- antigenic properties: agglutination reaction on slide or EIA (immunoenzymatic assay)
- PCR test – identification of species
B. Indirect diagnosis
a) Serologic diagnosis
- Wright (quantitative agglutination) => titter greater than 1/160
31

- EIA - detects specific IgM antibodies
Useful
Antibiotics: Tetracyclins, aminoglycosides, rifampin.
Optional : Indirect diagnosis:
Intradermal reaction (IDR) to brucelin => (+) if inflammatory reaction is greater than 6-8
cm (it demonstrate a late hypersensitivity reaction = Cellular mediated immunity)
Questions and reviewing
A patient who works as a veterinarian presents at the doctor complaining about
symptoms related with fever (weakness, sweets, loss of apatite), however his fever comes
and goes at different intervals. By blood culture a Gram negative coccobacillus is
isolated. The most probable bacterial agent to be the cause of these symptoms is:
a) E.coli
b) Bordetella pertusis
c) Helicobacter pylori
d) Brucella spp.
e) None of them
SEXUAL TRANSMITTED DISEASES
Laboratory diagnosis of infections with Treponema pallidum
Summary
Species
Infections
Laboratory diagnosis
Practical activities:
- observation and interpretation of silver impregnated smears
- performing antigen-antibodies reactions and interpretation (TPHA)
Educational objectives
Essential
3 genera => 2 families.
1. Family Spirochaetaceae:
Genus Treponema
Genus Borrelia
2. Family Leptospiraceae:
Genus Leptospira
GENUS TREPONEMA
General properties
- thin walled, flexible, spiral rods
- motile
- only by dark field microscopy, silver impregnation, immunofluorescence
- do not grow on culture media, grown only in tissue cultures
Species:
- Treponema pallidum – the most impotant
32

- Treponema carateum
- Treponema pertenue
Treponema pallidum - syphilis
Transmission:
- direct sexual contact
- transplacentally
- blood
- accidentally: clinical laboratory workers, hospital personnel, dentists, gynecologists,
blood transfusion recipients
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection
2. Microscopic examination
- dark field examination: live, highly motile organisms, bright white, motile spiral rods.
- immunofluorescence, Giemsa staining
- Fontana-Tribondeau = silver impregnation: black-brown against a yellow background
3. Isolation:
- only in animals- rabbit
- do not grow in bacteriologic media
B. Indirect diagnosis (serologic test):
- nonspecific (nontreponemal) - screening
- specific (treponemal) – confirmatory
Nonspecific test
- use cardiolipin as antigen
- phospholipid-rich fraction from bovine heart
- antiphospholipid antibodies produced by T. pallidum cross-react with cardiolipin
similar in antigenic structure
- false positive results - usually of a low titer because of viral diseases, autoimmune or
collagen disorders, pregnancy, elderly patients.
Techniques :
VDRL test (Venereal Disease Research Laboratory)
Specific test
T. pallidum = antigen
- detect specific antitreponemal antibody.
1. T. pallidum immobilization (TPI) test
2. Fluorescent treponemal antibody absorbtion test (FTA-ABS)
3. Microhemagglutination T. pallidum (MHA-TP); TPHA (Treponema pallidum
hemagglutination)
- positive for life after effective therapy
Treatment
Penicillin
Important
Adult syphilis: - 3 stages (primary syphilis, secondary or systemic, tertiary syphilis)
Congenital syphilis
Useful
33

Nonspecific test
Techniques:
Rapid plasma reagin (RPR) test
- flocculation tests
- detect both IgG and IgM antibodies.
- positive very early in the disease.
Complement fixation - Bordett-Wassermann test - IgG
- non-specific antibodies decrease with effective treatment
Optional
Nonvenereal Treponematoses
- transmitted by direct contact
Diseases:
- bejel in Africa,
- yaws in many humid tropical countries (caused by T.pertenue)
- pinta (caused by T.carateum ) in Central and South America.
Treatment: penicillin.
Questions and reviewing
Treponema pallidum
a. is the etiological agent of ........................................ disease transmitted by .......... way.
b. the disease has 3 stages...............................................................................….................
c. the diagnosis in the first stage is based on microscopy. Describe the morphology of
Treponema pallidum ………………………
and the methods used for this purpose. ……………………………..
d. For the serological diagnosis of syphilis serology tests are used; please mention only
the nontreponemical (non-specific) tests used for diagnosis :
GENUS LEPTOSPIRA
Laboratory diagnosis of infections with Leptospira spp.
Educational objectives
Essential
General properties
- highly coiled and motile
- hooked end
- grown in enriched artificial media
Representants
I. Leptospira interogans
II. Leptospira biflexa
I. Leptospira interogans
- several serotypes pathogenic for humans
- causes icteric leptospirosis (Weil’s syndrome)
Laboratory diagnosis
A. Direct diagnosis
1. Sample collection: first week - blood, CSF ; after : urine
34

2. Microscopic examination: immunofluorescence
3. Isolation on cultural media:
- media with rabbit serum
- 6 weeks (28-32˚C)
4. Identification based on:
- morphologic properties: dark field microscopy; staining: Burri, Giemsa, silver
impregnation
- culture: examined weekly
- antigenic properties: agglutination-lyses reaction with specific serum
- PCR
B. Indirect diagnosis (serologic test)
- complement fixation,
- agglutination
- Antibody titers: takes approximately 4 weeks to peak (will not give a quick diagnosis).
Treatment
Penicillin G
Important
Transmission
- contact with infected animals (rats, dogs, cats and livestock)
- contact - products contaminated with animal urine (soil, food, and water).
Useful
Prophylaxis:Avoid contact with contaminated environment.
Doxycycline – prevent disease in exposed persons.
Optional
- pathogenic properties: inoculation in guinea pig => spirochetemia, renal location and
elimination through urine.
Questions and reviewing
A worker from a unity providing animals for different laboratories was bitten by a rat
during cage cleaning. After several days he present at the doctor with a yellowish color of
the tegument and conjunctiva, renal and hepatic symptoms. The most probable causative
agent of his disease is:
a) Brucella abortus
b) Treponema pallidum
c) Trichinella spiralis
d) Leptospira spp.
e) Salmonella tiphymurium.
Summary
Species
Diseases
Laboratory diagnosis
RICKETTSIACEAE FAMILY
Laboratory diagnosis of infections with Rickettsia
35

