Colonies of B anthracis are round and have a “cut glass” appearance in transmitted light. Hemolysis is

uncommon with B anthracis but common with B cereus and the saprophytic bacilli. Gelatin is liquefied, and

growth in gelatin stabs resembles an inverted fir tree.

Bacillus anthracis

Anthrax is primarily a disease of herbivores goats, sheep, cattle, horses, and so on; other animals (e.g., rats) are

relatively resistant to the infection.

Humans become infected incidentally by contact with infected animals or their products. In animals, the portal

of entry is the mouth and the gastrointestinal tract. Spores from contaminated soil find easy access when

ingested with spiny or irritating vegetation. In humans, the infection is usually acquired by the entry of spores

through injured skin (cutaneous anthrax) or rarely the mucous membranes (gastrointestinal anthrax) or by

inhalation of spores into the lung (inhalation anthrax). A fourth category of the disease, injection anthrax, has

caused outbreaks among persons who inject heroin that has been contaminated with anthrax spores. The spores

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germinate in the tissue at the site of entry, and growth of the vegetative organisms results in formation of a

gelatinous edema and congestion. Bacilli spread via lymphatics to the bloodstream, and they multiply freely in

the blood and tissues shortly before and after the animal’s death.

Anthrax toxins are made up of three proteins, protective antigen (PA), edema factor (EF), and lethal factor

(LF). PA is a protein that binds to specific cell receptors, and after proteolytic activation, it forms a membrane

channel that mediates entry of EF and LF into the cell. EF is an adenylate cyclase; with PA, it forms a toxin

known as edema toxin. Edema toxin is responsible for cell and tissue edema. LF plus PA form lethal toxin,

which is a major virulence factor and cause of death in infected animals and humans.

When injected into laboratory animals (e.g., rats), the lethal toxin can quickly kill the animals by impairing both

innate and adaptive immunity, allowing organism proliferation and cell death.

In inhalation anthrax (woolsorters’ disease), the spores from the dust of wool, hair, or hides are inhaled;

phagocytosed in the lungs; and transported by the lymphatic drainage to the mediastinal lymph nodes, where

germination occurs. This is followed by toxin production and the development of hemorrhagic mediastinitis and

sepsis.

Figure shows Bacillus anthracis in broth culture

In humans, approximately 95% of cases are cutaneous anthrax, and 5% are inhalation. Gastrointestinal anthrax

is very rare; it has been reported from Africa, Asia, and the United States when people have eaten meat from

infected animals. The bioterrorism events in the fall of 2001 resulted in 22 cases of anthrax 11 inhalation and

11 cutaneous. Five of the patients with inhalation anthrax died.

Cutaneous anthrax generally occurs on exposed surfaces of the arms or hands followed in frequency by the face

and neck. A pruritic papule develops 1–7 days after entry of the organisms or spores through a scratch. Initially,

it resembles an insect bite. The papule rapidly changes into a vesicle or small ring of vesicles that coalesce, and

a necrotic ulcer develops. The lesions typically are 1–3 cm in diameter and have a characteristic central black

eschar. Marked edema occurs. Lymphangitis, lymphadenopathy, and systemic signs and symptoms of fever,

malaise, and headache may occur.

After 7–10 days, the eschar is fully developed. Eventually, it dries, loosens, and separates; healing is by

granulation and leaves a scar. It may take many weeks for the lesion to heal and the edema to subside.

Antibiotic therapy does not appear to change the natural progression of the disease but prevents dissemination.

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In as many as 20% of patients, cutaneous anthrax can lead to sepsis, the consequences of systemic infection

including meningitis and death.

The incubation period in inhalation anthrax may be as long as 6 weeks. The early clinical manifestations are

associated with marked hemorrhagic necrosis and edema of the mediastinum. Substernal pain may be

prominent, and there is pronounced mediastinal widening visible on chest radiographs. Hemorrhagic pleural

effusions follow involvement of the pleura; cough is secondary to the effects on the trachea.

Sepsis occurs, and there may be hematogenous spread to the gastrointestinal tract, causing bowel ulceration, or

to the meninges, causing hemorrhagic meningitis. The fatality rate in inhalation anthrax is high in the setting of

known exposure; it is higher when the diagnosis is not initially suspected.

Animals acquire anthrax through ingestion of spores and spread of the organisms from the intestinal tract. This

is rare in humans, and gastrointestinal anthrax is extremely uncommon. Abdominal pain, vomiting, and bloody

diarrhea are clinical signs.

Injection anthrax is characterized by extensive, painless, subcutaneous edema and the notable absence of the

eschar characteristic of cutaneous anthrax. Patients may progress to hemodynamic instability due to septicemia.

Laboratory Diagnosis

Specimens to be examined are fluid or pus from a local lesion, blood, pleural fluid, and cerebrospinal fluid in

inhalational anthrax associated with sepsis and stool or other intestinal contents in the case of gastrointestinal

anthrax. Stained smears from the local lesion or of blood from dead animals often show chains of large grampositive rods. Anthrax can be identified in dried smears by immunofluorescence staining techniques.

