Respiratory System Module

2021-2022

Dr. Mohammad Odibate

Department of Microbiology and Pathology

I

Laboratory Diagnosis of Group A Streptococcus

Steps of Laboratory Diagnosis of Group A

\: i,~~ Streptococcus ~ \~~ p

ro , li"J ---±;l> Specimen co 11 ecti on

J,«y,a >•• 2. Direct Antigen detection

3. Group A streptococci screening culture

4. Identification of GAS ,, & ro ~~ A s \-r~ p ~0 c 0 c, u.~ ''

5. Reporti ng results.

Laboratory Diagnosis of Group A Streptococcus

1- Specimen:

Throat swab of tonsillar area and/or posterior

pharynx (Avoid the tongue and uv~ la)

Swc1b

Throat is swabbed

in the area o f

the tonsi ls

Laboratory Diagnosis of Group A Streptococcus

~ . 2. Direct Antigen detection: 1. The pati ent's throat is first swabbed to collect a sampl e of mucu s. 2. The sample is applied to a strip of nitrocellulose film and, if GAS ant ige ns

are present, these will migrate along the film to form a vi sibl e line of antigen bound to labeled antibodies

3. Because a common probl em is the low se nsitivity. All negat ive res u lt s should be followed by culture.

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Laboratory Diagnosis of Group A Streptococcus L ~ - \il<: ¥V\ c) J ~ ,·c_

3. Group A streptococci screening culture Incubate cultures under atmospheric conditions (35°C for 18-24h) Examine the presence of hemolytic colonies on blood agar Re incubate negative cultures for an additional 18-24h ~ --==--------

~-hemolys is (Group A streptococci)

a -he mo lys is +-------iii~----.,._;.

cl e."r \ :: •

Z c' tt e q ro\.\" J ·. -hemolysis

Laboratory Diagnosis of Group A Streptococcus

4. 1ldentification of GAS:

Catalasetest - ve_

~acitracin susceptibilty (\(\ '--' b \·.)\-t'c_ - Pr inciple:

• Fo r identification of group A

• dist ingujsh between 5. pyogenes from other beta hemolytic streptococ€i

~ e,.,c; 1 h lJ e

• Strep. pyogenes is sensitive to Bacitracin giving zone of inhibition aro und disk

Group A streptococci is susceptible to

Ba cit ra c i n disk ( I eft); The right shows

resistance

Laboratory Diagnosis of Group A Streptococcus

5. Reporting results:

The results on the microbiology request form may include

- 5. pyogenes group A isolated

beta hemoltyic streptococci, not group A streptococci

isolated

No 5. pyogenes or beta hemolytic steptococci

DIAGNOSIS OF DIPHTHERIA

Diagnosis r')(/''7 br<H\~ J , u-tJ

1. The initial diagnosis of diphtheria is entirely cli nical ~ _)\ ~ ._3

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2. Laboratory diagnosis C· c.1_,l;

A. Specimen: from the nose and throat and any other mucocuta neous

lesion. A portion of membrane should be removed and subm itted for

culture along with underlying exudate

B. Direct smear:

Gram stain: club shaped Gram positive bacilli with chinese lette r

arrangment

C. Culture media: cysteine-tellurite plate (Tisdale agar)

Results:

• C. diphtheriae : produce grayish -black coloni es, surround ed by a

brown/black halo.

D. Urease and oxidase negative, Catalase positive

UIAUNOSIS O F DIPHTHERIA

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DIAGNOSIS OF DIPHTHERIA

F. Toxin demonstration. As the pathogenesis is due to diphtheria toxin, isol ation of bacill i dose not complete the diagnosis. Toxin demonstration should be done following isolation, which can be of two types, in vivo and in vitro

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DIAGNOSIS OF DIPHTHERIA L_ __ ~~:..:..:.~-=----=--=------------

Elek 's test: rapid diagnosis (16-24 hrs) Sterile filter paper with C. diphtheriae antitoxin

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(positive contro l)

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DIAGNOSIS OF DIPHTHERIA btl)JJ,::11 c? -

Elek 's test: rapid diagnosis (16-24 hrs)

DIAGNOSIS OF DIPHTHERIA t ~<~ ~ ), 1~~r

Elek 's t est: rapid d iagnosis (16-24 hrs)

Results:

Positive test: formation of four radiating lines resulting from the precipitation reaction between exotoxin and diphtheria antitoxin.