Practical activities:
- performing antigen-antibodies reactions and interpretation
Educational objectives
Essential
3 genera: Rickettsia, Coxiella and Ehrlichia
General characteristics
- small Gram negative cocobacilli
- obligate intracellular parasites (not grow on artificial media)
Diseases
- insect bite or contamination of abraded skin with fecal material from an infected insect
(ticks, fleas, lice, and other vectors)
In U.S.A: Rocky Mountain spotted fever (R.rickettisae), epidemic typhus (R.prowazekii)
In Romania: epidemic typhus (R.prowazekii), relapse typhus - several years later (Brill’s
disease), murine typhus, Q fever (Coxiella burnetti)
Laboratory diagnosis
A. Direct diagnosis - low utility (dangerous and difficult).
1. Sample collection: blood
2. Microscopic examination:
- direct immunofluorescence: identification of R.rickettsii in skin lesion biopsy samples
3. Isolation on embrionated egg, tissue culture and guinea pig
4. Identification based on:
- morphological characteristics: Giemsa stained smears
- antigenic properties:
- Immunofluorescence in infected tissue
- Complement fixation with standard serum
B. Indirect diagnosis
Serologic (specific and unspecific reactions)
Unspecific serologic reactions (Proteus OX19 or OX2 are used as an Ag)
1. Agglutination on slides (for screening)
2. Weil-Felix test
Specific serologic reaction
Ag - infected embrionated egg
1. Complement fixation: rise in the antibody titter
2. Indirect fluorescence (discrimination IgG/IgM)
3. Passive hemaglutination
4. ELISA
Treatment
Doxycycline, chloramfenicol
Important: Rocky Mountain spotted fever, epidemic typhus, Q fever
Useful:
• vaccine: formalin-killed R. prowazekii organisms - military during wartime.
•Persons at risk to contact C. burnetti (veterinarians, shepherds, abattoir workers and
laboratory personnel) - vaccine (killed organism).
Optional
Technique of Weil-Felix test
Biologic reaction (IDR for epidemic typhus).
36

Questions and reviewing:
1. The following diseases are transmitted via vectors:
a. Q fever
b. Rocky Mountains spotted fever
c. malaria
d. meningococcal meningitis
e. flu.
Bibliography
1. Monica Junie, Luciana Stănilă, Carmen Costache: “Medical Microbiology”,
“Iuliu Haţieganu” University Publishing House, Cluj-Napoca 2003.
2. Jawetz, Melnick & Adelberg’s, 22 - nd : Brooks G. F., Butel J.S. and Ornston L.N.:
“Medical Microbiology”.
Laboratory diagnosis of infections with parasites
Practical activities
- observation of adult worms preserved in jars
- microscopy for observation/drawing of eggs, adult worms
Laboratory diagnosis in parasitology
- morphologic criteria
- proper specimen collection
Feces
- gastrointestinal parasites
- alternate days
- total of three specimens within 10 days
- 1 every 5 days for 15 days)
- Macroscopic
- adult worms - e.g. Ascaris, Enterobius
- Tape worms proglottis
- blood, mucus
- Microscopic = coproparasitologic (CPZ) = (O& P = ova and parasites)
- O& P
- wet mount,
- stool concentrate
- permanently stained smear
- Detect:
- trophozoites / cyst of protozoan
- eggs and larvae for helminthes
Blood samples
- capillary: finger, ear lobe
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- malaria, trypanosomiasis, etc
Culture: rare, on specialized media
Serology
- toxoplasmosis, amoebiasis,
- trichinellosis, echinococcosis (hydatid disease), etc
Questions and reviewing
The definition of definitive host (in parasitology) includes:
a. organism in which the parasite live its whole life
b. organism in which the parasite develops its adult life
c. organism in witch the parasite undergo its sexual multiplication phase
d. organism in which the parasite is present as a larvae
e. organism in which the parasite undergo its asexual multiplication phase
Laboratory diagnosis of infections with protozoa
Summary
Digestive tract infections
Entamoeba hystolitica: Laboratory diagnosis
Giardia: Laboratory diagnosis
Cryptosporidium spp: Laboratory diagnosis
Microsporidium: Laboratory diagnosis
Congenital infections
Toxoplasma gondii: Laboratory diagnosis
Blood infections
Plasmodium: Laboratory diagnosis.
Sexual transmitted diseases
Trichomonas vaginalis: Laboratory diagnosis
DIGESTIVE TRACT INFECTIONS
ENTAMOEBA HYSTOLITICA/DISPAR
Essential
- amebiasis (amebic dysentery)
Laboratory diagnosis
Direct diagnosis
- complete examination for cysts includes a wet mount in saline, an iodine-stained wet
mount and a fixed, trichrome-stained preparation
- finding either trophozoites in diarrhea stools or cysts in formatted stool
- cysts are passed intermittently, at least 3 specimens should be examined
Important: Morphology: Trophozoite: the motile amoebae-pseudopode
Cyst: usually spherical, but may be ovoid, 4 nuclei
Useful: Serologic testing - diagnosis of invasive amebiasis
38

GIARDIA
Practical activities:
- wet smear from feces in saline, lugol observation and interpretation
- stained smear from culture to observ vegetative form
Educational objectives
Essential
Laboratory diagnosis
- on finding the distinctive cysts in formed stool
- cysts and trophozoites in liquid stool
For a certified diagnosis: 3 samples in alternate days (3 in 10 days or 5 in 15 days)
Important - Morphology of Giardia trophozoit and cyst.
1. Trophozoites
- roughly pear shaped
- length: 10 to 15 μm, and width: 5 to 7 μm.
- anterior portion of the ventral surface form a sucking disk,
- attachment - damage mucosa
- two nuclei: round or ovoid
- central nucleolus (kariosome)
- four pairs of flagella:
• 3 dorsal: anterior (X), lateral, and recurrent.
• 1 ventral
- median body
- axostyl
Cysts
- ovoid
- 8 -12 μm by 6-10 μm in width.
- four prominent nuclei
- flagella structures dispersed in a seemingly helter-skelter fashion.
- cyst wall: smooth and bright
- median bodies
CRYPTOSPORIDIUM
Essential
Laboratory Diagnosis
- schizonts containing merozoites and micro and macrogamets in intestinal biopsy
material and by
- detecting oocysts in stool specimens (in fresh stool)
Identifying organisms: stool exam requires special stains: oocysts may by stained using
the modified acid fast stain (Kinyoun)
1. Oocysts = infective stage
- round or spherical ( 5μ),
- contain 4 sporozoites,
2. Sporozoites
- released - enter epithelial cells (microvili )- undergo two asexual generations:
- become trophozoites.
3. Trophozoites
39