When grown on blood agar plates, the organisms produce nonhemolytic gray to white, tenacious colonies with

a rough texture and a ground-glass appearance. Comma-shaped outgrowths (Medusa head, “curled hair”) may

project from the colony. Demonstration of capsule requires growth on bicarbonate- containing medium in 5–7%

carbon dioxide. Gram stain shows large gram-positive rods. Carbohydrate fermentation is not useful. In

semisolid medium, anthrax bacilli are always nonmotile, but related organisms (e.g., B cereus) exhibit motility

by “swarming.”

Definitive identification requires lysis by a specific anthrax γ-bacteriophage, detection of the capsule by

fluorescent antibody, or identification of toxin genes by polymerase chain reaction (PCR). These tests are

available in most public health laboratories. A rapid enzyme-linked immunoassay (ELISA) that measures total

antibody to PA, but the test result is not positive early in disease.

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Lecture 13

Mycobacterium

Mycobacteria are slender rods with lipid-rich cell walls that are resistant to penetration by chemical dyes such as

those used in the Gram stain.

They stain poorly but, once stained, cannot be easily decolorized by treatment with acidified organic solvents.

Therefore, they are termed “acid-fast”. Mycobacteria survive and replicate intracellularly. Mycobacteria infections

generally result in the formation of slow-growing granulomatous lesions that are responsible for major tissue

destruction.

Mycobacterium tuberculosis causes tuberculosis, the principal chronic bacterial disease in humans and a leading

cause worldwide of death from infection. This organism has increasingly become a cause for special concern in

immunocompromised patients.

Members of the genus Mycobacterium also cause leprosy as well as several tuberculosis-like human infections. This

genus belongs to the order of organisms (Actinomycetales) that also includes the genera Actinomyces and Nocardia.

These organisms all cause granulomatous lesions with various clinical presentations.

Mycobacteria are nonmotile and do not form spores. Mycobacteria cell walls are unusual in that they are

approximately 60 % lipid, including a unique class of very

Long-chain (75-90 carbons), β-hydroxylated fatty acids (mycolic acids).

These complexes with a variety of polysaccharides and peptides, creating a waxy cell surface that makes

Mycobacteria strongly hydrophobic and accounts for their acid-fast staining characteristic. Their unusual cell walls

make Mycobacteria impervious to many chemical disinfectants and convey resistance to the corrosive action of

strong acids or alkalis, so decontaminating clinical specimens, such as sputum, in which non-Mycobacteria

organisms are digested by such treatments. Mycobacteria are also resistant to drying but not to heat or ultraviolet

irradiation. Mycobacteria are strictly aerobic. Most species grow slowly with generation times of 8 to 24 hours.

It is currently estimated that about one third of the world’s population is infected with M. tuberculosis (tubercle

bacillus), with 30 million people having active disease.

Figure shows Mycobacterium tuberculosis

(Acid-fast stain of sputum from a

patient with tuberculosis)

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Patients with active pulmonary tuberculosis shed large numbers of organisms by coughing, creating aerosol

droplet nuclei. Because of resistance to desiccation, the organisms can remain viable as droplet nuclei

suspended in room air for at least

30 minutes. The principal mode of contagion is person-to-person transmission by inhalation of the aerosol.

A single infected person can pass the organism to numerous people in an exposed group, such as a family,

classroom, or hospital ward without proper isolation.

After being inhaled, Mycobacteria reach the alveoli, where they multiply in the pulmonary epithelium or

macrophages. Within 2-4 weeks, many bacilli are destroyed by the immune system, but some survive and are

spread by the blood to extra - pulmonary sites.

The virulence of M. tuberculosis rests with its ability to survive and grow within host cells. Although the

organism produces no demonstrable toxins, when engulfed by macrophages, bacterial sulfolipids inhibit the

fusion of phagocytic vesicles with lysosomes. The ability of M. tuberculosis to grow even in immunologically

activated macrophages and to remain viable within the host for decades is a unique characteristic of the

pathogen.

M. tuberculosis stimulates both a humoral and a cell mediated immune response. Although circulating

antibodies appear, they do not convey resistance to the organism. Instead, cellular immunity (CD4+ T cells) and

the accompanying delayed hypersensitivity directed against a number of bacterial protein antigens develop in

the course of infection and contribute to both the pathology of and immunity to the disease.

Primary tuberculosis occurs in a person who has had no previous contact with the organism. For the majority

of cases (95%), the infection becomes arrested, and most people are unaware of this initial encounter.

The only evidence of tuberculosis may be a positive tuberculin test; the course of tuberculosis infection either

remaining dormant or progressing to clinical disease.

About 10 % of those with an arrested primary infection develop clinical tuberculosis at some later time in their

lives.

As primary tuberculosis is usually acquired by the respiratory tract, the initial lesion occurs in a small

bronchiole or alveolus in the mid lung periphery. The organisms are engulfed by local mononuclear

phagocytes, and their presence initiates an inflammatory reaction. The tubercle bacilli grow well in phagocytic

cells; the bacteria proliferate and are carried by lymphatic drainage to the lymph nodes and beyond to set up

additional foci.

This initial phase of the infection is usually mild or asymptomatic and results in exudative lesions in which

fluid and polymorphonuclear leukocytes accumulate around the bacilli. A specific immune response develops

after about 1 month, and this changes the character of the lesions.

Macrophages, activated by specific T lymphocytes, begin to accumulate and destroy the bacilli.

Tubercle formation: The productive (granulomatous) lesion that develops is known as a tubercle. It consists of

a central area of large, multinucleate giant cells (macrophage syncytia) containing tubercle bacilli, a mid-zone

of pale epithelioid cells, and a peripheral collar of fibroblasts and mononuclear cells.

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Tissue damage is produced by the destruction of both bacilli and phagocytes, which results in the release of

degradative enzymes and reactive oxygen species such as superoxide radicals. The center of the tubercle

develops a characteristic expanding, caseous (cheesy) necrosis.

Primary tuberculosis follows one of two courses:

1- If the lesion arrests, the tubercle undergoes fibrosis and calcification, although viable but non-proliferating

organisms may persist.

2- If the lesion breaks down, the caseous material is discharged, and a cavity is created that can facilitate

spread of the infection. The organisms are dispersed by the lymph and the bloodstream and can seeds the

lungs; regional lymph nodes; or various distant tissues, such as liver, spleen, kidneys, bone, or meninges.

In progressive disease, one or more of the resulting tubercles may expand, leading to destruction of tissue

and clinical illness of (chronic pneumonitis, tuberculous osteomyelitis, and tuberculous meningitis). In

the extreme instance, active tubercles develop throughout the body, a serious condition known as miliary

(disseminated) tuberculosis.

Secondary disease reactivation:

It caused by M. tuberculosis that has survived in a dormant primary tubercle lesion. Any of the preexisting

tubercles maybe involved, but pulmonary sites are most common, particularly the lung apices where high

oxygen tension favors Mycobacteria growth. The resulting pathology is known as ((caseation necrosis)).

Destruction of the lung tissue leads to air-filled cavities where the bacteria replicate actively. Bacterial

populations in such lesions often become quite large, and many organisms are shed (in sputum). The patient

again becomes capable of exposing others to the disease.

Reactivation is apparently caused by impairment in immune status, often associated with:

a. Malnutrition

b. Alcoholism

c. Advanced age

d. Severe stress

e. Immune-suppressive medication

f. Diseases (such as diabetes and, particularly, AIDS)

Tuberculin reaction

The tuberculin reaction test is a manifestation of delayed hypersensitivity to protein antigens of M. tuberculosis.

Although such tests can be used to document contact with the tubercle bacillus, they do not confirm that the

patient currently

has active disease.

In the Mantoux test, purified protein derivative (PPD) is prepared from culture filtrates of the organism and

biologically standardized. Activity is expressed in tuberculin units. In the routine procedure (Mantoux test), a

measured amount of PPD is injected intradermally in the forearm. It is read 48-72 hours later for the presence

and size of an area of induration (hardening) at the site of injection, which must be observed for the test to be

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positive. A positive reaction usually develops 4 to 6 weeks after initial contact with the organism. It remains

positive for life, although it may wane after some years or in the presence of immune-suppression by

medications or disease.

Laboratory Identification

A microscopic approach for acid-fast bacilli using techniques such as the Ziehl-Neelsen stain is the most rapid

test for Mycobacteria. However, M. tuberculosis cannot be reliably distinguished on morphologic grounds from

other pathogens in the genus, from some saprophytic Mycobacteria species that may contaminate glassware and

reagents in the laboratory, or from those Mycobacteria that may be part of the normal flora. Therefore, a

definitive identification of M. tuberculosis can only be obtained by culturing the organism or by using one of

the molecular methods. The conventional methods often require 6–8 weeks for identification.

Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, cerebrospinal fluid, joint fluid,

biopsy material, blood, or other suspected material.

Decontamination and concentration of specimens from sputum and other non-sterile sites should be liquefied

with NaOH (kills many other bacteria and fungi), neutralized with buffer, and concentrated by centrifugation.

Specimens processed in this way can be used for acid-fast stains and for culture. Specimens from sterile sites,

such as cerebrospinal fluid, do not need the decontamination procedure but can be directly centrifuged,

examined, and cultured.

Smears Sputum, exudates, or other material is examined for acid-fast bacilli by staining. Stains of gastric

washings and urine generally are not recommended because saprophytic Mycobacteria may be present and

yield a positive stain. Fluorescence microscopy with auramine-rhodamine stain is more sensitive than

traditional acid-fast stains, such as Ziehl-Neelsen, and is the preferred method for clinical material. If acid-fast

organisms are found in an appropriate specimen, this is presumptive evidence of Mycobacteria infection.

