DIAGNOSIS OF ORAL INFECTION

ORAL INFECTIONS

Bacterial infection•Impetigo •Erysipelas •Scarlet fever •Streptococcal phayngitis • Syphilis •Diphtheria •Gonorrhea •Leprosy •Tuberculosis•Actinomycosis •Cat scratch disease

Fungal and ProtozoaL infections•Candidiasis•Histoplasmosis•Blastomycosis•Coccidiomycosis •Cryptococosis•Zygomycosis •Paracoccidiomycosis•Aspergillosis •Toxoplasmosis

Viral infections•Human herpes viruse•Herpes simplex viruses •Varicella •Herpes zoster •Infectious mononucleosis •Enteroviruses •Rubeola •Rubella •Mumps •Human immunodeficiency virus

DIAGNOSIS OF IMPETIGO

Impetigo is usually diagnosed on the basis of clinical findings. A strong presumptive diagnosis can normally be made from clinical presentation

The Nonbullous impetigo appears as red macules or papules with subsequent development of the vesicles.amber colour rust form on the vesicles which is adherent and can be described as “cornflakes glued to the surface

Bullous impetigo lesions are categorized by superficial vesicles that enlarge to form flaccid bullae which are filled with clear serous fluid.later on the fluid becomes turbid and purulent.

Laboratory findings in impetigo Bacterial culture and sensitivity are recommended (1) in cases to

identify methicillin-resistant Staphylococcus aureus (MRSA), (2) if an outbreak of impetigo has occurred, or (3) if poststreptococcal glomerulonephritis is present. Exudate from underneath the crust is sent for culture.

Leukocytosis Antideoxyribonuclease (anti-DNAase) B antibody levels are often

elevated in persons with streptococcal impetigo. A bacterial culture of the nares may be obtained to determine whether

a patient is an S aureus carrier. If the nares culture is negative and the patient has persistent recurrent

episodes of impetigo, bacterial cultures should be obtained from the axillae, pharynx, and perineum.

Obtain serum IgM levels in cases of recurrent impetigo in patients with negative S aureus carrier status and no predisposing factors such as a preexisting dermatosis.

A biopsy may be necessary in doubtful or refractory cases of impetigo

DIAGNOSIS OF ERYSIPELAS

Clinical findings: commonly occurs in traumatic regions.most commonly affected area is the leg. In facial erysipelas increase prevalence is noted in the spring and winter months. The lesion appears as butterfly rash involving cheeks,eyelids and bridge of the nose.skin demonstrate a surface texture that resemble an orange peel (peau d’orange). High fever and lymph- adenopathy are often present. Culture are usually not beneficial therefore diagnostic confirmation is difficult.

Laboratory Studies In general, diagnosis of erysipelas is made clinically. Complete blood count (CBC): Increased WBC with a may be observed

but is not specific for the diagnosis. Blood cultures

Positive in only 5% of cases Antistreptolysin (ASO), streptozyme, and anti-DNAase titers may be

helpful.

Differential diagnosis of erysipelasContact dermatitis Angioneurotic edemaLupous erythematousHerpes zoster

Differential diagnosis of impetigoContact dermatitis by poison ivyTinea infectionHerpes simplex infecton

DIAGNOSIS OF SCARLET FEVER

Clinically disease occuras erythmatous and edematous tonsils ,soft palate and phaynx. Tonsillar crypts may be filleda yellowish exudates.

during first 2 days tongue demonstrate a white coating though which only the fungiform papilae can be seen.this has been called as white strawberry tongue.

By the fourth or fifth day white coating desquamates to teveal an erythematous dorsal surface and called as red strawberry tongue.

The pastia ‘s lines seen in the folds of the rash. Patient present with high fever.

A culture of throat secretion may be used to confirm the diagnosis, but this has been replaced by the several methods of detection of the antigens that are specific for group a beta hemolytic streptococci.

Laboratory Studies Throat culture remains the criterion standard for confirmation of group

A streptococcal upper respiratory infection. Throat cultures are approximately 90% sensitive for the presence

of group A beta-hemolytic streptococci in the pharynx. Vigorously swab the posterior pharynx, tonsils, and any exudate

with a cotton or Dacron swab under strong illumination, avoiding the lips, tongue, and buccal mucosa.

. Direct antigen detection kits (ie, rapid antigen tests [RATs], strep screens) have been

proposed to allow immediate diagnosis and prompt administration of antibiotics. Kits are latex agglutination or a costlier enzyme-linked immunosorbent assay

(ELISA). Several studies of RAT kits report results of 95% specificity but only 70-90%

sensitivity. Streptococcal antibody tests are used to confirm previous group A streptococcal

infection The most commonly available streptococcal antibody test is the antistreptolysin O

test. Complete blood count

White blood cell (WBC) count in scarlet fever may increase to 12,000-16,000 per mm3, with a differential of up to 95% polymorphonuclear lymphocytes.

During the second week, eosinophilia, as high as 20%, can develop

Differential diagnosis

Mononucleosis Kawasaki Disease

RoseolaStaphylococcal Scalded Skin Syndrome

DIAGNOSIS OF DIPTHERIA

Clinical features Initial systemic symptoms include low grade fever headache malaise,sore throat etc. pseudomembrane formation of one or both the tonsils as a patchy

yellow white thin film that thickens to form an adherent gray covering

attempts at removal results in bleeding.aithough the clinical presentation can be distinctive in severe cases but lab confirmation is sought in all instances.

Laboratory Studies The specimen for culture should be obtained from underneath the

surface of the membrane. Others areas for the culture are the nasal mucosa and swabs from the wounded surface.

Diagnostic tests used to confirm infection combine isolation of C diphtheriae on cultures with toxigenicity testing

Bacteriologic culturing is essential to confirm the diagnosis of diphtheria. Obtain specimens from the nose and throat (ie, nasopharyngeal

and pharyngeal swab) for culture. Obtain specimens from the membrane as well as from the nose

and throat. If possible, swabs also should be taken from beneath the membrane

isolation of C diphtheriae requires special culture media containing tellurite. C diphtheriae may be grown on various selective media, including tellurite agar or specially enriched Loeffler, Hoyle, Mueller, or Tinsdale medium.