4. Merozoites - liberate from the parent cell to begin a new cycle.
Useful
Trophozoites
- minute (2-5μm) intracellular spheres
- great numbers under the mucosal epithelium of intestine.
- Mature trophozoite (schizont)
- divides into 8 arc-shaped merozoites
MICROSPORIDIUM
Essential
Laboratory diagnosis
- Light microscopy – special staining (trichrome)
- oval spores
- Immunefluorescence Assay
- PCR
Questions and reviewing
1. Giardiosis may be eliminated as a diagnosis:
a. after one O&P examination that results negative.
b. in adults that have not giardiasis records.
c. after 3 O&P negative examinations, realized at a distance of 3-5 or even 7 days.
d. after 3 O&P examinations realized in succession of 3 days.
e. only after performing a duodenal test.
2. Diagnosis in criptosporidiasis mainly relies on:
a. clinical aspect
b. characteristics of the diarhea
c. O&P examination of the feces
d. special staining of a smear from feces (modified acid-fast staining- Kinyoun)
e. serologic examination
CONGENITAL INFECTIONS
TOXOPLASMA GONDII
Practical activities
- interpretation of different serologic profiles (ELISA)
- interpretation of Western Blott strips
Educational objectives
Essential
Laboratory Diagnosis
- Serologic assays for toxoplasmosis
– the most common parasitic serology tests
– the method of choice for diagnosing the infection.
- detection of Ig M against toxoplasma the pregnant women use to be diagnosed as
40

having an acute toxoplasmosis => NOW Ig G high avidity or presence of specific Ig A
are criteria for acute toxoplasmosis !
- PCR
- fetal blood analysis
- amniotic liquid analysis
Diagnosis of congenital toxoplasmosis
- serology
- Ig G antibodies may pass through placenta from mother !
- Ig M, IgA antibodies of the new-born present in case of congenital toxoplasmosis !!
Questions and reviewing
Acute Toxoplasma gondi infection acquired in the first semester of the pregnancy;
a. diagnosis of mother is made by the detection of specific Ig G antibodies in blood
b. diagnosis is made by the detection of T.gondii oocysts in mother’s feces.
c. The IgM test is more reliable than the IgG test for determination of past infections;
d. The test for Toxoplasma antibodies is highly nonspecific
e. induce the formation of Toxoplasma antibody
BLOOD INFECTIONS
PLASMODIUM
Practical activities: observation and interpretation of Giemsa stained thin and thick
smears
Educational objectives
Essential: Diagnosis
1. confirmed by demonstration of the parasites in thin and thick blood smears:
- Giemsa-stained blood smears
- each species has distinct morphologic characteristics during erytrocytic cycle
2. Quantitative Buffy Coat Assay (QBC®) modification of fluorescent microscopy.
3. Malaria antigen detection in blood
a. histidine-rich protein-2 (HRP-2)
b. parasite lactate dehydrogenase (pLDH)
c. immunochromatographic rapid diagnostic test (RDT).
4. PCR : identify parasite genetic material.
a. Benefits: high sensitivity. 0.05-0.1 parasite/µL, allows species differentiation.
identify mutation ~correlated to drug resistance acquired by the parasite.
b. Limits: large research labs, cost, contamination (false positive), ~ trained
personnel.
Useful
Plasmodium falciparum: malignant tertian malaria
- invades all red cells regardless of age: reticulocytes, young, old red cells which are
not enlarged and pale
- coarse stippling (Maurer’s clefts) on the surface
41

- one or multiple but small, delicate ring stage of trophozoites in peripheral blood
- only rings and gametocytes in peripheral blood; other forms in visceral blood
- pigment in developing trophozoites is coarse, black, few clumps
- older trophozoites are compact or rounded
- mature schizont: usually 8-32 merozoites
- gametocytes: banana - shaped
Plasmodium vivax: benign tertian malaria
- primarily invades reticulocytes, young red cells which are enlarged and pale
- fine brick-red stippling (Scüffner’s dots) on the surface
- large but delicate ring stage of trophozoites
- pigment in developing trophozoites is fine, long brown, scattered
- older trophozoites are very pleomorphic
- Mature schizont: more than 12 merozoites still 32 merozoites.
- gametocytes: round or oval
- Distribution in peripheral blood: all forms
Plasmodium ovale: benign tertian malaria
- primarily invades reticulocytes, young red cells which are enlarged and oval
- fine brick-red stippling (Scüffner’s dots) on the surface
- large but thick ring stage of trophozoites
- pigment in developing trophozoites is coarse, dark- yellow - brown, scattered
- older trophozoites are rounded and compact
- mature schizonts: more than 12 merozoites
- gametocytes: round or oval
Plasmodium malariae: quatrain malaria
- primarily invades older red cells which are not enlarged
- have not stippling on the surface
- large thick ring stage of trophozoites
- pigment in developing trophozoites is coarse, dark brown, abundant, scattered in
clumps
- older trophozoites are band form
- mature schizont: 8 merozoites (often in rosette as a “daisy”)
- gametocytes: round or oval
- distribution in peripheral blood: all forms are present in peripheral blood
QBS principle and technique
- blood centrifuged in AO-coated tubes.
- Both parasites and granulocytes take the dye.
- parasites concentrate below the granulocyte level in tube.
- quick => useful for screening large number of samples.
- identification and quantification of species is difficult.
42

