Examination of throat

Examination of throatand mouth specimens

Lecture 2

Possible pathogens Gram positive Streptococcus

pyogenes Corynebacterium diphtheriae Corynebacterium

ulcerans

Gram negative Vincent’s organisms

Note: Pathogens in the upper respiratory tract such as Bordetella pertussis, Streptococcus pneumoniae, and Neisseria meningitidis, are usually more successfully isolated from naso-pharyngeal secretions collected by aspiration

Notes on pathogens

S. pyogenes, Lancefield Group A beta-haemolytic Streptococcus is the commonest cause of bacterial pharyngitis (sore throat), especially in young children. Its association with rheumatic heart

The term scarlet fever is used when streptococcal pharyngitis is accompanied by a characteristic skin rash

Sore throat Fever Bright red tongue with a "strawberry" appearance

Notes on pathogens

● C. diphtheriae produces a powerful and often fatal exotoxin and

therefore when diphtheria is suspected, the patient is treated

immediately with antitoxin. The role of the laboratory is to

confirm the clinical diagnosis.

● Infection with Vincent’s organisms (Borrelia vincenti in

association with Gram negative anaerobic fusiformbacilli) causes

Vincent’s angina (Vincent’s gingivitis), an ulcerative tonsilitis with

tissue necrosis.

CommensalsGram negative Gram positive

Moraxella catarrhalis Viridans streptococci

Neisseria pharyngitidis Non-haemolytic streptococci

Fusobacteria Staphylococcus epidermidis

Coliforms Streptococcus pneumoniae

Bacteroides species Micrococci

Haemophilus influenzae Lactobacilli

DiphtheroidsAlso various spirochaetes, actinomycetes, aerobicand anaerobic spore forming.

COLLECTION AND TRANSPORT OF THROAT AND MOUTH SWABS

In a hospital with a microbiology laboratory

1 In a good light and using the handle of a spoon to depress the tongue, examine the inside of the mouth. Look for inflammation, and the presence of any membrane, exudate, or pus.

– With diphtheria, a greyish-yellow membrane (later becoming greyish green-black and smelly) can often be seen extending forwards over the soft palate and backwards onto the pharyngeal wall.

– With a streptococcal sore throat, the tonsils are inflamed and often covered in yellow spots.

– With Vincent’s angina, there is ulceration of the mouth, throat, or lips.

COLLECTION AND TRANSPORT OF THROAT AND MOUTH SWABS

2 Swab the affected area using a sterile cotton wool

swab. Taking care not to contaminate the swab with saliva, return it to its sterile container.

Important: For 8 hours before swabbing, the patient must not be treated with antibiotics or antiseptic mouth-washes (gargles).

Caution: It can be dangerous to swab the throat of a child with acute haemophilus epiglottitis because this may cause a spasm that can obstruct the child’s airway. Blood for culture should be collected instead.

3 Within two hours of collection, deliver the swab

with a completed request form to the laboratory.

In a health centre for dispatch to amicrobiology laboratory

1 Using a sterile swab (supplied in a tube of silica gel by the microbiology laboratory), collect a specimen from the infected area as described under the hospital collection of throat swabs.

2 Taking care not to contaminate the swab, return it to its tube. Seal with adhesive tape and label the tube.

Culture the specimenBlood agar

– Inoculate the swab on a plate of blood agar Use the loop to make also a few stabs in the agar (well area). Colonies of S. pyogenes growing below the surface will show more distinct zones of haemolysis because of the anaerobic conditions provided.

– When a swab is received in silica gel (e.g. from a health centre), moisten it first with sterile nutrient broth and then inoculate the plate.

– Add a 0.05 unit bacitracin disc (Reagent No. 15) to the plate. This will help in the identification of S. pyogenes (see subunit 7.18.2). Some workers also add a co-trimoxazole disc (as used for

susceptibility testing) which prevents the growth of other bacteria, making it easier to see betahaemolytic S. pyogenes colonies.

– Incubate the plate preferably anaerobically or, when this is not possible, in a carbon dioxide enriched atmosphere overnight at 35–37 C. Candle jar incubation will detect

Culture : Blood agar: S. pyogenes produces betahaemolytic colonies, i.e. the colonies are surrounded by a zone of complete

haemolysis with decolorization of the haemoglobin. Colonies are usually small (0.5–1 mm), colourless, dry, shiny or mucoid. Haemolysis is more marked under anaerobic conditions as seen in colonies growing below the agar surface (following stabs made in the culture medium.

Choice of blood To isolate beta-haemolytic streptococci, use sheep blood (1st choice),

horse, rabbit or goat blood to prepare blood agar plates. Do not use human blood because this may contain unwanted substances such as citrate (e.g. donor blood), antibiotics, or antibodies such as ASO or anti-M protein that could interfere with the growth or haemolytic activity of S. pyogenes.