Toxigenicity testing is also performed. Perform toxigenicity testing using the Elek test to determine if the

C diphtheriae isolate produces toxin. Toxigenicity tests are not readily available in many clinical

microbiology laboratories; send isolates to a reference laboratory with personnel proficient in performing the tests

Measurement of the patient's serum antibodies to diphtheria toxin before administration of antitoxin may help assess the probability of the diagnosis of diphtheria.

If antibody levels are low, diphtheria cannot be excluded, but if levels are high, C diphtheriae is less likely to produce serious illness. 

Differential diagnosisVincent anginaExudative pharyngitis due to Streptococcus pyogenes and Epstein-Barr virusMucositisHerpes Simplex Virus InfectionImpetigoCandidiasis

DIAGNOSIS OF GONORRHEACLINICAL PRESENTATION: Most common site of orophayngeal involvement is phaynx along with

tonsils and uvula. Mild to moderate sore throat Involved tonsils demonstrate edema and erythema with scattered

small punctate pustules. Rarely involve anterior part of oral cavityand infectiuos areas appear

as erythematous,pustular edematous and ulcerative simulating NUG. Cervical or submandibular lymphadenopathy may be seen.Laboratory Studies Males with a urethal discharge, a gram stain of the purulent material

can be used to demonstrate gram negative diplococcic within the neutrophils.

Although gram stains may be beneficial, confirmation of the diagnosis is recommended by culture of endocervical swabs if conditions are adequate to maintain the viability of the organisms. Nucleic acid amplification tests (NAATs) amplify and detect N.gonnorhea-speciic DNA or RNA sequences are recommended for the diagnosis when onditions are not adequate to maintain the viability of the organisms.

in spite of the availaibility of NAATs, culture remains the preferred diagnostic method for diagnosis of oropharyngeal infections.

Culture is the most common diagnostic test for gonorrhea, followed by the DNA probe, and then polymerase chain reaction (PCR) and ligand chain reaction (LCR).

The DNA probe is an antigen detection test that uses a probe to detect gonorrhea DNA in specimens.

PCR and LCR are gene amplification techniques that markedly increase the sensitivity of specimen testing. Both techniques amplify the genetic fingerprint of specimens with very few organisms present in order to more easily detect and identify the organisms

Perform a culture or nonculture detection test for N gonorrhoeae on endocervical, urethral, pharyngeal, or rectal discharge. Because organisms are intracellular, attempt to obtain specimen in a manner that will contain mucosal cells and not merely discharge

Culture is performed on Thayer-Martin plates that must be stored refrigerated but warmed to room temperature before obtaining sample. The plate is then incubated in a carbon dioxide atmosphere. Poor technique drastically reduces test sensitivity.

Histologic Findings A Gram stain of urethral or cervical discharge may show

gram-negative intracellular diplococci (diagnostic in the male) and polymorphonuclear cells.

This is very useful if the physician has easy access to a microscope because the diagnosis may be made without waiting for culture results.

The presumptive diagnosis is made by the medical history of the patient. The definitive diagnosis of tuberculosis is made by demonstration of positive delayed hypersensitivity skin reaction purified protein derivative and demonstration of acid fast bacilli in clinical specimen.

Clinical findings: Primary tuberculosis is usually asymptomatic. Secondary tuberculosis have a low grade fever,

malaise anorexia, weight loss and night sweats. Oral lesion of TB are uncommon, with most cases

appearing as a chronic painless ulcers. Less frequent presentations include nodular, granular

or firm leukoplakic areas. Primary oral TB usually involves the gingiva,

mucobuccal fold and ares of inflammation adjacent to teeth . Tese lesion are usually associated with enlarged regional lymph nodes.

TUBERCULOSIS

Secondary oral lesion present on tongue palate and lips.

Scrofula is a mycobacterial infection characterized by by enlargement of oropharyngael lymph nodes and cervical lymph nodes.

Caseous necrosis occurs in lymph nodes and form numerous sinus tract through overlying skin.

Hitopathologic features CMI is responsible is responsible for

classic histopatholgic presentation of TB. Areas of infection demonstrate granulomas formation which are circumscribed collections of epithileoid histiocytes, lymphocytes and multinucleated giant cells often with central caseous necrosis.

Special stains such as Ziehl-neelson or other acid fast stains are required to demonstrate the mycobacteria.

Lab tests Tuberculin tests or purified protein derivative skin tests.o A positive tuberculin tests result in dictates exposure and to the

organism and does not distinguish infection from active disease. Detection of mycobacteria by special stains Culture PCR

Differential diagnosis SarcoidosisBlastomycosis Catscratch diseaseActinomycosis AspergillosisNocardiosis ParacoccidiomycosisHistoplasmosisBronchiectasis

LEPROSY

The definitive diagnosis is based o the clinical presentation and supported by the demonstrated of the acid fast bacilli on a smear or in the tissue. The organism cannot be cultivated on artificial media , but M.leprae without developing the disease ; this creates difficulties in establishing the diagnosis and determining the prevalence of infection

Clinical presentation : clinically disease occurs in two forms paucibacillry and multibacillary

with distinction influencing the recommende formof therapy. oral lesion in the paucibacilary form is rare. The multubacillary lesion occurs mostly on face and skin

enlargements can lead to distorted facial appearances( leonine facies).nasal involvement results in nose bleed,stuffiness, and loss of sense of smell. The hard tissue of floor,septum and bridge of nose may be affected. Collapse of the bridge of nose is considered pathognomonic.

The oral location affected in order of frequency are hard palate ,soft palate, labial maxillary gingival, and buccal mucosa.

affected soft tissue initially appear as yellowish to red ,sessile ,firm, enlarging papules that develop ulceration and necrosis followed by attempted healing by secondary intention.

The infection creates a unique pattern of facial destruction that has been termed facies leprosa and demonstrate a triad of lesions consisting of atrophy of anterior nasal spine, atrophy of anterior maxillary ridge and endonasal inflammatory changes.

Granulomatous involvement of the nasal cavity can erode through the palatal tissues and result in perforation.

Laboratory StudiesLaboratory studies include the following: Skin biopsy, nasal smears, or both are used to assess for acid-fast

bacilli using Fite stain. Biopsies should be full dermal thickness taken from an edge of the lesion that appears most active.

Serologic assays can be used to detect phenolic glycolipid-1 (specific for M leprae) and lipoarabinomannan (commonly seen in mycobacteria)

Laboratory tests related to drug treatment follow-up include the following: CBC count Creatinine level Liver function tests Other Tests Immunologic tests include the following

Lepromin skin test. Phenolic glycolipid-1: This is a specific serologic test based on the

detection of antibodies to phenolic glycolipid-1. This test yields a sensitivity of 95% for the detection of lepromatous leprosy but only 30% for tuberculoid leprosy.

Polymerase chain reaction (PCR): PCR and recombinant DNA technology have allowed for the development of gene probes with M leprae –specific sequences. This technology can be used to identify the mycobacterium in biopsy samples, skin and nasal smears, and blood and tissue sections.

Lymphocyte migration inhibition test (LMIT): As determined by a lymphocyte transformation and LMIT, cell-mediated immunity to M leprae is absent in patients with lepromatous leprosy but present in those with tuberculoid leprosy.

Histologic Findings Findings vary but can include giant cells, infiltration of nerve bundles

with mononuclear cells, and granulomas. Lepromatous lesions generally contain numerous acid-fast bacilli and fat-laden macrophages with a paucity of lymphocytes. Histopathology of leprosy is seen in the image below. 

Histopathology of leprosy: Large numbers of acid-fast bacilli (in clusters) in histiocytes and within nerves. Fite-Faraco stain 500x

DIAGNOSIS OF SYPHILIS The presumptive diagnosis can be made by the

clinical features of syphilitic lesions. The definitive diagnosis of the disease can be confirmed by demonstration of the spiral organism and by various lab findings.

Clinical featuresPrimary syphlis: Characterize by chancre that develop at site of

inoculation May be solitary or multiple in numbers. External genitalia and anus are the most

common site. Oral cavity is most common extragenital site. Oral leions : commonly seen on lips, painless and clear based ulceration bilateral regional lymphadenopathySecondary syphilis: discovered after 4 to 10 weeks after initial

infection. A consistent sign is diffuse, painless

maculopapular rash and heals without scarring. Systemic symptoms are also there.

Elevated mucous patches may be centered over the crease of oral commisure and termed as split papules.

Papillary lesion resemble viral pappilomas known as condylomata lata may be occasionally present

Tertiary syphilis: Gumma : indurated, nodular or ulcerated

lesion that may produce extensive tissue destruction.

Tongue appear large , lobulated and irregularly shaped. This lobulated pattern is known as interstitial glossitis.

Leutic glossitis : diffuse atrophy and loss of dorsal tongue pappilae.

Congenital syphilis: three pathognomic diagnmostic features known as Hutchinson’s triad:

1. Hutchinson teeth, 2. Ocular intertstial keratitis and 3. Eight nerve deafness

Histopathologic findings :PRIMARY SYPHLIS: Histopathologic picture of the oral lesions in

syphilitic patient is not specific. The surface epithelium is ulcerated in primary lesion and the underlying lamina propria is highly vascular and chronic inflammatory reaction is present

SECONDARY SYPHILIS: in secondary syphilis ulceration may not be

present and surface epithelium show hyperplasia with significant spongiosis and exocytosis .

use of special stains such as Warthin-Starry or Steiner stains often show scattered corkscrew like spirochetal organisms that frequently found in surafce epithelium.

TERTIARY LESION: oral tertiary lesion show surface ulceration with

psuedoepitheliomatous hyperplasia. The underlying inflammatory infiltrate usually demonstrate foci of granulomatous formation with circumscribed collections of histiocytes and multinucleated giant cells.

Darkfield microscopy of chancre showing

– corkscrew-shaped organisms with tightly wound spirals – forward and backward motion with rotation – Soft side-to-side bending and twisting – Specific but not sensitive • Direct fluorescent antibody test of specimen (DFA-TP) Serologic tests – Non-treponemal • Venereal Disease Research Laboratory (VDRL) • Rapid Plasma Reagin (RPR) test – Tests for auto-antibodies to cardiolipin, a tissue lipid – Easy and cheap, used for screening – Used to follow treatment – Sensitive except in late syphilis, specific

Lab diagnosis of syphilis

Gold Standard: – Culture of T. pallidum by in vivo intratesticular inoculation of rabbits – Not done routinely Serologic tests Treponemal • Fluorescent treponemal antibody absorption (FTA-ABS) test • Microhemagglutination test for antibodies to Treponema pallidum

(MHA-TP) • Treponema pallidum particle agglutination assay(TPPA) – More

sensitive and more specific, even in late syphilis

Differentail diagnosis•Candidiasis (oral or genital)•Chancroid• Fungal infections• Herpes simplex• Herpes zoster• Infectious mononucleosis

• Leprosy• Rubella• Rubeola• Sarcoidosis• Tuberculosis

DIAGNOSIS OF ACTINOMYCOSIS

The diagnosis of actinomycosis is made by culture and the isolation of the causative organism. A presumptive diagnosis can be made by clinical features.

Clinical features Classic lesions of cervicofacial actinomycosis are chronic low grade

persistent infection . Submandibular region is the most affected site. Associated changes may be seen such as nonhealing socket,

exuberant granulomatous tissue,or periosteal thickening of alveolus. Board like induration of the skin. Multiple draining sinus. Skin discharging sinuses is purplish and there may be areas of

hypertropic granulation tissue. Sulphur granules are often present in the pus .

Laboratory Studies CBC count: Anemia and mild leukocytosis are common. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)

levels are often elevated. Chemistry results usually are normal, with the exception of a

frequently elevated alkaline phosphatase level in hepatic actinomycosis.

Organism cultures Because actinomycosis is difficult to diagnose based on the typical

clinical features, direct identification and/or isolation of the infecting organism from a clinical specimen or from sulfur granules is necessary for definitive diagnosis in most cases.

Acceptable specimen material is obtained from draining sinuses, deep needle aspirate, or biopsy specimens;

A Gram-stained smear of the specimen may demonstrate the presence of beaded, branched, gram-positive filamentous rods, suggesting the diagnosis of actinomycosis.

Nucleic acid probes and polymerase chain reaction (PCR) methods are being developed for more rapid and more accurate identification

Histologic Findings Actinomycosis is characterized by mixed suppurative and

granulomatous inflammatory reactions, connective-tissue proliferation, and the presence of sulfur granules. The sulfur granules are nearly pathognomonic for actinomycosis, although similar findings have been reported with infections caused by Nocardia brasiliensis, Streptomyces madurae, and Staphylococcus aureus presenting as botryomycosis. The granules are approximately 0.1-1 mm in diameter and may be seen with the naked eye as yellowish particles.

Microscopically, the granules manifest a cauliflowerlike shape at low magnification; at higher magnification (X100), when the particle has been pressed between slide and cover slip, a clump of filamentous actinomycete microcolonies surrounded by polymorphonuclear neutrophils (PMNs) can be observed.

Gram stain renders these microcolonies visible as gram-positive, intertwined branching filaments, with radially arranged, peripheral hyphae. Coexisting with them are the companion bacteria, which are gram-positive and gram-negative cocci and rods.

ACTINOMYCOSIS IN THE ENDOMETRIAL TISSUE, LOW-POWER VIEW

ACTINOMYCOSIS IN THE ENDOMETRIAL TISSUE, HIGH-POWER VIEW

Differential diagnosis Tuberculosis SyphilisHistoplasmosisOsteomyelitis

Cat scratch disease Lymphogranuoma venerumNeoplasm

Diagnosis of Cat scratch disease:

The diagnosis of cat scratch disease is established via serologic tests that demonstrate a high degree of sensitivity and specificity. The most widely used is an indirect florescent antibody assay for detecting antibodies to B.henselae.

Another method is an enzyme linked immunosorbent assay for IgM antibodies to the organism. Polymerase chain reaction techniques also are available but are not widely used.

Clinical features: the infection can appear as an intraoral mass in the buccal mucosa when lymphoid aggregates became involved from an adjacent cutaneous primary site. Primary lesion adjacent to eye can result in a conjuctival granuloma that is associated with preauricular lymphaenopathy (oculo-glandular syndrome of parinaud) this pattern is thought to occur when an individual touches fur moistened with cat’s saliva during grooming

Laboratory Studies Routine laboratory tests in patients with suspected catscratch disease

(CSD) are usually unremarkable and are very unlikely to aid in diagnosis. Findings such as mild leukocytosis and elevated erythrocyte sedimentation rate are common but are also nonspecific and of little value.

Until recently, diagnosis was based on the fulfillment of specific clinical criteria. The recent development of serologic testing has effectively provided laboratory confirmation of the diagnosis. The addition of PCR from lymph node biopsy provides an even more sensitive detection of disease. When such testing is performed, it is usually performed on an outpatient basis.

Indirect fluorescent antibody (IFA) testing for Bartonella is quite variable as many different tests are available. Test sensitivity (as low as 53%) is typically quite poor, but this can be improved by concurrent use of both IgG and IgM testing. Specificity of IgM and IgG testing ranges from 88-98% and 50-62%, respectively. Most populations have low (2-6%) background seropositivity rates, limiting false-positive test results. The IFA shows cross-reactivity between Bartonella species, Epstein-Barr virus, cytomegalovirus, Toxoplasma gondii, and Streptococcus pyogenes.5

Infection is best demonstrated by rising immunoglobulin G (IgG) titers; however, patients may already have high levels at presentation.

Histopathological features of lymph nodes are consistent but not pathognomonic for CSD. Features include granuloma formation, stellate abscesses, and lymphocytic infiltrates.

Brown-Hopp tissue Gram stain and Warthin-Starry silver staining show small, curved, gram-negative bacilli .

Warthin-Starry stained sections of lymph node showing chains and clusters of organisms.

Differential diagnosisSyphilisLymphogranuloma VenereumToxoplasmosisMononucleosisTuberculosisSarcoidosis

DIAGNOSIS OF CANDIDIASIS

CLIICAL FINDINGS: Psuedomembranous candidiasis characterized by adherent white

plaques that resmble cottage chese or curdled milk on the oral mucosa.scrapng the lesion with tongue blade or rubbing them with dry gauge can remove these plaques.

Eryhematous candidiasis:it may present as acute atrophic candidiasis or sore mouth in which burning sensation in the mouth accompanied by loss of filiform papillae of the dorsal resulting n a reddened bald appearance of the tongue.

Cental papillary atrophy of tongue or median rhomboid glossoitis.clinically it appers as well demarcated erythematous zone that effect the midline ,posterior dorsal tongue and often is asymptomatic.erythema is due to loss of filliform papilaein this area. It may also present as kissing lesion when dorsal erythmatous lesion of the tongue and the palatal lesion come in close proximity.

Angular chielitis is characterized by erythema fissuring and scaling it often occurs elder person as aresult of reduced vertical dimension.

Denture stomatitis it also classified as form of erythematous candidiasis .this lesion is characterized by petechial hemorage located o he denture bearing area it may present as pin point lesion or as large erytmaous area under the denture.

Chronic hyperplastic candidiasis whit plaques that are not removed on scraping. Such lesion are locted on the anterior buccla mucosa and often leukoplakic lesion is associated candidial infection has a fine intermingling of red and white areas resulting in spekled leukoplakia.

Mucocutaneous candidiasis white plaques some of which may be removed.this lesion may be seen as relatively rare group of immunologic disorders known as mucocutaneous candidiasis.

Lab diagnosis demonstration of candida in the culture and smear confirm the diagnosis.

A speimen for culture is obtained by rubbing a sterile cotton swab over the lesion and then streaking the swab on the surface of sabouraud’s agar slant.

The cytologic preparation demonstrates tubular appearing fungal hyphae and ovoid yeasts of candida albicans.

If the lesion is clinically suggestive of chronic hyperplastic candidiasis albicans but non responsive to anifungal therapy then a biopsy should be performed to rule out the possiblity of candida albicans superimposed on the epithelial dysplasia , squamous cell carcinoma or lichen planus .

Histopathological finding: On staining with PAS ,the candidial hyphae and

yeast can be readily identified( as bright magenta color)

These hyphae may are approximately 2 um in dia.

Elongation of epithelial ridges and increase thickness of parakeratin.chronic inflammatory cell infiltrate.neutrophil( micoabscess) are often identified in the parakertin layer.

Culture A specimen for culture is obtained by rubbing a

sterile cotton swab over the lesion and then streaking on surface of sabouraud’s agar slant.

Grow as creamy smooth surface colonie after 2 to 3 days of incubation at room temperature.

DIAGNOSIS OF HISTOPLASMOSIS

Histoplasmosis diagnosis can be made by histopathologic identification of the organism in tissue sections or by culture.

Microscopic examation of the lesional tissue shows a diifuse infiltrate of macrophages or more commonly collection of macrophages organized ito granulomas the causative organism can be identified with some diificulty in the routine hematoxylin and eosin .the special stains such as PAS and grocott-gomori methemine silver methods readily demonstate the characterstics 1-2 um yeasts of H. capsulatum.

Differential dianosis BlastomycosisPneumoniaSan Joaquin Valley fever (coccidioidomycosis)Aspergillosis

D IA G N O S IS O F B L A S T O M Y C O S IS

Rapid diagnosis of blastomycosis can be peformed by microscoic examination of either histopathologic sections or an alcohol fixed cytologic prepation. Most rapid diagnosis is the KOH prepartion which may be used for examining scrapings from suspected lesions.

The most accurate method of identifying B. dermatidis is by obtaining culture specimen from the sputum or fresh biopsy material and growing the organism on sabourard’s agar. This is slow technique taking as long as 3 to 4 weeks for the characteristics mycelium to yeast conversion to take place.

A specific DNA probe has been developed allowing immediate identification mycelial phase. Skin testing and sereologic studies are usually not helpful becuase of lack of reactivity and specificity.

Differentila diagnosisBacterial pneumonia Tuberculosis Other endemic mycosesSarcoidosis Cancer

HISTOPATHOLOGIC FINDINGS

Histopathological examination shows a mixture of acute inflammation and granulomatous inflammation surrounding variable number of yeasts(8 to 20 um in dia).

Yeast characyerized by doubly refractile wall and broad attachment between budding daughter and parent cells.

Induce pseudoepitheliomatous hyperplasia

DIAGNOSIS OF PARACOCCIDIOMYCOSIS microscopic evaluation of tissue obtained from an oral lesion may reveal

pseudoepitheliomtous hyperplasia in addition to ulceration of the overlying surface epithelium.

P .brasiliensis elicits a granulomatous inflammatory host response that is characterized by collection of epithileiod macrophages and multinucleated giant cells.

Scatterred ,large (upto 30 um in dia )yeasts are really identified after staining of the section with Grocott-gomorri methenamine silver or PAS method. Organism show multiple daughter buds on the parent cells resulting in an mickey mouse ears or the spokes of a ship steering wheel( mariner wheel)

Clinical findings : oral lesions appear as mulberry like ulceration that most commonly affect the alveolar mucosa, gingival and palate. The lips ,tongue, orophaynnx ,and buccal mucosa are rarely involved .

Specimens for culture can be obtained but P. brasileinsis grow quite slowly

Differential diagnosis Coccidioidomycosis (Infectious Diseases)HistoplasmosisLeishmaniasisSyphilisLeprosyTuberculosis

The diagnosis of coccidiomycosis can be confirmed by culture or identification of characteristics organism in biopsy material. If the organism do not have a classic microscopic appeaance, then in situ hybridization studies using specific complementary DNA probes for C.immitis cab be performed to definitively identify the fungus . cytologic prepartions from bronchial swabbing or sputum samples may reveal the organisms.

Serologic studies are helpful in supporting the diagnosis and they may be performed at the same time as skin testing

Diagnosis of cocciodiomycosis

Differential diagnosis CryptococcosisPneumoniaHistoplasmosisLung AbscessTuberculosisWegener Granulomatosis

DIAGNOSIS OF CRYPTOCOCCOSIS

microscopic sections of a cryptococcal lesion generally show a granulomatous inflammatory response to the organism. The yeast appear as a round to ovoid structure ,4 to 6 um in diameter surrounded by clear halo that represebnts the capsule staining with the PAS or Grocott- gomorri methanaminesilver method readily identifies the fungus moreover a mucicaramine stain uniquely demonstrate its mucopolysacchride capsule.

The diagnosis of cryptococcosis can be made by several methods including biopsy and culture. Detection of cryptococcal polysacchride antigen in the serum or cerebrospinal fluid is also useful as a diagnostic procedure.

Differential daignosis

Basal Cell CarcinomaSyphilis

HistoplasmosisToxoplasmosis

LipomasTuberculosis

Molluscum Contagiosum

DIAGNOSIS OF ASPERGILOSIS

the diagnosis of fungal infection can be established by identification of hyphae within tissue section, this finding is only suggestive of aspergilosis because other fungal organisms may appear similar microscopically.

Ideally diagnosis is supported by culture of the organism from the lesion. Culture specimen of sputum and blood are of limited value because they are often negative despite disseminated disease.

Histopathologic features : Tissue section show varying numbers of septate hyphae 3 to 4 um in dia .

these hyphae show a tendency to branch at an acute angle and to invade small blood vessels . the aspergilloma is characterized by a tangled mass of hyphae with no evidence of tissue invasion .

Differential diagnosis Mucormycosis and other zygomycoses (e.g., phycomycosis, Fusariosis

MalignancyMycobacterial infectionNocardiosis

TOXOPLASMOSIS

Diagnosis is usually established by Identification of rising serum antibody titres to T .gondii within10 to 14 days after infection

Diagnosis may rest on the clinical findings and response of patient to therapy . Biopsy of an involved lymph node may suggest the diagnosis and the causative

organisms sometimes be detected immunohistochemically using antibodies directed against T.gondii specific antigens. The diagnosis should also be confirmed by serologic studies, if possible

Laboratory Studies Results from basic laboratory studies such as complete blood cell count (CBC),

chemistries, and liver function tests (LFTs) are typically normal, although lymphocytosis may be present.

Indirect detection Indirect detection is performed in pregnant women and

immunocompromised patients. Detection of immunoglobulin G (IgG) is possible within 2 weeks of infection

using enzyme-linked immunoassay (ELISA), IgG avidity test, and agglutination and differential agglutination test. The presence of IgG indicates a likely past infection, while the presence of IgM usually indicates acute infection (particularly in the absence of IgG).

Sabin-Feldman dye test: Live organisms are used to demonstrate the presence of anti-T gondii antibodies. This test is primarily used as a confirmatory test at reference laboratories.

IgG avidity test: IgG produced early in infection is less avid and binds to T gondii antigens more weakly than antibodies produced later in the course of infection. High antibody avidity indicates an older, earlier infection. This test may be helpful in the setting of pregnancy, as the timing of infection has prognostic value.

Direct detection Polymerase chain reaction (PCR) amplification of T gondii genes is possible

(samples may be taken of the CSF, blood, lymph node, or tissue biopsies or aqueous humor).

Culture: T gondii may be isolated from the blood via either inoculation of human cell lines or mouse inoculation. Mouse inoculation may require a longer time to yield results and also is likely to be more expensive.

DIAGNOSIS OF ZYGOMYCOSIS

Zygomycosis: diagnosis of zygomycosis is usally based on the histopathologic findings. a neutrophic infiltrate usually predominates in the viable tissue , but the

host inflammatory cell response to the infection may be minimal , particularly if the patient is immunocompromised

Histopathologic examination of lesional tissue shows extensive necrosis with numerous large,branching, nonseptate hyphae at the periphery . the hypahe tend to branch at 90 degree angles .

Differential diagnosis ActinomycosisPeptic Ulcer DiseaseAspergillosisToxoplasmosisCryptococcosisNocardiosis

DIAGNOSIS OF VIRAL INFETION

MEASLES

The diagnosis of measles is made based on clinical findings, including the classic triad of cough, coryza, and conjunctivitis; the pathognomonic Koplik spots; and the characteristic cephalocaudal progression of the morbilliform exanthem. However, laboratory identification and confirmation of the diagnosis is necessary for purposes of public health and outbreak control.

The US Centers for Disease Control and Prevention (CDC) clinical case definition for reporting purposes requires only the following:

Generalized rash lasting greater than or equal to 3 days Temperature greater than or equal to 101.0°F (38.3°C) Cough, coryza, or conjunctivitis Available laboratory tests are as follows:1. Immunoglobulin M (IgM): Measles-specific IgM antibody titers become

positive around 3 days after the exanthem manifests and persist for at least 28 days.

2. Immunoglobulin G (IgG): A significant (>4-fold) rise in measles-specific IgG titers between acute and convalescent sera confirms the diagnosis of measles, although relying solely on rising IgG titers for the diagnosis delays treatment considerably.

IgG testing in atypical measles: In atypical measles, laboratory evaluation of serum/blood reveals very low titers of measles antibody early in the course of the disease, followed by extremely high measles IgG antibody titers (eg, 1:1,000,000).

Viral culture: 1. Throat swabs and nasal swabs 2. Urine specimens Polymerase chain reaction (PCR): Reverse transcription PCR is highly

sensitive at visualizing measles virus RNA in blood, throat, nasopharyngeal, or urine specimens and, where available, can be used to rapidly confirm the diagnosis of measles.

A complete blood cell count may reveal leukopenia with a relative lymphocytosis and thrombocytopenia.

Liver function test results may reveal elevated transaminase levels in patients with measles hepatitis.

Histopathologic Findings Koplik spots represents the area of focal

hyperparakeratosis in which uncerlying epithelium shows spongiosis and vesiculation in the epidermis with scattered dyskeratotic keratinocytes. Occasional multinucleated epithelial giant cells can be seen.

As the spots ages the epithelium exhibit exocytosis by neutrophils leading to microabscess formation, epithelial necrosis and ulceration.

within the hyperplastic lymphoid tissue, numerous multinucleated giant cells can be seen. These cells are known as warthin-finkeldey giant cells

Differential diagnosis of Rubeola •Drug reactions• Kawasaki's disease• Rocky Mountain spotted fever• Rubella (German measles)• Scarlet fever• Secondary syphilis

RUBELLA

The diagnosis of rubella solely based on clinical signs and symptoms is unreliable because there are many other causes of rash that may mimic rubella infection and up to 50% of rubella infections may be subclinical.

Clinical presentation; Exanthematous rash: first sign , occurs on face and neck spread to entire body within 1 to 3 days rash resolved completely within 3 days Rash form discrete pink papules and finally fades with flaky

desquamation .Oral lesions Known as Forchheimer’s sign These consist of small dark red papules that develop on soft palate

and may extend to hard palate

Available laboratory tests are as follows: Detection of rubella IgM IgG seroconversion or a fourfold or greater rise in titre to rubella virus

(where the second serum sample is collected at least 10 days after the first, acute sample)

Detection or rubella virus genome in an appropriate specimen (not routinely preformed for diagnosis as it is more difficult to perform than serologic techniques)

A positive culture for rubella virus (not routinely performed) Rubella virus can be isolated from nasal, blood, throat, urine and

cerebrospinal fluid specimens from rubella. Virus may be isolated from the pharynx 1 week before and up to 2 weeks after rash onset. Although isolation of the virus is diagnostic of rubella infection, viral culture is demanding and labour intensive. Nasal, throat, blood, urine and cerebrospinal fluid specimens can be used, together with tissues from biopsy or autopsy for laboratory confirmation of CRS cases.

DIFFERENTIAL DIAGNOSIS OF RUBELLA Allergic drug reactionRoseolaRubeola (measles)Scarlet fever

HERPES SIMPLEX

Clinical signs and symptons Microscopic diagnosis PCR Tissue culture Elevated Ig M titres followed by Ig G titres

1. Clinical presentation Most Herpes labialis cases are diagnosed clinically. Most episodes are

preceded by a prodromal phase that is characterized by pain, burning, itching, and erythema, lasting about six hours. These symptoms are usually followed by lesions on or near the lips. Over the next 72-96 hours these papules progress to vesicles (blisters) and then ulcers. As the lesions heal they form hard crusts. The lesions are generally completely healed by 8 to 10 days. Pain can be severe at the start of the infection and resolves over the next 4 - 5 days.

Most lesions occur on the lips. However, lesions can also occur on the nose, cheeks, or chin. Lesions occurring in the oral cavity or face are less common. Intraoral lesions are hard to locate and are difficult to distinguish from apthous ulcers or canker sores.

About 25% of all episodes do not progress beyond the papule stage. These are called "aborted" episodes. About one-half of these do not progress beyond the prodromal stage. HSV isolation by cell culture is the “gold standard” for hsv 1 since it grows readily in tissue culture .

Lab tests Viral isolation :most definitive diagnostic procedure Detect HSV antigens by fluorescent assay Viral DNA by PCRMost commonly diagnostic procedure Cytologic smear Tissue biopsy

Differential diagnosis Herpangina Hand Foot and Mouth DiseaseEryhthema multiforme Oral aphthous ulcers Chancroid Apthous ulcers Syphilis Cytomegalovirus

HISTOPATHOLOGIC FEATURES

Infected epithelial cells exhibit acantholysis,nuclear clearing and nuclear enlargement or ballooning degeneration.

The acantholytic cells are termed tzank cells. Intercellular edema appears and lead to formation of intarepithelial

vesicles.

VARICELLA ZOSTER( CHICKEN POX) The diagnosis of varicella can be made from the history of exposure to VZV

within last 3 weeks and presence of typical exanthem. Confirmation can be obtained through a demonstration of viral cytopathologic

effects present withinepithelial cells harvested from vesicular fluid. Viral isolation in cell culture or rapid diagnosis from fluorescein-conjugated

VZV monoclonal antibodies can be performed. Serum samples can be obtained during the acute stage and 14 to 28 days

later. Later sample demonstrate fourfold increase in antibody titers to VZV.

Histopthological findings The cytolgic alterationare virtually identical to those described to HSV.

Differential diagnosisContact DermatitisEnteroviral InfectionsHerpes Simplex Virus Infection

ImpetigoUrticaria

ENTEROVIRUS

Diagnosis of enterovirus infections is often clinical.Clinical presentation:Herpangina: acute onset of sore throat, dysphagia,

fever,vomiting, myalgia and headache. Oral lesions: few in numbers(two to six), usually on

the posterior area.(soft palate and tonsillar pillars) ,appear as red macules which form fragile vesicle that rapidly ulcerate.(2 to 4 mm in dia). Ulcerations take 7 to 10 days to heal

Hand –foot and mouth disease: Skin rash and oral lesions are associted with flu like

symptoms. the cutaneous lesion range from few to dozens and

primarily affect the borders of palms and soles and ventral surface and sides of fingers.

The oral lesion resemble those of herpangina but more numerous and not confined to posterior areas of mouth.

The labia mucosa, buccal mucosa and tongue are the most common sites acute lymphonodular pharyngitis: characterized by sore throat, fever and mild

headache. Low numbers of yellow to dark pink nodules develop on soft palate or tonsillar pillars. Nodules resolved within 10 days without vesiculations or ulceration

Histopthological findings In pateints with herpangina and hand –foot and mouth disease, the areas of

affected epithelium exhibit intracellular and intercellular edema, which leads to extensive spongiosis and formation of an intraepithelial vesicle. The vesicle enlarge and rupture through epithelial basal layer ,with resulatnt formation of a subepithelial vesicle. Epithelial necrosis and ulceration soon follows.inclusion bodies and multinucleated giant cells are absent

Laboratory diagnosis can be achieved with serological tests, viral isolation by cell culture, and polymerase chain reaction (PCR).

Serology: The microneutralization test is the most widely used method for detecting antibodies to enteroviruses.

Viral isolation: The virus can be isolated from CSF, blood, or feces, depending on the site affected, and the yield is increased if multiple sites are sampled. Enterovirus proPCR: This rapid test is highly sensitive and specific for detecting enteroviral RNA in CSF specimens, with a sensitivity of 100% and specificity of 97%.

Cardiac enzyme levels may be elevated in persons with myopericarditis, indicating myocardial damage.

CSF analysis: The CSF profile in patients with aseptic meningitis usually reveals a mildly elevated white blood cell count, and the differential invariably shifts to a predominance of lymphocytes during the initial 1-2 days of illness. Glucose levels are normal or mildly decreased, while the protein level is normal or slightly increased.

INFECTIOUS MONONUCLEOSIS

The diagnosis of infectious mononucleosis is suggested by the clinical presentation and should be confirmed through lab procedures.

Clinical presentation: In classic infectious mononucleosis in a young adult the prodromal

fatigue,maliase, and anorexia occur up to 2 weeks before development of pyrexia.