SEXUALLY TRANSMITTED DISEASES
TRICHOMONAS VAGINALIS
Educational objectives
Essential
Laboratory Diagnosis of trichomoniasis
1. Sample collection:
- vaginal, urethral secretions/discharge (vaginal posterior fold where the microorganism
is most likely to be recovered)
- In males: urethral discharge, prostatic secretions or centrifuged urine
2. Direct microscopy:
- direct wet mount => characteristic motile trichomonads.
Phase contrast microscopy is especially desirable for observing the flagella and
undulating membrane
- Smears: Gram, Giemsa or other stains (Papanicolau –Pap smear, acridine orange).
Examination of urethral discharge (female) may yield positive results when no organism
can be found in vaginal secretions
3. Rapid antigen testing.
Important
Morphology of trophozoit (Does NOT have a cyst form)
- 5-15 μm long - up to 30 μm.
- move very quickly (jerky and non-directional motion).
- four anterior flagella plus a recurrent flagellum (anterior flagella serve for propulsion)
- undulating membrane - rotating motion
- joins the body along a line marked by the presence of a curved thin rod, called costa.
- costa is about the same length as the undulating membrane and is an important
diagnostic characteristic
- single nucleus
- axostyl: a slender rod, extends through the body from the anterior to the posterior end.
sharply pointed and protrudes prominently beyond the posterior end of the body.
Useful
Overnight culture
- Diamond’s medium standard for culture, manufactured culture kit (the InPouch TV kit)
sensitivity range of 75-95%.
Optional
Transcription-mediated amplification
PCR, using the primers L23861 Fw and Rev
Questions and reviewing
Plasmodium falciparum:
a. Only ring forms of early trophozoites and the gametocytes are seen in the peripheral
blood
b. Schüffner stippling is routinely seen in red blood cells that harbor parasites
c. has a particular shape (banana or sausage-like) of the gametocyte
d. is responsible for quartian fever
e. all evolutionary forms are usually found in capillary blood
43

The diagnostic characteristics of Plasmodium vivax are best described by which one of
the following statements?
a. A period of 72 h is required for the development of the mature schizont, which
resembles a rosette with only 8 to 10 oval merozoites
b. Infected red blood cell has irregular appearance of the edges
c. The signet-ring–shaped trophozoite is irregular in shape with ameboid extensions of
the cytoplasm
d. all evolutionary forms are usually found in capillary blood
e. Schüffner stippling is routinely seen in red blood cells
Trichomonas vaginalis:
a. is a helminth
b. is a protozoan living inside erythrocytes
c. is a protozoan living inside duodenum
d. is a protozoan present at the level of vagina and cervix
e. exists only as a vegetative form (trophozoite).
Laboratory diagnosis of infections with nematodes
Summary
Representatives
Life cycle
Infections
Laboratory diagnosis
Treatment
Practical activities
- observation of adult worms preserved in jars
- microscopy for observation/drawing of eggs, adult worms
Educational objectives
Essential
General features
– worm-like,
– smooth,
– unsegmented,
– mm.- cm
– separate-sexed:
• Female: bigger than male,
• Male: small with curved tail,
Reproductive modality
- by eggs
- embryonated eggs = infectious at emission
- non-embryonated eggs (noninfectious, need soil for embryonation = geohelminths
- by larvae: e.g.Trichinella spiralis
44

ASCARIS LUMBRICOIDES
General properties
- resemblance to common earthworm (creamy-white or pinkish)
- Female: 20 to 35 cm
- Male: smaller, seldom over 30 cm, incurved tail
Life cycle: www.dpd.cdc.gov/dpdx
Transmission: eggs
- any kind of food contaminated with soil or
- drinking water
Ascaridosis
most infections are light and symptomless
transient pneumonitis Löffler’s syndrome
Hypersensitive patients: urticaria, asthma
Ectopic sites
Complications: heavy infections
Laboratory diagnosis
- detection of larvae in sputum
- adult worms passed or vomited
a) Fertilized egg features
- round/oval
- 60x45μm
- golden-brown
- bumpy (mammillated) surface = outer cover
- thick smooth shell = inner shell
- embryo in the middle
b) Infertile egg: features
- longer
- narrower than the fertile ones,
- both inner and outer coat may be thin
Important: Treatment:Albendazole, Levamisole
Useful: Distribution
Optional: Treatment details
- Bowel obstruction, Hepatobiliary ascariasis
may require invasive intervention (eg, ERCP).
TRICHURIS TRICHIURA (WHIPWORM), TRICHOCEPHALUS
Educational objectives
Essential
General properties
- roundworm
- lives in colon:
- the thinner, almost colorless, anterior part is embedded in the mucosa
- the thicker, pinkish-gray, posterior end is free in the lumen
45

Transmission: ingestion of eggs from contaminated vegetables or soil
Tricocephalosis
- > 2 weeks after incubation
- most infections: asymptomatic
- heavier infections: Diarrhea, anorexia, nausea, abdominal pain, mucosa hemorrhage,
anemia and rectal prolaps
Diagnosis: O&P/CPZ examination => characteristic eggs
- oval, 50 x 25μm (lemon shape)
- brown
- distinctive protruding polar plugs
Treatment: Albendazole, Mebendazole (only moderately effective)
Important
Soil is necessary for fully embryonation = geohelminth
moderate eosinophilia
Optional
3000-7000 eggs daily
ENTEROBIUS VERMICULARIS (pinworm)
Educational objectives
Essential
General properties
- small, yellowish-white
- female: 1 cm with sharply pointed tail, male: 3 mm
- direct life cycle with no tissue migration phase
Transmission :
- eggs deposited in sticky secretion on the perianal skin by wandering female worm =>
contaminate bedclothes and air during bed making.
- ingestion or inhalation of eggs
- fecal-oral route
- scratching
- transportation under nails.
Life cycle: www.dpd.cdc.gov/dpdx
oxiuriasis 1/3 asymptomatic
- irritation and pruritus ani = most common presentation
- ectopic sites migration - appendix and female genitourinary tract => ~ symptoms
Laboratory diagnosis:
1. cellophane tape test (scotch test, Graham test)
2. O&P eggs only rarely seen in stool, but adult female worms may be seen
Eggs features:
- oval, plan-convex, 50 x 25μm
- translucent shell
- an incurved embryo inside the egg called giriniform embryo.
Treatment: Albendazole, repeated after 10-14 days
Prevention of reinfection:
- treating the patient's entire family
46

