Principle:

• Soil is the prime storage of the microorganisms producing antibiotics which are able to inhibit the growth of other microorganisms. Antibiotics have been implemented in one or other forms for centuries. The screening of wild isolates from the soil has yielded the broad majority of new antibiotics. Even if the purification of several hundred naturally produced antibiotics has been performed, only a few have been proved to be successful to be used in medical practice. Those which are presently of greatest use were derived from a comparatively small group of microorganisms belonging to the genera Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus. In this era, the continuous efforts to develop new antibiotics are the emerging trends.
• Even if soils from various parts of the world are continually screened in industrial laboratories in order to isolate new antibiotic-producing microorganisms, industrial microbiology is directing its efforts toward chemical modification of existing antibiotic substances. This is completed by adding or replacing chemical side chains, reorganizing intramolecular bonding, or producing mutant microbial strains able to excrete a more potent form of the antibiotic. The establishment of chemical congeners accounts for the overcoming of antibiotic resistance, reducing adverse side effects in the host and increasing the effective spectrum of a given antibiotic.
• I: We will use the crowded-plate technique for the isolation of antibiotic producing microorganisms from two soil samples, one of which is seeded with Streptomyces griseus to serve as a positive control.
• II: For the determination of anti-microbial spectrum of isolates, isolates manifesting antibiotic activity will be screened against several different microorganisms to establish their effectiveness.

I. Isolation of antibiotic producing microorganisms

Requirements:

1.  Soil Suspensions:
   • – 1:500 dilution of soil sample suspension (0.1 g of soil per 50 ml of tap water) to serve as an unknown
   • – 1:500 dilution of soil sample seeded with S. griseus (0.1 g of soil per 50 ml of tap water) to serve as a positive control.
2. Media:
   • Six 15-ml Trypticase soy agar deep tubes, and two Trypticase soy agar slants.
3. Equipment:
   – 500-ml beaker
   – test tubes
   – test tube rack
   – sterile Petri dishes
   – inoculating needle
   – hot plate
   – thermometer
   – 1-ml and 5-ml pipettes
   – mechanical pipetting device
   – magnifying hand lens.

Procedure for Isolation of antibiotic producing microorganisms

• Label two sets of three sterile Petri dishes with the types of soil samples being used and dilutions (1:1000, 1:2000, and 1:4000).
• Place six Trypticase soy agar deep tubes into a beaker of water and bring to 100°C on a hot plate. Once agar is liquefied, add cool water to the water bath. Cool to 45°C, checking the temperature with a thermometer.
• Prepare a serial dilution of the unknown and positive control 1:500 soil samples as follows:
  – Label three test tubes 1, 2, and 3. With a pipette, add 5 ml of tap water to each tube.
  – Shake the provided 1:500 soil sample thoroughly for 5 minutes to effect a uniform soil-water suspension.
  – Using a 5-ml pipette, transfer 5 ml from the 1:500 dilution to Tube 1 and mix. The final dilution is 1:1000.
  – Using another pipette, transfer 5 ml from Tube 1 to Tube 2 and mix. The final dilution is 1:2000.
  – Using another pipette, transfer 5 ml from Tube 2 to Tube 3 and mix. The final dilution is 1:4000.
  – Using separate 1-ml pipettes, transfer 1 ml of the 1:1000, 1:2000, and 1:4000 dilutions to their appropriately labelled Petri dishes.
  – Pour one tube of molten Trypticase soy agar, cooled to 45°C, into each plate and mix by gentle rotation.
  – Allow all plates to solidify.
• Incubate all plates in an inverted position for 2 to 4 days at 25°C.
• Examine all crowded-plate dilutions for colonies exhibiting zones of growth inhibition. Use a hand magnifying lens if necessary. Record in the Lab Report the number of colonies showing zones of inhibition.
• Aseptically isolate one colony showing a zone 34 of growth inhibition from each soil culture
  with an inoculating needle and streak onto
  Trypticase soy agar slants labelled with the soil sample from which the isolate was obtained

• Incubate the slants for 2 to 4 days at 25°C. These will serve as stock cultures of antibiotic-producing isolates to be used in Part B.

II. Determination of antimicrobial spectrum of isolates

Requirements:

1. Cultures:
   • – 24-hour Trypticase soy broth cultures of Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis, and Pseudomonas aeruginosa.
2. Media:
   Two Trypticase soy agar plates.
3. Equipment:
   – Bunsen burner
   – inoculating loop
   – glassware marking pencil.

Procedure  for determination of antimicrobial spectrum of isolates.

• Label the Trypticase soy agar plates with the soil sample source of the isolate.
• