SPECIAL TECHNIQUES FOR DIAGNOSING INFECTIOUS
Techniques Infectious Agents
Gram stain Most bacteria
Acid-fast stain Mycobacteria,
Silver stains Fungi, legionellae,
Periodic acid-Schiff Fungi, amebae
Antibody probes All classes
Culture All classes
DNA probes All classes
• A, Gram stain of sputum from a patient with
pneumonia. There are gram-positive cocci in
clusters (Staphylococcus aureus) with
• B, Gram stain of sputum from a patient with
pneumonia. Gram-positive, elongated cocci in
pairs and short chains (Streptococcus pneumonia)
and a neutrophil are seen.
• C, Gram stain of Clostridium sordellii grown in
culture. A mixture of gram-positive and gram-
negative rods, many of which have sub terminal
spores (clear areas), are present.
• Clostridia species often stain as both gram-positive and gram-
negative, although they are true gram-positive bacteria.
• D, Gram stain of a bronchoalveolar lavage
specimen showing gram-negative intracellular rods
typical of Enterobacteriaceae such as Klebsiella
pneumonia or Escherichia coli.
• E, Gram stain of urethral discharge from a patient
with gonorrhea. Many gram-negative diplococci
(Neisseria gonorrhoea) are present within a
• F, Silver stain of brain tissue from a patient with
Lyme disease meningoencephalitis.
• GRAM-POSITIVE BACTERIAL INFECTIONS
• Common gram-positive pathogens include
Staphylococcus, Streptococcus, and Enterococcus,
each of which causes many types of infections.
• Four less common diseases caused by gram-
positive rod-shaped organisms are also discussed
here: diphtheria, listeriosis, anthrax, and
nocardiosis. Clostridia, which are gram-positive,
are discussed with the anaerobes.
• All these infections are diagnosed by culture and
some special tests mentioned below.
• Staphylococcal Infections
• Staphylococcus aureus are pyogenic gram-positive cocci
that form clusters like bunches of grapes.
• These bacteria cause skin lesions (boils, carbuncles,
impetigo, and scalded-skin syndrome) as well as
abscesses, sepsis, osteomyelitis, pneumonia,
endocarditis, food poisoning, and toxic shock syndrome
• S. epidermidis, a species that is related to S. aureus,
causes opportunistic infections in catheterized patients,
patients with prosthetic cardiac valves, and drug addicts.
S. saprophytic us is a common cause of urinary tract
infections in young women.
Toxins of S. aureus
– lyse RBCs
– damages cell membranes
– act in the gastrointestinal tract
• Exfoliative toxin
– separates epidermal layers
• Toxic shock syndrome toxin (TSST)
The many consequences of staphylococcal infection
• Whether the lesion is located in the skin, lungs,
bones, or heart valves, S. aureus causes
pyogenic inflammation that is distinctive for its
• A furuncle, or boil, is a focal suppurative
inflammation of the skin and subcutaneous
tissue, either solitary or multiple or recurrent in
• Furuncles are most frequent in moist, hairy
areas, such as the face, axillae, groin, legs, and
• A carbuncle is a deeper suppurative infection that
spreads laterally beneath the deep subcutaneous
fascia and then burrows superficially to erupt in
multiple adjacent skin sinuses.
• Carbuncles typically appear beneath the skin of
the upper back and posterior neck, where fascial
planes favor their spread.
• Hidradenitis is a chronic suppurative infection of
apocrine glands, most often in the axilla. Infections
of the nail bed (paronychia) or on the palmar side
of the fingertips (felons) are exquisitely painful.
• Staphylococcal lung infections have a polymorph
nuclear infiltrate similar to that of pneumococcus
but cause much more tissue destruction.
• S. aureus lung infections usually occur in people
with a haematogenous source, such as an infected
thrombus, or a predisposing condition such as
• Staphylococcal scalded-skin syndrome, also called
Staphylococcal abscess of the lung with extensive neutrophil
infiltrate and destruction of the alveoli .
• Antibiotic resistance is a growing problem in treatment of
S. aureus infections.
• Methicillin-resistant S. aureus (MRSA) are resistant to all
currently available beta-lactam cell-wall synthesis
inhibitors (which include the penicillins and
• Until recently, MRSA was mainly found in healthcare-
associated infections, but community-acquired MRSA
infections have now become common in many
• Community-acquired MRSA commonly produce a potent
membrane damaging toxin, which kills leukocytes and
may make these strains of S. aureus particularly virulent.
• Enzymes. Bacteria secrete a variety of enzymes
(proteases, hyaluronidases, coagulases, fibrinolysins)
• Neurotoxins produced by Clostridium
botulinum and Clostridium tetani inhibit release of
neurotransmitters, resulting in paralysis.
• Super antigens are bacterial toxins that stimulate
very large number of T lymphocytes by binding to
conserved portions of the T-cell receptor, leading to
massive T-lymphocyte proliferation and cytokine
• The high levels of cytokines can lead to capillary leak
and shock. Super antigens made by S. aureus and S.
pyogenes cause toxic shock syndrome (TSS).
SPECTRUM OF INFLAMMATORY
RESPONSES TO INFECTION
• There are five major histologic patterns of tissue
reaction in infections.
• Suppurative (Purulent) Inflammation
• Mononuclear and Granulomatous Inflammation
• Cytopathic-Cytoproliferative Reaction
• Tissue Necrosis
• Chronic Inflammation and Scarring.
Pneumococcal pneumonia. Note the intra-alveolar
polymorph nuclear exudate and intact alveolar sept
• Small, lancet-shaped cells
arranged in pairs and short
• Culture requires blood or
• Growth improved by 5-10%
• Lack catalase & peroxidases
– cultures die in O2
• Gram-positive spherical/ovoid
cocci arranged in long chains
• Non-spore-forming, nonmotile
• Can form capsules & slime layers
• Facultative anaerobes
• Sensitive to drying, heat
• & disinfectants
• 25 species
• Impetigo (pyoderma)
– superficial lesions that break and
form highly contagious crust
– often occurs in epidemics in school
– pathogen enters through a break in
• eventually spreads to the dermis and
– can remain superficial or become
• Streptococcal pharyngitis
– strep throat
• Causes streptococcal
– Strep throat
– enters the upper
respiratory tract through
• Some patients with strep
throat may develop
– exhibiting a rash caused by
• Rheumatic fever
– serious complication
affecting the joints and
– causes permanent heart
damage called rheumatic
• Acute glomerulonephritis
– rare inflammatory response
to M proteins
– may lead to renal damage
• Diphtheria is caused by Corynebacterium
diphtheriae, a slender gram-positive rod with
clubbed ends, that is passed from person to person
through aerosols or skin exudate.
• C. diphtheria may be carried asymptomatically or
cause illnesses ranging from skin lesions in neglected
wounds of combat troops in the tropics, and a life-
threatening syndrome that includes formation of a
tough pharyngeal membrane and toxin-mediated
damage to the heart, nerves, and other organs.
• C. diphtheriae produces only one toxin, which is a
phage-encoded A-B toxin that blocks host cell
Membrane of diphtheria (arrow) lying within a
• Listeria monocytogenes is a gram-positive, facultative intracellular
bacillus that causes severe food-borne infections. Mini-epidemics
of L. monocytogenes infection have been linked to dairy products,
chicken, and hot dogs.
• Pregnant women, their neonates, the elderly, and
immunosuppressed persons (e.g., transplant recipients or AIDS
patients) are particularly susceptible to severe L. monocytogenes
• In pregnant women (and pregnant sheep and cattle), L.
monocytogenes causes an amnionitis that may result in abortion,
stillbirth, or neonatal sepsis. In neonates, L. monocytogenes may
cause disseminated disease (granulomatosis infantiseptica) and an
exudative meningitis, both of which are also seen in
• Listeria monocytogenes has leucine-rich proteins on its surface
called internalins, which bind to E-cadherin on host epithelial cells
and induce internalization of the bacterium.
• large, block shaped
• central spores
– develop under all
conditions except in the
• virulence factors
– capsule & exotoxins
• Cutaneous anthrax, which makes up 95% of naturally occurring
infections, begins as a painless, pruritic papule that develops into a
vesicle within 2 days. As the vesicle enlarges, striking edema may form
around it, and regional lymphadenopathy develops.
• Inhalational anthrax occurs when spores are inhaled. The organism is
carried by phagocytes to lymph nodes where the spores germinate, and
the release of toxins causes haemorrhagic mediastinitis.
• Gastrointestinal anthrax is an uncommon form of this infection that is
usually contracted by eating undercooked meat contaminated with B.
anthracis. Initially, the person has nausea, abdominal pain, and vomiting,
followed by severe, bloody diarrhea. Mortality is over 50%.
• Anthrax lesions at any site are typified by necrosis and exudative
inflammation with infiltration of neutrophils and macrophages.
• The presence of large, boxcar-shaped gram-positive extracellular bacteria
in chains, seen histopathologically or recovered in culture, should suggest
Bacillus anthracis in the sub capsular sinus of a hilar
lymph node of a patient who died of inhalational
Nocardia asteroides in a Gram-stained sputum
sample. Note the beaded, branched gram-positive
organisms and leukocytes
• Nocardia are aerobic gram-positive bacteria that grow
in distinctive branched chains.
• In culture, Nocardia form thin aerial filaments
• Nocardia appear in tissue as slender gram-positive
organisms arranged in branching filaments
• Irregular staining gives the filaments a beaded
appearance. Nocardia stain with modified acid-fast
stains (Fite-Faraco stain), unlike Actinomyces, which
may appear similar on Gram stain of tissue.
• At any site of infection, Nocardia elicit a suppurative
response with central liquefaction and surrounding
granulation and fibrosis. Granulomas do not form.
• GRAM-NEGATIVE BACTERIAL INFECTIONS
• Only a few gram-negative bacteria are considered
in this section.
• A number of important gram-negative pathogens
are discussed in the appropriate chapters of organ
systems, including bacterial causes of
gastrointestinal infections and urinary tract
• Gram-negative bacterial infections are usually
diagnosed by culture.
Typhoid fever, also referred to as enteric fever, is
caused by Salmonella typhi and Salmonella paratyphi.
It affects up to 30 million individuals worldwide each
The majority of cases in endemic countries are due to
S. typhi, while infection by S. paratyphi is more
common among travellers,
• caused by Salmonella enterica
– (S. typhi)
– typhoid fever
– ingested bacilli adhere to small
• cause invasive diarrhea that leads to
• S. Typhi is transmitted by the five
• Widal in 1896, and
Widal & Sicard in 1896
described the Widal
reaction, and this test
has proved of value in
cases where positive
cultures have been
Georges- Fernand- Isidor Widal
• invades villus of large intestine
– can perforate intestine or invade blood
• enters Peyer’s patches & instigates inflammatory response
– endotoxin & exotoxins
• Neisserial Infections
• Neisseria are gram-negative diplococci that are
flattened on the adjoining sides, giving the pair the
shape of a coffee bean
• These aerobic bacteria have stringent nutritional
requirements and grow best on enriched media
such as lysed sheep's blood agar ("chocolate"
agar). The two clinically significant Neisseria are N.
meningitidis and N. gonorrhoeae.
• N. meningitides is a significant cause of bacterial
meningitis, particularly among children younger
than 2 years of age.
• Meninges of brain and
– cross the blood-brain barrier
• can be caused by several
– Neisseria meningitides
– Streptococcus pneumonia
– Haemophilus influenzae
• Gram-negative diplococci
• none develop flagella or spores
• capsules on pathogens
• Strict parasites
– do not survive long outside of the host
• Residents of mucous membranes of warm-blooded animals
• Aerobic or microaerophilic
• Pathogenic species require enriched complex media and CO2
• 2 primary human pathogens
– Neisseria gonorrhoeae
– Neisseria meningitidis
• Virulence factors
– capsule, pili, IgA protease
• 12 strains
– serotypes A, B, C, cause most cases
• Disease begins when bacteria enter bloodstream, pass
into cranial circulation, multiply in meninges
– very rapid onset
– endotoxin causes hemorrhage and shock
– can be fatal
• Causes gonorrhea
• Virulence factors
– pili, other surface molecules, IgA protease
• Strictly a human infection
• In top 5 STDs
• Infectious dose 100-1,000
• Does not survive more than 1-2 hours on fomites
• Infection is asymptomatic in 10% of males and
50% of females
– vaginitis, urethritis,
salpingitis (PID) mixed
– common cause of
sterility & ectopic tubal
Gonorrhea in Newborns
• Infected as they pass
through birth canal
• Eye inflammation,
• Prevented by
prophylaxis after birth
• Gram stain
• 20-30% of new cases are
PPNG or tetracycline
• Recurrent infections can
• Reportable infectious
• Whooping Cough
• Whooping cough, caused by the gram-negative
coccobacillus Bordetella pertussis, is an acute,
highly communicable illness characterized by
paroxysms of violent coughing followed by a loud
• B. pertussis vaccination, whether with killed
bacteria or the newer acellular vaccine, has been
effective in preventing whooping cough
• The diagnosis is best made by PCR, because
culture is less sensitive.
Whooping cough showing a haze of bacilli (arrows)
entangled with the cilia of bronchial epithelial cells
• Pseudomonas Infection
• Pseudomonas aeruginosa is an opportunistic aerobic gram-negative
bacillus that is a frequent, deadly pathogen of people with cystic
fibrosis, severe burns, or neutropenia.
• 93 Many people with cystic fibrosis die of pulmonary failure
secondary to chronic infection with P. aeruginosa. P. aeruginosa can
be very resistant to antibiotics, making these infections difficult to
• P. aeruginosa often infects extensive skin burns, which can be a
source of sepsis. P. aeruginosa is a common cause of hospital-acquired
infections; it has been cultured from washbasins, respirator tubing,
nursery cribs, and even antiseptic-containing bottles.
• P. aeruginosa also causes corneal keratitis in wearers of contact
lenses, endocarditis and osteomyelitis in intravenous drug abusers,
external otitis (swimmer's ear) in healthy individuals, and severe
external otitis in diabetics.
• Yersinia pestis is a gram-negative facultative
intracellular bacterium that is transmitted from
rodents to humans by fleabites or, less often, from
one human to another by aerosols.
• It causes an invasive, frequently fatal infection
• Plague, also named Black Death, caused three
great pandemics that killed an estimated 100
million people in Egypt and Byzantium in the sixth
• bubonic plague the infected fleabite is usually on
the legs and is marked by a small pustule or ulcer.
The draining lymph nodes enlarge dramatically
within a few days and become soft, pulpy, and
plum colored, and may infarct or rupture through
• In pneumonic plague there is a severe, confluent,
haemorrhagic and necrotizing
bronchopneumonia, often with fibrinous pleuritis.
• In septicemic plague lymph nodes throughout the
body as well as organs rich in mononuclear
phagocytes develop foci of necrosis.
• Fulminant bacteremias also induce DIC with
widespread hemorrhages and thrombi.
• Chancroid (Soft Chancre)
• Chancroid is an acute, sexually transmitted,
ulcerative infection caused by Hemophilus
• The disease is most common in tropical and
subtropical areas among lower socioeconomic
• Chancroid is one of the most common causes of
genital ulcers in Africa and Southeast Asia, where
it probably serves as an important cofactor in the
transmission of HIV infection.
• Granuloma Inguinale
• Granuloma inguinale, or donovanosis, is a chronic
inflammatory disease caused by Klebsiella
granulomatis (formerly called
Calymmatobacterium donovani), a minute,
• The organism is sexually transmitted.
• Clostridial Infections
Clostridium species are gram-positive bacilli that grow under anaerobic conditions
and produce spores that are present in the soil.
• C. perfringens, C. septicum, and other species cause cellulitis and myonecrosis of
traumatic and surgical wounds (gas gangrene), uterine myonecrosis often
associated with illegal abortions, mild food poisoning, and infection of the small
bowel associated with ischemia or neutropenia that often leads to severe sepsis.
• C. tetani, the cause of tetanus, proliferates in puncture wounds and in the
umbilical stump of newborn infants and releases a potent neurotoxin, called
tetanospasmin, that causes convulsive contractions of skeletal muscles (lockjaw).
Tetanus toxoid (formalin-fixed neurotoxin) is part of the DPT (diphtheria,
pertussis, and tetanus) immunization, which has greatly decreased the incidence
of tetanus worldwide.
• C. botulinum grows in inadequately sterilized canned foods and releases a
potent neurotoxin that blocks synaptic release of acetylcholine and causes a
severe paralysis of respiratory and skeletal muscles (botulism).
• C. difficile overgrows other intestinal flora in antibiotic-treated people, releases
toxins, and causes pseudomembranous colitis
• Chlamydia trachomatis is a small gram-negative
bacterium that is an obligate intracellular parasite.
C. trachomatis exists in two forms during its
unique life cycle.
• The infectious form, called the elementary body
(EB), is a metabolically inactive, spore like
• Members of the order Rickettsiales are vector-
borne obligate intracellular bacteria that cause
epidemic typhus (Rickettsia prowazekii), scrub
typhus (Orienta tsutsugamushi), and spotted fevers
(Rickettsia rickettsii and others).
• These organisms have the structure of gram-
negative, rod-shaped bacteria, although they stain
poorly with Gram stain. Epidemic typhus, which is
transmitted from person to person by body lice,
Viruses are the cause of many
clinically important acute and
chronic infections affecting virtually
every organ system
Organ System Species Disease
Influenza viruses A, B
Upper and lower
Upper respiratory tract
1 Digestive system. Mumps virus
Hepatitis A virus
Hepatitis B virus
Hepatitis D virus
Hepatitis C virus
Hepatitis E virus
Acute viral hepatitis
Acute or chronic
With HBV, acute or
Acute or chronic
Enteric ally transmitted
Systemic with Skin
Oral herpes ("cold
HIV-1 and HIV-2
Systemic with Skin
Oral herpes ("cold
HIV-1 and HIV-2
Dengue virus 1-4
Yellow fever virus
Measles giant cells in the lung. Note the glassy
eosinophilic intranuclear inclusions.
• Measles (rubeola) virus is a leading cause of vaccine-
preventable death and illness worldwide.
• More than 20 million people are affected by measles
each year. In 2005, there were an estimated 345,000
• the majority of them in children in developing
• Because of poor nutrition, children in developing
countries are 10 to 1000 times more likely to die of
measles pneumonia than are children in developed
Measles giant cells in the lung. Note the glassy
eosinophilic intranuclear inclusions
• The blotchy, reddish brown rash of measles virus
infection on the face, trunk, and proximal
extremities is produced by dilated skin vessels,
edema, and a moderate, nonspecific, mononuclear
• Ulcerated mucosal lesions in the oral cavity near
the opening of Stensen ducts (the pathognomonic
Koplik spots) are marked by necrosis, neutrophil
exudate, and neovascularization.
• The lymphoid organs typically have marked follicular hyperplasia,
large germinal centers, and randomly distributed multinucleate
giant cells, called Warthin-Finkeldey cells, which have eosinophilic
nuclear and cytoplasmic inclusion bodies
• mumps parotitis, which is bilateral in 70% of cases, affected glands are
enlarged, have a doughy consistency, and are moist, glistening, and reddish
brown on cross-section.
• On microscopic examination the gland interstitium is edematous and
diffusely infiltrated by macrophages, lymphocytes, and plasma cells, which
compress acini and ducts. Neutrophils and necrotic debris may fill the ductal
lumen and cause focal damage to the ductal epithelium.
• In mumps orchitis testicular swelling may be marked, caused by edema,
mononuclear cell infiltration, and focal hemorrhages. Because the testis is
tightly contained within the tunica albuginea, parenchymal swelling may
compromise the blood supply and cause areas of infarction.
• Sterility, when it occurs, is caused by scars and atrophy of the testis after
resolution of viral infection.