Note: Other beta-haemolytic streptococci belonging to other Lancefield groups) also produce colonies similar to S. pyogenes. Betahaemolysis may also be seen with some strains of S. aureus, Haemophilus (particularly from throat swabs), Corynebacterium and Moraxella. It is therefore important to examine a Gram stained smear of the culture.

A catalase test can be used to differentiate streptococci (negative) from staphylococci (positive).

Group B: Streptococcus

Immunological detection of S.pyogenes.

Direct detection of antigen A from throat swab extracts

Several tests have been developed to detect antigen A directly

extracted from throat swabs without the need to culture the

specimen, thus providing an early presumptive diagnosis of

streptococcal sore throat.

Direct antigen A detection tests are highly specific but sensitivity

varies between manufacturers.

Test is also influenced by the quality of the specimen. Culturing

is required when infection with S. pyogenes is suspected and a

direct antigen test is negative

PYR (pyrrolidonyl) test: This detects pyrrolidonyl peptidase enzyme

activity. Besides S. pyogenes, Enterococcus species and occasionally streptococci belonging to groups C and G are also PYR positive.

The test can be rapidly and simply performed using PYR impregnated strips

Culture of specimen when diphtheria is suspected

When diphtheria is suspected and culture is specifically requested, inoculate the swab on Tinsdale medium or tellurite blood agar Incubate the plate aerobically at 35–37 C for up to 48 hours, examining for growth after overnight incubation.

Tellurite blood agar: This medium is widely

used as a primary medium for isolating C.diphtheriae

from throat and nasopharyngeal swabs. C. diphtheriae reduces tellurite and produces grey or grey-black colonies measuring 0.5–2 mm in diameter after 24–48 h incubation

Corynebacterium diphtheriae cultivated on Tinsdale agar.Cultivation 72 hours, 37 °C in an aerobic atmosphere

Tinsdale medium

Tinsdale medium: After 24–28 h incubation,

C. diphtheriae colonies are grey-black, raised,

and surrounded by a dark brown area. The brown color is due to the hydrogen sulphide produced from the cystine interacting with the tellurite.

Occasionally commensal diphtheroids and other respiratory tract commensals may grow on Tinsdale’s medium but the colonies are not surrounded by a brown halo like those of C. diphtheriae.

Bordetella pertussis Specimens: Preferably nasopharyngeal secretions collected

by aspiration or a correctly taken pernasal swab Cultur: Bordetella species are strict aerobes. Specimens for

the isolation of B. pertussis must be cultured as soon as possible after they are collected. A selective and enrichment medium such as charcoal cephalexin blood agar is recommended for the primary isolation of B. pertussis.

Charcoal cephalexin blood agar: When incubated for 2–6 days at 35–37 ºC in a moist aerobic atmosphere, B. pertussis produces small pearly-grey, shiny (mercury-like), usually mucoid colonies.

B. parapertussis grows more rapidly and forms larger colonies than B. pertussis. It produces a pigment in the medium and is able to grow aerobically on blood agar and nutrient agar

Examine the specimen microscopicallyGram smear

spread smear of the specimen on a slide. Allow the smear to air-dry in a safe place. Fix, and stain by the Gram technique . Use dilute carbol fuchsin (1 in 10 dilution) as the counter stain in preference to safranin or neutral red (stains Vincent’s organisms better). Examine the smear for pus cells and Vincent’s organisms:Vincent’s organisms: These are seen as Gram negative spirochaetes (B. vincenti) and Gram negative fusiform rods

Other bacteria: No try should be made to report routinely other bacteria in a Gram stained smear from a throat swab because the throat contains a wide variety of commensals that cannot be distinguished morphologically from pathogens.

Pathogenesis Vincent angina is caused by Borrelia vincenti. Borrelia vincenti is anaerobic spirochaete which lies

commensal in healthy human mouth. Under certain conditions which cause injury of the

mucous membrane, Borrelia vincenti together with anaerobic cigar shaped fusiform bacilli (fusobacterium) multiply in the lesions causing ulcers.

These ulcers may become covered by pseudomembrane containing pus cells and necrotic tissue.

Laboratory diagnosis Specimen: smears prepared from the pseudomembrane. Direct microscopic examination stained with Gram:

Gram negative borrelia in large numbers +

Gram negative cigar shaped fusiform bacilli

+ pus cells.

Albert stain Albert stained smear when diphtheria is suspected, we can see

pleomorphic rods containing dark-staining volutin granules . The pleomorphic rods tend to join together at angles giving the appearance of Chinese letters.

Pleomorphism and granule formation are best seen in smears from a Loeffler serum or Dorset egg medium culture.

Smears directly from specimens may not show these features.

Volutin granules It is also possible for commensal diphtheroids to contain volutin

granules but the commensals are not pleomorphic like C. diphtheriae. When C. diphtheriae is cultured on tellurite blood agar and modified

Tinsdale medium, granule formation is usually limited. Note: In Gram stained smears, C. diphtheriae stains variably and

weakly Gram positive, whereas commensal diphtheroids appear strongly Gram positive.

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