80% of affeted young adults have oropharyngeal tonsillar enlargement. Lingual tonsils become hyperplastic and compromise the airway. Oral lesion include petechiae on the hard and soft palate. the petechiae are

transient and usually disappear within 2 to 48 hours.

Lab diagnosis: Serological findings: Presence of paul bunnel heterophil antibodies Positive in 90% of individual In case of negative paul bunnel test, indirect immuofluorecsence

testing to detect EBV antibodies should be done ELISA and recombinant DNA derived antigens also may be used

Blood profile Increased WBC COUNT Lymphocytosis as high as 70%during second week Atypical lymphocytes in peripheral blood.

CYTOMEAGALO VIRUS The diagnosis of CMV is made by considering a

combination of clinical features and by conducting other examinations.

Clinical features: Often begin with prolonged fever, malaise, anorexia,

fatigue, night sweats, myalgia and arthralgia. Liver function abnormalities, leucopaenia, thrombocytopaenia and atypical lymphocytosis may be observed during these episodes

Oral finding: chronic oral ulcers in immunocompromised patient. Coinfection usually occurs( CMV combined with HSV)

LABORATORY_DIAGNOSIS 1. Virus Isolation ;- Urine, saliva, blood and biopsy samples can be

used for virus isolation.. Tissue biopsies should be placed in sterile plastic containers. The specimens can be treated in the following ways ;-

Cell culture - DEAFF ( Detection of early antigen fluorescent foci ) ;- This is a

method used for the early diagnosis of CMV infection. In immunocompromised patients, a sensitivity of 78% and a specificity of 100% has been claimed.

Tissue immunofluorescence - Infected lung and liver cells may be stained by specific anti-CMV antibodies. Broncheolavage specimens can also be examined in this manner. Results of high sensitivity and specificity are possible.

Electron microscopy - Virions in the urine of congenitally infected infants may be visualized by EM in up to 80% of cases.

ELISAs for CMV antigen in the urine - these tests carry low sensitivity as CMV is complexed to ß2-microglobulin in the urine  

Detection of CMV DNA by PCR - the use of PCR in the diagnosis of CMV infection had been

widely studied. PCR offers the advantages of being rapid and sensitive. However, its inherent sensitivity poses a problem since latent CMV genomes, which are present in practically all seropositive individuals, may be detected. Therefore, it is critical to adjust the sensitivity of the PCR so that latent genomes are not detected.

CMV antigenaemia test - this test is based upon the detection of pp65, a structural protein expressed on the surface of infected polymorphonyclear lymphocytes. The number of infected leucocytes present had been reported to correlate with the severity of infection. The main advantage of this test is that it is very rapid so that a result can be available within the same day. As a result, this test is now widely used especially in the monitoring of transplant recipients.

Histopthological features Biopsy specimen of intraoral lesin dempnstrate changes within vascular endothelial cells.

Scattered infected cells are extrmely swolen showing both intracytoplasmic and intranuclear inclusions and prominent nuclei. This enlarged cell has been termed as “owl eye” cell. Gomori methenamine silver and PAS demonstrate cytoplasmic inclusion.

Serelogical tests: CMV IgM antibodies are detected in primary infection and lasts 3 - 4 months. It

is not detectable in recurrent infection except in immunocompromised patients where it is detectable in about a third of the cases.

CMV IgM may be undetectable in primary infection in immunocompromised individuals.

Solid phase sandwich or antibody capture ELISAs or RIAs are now in routine use. CMV IgG is produced early in primary infection and persists lifelong. The

detection of CMV IgG is useful as an "immune status screen" (Seropositive individuals are not protected from reactivation of reinfection). Rising titres of IgG can be used as markers of acute infection.

This is particularly useful in diagnosing recurrent infections in normal individuals, and in immunocompromised patients who may not develop a IgM response to primary infection. Various methods are used for detecting CMV IgG including CFT, IFT, latex agglutination, ELISAs and RIAs. The test used at the RVL is a LA.

Where possible, serological investigation should be backed by virus culture, especially in the case of immunocompromised patients who may fail to mount an immune response. CMV IgG may also be transferred by blood products which may produce false positive results. The following are recommended methods for use in the diagnosis of CMV infection.

MUMPS

The diagnosis of mumps can be made from the clinical presentation Clinical; presentation: 30% of the infection are subclinical.in symptomatic cases prodromal

symptoms arise first Discomfort and swelling ( reaches peak wihtin 2 to 3 days) Chewing tend to increase the pain Second finding is epididymorchitis Rapid swelling with significant pain and tenderness Most common oral manifestation is redness and enlargement of

wharton’s duct and stenson’s salivary duct.

Lab findings : Demonstration of mumps-specific IgM or a fourfold rise of mumps

specific IgG titers during acute phase . Viral isolation Reverse transcriptase PCR testing.

LAB DIAGNOSIS OF HIV

Antibody detection Antigen detection Detection of viral nucleic acid Viral isolation Indirect predictors of HIV infection

THREE TYPES OF TESTS

(i) Screening tests - ELISA and simple/rapid tests.

(ii) Confirmatory or supplemental tests- Western Blot assay. (iii) Nucleic acid and antigen screening

tests. Polymerase chain reaction (PCR), Ligase chain reaction (LCR), Nucleic acid based Sequence assays (NASBA) and some ELISA tests.

SPECIMENS TO BE COLLECTED FOR ANTIBODY DETECTION

Blood / Serum / Plasma Saliva / Urine

P24 antigen assay (<40%) Viral Culture PCR

LAB DIAGNOSIS DURING WINDOW PERIOD

BLOOD DETECTION TESTS

Enzyme-Linked Immunosorbent Assay/Enzyme Immunoassay (ELISA/EIA)

Radio Immunoprecipitation Assay/Indirect Fluorescent Antibody Assay (RIP/IFA)

Polymerase Chain Reaction (PCR) Western Blot Confirmatory test

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