• In the enzyme-rich pancreas, lesions may be destructive, causing
parenchymal and fat necrosis and neutrophil-rich inflammation. Mumps
encephalitis causes perivenous demyelination and perivascular
• Poliovirus is a spherical, unencapsulated RNA virus
of the enterovirus genus.
• Other enteroviruses cause childhood diarrhea as
well as rashes (coxsackievirus A), conjunctivitis
(enterovirus 70), viral meningitis (coxsackieviruses
and echovirus), and myopericarditis
• There are three major strains of poliovirus, each
of which is included in the Salk formalin-fixed
(killed) vaccine and the Sabin oral, attenuated
• West Nile Virus
• West Nile virus is an arthropod-borne virus
(arbovirus) of the flavivirus group, which also
includes viruses that cause dengue fever and
• West Nile virus has a broad geographic
distribution in the Old World, with outbreaks
in Africa, the Middle East, Europe, Southeast
Asia, and Australia.
• Viral haemorrhagic fevers (VHFs) are systemic infections.
• VHF viruses produce a spectrum of illnesses, ranging from
relatively mild acute disease characterized by fever, headache,
myalgia, rash, neutropenia, and thrombocytopenia to severe,
life-threatening disease in which there is sudden hemodynamic
deterioration and shock.
• Herpes Simplex Virus (HSV)
• HSV-1 and HSV-2 differ serologically but are
genetically similar and cause a similar set of
primary and recurrent infections.
• These viruses produce acute and latent
• Both viruses replicate in the skin and the
mucous membranes at the site of entrance of
the virus (usually oropharynx or genitals), where
they produce infectious virions and cause
vesicular lesions of the epidermis
High-power view of cells from the blister in Figure 8-9 showing
glassy intranuclear herpes simplex inclusion bodies.
CHRONIC LATENT INFECTIONS
• Fever blisters or cold sores favor the facial skin around
mucosal orifices (lips, nose), where their distribution is
frequently bilateral and independent of skin dermatomes.
Intraepithelial vesicles (blisters), which are formed by
intracellular edema and ballooning degeneration of
epidermal cells, frequently burst and crust over, but some
may result in superficial ulceration
• Gingivostomatitis, which is usually encountered in
children, is caused by HSV-1. It is a vesicular eruption
extending from the tongue to the retro pharynx and
causing cervical lymphadenopathy.
• Swollen, erythematous HSV lesions of the fingers or palm
(herpetic whitlow) occur in infants.
• Trans placental transmission can occur from a newly acquired or
primary infection in a mother who does not have protective
antibodies ("congenital CMV").
• Neonatal transmission can occur through cervical or vaginal
secretions at birth or, later, through breast milk from a mother
who has active infection ("perinatal CMV").
• Transmission can occur through saliva during preschool years,
especially in day care centers. Toddlers so infected readily
transmit the virus to their parents.
• Transmission by the venereal route is the dominant mode after
about 15 years of age, but spread may also occur via respiratory
secretions and the fecal-oral route.
• Iatrogenic transmission can occur at any age through organ
transplants or blood transfusions.
An owl’s eye cell diagnostic for
• Hepatitis B Infections
– Causes hepatitis
• Inflammation of the liver
– HBV is the only DNA virus that causes hepatitis
– Viral infection can cause severe liver damage
– Various symptoms
• Jaundice, liver enlargement, abdominal distress, and
bleeding into the skin and internal organs
– Coinfection with hepatitis D virus increases risk of
permanent liver damage
Three types of viral particles
produced by hepatitis B virus
Diseases associated with Epstein-Barr virus
• Morphology. The major alterations involve the blood,
lymph nodes, spleen, liver, CNS, and, occasionally,
• The peripheral blood shows absolute lymphocytosis;
more than 60% of white blood cells are lymphocytes.
• Between 5% and 80% of these are large, atypical
lymphocytes, 12 to 16 μm in diameter, characterized
by an abundant cytoplasm containing multiple clear
vacuolations, an oval, indented, or folded nucleus,
and scattered cytoplasmic azurophilic granules
• These atypical lymphocytes, most of which express
CD8, are sufficiently distinctive to strongly suggest the