4%

MYCOLO2XGY

\Attus coPage| 111

all
 COLLECTION, TRANSPORT and EXAMINATI
ON of CLINICAL SPECIMEN ne 2%
| CULTURE
| net sis
INFECTIONS are no icable j
| || ne
beco in the usual sense of person-person or animal - to - person transfer; humans
th Maher of spores
accidental host i ¥ iInnalation or by their introduction into tissue by trauma.
TAXONOMY OF FUNGI
1 Presence of chitin in the cell wall
2 Presence of ergosterol in the cell membrane
| 3. Reproduction by means of spores, either asexually or sexually
| 4. Lack of chlorophyll
| 5 Lack of susceptibility to antibiotics
GENERAL FEATURES
| YEAST
| 1. Produce moist, creamy, opaque, or pasty colonies on media.
2. Invasive in tissues at 37 °C.

MOLDS/FILAMENTOUS FUNGI
1. Produce fluffy, cottony, woolly, or powdery colonies.
2. _ Infective to man at 25°C.

DIMORPHIC FUNGI
1. Exhibit either a yeast or mold form
2. Produce a mold form at 25°C -30°C and a yeast for at 35°C — 37°C under certain circumstances.

| DIPHASIC FUNGI
1. _ Exists as mold in vitro (25-37 °C) and yeast in vivo
1

| SAMPLE COLLECTION AND TRANSPORT

Sample Remarks
| Respiratory Tract Secretions Samples: Sputum, induced sputum, bronchial washing, bronchial lavage, tracheal
aspirations.
| Specimens may be stored at room temperature if processing is completed within
| 2 hours.
If processing is delayed, specimens should be refrigerated at 4°C.
| | Cerebrospinal fluid (CSF) Should be filtered through a: 00> wembane bias
If less than 1 mL of specimen is submitted for culture, it should be centrifuged,
and 1-drop aliquots of the sediment should be placed on several areas on the
agar surface.
CSF specimens should never be refrigerated.
Blood Use of automated blood culture systems such as BACTEC.
The optimal temperature for fungal blood cultures is 30°C, and the suggested
ee incubation time is 21 days.

3 A2A3B846050607888 9, D, 10.6 11.4 12 A, 13. B, 14. C, 15. B

SAMPLE COLLECTION AND TRANSPORT (Contd) arenes
it inna
Sample Remarks
directly onto
ea penned
Eye (Comeal Scrapings or Vitreous / Comeal scrapings collected by a physician should be placed
Humor) microscopic slides and inoculated onto non-inhibitory media
Vitreous humor aspiration should be concentrated by centrifugation, vg
processing a CSF sample, and the sediment should be used for smears =
Culture.
ih
Hair, Skin, and Nail Scrapings Samples collected from lesions may be obtained by scraping the skin or nails with |
| 2 Scalpel blade or microscope slide.
Infected hairs are removed by plucking them with forceps.
These specimens should be placed in a sterile container, they should not be
refrigerated.
If infection with Malassezia furfur is suspected, olive oil or an olive oil- saturated
paper disk should be placed in the first quadrant of the agar plate.
Vaginal Transported to the laboratory within 24 hours of collection using culture transport
swabs,
Vaginal cultures should be screened for yeasts and incubated at 30°C for 7 days.
Urine The 24-hour urine sample is unacceptable for culture.
All urine samples should be centrifuged and the sediment cultured using a loop
to provide adequate isolation of colonies.
Tissue, Bone Marrow, and Sterile All tissues should be processed before culturing by mincing not grinding.
Body Fluids Tissue pieces should be pressed into the appropriate culture media.
Bone marrow may be collected in a heparinized syringe.
Sterile body fluids should be concentrated by centrifugation before culturing, and
at least 1 mL of specimen should be placed on the surface of appropriate culture
media.

LABORATORY DIAGNOSIS OF MYCOSES

Fungal Culture Media; Indication for Use
Media | Indication for Use
PRIMARY RECOVERY MEDIA
Brain-heart infusion agar Primary recovery of saprobic and pathogenic fungi
Brain-heart infusion agar with antibiotics _| Primary recovery of pathogenic fungi exclusive of dermatophytes
Chromogenic agar Isolation and presumptive identification of yeast and filamentous fungi
Dermatophyte test medium Primary recovery of dermatophytes; recommended as screening medium.
Potato flake agar Primary recovery of saprobic and pathogenic fungi
|_Mycosel agar Primary recovery of dermatophytes
Sabouraud Dextrose with Brain Heart | Primary recovery of saprobic and pathogenic fungi
| Infusion (SABHI) agar
 Fungal Culture Media; Indication for Use Cont'd)
Media on ey
Indication for Use —

DIFFERENTIAL TEST MEDIA

Fpoette Ascospore agar
Christensen’s urea agar
Detection of ascospores in ascosporogenous yeasts (Saccharomyces Spp.)
Identification of Cryptococcus, Trichosporon, and Rhodotorula spp
Commeal agar with Tween 80 and trypan
|

production
[i

identification of Candida albicans by chtamydospore

blue
Cottonseed conversion agar Conversion of the dimorphic fungus Blastomyces sp . from mold to yeast