- individual hygiene
- bed lines and towels should be washed with warm water.
Important: Pathogenesis: eggs hatch in the large intestine
- worms mature in 2-4 weeks and live for 2 months (but continuous reinfection is
common).
Particular type of autoinfection: retrofection = hatching of the embryonated eggs after
their deposition in the perianal area and subsequent migration back into the rectum and
large intestine.
Useful: Distribution: cosmopolitan, but most common in:
- temperate areas,
- children
- communities
- more than 1 billion cases worldwide/year
- very contagious (10.000 eggs daily
Treatment: single doses of mebendazole and Pyrantel pamoate are highly effective
Optional: Adults treatment: warm tap water enemas are sometimes enough
TRICHINELLA SPIRALIS
Essential
General properties
- small,
- white (pinkish)
- female 2,5-4 mm long
- male 1-2 mm
Life cycle: www.dpd.cdc.gov/dpdx
Transmission: through consumption of infected meat (usually pork or bear and seldom
by infected horse meat) raw or insufficiently cooked.
Laboratory findings: serologic tests for humans
Important: Pathogenesis: depend on the:
- number of worms present
- stage
a) Intestinal stage: worms are mature 5 days postinfection and live for 2-3 weeks before
being expelled by the host immune response
- intestinal pathology includes villous atrophy and inflammation
- symptoms: nausea, vomiting, diarrhea, abdominal pains
- heavy infections can be fatal
b) Migratory stage-involve newborn larvae and lasts 1-4 weeks
- pneumonitis, neurologic symptoms, conjunctivitis and circumorbital edema
- hemorrhages spots under the nails
- muscle tenderness
c) Muscle stage starts 3 weeks after infection and lasts up to several months (2-3)
- the larvae growth and encyst leading to inflammation =>
- muscle tenderness, spasm and edema
- hypersensitivity reactions can occur: eosinophilia and increased levels of IgE are
pronounced during migratory and muscle stages
47

- larvae only encyst in striated muscle (not the heart): tongue, diaphragm, intercostals,
orbitals (death may occur from respiratory and cardiac failure)
Laboratory findings
- Stroubli (Knott, Nuclepore) test during migratory stage
- Muscle biopsy samples for animals => cyst:
- Oval, containing 1-2 larvae (300-800μm)
- 2 albuminoid poles
- a three-layer cover
Treatment: Mebendazole (kills migrating larvae and encysting larvae)
Prevention: meat (esp. pork) should be veterinary analyzed and well cooked before
eating.
Useful: zoonosis
- pigs and rats worldwide
- sporadically acquired by humans in all regions where pork meat is consumed.
Optional: Digestion method for the diagnosis of trichinelosis in animals
ANCYLOSTOMA DUODENALIS (HOOKWORM)
Essential
General properties: creamy white to pinkish, 1 cm long, live mostly in jejunum
Life cycle: adult hookworm live in intestine and eggs are passed in the feces, hatch in soil
releasing larvae (molt twice), become infective filariform larvae survive 2-5 weeks in
warm, moist soil (esp. mines, tunnels) www.dpd.cdc.gov/dpdx
Transmission: larval penetration of skin, usually through the feet
Clinical disease: most infections are asymptomatic because the worm burden is low.
-“ground itch” is a pruritic inflammation at the site of penetration
-Loeffler's syndrome (less severe than in Ascaris infection).
-blood loss can lead to anemia in moderate to heavy infections
-abdominal pain (rare)
Laboratory diagnosis:
-anemia and blood in feces
-O&P : eggs- embryonated, 60 x 40 μm
- thin shelled with 6-8 blastomers inside
Important
Distribution: worldwide
Pathogenesis: larvae migrate in a patter similar to that of Ascaris
A very similar worm lives in the American continent: Necator americanus.
(small differences in morphology)
The term hookworms refer to both of them.
Useful: Treatment: Mebendazole and Pyrantel pamoate
Prevention: proper disposal of garbage to avoid contamination of soil. Patients should be
advised to wear shoes when walking on soil.
Essential
STRONGYLOIDES STERCORALIS
48

General properties: life cycle and distribution similar with hookworm,
May exist for some time as a free-living nematode.
Reproduction both of the free-living and the parasitic female occurs mostly by
parthenogenesis. www.dpd.cdc.gov/dpdx
Transmission: skin, autoinfection
Clinical disease:
a) In immunocompetent patients: 50% are asymptomatic
- ground itch
- pneumonitis
- epigastric pain, watery diarrhea, eosinophilia
b) In immunodeficiency patients, autoinfection leads to a disseminated form of
strongyloidiasis, which may be fatal if untreated.
Laboratory diagnosis: O&P – eggs in feces, similar to those of hookworm (rare)
- usually larvae hatched when passed in the feces
Treatment: Thiabendazole or mebendazole
Prevention: Proper disposal of sewage and wearing of shoes.
Important
Pathogenesis: adult worms burrow deep into the crypts of the intestine => mucosal
breakdown and increase amount of mucus
Difference between:
Rhabditiform – Filariform larvae
hookwarm larvae - Strongiloides larvae www.dpd.cdc.gov/dpdx
Useful: The Harada-Mori test: incubation of feces to allow larvae to hatch and to make
differentiation from hookworm based on identification of the tail of the filariform larvae.
Duodenal content may be examined for larvae as well.
Optional
Other diseases caused by Nematodes
Toxocarosis
- caused by Toxocara canis
- diagnosis: serology
Anisakiasis
- caused by Anisakis marina
Questions and reviewing
1. An outbreak of mild intestinal distress, sleeplessness, itching, and anxiety has broken
out among preschool children in a private home. The single most likely parasitic cause of
the outbreak is:
a. Trichomonas vaginalis
b. Enterobius vermicularis
c. Ancylostoma duodenale
d. Plasmodium vivax
e. Ascaris lumbricoides
2.Oxiurasis
a. represents a leading cause of parasitic diseases in children colectivities
b. is frequent in immune compromised persons.
49

c. is present especially in tropical countries.
d. represents the cause of chronic enteritis in AIDS patients.
e. produces an important pruritus ani during the night
3. In which of the following diseases, bloody-mucous-violent stools, may appear along
with low-grade fever, anemia, sometimes-rectal prolaps, appendicitis and moderate
hipereozinofilia:
a. oxiurosis
b. botriocephalosis
c. tricocephalosis
d. criptosporidiasis
e. none
4. Trichinelosis
a. is produced by Trichomonas species
b. is produced Trichuris species
c. is produced by Trichinella species
d. evolves with febrile enteritis in the first weak of infection
e. evolves with muscular pain at the end of disease course.
PLATYHELMINTHS/ FLATWORMS
Laboratory diagnosis of infections with platyhelmints
Summary
Flatworms
- representatives
- life cycle
- infections
- laboratory diagnosis
- treatment
Practical activities
- observation of adult worms preserved in jars
- microscopy for observation/drawing of eggs
Educational objectives
FLATWORMS
Essential
Include:
1. Trematodes or Flukes
General features
Leaf shaped bodies
Ventral and oral suckers used for attachment and sucking nutrient fluids
Some can absorb nutrients through their cuticle.
Reproductive modality: hermafroditic
50

TREMATODES
FASCIOLA HEPATICA
Life cycle: www.dpd.cdc.gov/dpdx
Transmission:
Fascioliasis (sheep liver fluke disease)
- Acute: fever, anemia, abdominal pain
- nausea, vomiting, pruritus
- Chronic infections: symptoms are related to biliary duct damage:
- thickening, fibrosis,
- cholecystitis, cholelitiasis,
- fibrotic changes => obstructive jaundice may result.
- In serious cases, liver atrophy and cirrhosis may occur.
Suggestive diagnosis
- fever,
- liver enlargement,
- eosinophilia
- elevated serum transaminase level.
Laboratory diagnosis = Definitive
- O&P feces: large (150 x 80 μm) operculated eggs
- Serology: complement fixation, immunofluorescence, indirect hemagglutination,
counterimmunoelectrophoresis, and enzyme-linked immunosorbent assay (ELISA).
Important
Morphology- adult worm
Pathogenesis: metacercariae hatch => larvae burrows through the gut wall, penetrate the
liver, and eat their way through the parenchyma to the bile ducts leading to necrotic
lesions.
Useful
Distribution
- adult or larval stage of worm adheres to the posterior pharyngeal wall, causing severe
pharyngitis and laryngeal edema
Treatment: Bithionol, triclabendazole, Praziquantel (recommended only if bithionol or
triclabendazole is unavailable0.)
Optional
F.hepatica contamination also:
- eating fresh liver of goat or sheep => halzoun in Lebanon and marrerra in the Sudan
- consuming sashimi of bovine liver served in "Yakitori" bars in Japan
The Falcon screening test-ELISA is the most reliable diagnostic study and is the test of
choice because of its routine availability, cost, sensitivity, and specificity.
serum ELISA test result may become positive months before stool examination for ova
because flukes do not produce eggs until the chronic stage (ie, 4 mo after infection
[range, 3-18 mo])
Flukes named for host tissues in which adult lives.
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- Blood Fluke (Schistosoma spp.)
- Asian Liver Fluke (Clonorchis sinensis)
- Lung Fluke (Paragonius westermani)
- Liver Fluke (Fasciola hepatica)
CESTODES (TAPEWORMS)
Essential
- Long flat bodies
- Intestinal parasites
- Lack a digestive system, absorb food through cuticle.
Body organization:
- Head or scolex has: suckers, hooks for attachment.
- Neck: non-differentiated proliferative tissue
- Body is made up of segments called proglottids.
- Each proglottid has both male and female reproductive organs.
- Proglottids from the end contain many fertilized eggs.
DIPHYLLOBOTHRIUM LATUM
Essential
Morphology of adult worm
- up to 10-15 m length -3,000 proglottids.
The mature and gravid proglottids
- broader than long,
- typical rosette-shaped uterus.
- measure up to 2 cm in width.
Eggs are oval and operculated, measure 65-70 by 45-50μm
Life cycle: www.dpd.cdc.gov/dpdx
Transmission: eating infected host fish raw or undercooked .
diphylobothriasis
- After ingestion of the infected fish, the plerocercoid develop into immature adults and
then into mature adult tapeworms which will reside in the small intestine.
- The adults of D. latum attach to the intestinal mucosa by means of the two bilateral
groves (bothria) of their scolex .
- Immature eggs are discharged from the proglottids
- Eggs appear in the feces 5 to 6 weeks after infection.
Most infections are asymptomatic
- occasionally:
- diarrhea and weight loss may occur.
- 10% of patients develop a megaloblastic anemia because of vitamin B12
absorption by the parasite.
Laboratory diagnosis
- identification of proglottid or/and eggs in feces.
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- oval
- 70-x 45 μm,
- operculated,
- yellowish-brown.
Important: Scolex: elongated, spoon shaped, two long sucking grooves, measures 1 mm
/2.5 mm.
Useful: Many other mammals can also serve as definitive hosts for D. latum.
Treatment: Praziquantel
Optional : 1,000,000 eggs per day per worm) and are passed in the feces .
HYMENOLEPIS NANA
Essential
Life cycle: www.dpd.cdc.gov/dpdx
hymenolepiasis mostly asymptomatic
- gastrointestinal symptoms occur in heavy infections
- epileptiform fits may be allergic responses to the released antigens of the worm.
Diagnosis:
O & P examination of feces: eggs
Morphology-egg
- oval or subspherical
- size 40 - 60 µm X 30 - 50 µm.
- the inner membrane - two poles, from which 4-8 polar filaments spread out
between the two membranes.
- The oncosphere has six hooks
Important
~ specific for humans ( H. diminuta – rodents, rarely humans)
Pathogenesis: attaches to the gastrointestinal mucous using four sucking disks and a
hooker rostrum.
Useful
Possible contamination with H.diminuta from pastry cooked with floor contaminated
with rodent excretes !
Treatment : Praziquantel
Optional
High prevalence rate: Portugal, Spain, Sicily, Egypt, Sudan, India
Morphology-adult worm
Scolex 4 sukers, Hooked rostrum
Small: 1-5 cm , 3 tests
TENIA SAGINATA
TENIA SOLIUM
Essential
• Beef teniasis Pork teniasis
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• 6-8 m, 2-3 m
• Opac white Transparent white
• Pear-shaped scolex globular scolex
• Four suckers four suckers and
• No hooks circular row of hooks (rostellum)
• L~5l L=2l
Life cycle: www.dpd.cdc.gov/dpdx
Diagnosis
- history, clinic
- examination of proglots
T.solium:
- chain of proglots liberated at defecation
- wider than those of T.saginata
- few ramification (less than 15)
T.saginata: independent proglots liberated while sleeping
- Narrower than those of T.solium
- More than 15 uterus ramifications
Eggs of T. saginata and T. solium are indistinguishable morphologically
(morphologic species identification will have to rely on the proglottids or scolices).
rounded, diameter 31 to 43 µm, with a thick radially striated brown shell.
Inside each shell is an embryonated oncosphere with 6 hooks.
Optional
1000-2000 proglote 1000 proglote
100000 eggs/proglota 50.000 eggs/proglota
Questions and reviewing
1. Botriocephalosis:
a. evolves as a feverish enteritis, with watery stools
b. often has non-symptomatic evolution
c. is transmitted through consumption of cercariae infested water or aquatically plants.
d. represents the cause of some serious complication due to worm migration to other
sites.
e. is transmitted through consumption of raw fish.
2. Human infection with the beef tapeworm, Taenia saginata, usually is less serious than
infection with the pork tapeworm, T. solium, because
a. Acute intestinal stoppage is less common in beef tapeworm infection
b. Larval invasion does not occur in beef tapeworm infection
c. Toxic by-products are not given off by the adult beef tapeworm
d. The adult beef tapeworms are smaller
e. Beef tapeworm eggs cause less irritation of the mucosa of the digestive tract.
3. Analysis of a patient’s stool reveals small structures resembling large rice grains;
microscopic examination shows these to be proglottids. The most likely organism in this
patient’s stool is
a. Enterobius vermicularis
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. Ascaris lumbricoides
c. Necator americanus
d. T. saginata
e. Trichuris trichiura.
4. Which one of the following parasites has as an intermediary host fishes or snails:
a. Tenia solium;
b.Trichinella spiralis;
c. Botriocephalus;
d. Toxocara cannis;
e. Fasciola hepatica.
CISTICERCOSIS, HYDATIC CYST, ALVEOLAR ECHINOCOCCOSIS
Diseases produced by nematodes larvae
Summary
Cisticercosis
- cysticercus cellulose vs. cysticerus bovis
- degrees of severity
Hydatic cyst
- the parasite
- human contamination
o risk groups
- location
- laboratory diagnosis
Alveolar echinococcosis
- human contamination
o risk groups
- laboratory diagnosis
Educational objectives
CISTICERCOSIS
Essential
- disease produced by development of cysticercus cellulose = the larval stage of T
solium that can also infect humans
- humans are intermediary host
- larval cysts develop in lung, liver, eye and brain => blindness and neurological
disorders.
Human contamination: 3 degrees of severity
Optional: incidence of cerebral cysticercosis can be as high 1 per 1000 population and
may account for up to 20% of neurological case in some countries (e.g., Mexico);
- cysticercosis ocular involvement occurs in about 2.5% of patients and muscular
involvement is as high as 10% (India).
Educational objectives
Essential
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HYDATIC CYST
Hydatic cyst is produced by Echinococcus granulosus
Morphology – adult worm
- 3 to 9 mm long
- only 3 proglottids
Cyst structure
- round
- measures 1 to 7 cm in diameter (may grow to 30 cm)
- outer anuclear hyaline cuticula
- inner nucleated germinal layer
- containing clear yellow fluid.
- Daughter cysts attach to the germinal layer,
- some cysts, known as brood cysts, may have only larvae (hydatid sand).
Life cycle
- adult worm lives in domestic and wild carnivorous animals ( definitive host) => Eggs
- ingested by the grazing farm animals or man,
- embryo hatch => larvae = hydatid
- penetrate the small intestine
- enter the circulation form cysts in liver, lung, bones, and sometimes, brain.
- hydatid cysts containing many larvae (proto-scolices or hydatid sand)
When other animals (intermediary host) consume infected organs of these animals, protoscolices
escape the
Man is a dead end host (IH)
Echinococcosis (hydatid disease)
- hydatid cysts take several years to develop sufficiently to cause symptoms, depending
on the size and their location.
- inflammation (with eosinophilia in 25% of cases) led to fibrosis and pressure on the
affected organ.
- may creak or break => anaphylactic shock
- most cysts occur in the liver or lungs
Diagnosis
- history, symptoms & signs
- imaging (XR, CT, Ultrasonography,)
- serologic
Important
Hepatic hydatidosis
- asymptomatic for 10-15 years
- pseudotumoral symptoms: dyspepsia and hepatic pain
- complications: compression on bile ducts and portal or cavum system lead to
hypertension and digestive hemorrhage.
Pulmonary hydatidosis
- asymptomatic for years
- caught, dyspnea, hemoptizia, thoracic pain
- complications: pressure on bronchia, breaking in a bronchia => cough which eliminate a
clear, salty liquid with small vesicles and cystic membrane fragments = vomit
Other location
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- bone, CNS, heart, kidney
o serious prognosis.
Some hydatid may
- become infected with bacteria due to a small breaking in the wall;
- die and become calcified.
Hydatic disease has professional risk: veterinarians, shepherds, pet store workers
Optional
When the hydatid is formed in bone:
- development is markedly abnormal (the limiting membrane is not formed)
- develops first in the marrow cavity, from which it expands and frequently erodes large
areas of bone.
Biologic diagnosis of hydatid cyst by Casoni IDR is obsolete.
ALVEOLAR ECHINOCOCCOSIS
Essential
Alveolar echionococcosis is produced by E. multilocularis.
E.multilocularis is similar in morphology with E.granulosus.
Their larval form (hydatid) is different !
- Human contamination:
- eating wild berries
- contact with foxes (fur)
- Humans, develop multilocular (many chambers) cyst = alveolar echinococcosis
(alveolar cyst)
- Only in liver
Important
Professional at risk for alveolar echinococcosis are: forest workers, workers in fur factory
Treatment:
- resistant to praziquantel;
- high doses of Albendazole has some anti-parasitic effect.
- Surgery is the means of removing the cyst.
Rodent control is the means of prevention
Bibliography
1. Medical Parasitology – Markell, Voge, John, 9-th edition, 2006
2 Diagnostic Medical Parasitology - Lynne Shore Garcia, 5th Edition, ASM Press, 2006
3. www.dpd.cdc.gov/dpdx
Questions and reviewing
Cisticercosis
For which one of the following parasites human beings can to be an intermediary host:
a. Tenia solium
b. Toxoplasma gondii
c. Echinococcus granulosus
d. Tricocephalus
e. Fasciola hepatica.
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Human contamination with Echinococcus granulosus is realized through :
a. Contact with dogs.
b. Consumption of infested pork meat
c. Eating non-properly washed vegetables
d. Direct human-to-human contact.
e. Drinking contaminated water.
Hydatic cyst:
a. is produced by Echinococcus multilocularis
b. human contamination is realized through contact with foxes.
c. can crack or break spontaneously or traumatically.
d. can be diagnosed by serological reactions.
e. human contamination is realized through contact with dogs
LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS
Summary
General characteristics
Pneumocistis jirovecii
- laboratory diagnosis
Aspergillus sp.
- laboratory diagnosis
Candida sp.
- laboratory diagnosis
Cryptococcocus neoformans
- laboratory diagnosis
Dermatophytes
- laboratory diagnosis in infections with dermatophytes
Practical activities:
- wet mount, observation & interpretation
- Gram staining, observation & interpretation
- Colonies of Candida: observation & interpretation
- stained smears, observation & interpretation
- performing antigen-antibodies reactions and interpretation
Educational objectives
Essential
General characteristics
- widely distributed in nature (air, water, soil, decaying organic debris)
- ~400,000 types
- Eukaryotic cells
- highly developed cellular structure
- Aerobic conditions
- Chemotropic, nutrition: by absorption
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- Nonphotosynthetic (lack chlorophyll)
Classification:
- Yeast = unicellular growth form of fungi, spherical to elipsoidal, reproduces by
budding: Candida sp.
- Mould = a filamentous fungus composed of filaments that generally form a colony:
Aspergillus, Zygomycosis (Rhizopus, Mucor, Rhizomucor)
- Dimorphic = 2 distinct morphological forms: yeast in tissue (in vivo) and molds in
culture (in vitro) (ex. Histoplasma capsulatum, Coccidioides)
MYCOSES
- Superficial (Hair, skin, nail, cornea)
- Subcutaneous
- Systemic
Laboratory diagnosis in infections with Pneumocystis jirovecii
Practical activities:
- Giemsa staining, observation and interpretation.
Essential
Pneumocystis jirovecii
Morphology
Trophozoit:
- ovoid, 2- 4μm
- single nucleus
Cysts:
- 7-10μm in diameter,
- eight nuclei (also called intracystic bodies),
- rosette, containing the sporozoites
- The identification of P.jirovecii in pulmonary tissue or in lung fluids
- Specimens (sample): sputum, induced sputum, bronchoalveolar lavage, lung biopsy
or needle aspiration of the lung; bronchial brushing; transbronchial aspirates.
Important
- Microscopic morphology: diagnosis still relies on morphologic identification of
Pneumocystis from respiratory specimen.
- Stains used to identify Pneumocystis: Gomori- Grocott (silver methenamine
technique), Giemsa, fluorescence-labeled antibody (imunofluorescence) or toluidine
blue.
Useful
- NO reliable techniques for cultivating
- NO serologically diagnosis
Optional
- Molecular methods (high sensitivity and specificity).
- Interpretation of molecular techniques
Laboratory diagnosis in infections with Aspergillus
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Practical activities:
- wet mount, observation and interpretation
- Gram staining, observation and interpretation
- Colonies of Aspergillus: observation and interpretation
- stained smears, observation and interpretation
- performing antigen-antibodies reactions and interpretation
Essential
Microscopic, cultural and molecular evidence for aspergillosis
Susceptibility testing for antifungal drugs
A. Direct diagnosis
1. Sample collection
Important
2. Direct microscopic examination from the sample
- wet mount: potassium hydroxide (KOH),
- staining: Grocott (silver staining)
- fungal hyphae may be seen in the sputum.
3. Isolation on culture media:
- culture media: Sabouraud medium, in 3-4 days.
Useful
4. Identification based on:
- morphological characteristics
- cultural characteristics
Optional
Serology: antigen and antibody detection
Aspergillus specific tests:
- precipitating antibodies to Aspergillus species in >90% of cases
- aspergillus-specific IgE RAST test
Laboratory diagnosis in infections with Candida
Practical activities:
- wet mount, observation and interpretation
- Gram staining, observation and interpretation
- Colonies of Candida: observation and interpretation
- stained smears, observation and interpretation
- performing antigen-antibodies reactions and interpretation.
Essential
- Candida species
- Candidiasis
A. Direct diagnosis
1. Sample collection
2. Direct microscopic examination
- wet mount: potassium hydroxide (KOH)
- staining: Gram
Important
3. Isolation on culture media:
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- culture media: Sabouraud dextrose agar, chromogen media
4. Identification based on:
- microscopic characteristics
- cultural characteristics
- biochemical tests
- antigen detection
Susceptibility testing for antifungal drugs
Optional
B. Indirect diagnosis
- Serology: antibody detection
Laboratory diagnosis in infections with Cryptococcus
Practical activities:
- wet mount, observation and interpretation
- China ink staining, observation and interpretation
- Colonies of Criptococcuss: observation and interpretation
- performing antigen-antibodies reactions and interpretation
Essential
A. Direct diagnosis
1. Sample collection: CSF, blood, urine
2. Direct microscopic examination
- wet mount from CSF with India ink
- staining: Gram, Giemsa
Important
3. Isolation on culture media:
- culture media: Sabouraud dextrose agar
4. Identification based on:
- microscopic characteristics: encapsulated yeast
- cultural characteristics: S type colonies, very mucous colonies
- biochemical tests
- antigen detection in CSF, serum
Optional
- biochemical tests
- antigen detection in CSF, serum
Laboratory diagnosis in infections with dermatophytes
Practical activities:
- KOH preparation, wet mount - observation and interpretation
- Colonies of dermatophytes: observation and interpretation
Essential
General properties
Produces superficial fungal infections : skin, hear, nails
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Species
Trichophyton spp. – anthropophilic, zoophilic, geophilic species
Microsporum spp.: - anthropophilic, zoophilic, geophilic species
Epidermophyton flocossum
Laboratory diagnosis
1. Sample collection: hear, nails, skin
2. Microscopical examination
3. Inoculation on culture media:
- Sabouraud agar and Sabouraud with cycloheximide
- incubation at 27°C for several weeks (up to 4 weeks)
4. Identification based on microscopic and cultural properties
Important
Diseases:
Tinea capitis, Tinea favosa, Tinea corporis, Tinea pedis, Tinea manuum, Tinea cruris,
Tinea barbae, Tinea ungium (onychomycosis)
Bibliography
1. Monica Junie, Luciana Stănilă, Carmen Costache: “Medical Microbiology”
“Iuliu Haţieganu” University Publishing House, Cluj-Napoca 2003
2. Jawetz, Melnick & Adelberg’s, 22 - nd : Brooks G. F., Butel J.S. and Ornston L.N.:
“Medical Microbiology”
Questions and reviewing
Aspergillus:
a) produces aspergilloma (fungus ball)
b) is an yeast
c) is a mould (filamentous fungi)
d) is a protozoan
e) it can not be cultivated in laboratory using culture media.
Cryptococcus neoformans:
a) is an encapsulated yeast
b) is a bacteria
c) on Sabouraud medium forms S type colonies, very mucous.
d) on Sabouraud medium forms R type colonies
e) the cryptococcus antigen can be identified from CSF and serum from the patients.
Candida :
a) is an yeast
b) is a bacteria
c) it can be cultivated on Chapman medium
d) it can be cultivated on Sabouraud medium
e) is a mould.
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