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Laboratory Diagn osis Lower Respiratory Infecti on

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Re» f'•,.,,r~putum culture 1' \ ~ C'c. ~.·v"' Th I · · . I e sputum cu ture 1s an important part of the diagnostic evalu at ion of potent1 a

lower respiratory tract infections . However, expectorated sputum specimens are

variably contaminated by colonizing oropha ryngeal flora , ma king resu Its hard to

interpret. Proper collection of the specimen is crucial to the re covery of the

etiological agent.

Specimen criteria: • If possible, specimen should be collected before antimicrobial treat m ent.

• First morning specimen is best.

• Spec imen must be collected in a sterile container.

• If multiple cultures are ordered they should be collected at least 24 hours

a pa rt.

Laboratory Diagnosis Lower Respiratory Infection

~ ~ xpectorated sputum ;.::,. l; • Specimen collection should be supervised by a trained

()e.,q> _h req H-, professional. 0 \.:_\ ~

~\ ~_J_) • Request the patient to remove any dentures and to

rinse the mouth or gargle with plain water before

specimen collection.

• Tell the patient to provide a specimen fro m a deep

cough, avoiding, as much as possible, mixing the

specimen with saliva or nasal secretions.

• Make sure the patient understands the difference

between saliva (from mouth) and sputum (from chest).

Laboratory Diagnosis Lower Respiratory Infection

Specimen Quality

Poor quality sputum Better quality

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Laboratory Diagnosis of H. lnfluenzae 1. Specimen collection and transport

D Depending on the site of infection, various specimn s m ay be collected such as CSF, blood, respiratory tract sputum, throat swabs, midd le ear, and sinuses

□ As H. influenze is highly sensitive to low tempertures, the specimen should neve r be refragutrated

□ Sample should be trasported and proscessed immdediatly w it hout any delay.

2. Direct detection: □ Gram staining : preparation from different sampls may show gram -negat ive

coccobacilli z;, \S \ )\ --0

c,,rs,, h ra--J 0 Capsule detection (Quellung reaction)

Antigen detection: The t yp e b ca psular anti gen can be detcted in CSF, urine , or other bdy fuids by

latex agg lutin ation using particl es co at ed with anti bodi es to type b antige n o r Direct im mun oflurcscnce tes t .

Laboratory Diagnosis of H. lnflu~r,_zpe . .. - - - ~- ,, .. . , .. . . ._.., - . . . .. "" .. ,.... ..,

Capsule detection (Quellung reaction}

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Laboratory Diagnosis of H. lnfluenzae

Culture: L~ ~ L'\ $ ~ i J I ,., \,\ )

A.

B.

Culture conditions: aerobic with 5-10 % CO2.

Culture media used are as follows:

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Blood aga r wi th 5. auru es strea k line : Co lo nies of H. influenzae grow adjacent to). RB <..

aurues strea k line (ph enomenon is known as sat elli ti srn)

Choclet agar

H. !nfluenzae grow around 5. aureus utilizing

X & V factors released from hemolyzed RBCs H. lnflu enzae grown on Choclet agar

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Laboratory Diagnosis of H. lnfluenzae ~ ,

Growth requirements

Laboratory Diagnosis of H. I nfl uenzae

4. Biochemical tests:

• Reduces nitrate to nitrite.

• Catalase and Oxidase positive

• Fermentation of sugars: Glucose(+),

Sucrose(-), Lactose(-) Mannitol (-).