Culture, identification, and susceptibility testing processed specimens from non-sterile sites and centrifuged

specimens from sterile sites can be cultured directly onto selective media. The selective broth culture often is

the most sensitive method and provides results most rapidly. A selective agar media (e.g., Löwenstein-Jensen

or Middle brook 7H10/7H11 biplate with antibiotics) should be inoculated in parallel with broth media

cultures.

Incubation is at 35-37°C in 5-10% CO2 for up to 8 weeks. If culture results are negative in the setting of a

positive acid-fast stain, then a set of inoculated media should be incubated at a lower temperature (e.g., 24-

33°C) and both sets incubated for 12 weeks.

Blood for culture of Mycobacteria should be anticoagulated and processed by one of two methods:

(1) Commercially available lysis centrifugation system

(2) Inoculation into commercially available broth media specifically designed for blood cultures

Conventional methods for identification of Mycobacteria include observation of:

1. Rate of growth

2. Colony morphology

3. Pigmentation

4. Biochemical profiles

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Nucleic acid amplification by molecular techniques is increasingly important in the diagnosis of tuberculosis

because they have the potential to shorten the time required to detect and identify M. tuberculosis in clinical

specimens. The amplified M. tuberculosis direct test uses enzymes that rapidly make copies of M. tuberculosis

16S ribosomal RNA, which can be detected using genetic probes. The sensitivity of the test ranges from 75-100

%, with a specificity of 95-100 %, and it is used for patients whose clinical smears are positive for acid-fast

bacilli and whose cultures are in progress.

The polymerase chain reaction (PCR), amplifies a small portion of a predetermined target region of the M.

tuberculosis DNA. Using human sputum, commercial PCR kits can confirm the diagnosis of tuberculosis

within 8 hours, with a sensitivity and specificity that rivals culture techniques.

Also, PCR analysis facilitates DNA fingerprinting of specific strains, allowing studies of the progress of

epidemics.

Mycobacterium tuberculosis colonies grown on LowensteinJensen medium

The four groups of Mycobacteria (M tuberculosis complex, M avium complex, M kansasii, and M gordonae)

make up 95% or more of clinical isolates of Mycobacteria. For species that cannot be identified by DNA

probes, many laboratories with molecular capabilities have implemented 16S rRNA gene sequencing to rapidly

identify probe-negative species or send such organisms to a reference laboratory with sequencing capability.

High-performance liquid chromatography (HPLC) has been applied to identification of Mycobacteria. The

method is based on development of profiles of mycolic acids, which vary from one species to another.

Several chemotherapeutic agents are effective against M. tuberculosis. Because strains of the organism resistant

to a particular agent emerge during treatment, multiple drug therapy is employed to delay or prevent

emergence. Isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide are the principal or “first-line”

drugs because of their efficacy and acceptable degree of toxicity.

Mutants resistant to each of these agents have been isolated even prior to drug treatment. Therefore, the

standard procedure is to begin treatment with two or more drugs to prevent outgrowth of resistant strains.

Sensitivity tests, administered as soon as sufficient cultured organisms are available, are an important guide to

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modifying treatment. In most parts of the United States, 8 to 14 % of M. tuberculosis strains are resistant to one

or more of the primary drugs.

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Lecture 14

Central Nervous System Diagnostic Microbiology

Meningitis is an infection of the membranes (meninges) surrounding the brain and spinal cord.

Meningitis is usually of multiple etiologies; bacterial, fungal or viral yet bacteria remain the common

etiological agent.

Meningitis can be acute, with a quick onset of symptoms, or chronic, lasting a month or more, or can be

mild or aseptic.

Bacterial meningitis continues to be a potentially life threatening emergency with significant morbidity

and mortality throughout the world and is an even more significant problem in many other areas of the

world, especially in developing countries

Types of bacteria that cause bacterial meningitis vary by age group. Currently, the average age of

contracting meningitis is above 25 years with Streptococcus pneumoniae, Neisseria meningitidis and

Haemophilus influenzae being the most common pathogens.

Trauma to the skull gives bacteria the potential to enter the meningeal space. Similarly, individuals with

a cerebral shunt or related device are at increased risk of infection through those devices. In these cases,

infections with Staphylococci, Pseudomonas aeruginosa and other gram-negative bacilli are more

likely.

Recurrent bacterial meningitis may be caused by persisting anatomical defects, either congenital or

acquired, or by disorders of the immune system.

Tuberculous meningitis is common in those from countries where tuberculosis is common.

Viral meningitis is generally less severe and clears up without specific treatment. Viral (Aseptic)

meningitis is serious but rarely fatal in people with normal immune systems. Usually, the symptoms last

from 7-10 days and the patient recovers completely. Often, in early stages of viral meningitis and

bacterial meningitis, the symptoms are almost similar

Fungal meningitis is rare, but can be life threatening. Although anyone can get fungal meningitis,

people at higher risk are those who have AIDS, leukemia, or other forms of immunodeficiency.

The most common cause of fungal meningitis in HIV is Cryptococcus spp. In the last two decades,

advancements in transplant procedures and concomitant use of immunosuppressive therapies as well as

the pandemic spread of HIV, have increased the incidence of central nervous system (CNS) fungal

infections. The clinical picture may mimic tuberculous meningitis.

The causes of non infectious meningitis include cancers, systemic lupus erythematosus, drug induced,

head trauma, brain surgery.

Collection, transportation, receipt and storage of cerebrospinal fluid (CSF)

Direct testing of CSF is the most accurate way to confirm the diagnosis of bacterial meningitis.

CSF should be collected from all the cases with suspected meningitis before the antimicrobial therapy.

Petechial fluid can be another specimen in cases with meningococcal meningitis. Petechial lesions, if

present, may be gently irrigated by injecting 0.2 ml of sterile saline solution using a small syringe with

a fine needle and the fluid collected for smear and culture.

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Early diagnosis is essential and is best established by laboratory examination of CSF. However, therapy

should not be dependent or delayed pending lumbar puncture or laboratory results.

To initiate the definitive identification of a bacterium responsible for meningitis, CSF specimens should

be obtained from patients with clinical signs and symptoms of meningitis and should be transported to

the laboratory without delay.

“N.meningitidis, S.pneumoniae, and H.influenzae are fastidious organisms that may not survive

long transit times”

The processing of a CSF specimen is one of the few clinical microbiology procedures that must be done

immediately.

Laboratorians should always record the date and time a specimen was received. Usually, three or more

tubes of CSF are collected during a lumber puncture procedure .

The tubes should be numbered in sequential order with tube number one containing the first sample of

CSF obtained.

The CSF in tubes 1, 2, and 3 most often are examined for chemistry, microbiology, and cytology .

However, the contamination with skin flora and disinfectant will be ruled out after the first tube of CSF

is collected.

The probabilities of detecting microorganisms by staining and by culturing are related to the volume of

specimen that is concentrated and examined.

CSF volumes of 2 to 3 ml are usually sufficient to detect bacteria, but for mycological and

mycobacterial investigations a minimum of 5 ml (preferably 10 to 15 ml) of CSF is required .

If only a small amount of CSF is received with requests for multiple assays, the order of priority of the

tests is determined after discussion with the physician

“The specimen should not be refrigerated before subjecting to microbiological tests as it may prevent

the recovery of the organisms; fastidious organisms may not survive variations in temperature”

CSF specimens should be stored at room temperature or at 37 °C if they cannot be processed

immediately or till microscopy and bacterial cultures are performed, after which it can be refrigerated

for further use.

Bacterial meningitis is a significant cause of mortality and morbidity worldwide,

neurological outcome and survival depend largely on damage to CNS prior to effective antibacterial

treatment

“Quick diagnosis and effective treatment is the key to success”

The diagnostic dilemma in acute pyogenic meningitis is due to large spectrum of signs and symptoms

Examination of CSF

The CSF should arrive still warm and either be examined immediately or placed in an incubator for

examination within an hour, If delay is anticipated either in transportation to the laboratory or for

examination, CSF should be divided into two tubes: one in a plain tube and the other in a tube having a

few drops of glucose broth.

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In the laboratory, CSF from the plain tube can be used for making smears for staining whereas cultures

are done from tube having CSF in glucose broth

The residual CSF should be preserved frozen for further assessment and evaluation

An examination of CSF involves the following

1.Macroscopic examination

2.Cytological examination

3.Examination of Gram stained smear

4.Culture and antimicrobial susceptibility testing

5.Latex agglutination test for antigen detection

Macroscopic examination

By appearance, the CSF is normally clear like water; cloudy, purulent, bloody or pigmented CSF as per

the disease states

Hazy, cloudy, turbid CSF indicates either metastatic spread of tumors into the CNS; Opalescent CSF

may be suggestive of Cryptococcal meningitis

“The CSF is attributable to both the bacteria and leukocytes present”

In evaluating patients with suspected meningitis or encephalitis, a careful history along with

biochemical and cellular analysis of CSF is required

CSF glucose

CSF glucose concentrations <45 mg/dl are indicative of bacterial meningitis. CSF glucose

concentrations depend on serum concentrations and “should always be tested on paired samples”

A CSF/serum ratio cut-off of <0.4 is helpful in distinguishing between bacterial and aseptic meningitis

with a sensitivity and specificity of 91% and 96%, respectively

CSF protein

Despite typical CSF findings, the spectrum of CSF values in bacterial meningitis is so wide that the

absence of one of more of the typical findings may not affect the diagnosis

In community-acquired bacterial meningitis only 50 % may have CSF glucose above 40 mg/dl, less

than half cases may have a CSF protein below 200 mg/dl, CSF protein measurements of >55 mg/dl are

diagnostic of bacterial, fungal and tubercular meningitis

Cytological examination

In untreated bacterial meningitis, the WBC count is elevated, usually in the range of 1000–5000

cells/mm3

, although this range can be quite broad (<100 to >10, 000 cells/mm3

)

Bacterial meningitis usually leads to a neutrophils predominance in CSF, typically between 80% and

95%; 10% of patients with acute bacterial meningitis present with a lymphocyte predominance (defined

as >50% lymphocytes or monocytes) in CSF.

It is important to note that a false-positive elevation of the CSF WBC can be found after traumatic

lumbar puncture, or in patients with intracerebral or subarachnoid hemorrhage in which both red blood

cells and white blood cells are introduced into the subarachnoid space.

Examination of Gram stained smear

It is preferable to make a smear from CSF at the time of collection itself, for direct

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demonstration of organisms.

The Gram stained smear made either directly from the CSF or from the centrifuged deposit can reveal

not only the Gram character of the causative organism, but can also clinch the diagnosis in some cases.

“Gram stain may not be interpretable in grossly blood stained samples”

Although Gram staining of CSF sediment is a very useful, cheap and fairly rapid method of

identification of organism, the sensitivity in developing countries is only 25-40% when compared to 80-

85% in developed countries.

CSF Gram staining may identify the causative microorganism for patients with suspected bacterial

meningitis.

In 1/3 of the cases with bacterial meningitis defined by CSF parameters may have negative CSF

cultures; around 50% of the CSF culture negative patients have a positive Gram stain with equal percent

of patients being pretreated with antibiotics.

The Gram stain is positive in 10-15 percent of patients who have bacterial meningitis but with negative

CSF culture.

In developing countries among suspected meningitis cases, CSF Gram staining can identify the

causative organisms in 2/3rd, and CSF culture is positive in 1/10th of the pretreated patients.

“Gram staining correctly identifies the pathogen in 69 to 93% of patients with pneumococcal

meningitis”

The reported sensitivities of CSF Gram staining vary considerably for different microorganisms. CSF

Gram staining correctly identifies the organism in 50-65% of children and in 25-33% in adults with H.

influenzae meningitis

The chances of recovery of bacteria in CSF Grams up to 100-fold, can be intensified by replacing

conventional centrifugation with cytospin centrifugation

This increase is comparable to the concentration of 100 ml of CSF to a volume of 1.0 ml by

conventional centrifugation

Cytospin-prepared smears not only increased the positivity of the smears, the morphology of the cells

was well preserved with uniform distribution of the cells

Culture and antimicrobial susceptibility testing

Culture is the gold standard for determining the causative organism in meningitis.

After the receipt, specimen should be cultured at the earliest. CSF should also be inoculated into

“enrichment medium like sodium thioglycollate broth” along with solid media like enriched, selective

or differential media. Incubate in air plus 5-10% carbon dioxide.

Anaerobic culture may be important for post neurosurgical or posttraumatic meningitis or for the

investigation of CSF shunt meningitis.

“Negative or inconclusive culture results may be seen in patients with partially-treated meningitis

and those with atypical bacteria, and Mycobacterium tuberculosis”

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Figure shows Gram stain of N. meningitidis in CSF with associated PMNs

Figure shows Gram stain of S. pneumoniae with WBCs

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Figure shows H. influenzae are small, pleomorphic gram-negative rods or coccobacilli with random

arrangements

Figure shows proper streaking and growth of N. meningitidis on a blood agar plate

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Figure shows proper streaking and growth of S. pneumoniae on a blood agar plate

Figure shows proper streaking and growth of H. influenzae on a chocolate agar plate

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Figure shows H. influenzae colonies on a chocolate agar plate

Figure shows N. meningitidis colonies on a blood agar plate

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Lecture 15

Gastrointestinal Tract Diagnostic Microbiology

1- Food and untreated water borne infectious diseases

A. Salmonella, shigella, cholera

B. Hepatitis A / E, Caliciviruses / Norwalk virus, poliomyelitis

C. Giardia, cryptosporidium, Cyclospora

2- Dairy (unpasteurized)

A- Brucella, Listeria, Salmonella, Shigella

3- Raw or undercooked food

A. Enteric bacteria (Salmonella, Shigella, C. jejuni)

B. Helminths (Ascaris, Trichinella, Taenia)

C. Protozoa (Amoebiasis, Toxoplasma)

Stool cultures are performed to detect enteric pathogens. Routine stool cultures should be examined

for the presence for Salmonella, Shigella, and Campylobacter spp at minimum.

It should be noted that media routinely set to detect these pathogens will also detect Aeromonas

hydrophila and Plesiomonas shigelloides. Vibrio, Yersinia and E. coli O157:H7.

Bloody stool specimens are routinely screened for E. coli O157:H7 or shiga-like producing strains of

E. coli.

Recently, the CDC has recommended routine screening of stool culture for the presence of E. coli

strains that produce a Shiga-like cytotoxin

Enterohemorrhagic E. coli (EHEC) has been isolated from patients who have hemorrhagic colitis and

hemolytic-uremic syndrome (HUS).

One virulence trait of all EHEC strains is the ability to produce cytotoxin(s) called Shiga-like toxin

(SLT) or verotoxin (VT)

SLT-I and SLT-II are the two most common toxins and individual EHEC strains have the ability to

produce both or either, in varying quantities. Therefore, SLT production and not individual (O157:H7)

serotype identification is a better diagnostic strategy for the determination of EHEC associated disease

Specimen Collection, Transport and Handling

Feces collected in sterile or non sterile clean container and sent to the laboratory for immediate processing. The

pH of the specimen will begin to drop after collection and this could reduce the quantity of pathogens

present.

The number of stool specimens submitted to the laboratory is controversial. It is recommended if more than one

specimen is submitted that it should be on different days

Specimen transport and handling

Stool held at room temperature should be cultured within 1 hour of collection.

Refrigeration of stool specimens is not recommended as some pathogens, especially Shigella species, are very

susceptible to lower temperatures and will die rapidly.

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Specimens that cannot be sent immediately to the laboratory or processed shortly after collection should be

placed in an appropriate enteric transport media (Cary-Blair transport swab or Amie's transport swab).

Specimens containing barium, mineral oil or urine should be rejected

Stool direct examination

Leukocyte evaluation

1- Perform a Gram stain on the stool specimen to evaluate for the presence of leukocytes.

Direct Gram stain is not useful beyond its determination of the presence of leukocytes, as it will not

differentiate suspected pathogens from normal microbial flora.

Place a drop of feces on a slide, spread out, and allow to air dry. Stain slide using Gram’s stain procedure.

Evaluate slide under oil immersion for PMN’s

2- Latex agglutination for detection of lactoferrin released by fecal leukocytes in diarrheal stool specimens.

Not appropriate for detection of leukocytes in breastfeeding babies. Lactoferrin present in breast milk

Culture Setup

A. The purpose of a stool culture is to use selective and routine media to screen for the presence of stool

pathogens.

B. In many cases, full identification of a suspicious colony is not performed. Instead, screening tests are

performed to rule out potential stool pathogens. If suspicious colonies are noted then full identification

performed.

C. Routine stool cultures should always include testing for the presence of Salmonella species, Shigella

species, and Campylobacter jejuni

D. It is recommended that routine stool cultures include testing for the presence of Aeromonas species,

and Plesiomonas species. The CDC recommends routine screening of stool for E. coli strains that

produce a Shiga-like cytotoxin.

E. When required by the physician, media may also be included to detect Yersinia species or Vibrio

species. However, in areas where these pathogens are common including media for these organisms is

part of the routine culture

F. The CDC recommends testing to detect shiga-like toxin producing strains of E. coli in addition to

culture for Escherichia coli O157:H7.

G. It is no longer recommended to do culture alone. Clinical sites may perform culture and toxin testing

or toxin testing alone.

H. A macroscopic exam is reported with every culture

Pathogens commonly isolated in stool

a. Salmonella species

b. Shigella species

c. Campylobacter jejuni

d. Vibrio cholera

e. Vibrio parahaemolyticus

f. Yersinia enterocolitica

g. Clostridium difficile

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h. Staphylococcus aureus

i. Aeromonas species

j. Plesiomonas species

k. Hemorrhagic Escherichia coli O157:H7

l. Shiga-like toxin producing strains of Escherichia coli

Normal flora commonly isolated in stool

a. Enteric organisms

b. Staphylococcus species

c. Streptococcus / Enterococccus species

d. Anaerobic organisms

Inoculate media

Use a swab to inoculate plates using representative areas of the specimen. Inoculate the media making the first

streak then use a sterile loop to streak for isolation. Non inhibitory media should always be inoculated first

Routine stool culture

a. Blood agar

b. MacConkey’s agar

c. Hektoen enteric (HE) agar or xylose-lysine deoxycholate (XLD) agar

d. Selective agar for Campylobacter (i.e., CVA, Campy BAP, Skirrow’s)

e. GN broth (or Selenite F) is a selectively enhances the growth of Salmonella and Shigella while suppressing

the growth of normal bowel flora. Also, used for the detection of shiga-like toxin producing strains of E. coli.

This will increase the chances of isolating these pathogens when they are present in small numbers.

Yersinia culture

1. Cefsulodin Novobiocin (CN) agar or Yersinia Selective agar

2. Phosphate buffered saline (PBS) broth – suppresses the growth of normal bowel flora allowing easier

detection of Yersinia species

Vibrio culture

a. TCBS agar

b. Alkaline peptone broth

Incubate media Temperature

a. Campy BAP: 42ºC

b. PBS broth: 4ºC

c. CN plate: room temperature

d. All other plates: 35ºC

Atmosphere

Campy BAP: microaerophilic (increased CO2)

b. BAP: either ambient air or CO2

c. All other plates: ambient air Time: 24- 48 hours

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Culture Interpretation

A. Evaluation for Salmonella and Shigella

After 24 hours incubation, examine the HE and MAC plates for non-lactose fermenting colonies and H2S

producing colonies.

These colony types are suspicious for Salmonella species and Shigella species, but may also be normal enteric

flora; so biochemical screens must be performed to rule in or out the presence of these pathogens.

For colonies that are suspicious for Salmonella or Shigella perform KIA slants.

If screen is positive perform biochemical ID panels such as API 20 E.

Colonies that appear to be Salmonella species or Shigella species are confirmed by performing serological

agglutination tests. If enrichment broth (i.e., GN broth or Selenite F) is used, they are subcultured to HE at 24

hours.

Suspicious colonies from the enrichment broth subculture plates are screened biochemically.

A susceptibility test is performed only on any confirmed colonies of Salmonella and Shigella species when

requested

Treatment with antibiotics is not recommended for Salmonella species because it may induce a carrier state in

the patient. In most case treatment of the clinical symptoms such as dehydration is sufficient.

If no suspicious colonies are found or if all biochemical screens are negative, the report is sent out as: “No

Salmonella or Shigella isolated”

B. Campylobacter jejuni

Campy plates are examined at 48 hours and 72 hours. Colonies growing on the original Campy BAP are tested

for oxidase. Any oxidase positive colonies are Gram stained to look for the typical curved rods of

Campylobacter species.

Oxidase positive curved gram-negative rods should be further tested to confirm the presence of Campylobacter

species. A positive Sodium Hippurate test will confirm the species Campylobacter jejuni.

C. Aeromonas and Plesiomonas

After 24 hours incubation, examine the BAP for large gray colonies that are gram-negative rods in

predominance.

Do an oxidase on each different colony type of gram-negative rods. If a colony type is oxidase positive and has

a smooth morphology, perform a spot indole test. An oxidase positive indole positive gram-negative rod is

suspicious for Aeromonas species or Plesiomonas species.

Confirm identification of suspicious organisms with various biochemical ID panels.

Aeromonas species are slightly beta hemolytic on the BAP but Plesiomonas species are nonhemolytic.

Do not use the MAC to screen for Aeromonas or Plesiomonas as these organisms can be lactose variable

D. Yersinia

After 24 hours incubation, examine the CN plate for dark red colonies with a “bull’s eye” center surrounded by

transparent border colonies that indicate mannitol fermentation and is suspicious for Yersinia.

Confirm identification of suspicious organisms with various biochemical ID panels.

CN plates are held for 72 hours at room temperature.

E. Vibrio

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After 8-24 hours of incubation, subculture alkaline peptone broth to a TCBS agar. Examine the TCBS agar for

yellow colonies that indicate sucrose fermentation. Screen suspicious colonies biochemically.

F. Staphylococcus aureus or Yeast

After 24 hours incubation, examine the BAP for Staphylococcus aureus or yeast. If organism is in moderate to

many amounts or as the predominant organism, work up the organism

G. Escherichia coli O157:H7

Setup stool culture with a MacConkey’s agar with 1% D-sorbitol (instead of lactose).

After 24 hours of incubation, examine Mac-Sorbitol plate for colorless colonies (E. coli O157:H7 is sorbitol

negative and most other normal flora strains of E. coli are sorbitol positive).

Figure shows E coli O157:H7 on a sorbitol-MacConkey’s agar plate Arrow indicates distinctive colorless

Confirm identification of suspicious organisms with various biochemical ID panels and O157:H7 antisera.

Shiga-like toxin producing strains of E. coli carry out EIA or molecular testing is performed to detect

these strains.

Serological Testing for Salmonella and Shigella

A. All isolates that biochemically resemble either Salmonella species or Shigella species must be confirmed by

serological methods following site specific procedures.

B. For suspected Shigella species, test the following somatic (“O”) antigens:

1. Antigen A = Shigella dysenteriae

2. Antigen B = Shigella flexneri

3. Antigen C = Shigella boydii

4. Antigen D = Shigella sonnei

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C. For suspected Salmonella species, test the following somatic (“O”) antigens:

1. Polyvalent A-E, Vi serum

2. Polyvalent F-I serum

Clostridium difficile

A. Clostridium difficile is an anaerobic gram-positive spore-forming rod that causes antibiotic-associated

pseudomembranous enterocolitis.

Since C. difficile is found as part of the normal fecal flora in many individuals, isolation of the organism does

not prove the presence of disease. In cases of C. difficile diarrhea, the patient’s normal fecal flora is suppressed

by prolonged antibiotic therapy allowing the C. difficile to multiply and produce a toxin

B. Detection of the presence of the toxin is used to diagnose the disease. Toxin may be detected by tissue

culture assay, a latex agglutination test, or an ELISA test.

Stool specimens collected for toxin studies should be refrigerated until testing to preserve the toxin that is

rapidly labile at higher temperatures.

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Lecture 16

Diagnostic Microbiology

Urinary tract infections (UTIs)

Urinary tract infections (UTIs) are among the most common bacterial infections and account for a significant part of the workload

in clinical microbiology laboratories.

Enteric bacteria (in particular, Escherichia coli) remain the most frequent cause of UTIs, although the distribution of pathogens

that cause UTIs is changing.