— Differential identificati on of Aspergillus spp.

irdseed ag Cryptococcus gattii __]
Identification of Cryptococcus neoformans and
Demonstration of pigment production by Trichophyton rubrum
ange —
ice medium
vas — agar Identification of yeasts by determining fermentation |
assimilation
ves Identification of yeasts by determining carbohydrate
MICROSCOPIC DETECTION OF FUNGI IN CLINICAL SPECIMENS
Method Use
j i
Calcot uor = Stain Detection of fungi
India ink stain Detection of Cryptococcus spp. in CSF
Lactophenol cotton or aniline blue wet Most widely used method of staining and observing fungi
mount
10% KOH Clearing of specimen to make fungi more readily visible
Masson-Fontana stain Examination of melanin pigment in fungal cell walls
Methenamine silver stain Detection of fungi in histologic section
Huckert’s Modification of Gram Stain Fungi stain Gram Positive

Culture and Microscopic characteristics of Medically important Fungi

Culture characteristic Microscopy
Microsporum audouinii Downy white to salmon-pink colony Sterile hyphae, terminal chlamydoconida,
Reverse: Tan to salmon pink. FAVIC CHANDELIERS, and pectinate
Disease: ica caps
bodies.
Membranous with feathery periphery; | Thick walled, spindle ~— shaped,
Microsporum canis
capitis | center of colony is white to buff over | multiseptated rough walled macroconidia.
Disease: Tinea
associated with alopecia orange yellow.
Reverse: Lemon-yellow or yellow orange
apron
Cinnamon-colored, powdery colony Thick walled, rough, elliptical multiseptated
Microsporum gypseum
Reverse: Light tan macroconidia
Disease: Tinea capitis
| Macroconidia are large, smooth walled,
Epidermophyton floccosum | Center of colony tends to be folded and multi-septate, clavate.
Disease: Tinea pedis | is khaki green
(Athlete's foot) Reverse: Yellowish brown with folds
White downy to pink granular Microconidia usually teardrop-shaped.
Tricophyton rubrum
Disease: Tinea pedis Reverse: Yellow when colony is young,
|
(Athlete's foot) red color when old culture.
club-shaped
Trichophyton tonsurans White, tan to yellow or rust, suedelike to Microconidia are teardrop or
with flat bottoms
Disease: Ring worm (infection | powdery
of the scalp) Reverse: Yellow to tan to rust red,

\lActus coPage| 114

 culture and Microscopic characteristics of Medical important Fungi
Culture characteristic
Trichophyton schoenleinjj
Aspergillus fumigatus
Disease: Aspergillosis
Aspergillus flavus
Disease: Aspergillosis
Aspergillus niger
Disease: Aspergillosis
Blastomyces dermatitidis
Disease: North American
Blastomycosis, —Gilchrist's
diseass, Chicago disease
Coccidioides immitis
Disease: San Joaquin Valley
Fever, Dessert Fever, Valley
Fever
Histoplasma capsulatum
Disease: Darling's disease,
Spelunker's Lung, Cave
disease
Paracoccidioides brasiliensis
Disease: Paracoccidiomycosis
American-Blastomycosis,
Brazillian-blastomycosis
Sporothnix schenckii
Disease; “oee-Garveners
disease
Hortea werneckii
Disease: tinea nigra
Piedraia hortae
Disease: black Piedra
Cont'd
sm doth, white to Microscopy
cream colony
Reverse: White Hyphae: FAVIC CHANDELLERS
fluffy to granular,
white to blue-green Presence of septate hyphae and short or
colony,
eat Sporulating colonies long conidiophores with a characteristic
most often “foot cell’ at their base
avea blue-green, Owd
ery appearance,
Yellow-green colony Vesicles are globose, and phialides are
produced directly from the vesicle surface
(uniseriate) or from a primary row of cells
Vy
called metulae (biseriate).
Yellow Colony that soon develo
ps a black, Hyphae are hyaline and septate, as are
dotted surface as conidia are
produced, those of other aspergilli.
Glabrous Or Waxy-appearing colo
ny and Tufts of hyphae often project upward from
IS off-white to white, With age,
the aerial the colonies, and this has been referred to
hyphae often turn gray to brow
n, as the “prickly state” of the organism.
Fluffy white molds; pink to yellow
to Hyphae form arthroconidia that are
purple and black colonies characteristically rectangular to barrel-
shaped. The arthroconidia are larger than
the hyphae from which they were produced
and stain darkly with lactophenol cotton or
aniline blue.
White, fluffy mold that turns brown to buff The hyphae are small (approximately 2 mm
with age. in diameter) and are often intertwined to
H. capsulatum and B. dermatitidis cannot form ropelike strands.
be differentiated + using —_ colonial
morphologic features.
Heaped, wrinkled, moist, and yeastlike. Small hyphae (approximately 2 mm in
diameter) are seen, along with numerous
chlamydoconidia. Small (3-4 mm),
delicate, globose or pyriform conidia may
be seen arising from the sides of the
hyphae or on very short conidiophores
Small, moist, and white to cream-colored. Hyphae are delicate (approximately 2 mm
in diameter), septate, and branching,
Single-celled conidia 2 to 5 mm in
diameter are borne in clusters from the tips
of single conidiophores _(flowerette
arrangement).
Initial colonies may be olive to black, | One to two celled conidia form from the
shiny, and yeastlike and usually grow apical regions of conidiophores or small
within 2 to 3 weeks. projections on the hyphae.
Appear dark brown to black, and also | Produces short, dark hyphae containing
produce aerial mycelium. Some isolates thick-walled resting cells. The ascomata
may produce a red to brown diffusible consist of irregular shaped pseudothecia
pigment. that are black in color.
\Attus coPage| 115
cuture and Microscopic characteristics of Medical ly important Fungi (Cont'd
Culture characteristic Microscopy
eee
a Non-culturable Silver-staining to demo nstrate

-Gyyplococcus !neoformans
trophozoite/oyst form.
The growth begins as a smooth, white to | Appears as a spherical, iple-
pisease: Cryptococcosis tan colony that may be mucoid to cre sing 2 to ane in:
amy. budding, thick-walled yeast
diameter. It usually is surrounce e.
i ca sul
de
wide, refractile polysacchari
a n of clinicinal
io
Candida albicans | The colonial and microscopic Direct microscopic examinat a
Disease: Candidiasis, | morphologic features of Candida spp. specimens containing Candida ocon ae
Moniliasis, Thrush have little value for making a definitive reveals budding yeast cells (blast
meter and/o
identification. 2 to 4 mm_ in dia
pseudohyphae
Malassezia furfur Recovery of the organism is not required Detected through direct a
. °
Disease: "122 versicolor to examination of skin scrapings
establish a diagnosis in skin infections, organism is easily recognized as ova l- a
and it is seldom attempted for this bottle-shaped cells that exhibit ee
wit
purpose. budding in the presence of a cell wall
The colonies are small compared with the a septum at the site of the bud scar.
colonies of C. albicans and are creamy
and white to off-white.
VIROLO2%GY
4%

\ALTUS coPage| 118

_il
 oo CHARACTERISTICS, TRANSMISSION AND DISEASE ye ai
MEDICAL VIROLOGY
Viruses are obligate intracellular parasites; the y cannot multiply by binary fission
e Contain either RNA or DNA (never both)
e They lack Ribosomes, ATP ( needs host cell to be metabolically active)
e The largest viruses are the Poxviruses while the smallest are Picomaviruses

VIRAL REPLICATION/ INFECTIOUS CYCLE

1. | Attachment .
2, | Penetration Fusion of viral envelope with host cell membrane

3. | Uncoating Release of genome from the capsid during or after penetration

ication; Late RNA a nd
Vi genome replication;
4. | Macromolecular Synthesis Early mRNA protein synthesis;ice Viral
protein synthesis
Capsid assembly refers to the formation of thecapsid shell. |
». Assembly
Packaging: Refers to the viral genome placement inside a capsid.
6. Release Budding — for enveloped viruses; envelope comes from host cell
Lysis — for naked viruses; host cell lyses to release virus

THE DNA VIRUSES

Enveloped _ Herpesviridae

| Double stranded = ot Papillomaviridae

Circular +. oe SAC
——
rs, * eo. Ff : Polyomaviridae
” Naked sae
Lec, = — ee
@®
— Linear ee Adenoviridae
fae

he le
|
al
=|
- nian Sceepsieeaiorsiaan
Single stranded sect
nvel et

See
GROUP II oe ' se
S ———— 7 Complex — Poxviridae

2 | Double stranded DNA (RT) — Hepadnavinidae

GROUP Vil

Baltimore's Classification Group | DNA Viruses Transmission and Diseases

Virus Transmission Disease
Family
ALPHA GROUP Alphaherpesvirinae Direct contact with infected | Gingivostomatitis, pharyngitis,
Herpesviridae Human Simplex Virus1 | secretions herpes labialis, conjunctivitis,
keratitis
Latency: ovrons | Alphaherpesvirinae Direct contact with infecte d | Genital infection, herpetic
secretions whitlow, disseminated disease
Human Simplex Virus2
Alphaherpesvirinae Close personal contact, especially | Chicken pox _(varicella),
ea, Varicella-zoster virus respiratory shingles (zoster)
1 AZABD4DSEDEBR7DBAGDC 104A

Actus coPage| 119

patimore's Classification
Group I! DNA Viruses
rfaniy |ROUP
Virus
erpesv
Transmission and Diseases
Transmission Disease
ean oo 4 Close contact with infected | Congenital disease of newborn
heterophile-negative
herpes virus 5 uman | secretions, blood transfusions | and
haa 1 transplants, | infecti ous mononu cleosis
Latency: © o0ys
erpesvin ransplacenta
— hated 6 Most likely close contact via | Roseola infantum (Heaton
inclusion bodi — respiratory route, almost all subitem, 6° disease)
8S: -owiry A | children infected by age 2-3 | redAlert: Most common
a - years
Most likely close contact via | Can also cause infantum
i : rinae 6"
respiratory route; almost all | (Exanthema subitem;
en TeDOR WHET
children infected by age 2-3 | disease)
years |
FAL
Gammaherpesvirinae Close contact with infected | Infectious mononucleosis,
GAMMA GROUP
progressive — lymphoreticular
eiperie Epstein-Barr virus/ Human | saliva
disease, oral hairy leukoplakia
a ial
in patients with HIV
—— Human herpes virus 8 Not known; much less widely | !2pos) sarcors
disseminated than other
herpes viruses
Papillomaviridae Human Papilloma virus Direct contact, sexual contact Skin and genital warts, benign
Latency cell: Epithelial tissue | for genital warts head and neck tumors,
anogenital warts
Adenoviridae Human adenoviruses Respiratory, fecal-oral, and | Pharyngoconjunctival fever,
direct contact (eye) keratoconjunctivitis,
hemorrhagic cystitis, | and
gastroenteritis in children
Parvoviridae Parvovirus B-19 Close contact, probably | Eryinema infectiosum (fh
respiratory disease), aplastic crises in
patients with chronic hemolytic
anemias
Poxviridae Smallpox (Variola, Vaccina) | Respiratory droplets | Smallpox disease
(smallpox); direct contact
(molluscum contagiosum, orf,
monkeypox)

Baltimore's Classification Group VI! DNA Virus Transmission and Diseases

Family Virus Transmission Disease
Hepadnaviridae Hepatitis B virus Humans are reservoir and vector, | Acute liver infection, Fulminant
spread by direct contact, including | hepatitis.
Hepadnavirus the exchange of body secretions,
HEPA-DNA-VIRUS recipient of contaminated blood
(Virus that infects the products, percutaneous injection
liver cells) of virus, and perinatal exposure.

\ Actus coPage| 120

 THE RNA VIRUSES
Double stranded
Group tt 4 Naked
|
rd \cosahedral | Reoviridae
/ as

Flaviviridae

Enveloped ion at: Togaviridae

Single stranded positive sense ’ Coronaviridae

Helical —
ry Group lV
seamen etiam id dasiianaehenens nce
nn
wo — Picornaviridae
”
A sarees rt lcosahedral .
>
- on" 11 . Caliciviridae
S gi

— Orthomyxoviridae
=. anaes
ir
— Paramyxoviridae
| cece

a Rhabdoviridae
| Single stranded negalive sense i
Helical + ae
GroupV 4 Enveloped —
— et Filoviridae
Single stranded negalive saris
~ ee
a (RT) 4 Retroviridae
_____GroupVI_ Ee et Pla
ny — Bunyaviridae

_— Arenaviridae

Baltimore's Classification Group II] RNA Viruses Transmission and Diseases

Family Virus Transmission Disease
Reoviridae Rotavirus Fecal-oral; survives well on | Gastroenteritis in infants and
inanimate objects children 6 months to 2 years

Baltimore's Classification Group IV RNA Viruses Transmission and Diseases

Fami Virus Transmission Disease
Flaviviridae Yellow fever virus Arthropod vector, —usually Yellow fever
|
mosquito
Dengue virus Arthropod vector, — usually | Dengue hemorrhagic fever
mosquito
West Nile virus Arthropod vector, —_—usually | West Nile encephalitis
mosquito
ee
Japanese encephalitis virus Arthropod vector, —usually | Japanese encephalitis
mosquito
St. louis encephalitis virus Arthropod —_veclor, usually | St. Louis encephalitis
mosquito
fee oe Bee

Actus coPage| 121

paimore's Classfierion Group IV RNA Viruses Transmission and Diseases (Cont'd)
Family mus _ Transmission Disease chronic hepatitis;
,
Flaviviridae Hepatitis C Parenteral or sexual Acute and
strong _ correlation between
chronic HCV infection and
. hepatocellular carcinoma
re
Togavridae Alphaviruses Arthropod vector, usually | Eastern, Western. and
mosquito Venezuelan equine encephalitis |
Rubella virus Respiratory, transplacental Rubella (mild exanthematous

Coronaviridae Coronavirus Unknown, probably direct | Common —_ cold, possibly

contact or aerosol gastroenteritis, especially | In
children; SARS, MERS-COV,
COVID-19
Picornaviridae Polio virus Fecal-oral Polio disease
Inclusion bodies: © owery 4
ero —" Coxsackie A virus Fecal-oral — Hand-foot-mouth
isease _—
Pico- small
RNA virus Coxsackie B virus Fecal-oral Pleurodynia, pericarditis,
myocarditis
Echovirus Fecal-oral Neonatal disease
Enterovirus 68-71 Fecal-oral Aseptic meningitis
Enterovirus 72 Fecal-oral Hepatitis with short incubation,
Other name: hepatitis 4 abrupt onset, and low mortality,
no carrier state
Rhinovirus Contact with —_ respiratory | Common cold
*the common cold virus secretions
Caliciviridae Norovirus Fecal-oral Infectious gastroenteritis
Hepatitis E virus Fecal-oral Hepatitis similar to that caused
by hepatitis A virus except for
extraordinarily high case fatality
rate (10%-20%) among
pregnant women

Baltimore's Classification Group V RNA Viruses Transmission and Diseases

Family Virus Transmission Disease
Orthomyxoviridae | InfluenzaA Contact with respiratory | Influenza (fever, —_ malaise,
secretions headache, myalgia, cough);
primary influenza pneumonia;
in children, — bronchiolitis,
croup, otitis media
Influenza B Contact with respiratory | Similar to “mild” influenza
secretions
Influenza C Contact with —_—respiratory | Mild form of influenza causing
secretions URTIs
Paramyxoviridae | Parainfluenza virus Contact with —respiratory | Adults: upper —_ respiratory
secretions disease, rarely pneumonia
Children: respiratory including
croup, bronchiolitis, and
—— pneumonia

\Attus coPage| 122 6

al
sunt CT Vins V RNA Viruses Taraision and Diseases (Cont'd) ania |
Fay
paramyxoviridae Mumps virus
7 Transmission Disease
Person-to-person contact, | Mumps
presumably respiratory droplets
Measles virus Contact with nin Measles, atypical
Oat
secretions; extremely contagious | (occurs in those with wa :
vaccine immunity), 4"
sclerosing
subacute
: panencephalitis_____—__-
Respiratory Syncytial Virus | Person-to-person by hand and | Primarily in infants an

ee ae bronchiolitis,
pneumonia, and croup
Children: upper respiratory
— disease —
FRabdoviridae__| Rabies virus Bite of rabid animal Rabies zncal
Filoviridae Ebola Transmissible to humans from | Severe hemorrhage and liver
monkeys and, presumably, other | necrosis; mortality as high as
wild animals; human-to-human | 90%
transmission via body fluids and
respiratory droplets -
Marburg hemorrhagic | Transmissible to humans from | Severe hemorrhage and liver
fever virus monkeys and, presumably, other | necrosis; mortality as high as
wild animals; human-to-human | 90%
transmission via body fluids and
respiratory droplets
Bunyaviridae Arboviruses Mosquito, tick, and sandfly | Encephalitis for arboviruses
vectors
Arenaviridae Lymphocytic From rodent to human through | Asymptomatic to influenza-like
choriomeningitis virus contamination of human | to aseptic meningitis-type
environment with rodent urine disease;
Lassa fever virus From rodent to human through | influenza-like disease to severe
contamination of human |} hemorrhagic fever
environment with rodent urine

Baltimore's Classification Group VI RNA Viruses Transmission and Diseases

Family Virus Transmission Disease
Retroviridae HIV-1 and 2 Sexual contact, blood and blood | Acquired Immunodeficiency
M-tropic (R5): CORS product exposure, and perinatal | Syndrome (AIDS)
redAlert: M-tropic (Macrophage) | exposure
T-tropic (X4); CXCR4
redAlert: T-tropic (CD4 cells)
 COLLECTION, TRANSPORT and EXAMINATION
of CLINICAL SPECIMEN
i

} | 0 ye ea
Summary of Specimns
of Specime for Diagna
ens for
1

Dia nosis of Vj
='S Of Viral Diseases
Throat/ —
Nas: Stool Urine CSF Others
RESPIRATORY “asopharynx_|
Adenovirus 357 Seal
- S97 J
Influenza virus
Parainfluenza virus oe oe
Respiratory Syncytial Virus =.
Rhinovirus OO
Coronavirus (MERS-Cov,
COVID-19) aoko
Dermatologic and Mucous Membrane
Vesicular
Enterovirus OO OO
Vesicle fluid
Herpes simplex virus
Varicella-zoster virus OO
Exanthematous
Enterovirus OO 00
OO OO Serum
Measles
Rubella OO Serum

Parvovirus Serum,
Amniotic fluid
Pustular/Nodular
Tissue
Molluscum contagiosum
Papillomavirus Tissue, _ thin-
prep cervical
MENINGOENCEPHALITIS/ENCEPHALITIS
Arbovirus CSF/Serum
Enterovirus OO OO OO
Herpes simplex virus Serum
Mumps Serum
Polyomavirus (JC virus) Brain biopsy
Corneal cells,
Rabies virus
brain
GASTROINTENSTINAL DISEASE
Adenoviruses (serotype 40-41) OO
Norovirus OO
Rotavirus OO
DERMATOLOGIC and MUCOUS MEMBRANE
| Congenital and Perinatal
Cytomegalovirus OO
Rubella OO Serum
| Parvovirus Amniotic fluid
 ry ooff SpeSpcimec
|
summary
forforen
ensim DiaDiagsgncnosisetof Viral Diseases Cont'd
| ane so Urine CSF others
| (Ocular
Eye disease) een >
svi
OO | | )
| Adenovirus
| Post-transplantation Syndrome all
| | Epstein-Barr virus Serum i
| | HHG Blood
| | HEMORRHAGIC FEVER
| | Ebola/Marburg viruses Respiratory
| secretions,
—— serum
| Lassa fever virus OO Serum
| [HEPATITIS
| | Hepatitis | | Blood

Laboratory Processing
of Viral Specimens
|_ Source Specimen
| Blood Anticoagulated blood
_ CSF 1 mL CSF
| | Stool or rectal swab Pea sized or aliquot of feces
| | Genital, skin Vesicle fluid or scraping
| Respiratory tract Nasopharyngeal secretions, throat swab, respiratory tract
washings, sputum
| | Tissue Tissue in sterile container
| | Urine Midstream specimen
| Miscellaneous Swab, fluids

__redAlert: Serology Tests for Hepatitis Viruses

| Disease Virus Diagnostic tests
| | Hepatitis A Enterovirus 72 Antibody to Hepatitis A virus (IgG and IgM)
| Hepatitis B Hepadnavirus Hepatitis B surface- antigen (HBsAg) '
| Hepatitis B early antigen (HBeAg)
Anti-HBsAg
| Anti-HBeAg
| Anti-HB core antigen
|_ Hepatitis C Flavivirus Antibody to hepatitis C virus
Hepatitis D Delta agent (Hepatitis D virus) Antibody to delta agent
Hepatitis E Calicivirus-like (herpes virus) Antibody to hepatitis E virus

_ redAlert: Quantitation of Cell Culture Cytopathic Effects

| Quantitation Interpretation
| | Negati Uninfected monolayer
| Equivocal Atypical alteration of monolayer involving few cells
| 1%-25% of monolayer exhibits cytopathic effects (CPE)
| 25%-50% of monolayer exhibits CPE
| of monolayer exhibits CPE
50%-75%
76%-100% of monolayer exhibits CPE c
|

Actus coPage| 125 3

«il
 ort: The common childhood diseases
f
fu(* me or Other name ig ge el
disease Ca4, Meas] @, 14-4
, |eee ;
eee
| a
disease
scale lee , Seating
» German M
measles nana
Filalow-Duke’s sees 3-day
[a disease Scalded Skin Syndrome
usumoccalScala
| ymat
Erthe
ra ctioc
infeo
a infec
7 See

0) disease —
Exanthem subitum HHV-a
| @” disease__|

jt
LIV MastE-Acommon TTENUAVACGINE
TED .
ypoi d ( T y ) Influenza
[Rotavirus _
Epidemic typhus
| [Sabin_ |
BCG (Bacillus Calmette-Guerin) ad
| Yellow fever Chicken pox
| | Cholera we
Measles, Mumps, Rubella (MMR)
|
SES
| | yedAlert: List of ONCOVIRU

L| Hepatitis C
i Human Papillomavirus
HIV ce
Additional inclusion
EBV
ated herpesvirus
Kaposi sarcoma-assosius
| Merkel cell polyomavir
Human T-cell lymphotropic virus type 1

redAlert Common cell lines Application

Morphology/Species
Cell Line
Epithelial/Human Tumorigenicity and virus studies
HeLa B
Lymphoblast/Human Differentiation studies
s
HL60
Fibroblast/Mouse Tumorigenicity and virus studie
pl ication
312 clone A31
Fibroblast/Monkey Gene expression and virus re
COS-7 studies
Nutritional and gene expression
Epithelial/Hamster
| CHO studies
Transformation studies
Lt. Epithelial/Human
HEK293 n Signaling studies
Lymphoblast/Huma on,
Jurkat Epithelial/Canine Cell polarity, cell-to-cell adhesi
‘“WDCKK SCS response to growth factors studie
s
Canine Kid veolar
Madin-Darby
Epithelial/Human in vitro model of type Il al
ine
anne epithelial cells for drug metabolism
pci Daty Cani Mncltal
4 and as a transfection host.
uman Alvenoolmaar Basal Epithelia
Adenocarci cells

\Autus coPage| 126

 MOLECULAR DIAGNosTICs
{ Molecular diagnostics is a broad term describing a class of diagnostic tests that assess a person's health literally at a
molecular level, detecting and measurin if ibonucleic acid
(RNA) or the proteins they express, 9 Specific genetic sequences in deoxyribonucleic acid (DNA) or r!
2, Molecular diagnostics identify gene
» RNA, and protein variations that shed light on whether a specific person Is
predisposed to have a disease, whet
be effective for a specific disease. her they actually have a disease, or whether a certain treatment option is likely to
3, These tests also can detect and quantify the presence of specific viruses, bacteria, or types of cells.

AMPLIFICATION
Many molecular assays Use amplification to allow for the detection of the target or to ensure adequate copies of the target
for identification. Amplification enables detection of the target even when sample size is small or when there are very few
strands of DNA or RNA present. There are three types of amplification:

1. Target Nucleic Acid Amplification

The assay makes more copies of target nucleic acid before detection. Polymerase chain reaction (PCR) is the
typical target amplification method.

2. Signal Amplification
The assay increases the number of labels and signal from labels. Target number remains unchanged.

3. Probe or Probe Product Amplification

The probe specific to the target or probe product is amplified only in the presence of a target (target number
remains unchanged). Ligase chain reaction is an example of a probe amplification method.

Comparison of Nucleic Acid Amplification Methods

Method | Category Enzyme used Temp. Nucleic acid target
Requirement
PCR Target Tag DNA polymerase Thermal cycler DNA (RNA)
TMA Target Reverse transcriptase/RNA polymerase, | Isothermal RNA/DNA
ribonuclease H
NASBA Target AMV Reverse transcriptase, isothermal RNA/DNA
RNA polymerase, ribonuclease H
SDA Target Restrictive endonucleonase, DNA | Isothermal DNA
polymerase
LCR Probe DNA ligase Thermal cycler | DNA
bDNA Signal None \sothermal DNA/RNA
*TMA Transcription mediated amplification, NASBA Nucleic Acid Sequence-Based Amplification, SDA Strand Displacement
Amplification, LCR Ligase Chain Reaction, DDNA branched DNA
 common Types of Polymerase Chain Reaction (PCR)
erase Chain Reacti
. ee a specific DNA aot “an enzyme-criven, primer-mediated, temperature-dependent process or
reactions, the conditions of which va " only
Vito. The principle of PCR is based
in the tem eratur
on the repelitive cycling of three simple
Descriotion e of incubation.
nen

Temperature:
Purpose: 4) 95°C, template DNA double strand is separated
to two single strands.
Temperature: ©.
Purpose: Al 55°C f the two specific oligonucleotide
Se primers bind to the DNA template
Temperature: 2 © : 7
Purpose:
At 72°C, DNA polymerase extends the primers at the 3° of each primer and
Synthesizes the complementary strands along Sto 3’ of each template DNA. oat

3
. ; 2
Nucleotide

e% ! re adi
wa. » 5 a
e ; a

5S es 3 :
2 ee 5 Denaturation Annealing Extension
& eS = ™A
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"55a nsier ES ‘i
5
Original DNA

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Illustration of PCR process

seam A seans
2. Multiplex PCR
technique which allows an © Se B 26
a. ibis PCR is a type of PCR
86 Ss S ee
amplification of many target sequences concurrently in the same _ b 1 ;
. .
reaction mixture. == =
for different =—S_= =
b. A single reaction mixture includes sets of primer pairs
at ae a
DNA targets. It reduces the consumption of PCR reagents, and, i
the same time, imposes restrictions on used primers. a
be
¢. To work properly within one reaction, sets of primers must
optimized. They must have similar annealing temperatures and \y
produce amplicons of different sizes to form distinct gel _
analysis.
electrophoresis bands for the followed PCR

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i Attus coPage| 130

 | 3, Nested/semi-nested PCR
EEE

| In thistwo app
a. vith satroach,
of prgen
iseomic t emplate DNA ijs amplified
Ret Hnnninnnnnniin nae 3
| b. The first PCR set Produces ,

HH
. =
al i

that in second PCR set. :

pei rs
C. The second PCR set —_ -
uses the first POR pro
duct as
AS RE

=
iain ta oe wd et an internal region of DNA 4 oun
stina, econ . on
(nested/semi-nested) amplification
The primers in the second
PCR set can be different to
the first set (neste d) or one of the primer
s can be the
same as the first set (semi-nested),
—
1. The first round of PCR is the same procedure in
This method can be u increase we eneral PCR.
detection or to setts pi ek <i eaimautan 2. the 1* PCR product will be used as a template
the primers in the second PCR reaction are species- for second PCR with the use of specific primers.
specific,

4. Quantitative PCR (qPCR)

a. The amount of product that is synthesized during a set numbe
r of cycles
on the number of DNA molecules that are present in the starting mixture. ofThisa polym erase chain reacti
enables PCR to be usedonto depen
quantds ify
the number of DNA molecules present in an extrac
t.
In quantitative PCR the amount of product synthesized during a test PCR is compared with the
amounts synthesized
during PCRs with known quantities of starting DNA.

Real-time PCR
Quantification is carried out by
real-time PCR - a modification of the - &
standard PCR technique in which v
synthesis of the product is measured
over time. RNA extract Well reaction mixture

Transcription-Mediated Amplification (TMA)

| 1 Is an RNA transcription-mediated amplification system using three
enzymes to drive the reaction:
a. RNA polymerase
b. Reverse transcriptase (RT)
¢. RibonucleaseH
2. Principle:
One primer contains a 1/ promoter sequence for RNA
polymerase that hybridizes to the targer RNA.
b. RT creates a cDNA of the target RNA by extension from 3° end
of the promoter-primer
c. The RMA of the resulting RNA:DNA duplex is degraded by the
[tte got smhreuou

RNAse H activities of the RT.

d. Asecond primer then binds to the cDNA containing the promoter |
sequence fro the T7 promoter-primer. ———

\J Actus coPage| 131

 Nucleic Acid Sequence-Based
Amplification (NASBA)
1. RNA detection is commonly d one due
using RT-PCR, a time-consuming process often resu iting in false positives
to cross contamination.
7
a
Alternatively, nucleic acid sequence-based amplification (NASBA) is a one-step isothermal process for amplifying
RNA.
3. NASBA has proven to be Successful in detection of both viral and bacterial RNA in clinical samples.

COMPONENTS:
A NASBA reaction consists of Avian M
yeloblastosis
RNase H with two oligonucleotide primers,
virus (AMV) reverse transcriptase (RT), T7 RNA polymerase and
The amplification is more than 1000 fold in 90 to 120
minutes.
wp

on of dsDNA does not occur.

a SSRNA is possible only when the denaturati , as in the case of RT-PCR.
The NASBA reaction does not get false positives caused by genomic dsDNA
Ma anna
Molecular
Beacon
C) Z RNaseP2
H Reverse
Sense RNA Transcriptase
seaidbanins NT Mei
any =
P2 + —
T7 RNA Polymerase
i.

Amplification —
ac Process

eS
Light

NASBA Reaction Process (hitp:/Awww.premierbiosoft.com/tech_notes/NASBA)

This mechanism requires two short single stranded DNA fragments primers and three enzymes.
1. The viral RNA strands are represented as the sense strand present in the original samples.
2. Primer P1 binds to the RNA and is elongated by reverse transcriptase (AMV-RT). The RNA strand of the yielded
DNA:RNA hybrid is hydrolyzed by RNase H.
3. After the binding of P1, primer P2 can also bind. Primer P2 is then elongated by AMV —RT, yielding a double-stranded
DNA molecule.
redAlert: Primer P1 is designed in such a manner that when it forms a double- stranded DNA, its codes fora T7 RNA
polymerase Promoter site. This helps in generating antisense RNA copies using a DNA template.
4. The new copies of DNA are generated using RNA. The process is same as followed for sense strand. Here in this case,
P2 will bind first.

redAlert!
1. Ina NASBA reaction, DNA is the final product formed. It has a promoter region. This allows T7 RNA polymerase to
use it as a template. The reaction sequences starting from the sense and antisense RNA strands are different, but they
} are kinetically identical. They both are referred to as copy DNA (cDNA).
| 2 Molecular Beacon?! Molecular beacons are single stranded hairpin shaped oligonucleotide probes. Their 5' end has
a fluorescent label and the quencher is present at 3' end. These molecular beacons are highly specific to their targets.
| They form a stable hybrid with their amplified target RNA, when present in a NASBA amplification reaction.

\Avtus coPage| 132 6

 ~ grandSpA displacement amplification (SDA)
is an isothermal, in vitro DNA - ee
FS
)
technique that is | —————-#=——AAR-
| * smplification

—-* ete - "Fenn

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| a. The ability of a restriction ep | ht aes ;
to nick the unmodified |,, ..
enzyme a" ™™
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strand of |
of | mittee | / Cae
| hemiphosphorothioate form Falie ;
its recognition site = |
| aioe ms - Terget DNA
|
ba * [a;
i
“ a am
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replication at the nick and
|
y |
coe |
displace the downstream non-
template strand. www. researchqate.net/figure/Strand-displacement-amplification-SDA
9, Primers containing recognition sites for the nicking restriction enzyme bind to opposite strands of target DNA at
positions flanking the sequence to be amplified,
in which strands displaced
, The target fragment is exponentially amplified by coupling sense and antisense reactions
from the sense reaction serve as a target for the antisense reaction and vice versa.

Ligase Chain Reaction Process Primer | Primer 2

1. Denaturation- DNA is heated so that the double- Target Target

stranded structure becomes single-stranded.

J Annealing
2. Annealing- Two sets of DNA probes attach to each end
of the single strands of DNA. This step marks where the
duplication of the DNA will begin. From this point, Taq
polymerase will add nucleotides based on the already | Ligation
existing DNA strands.
3. Ligation- The DNA ligase will fill in the gap in the DNA
strand between the probes by filling in the appropriate
primer. Ligase will only recognize and ligate primers that Additional cycles of
properly anneal to the probe. denaturation, annealing, ligation

Branched DNA (bDNA)

1. Target RNA is captured with a bifunctional
Capture Extender oligonucleotide probe that
hybridizes to the target molecule and a Capture
Probe that is covalently attached to a substrate.
_ & A Signal Amplification complex (Preamplifier
| and Amplifier with labeled probes) containing
Several alkaline phosphatase enzymes is then
hybridized to the target molecule via a Label
Extender probe.

Tsongalis 2006

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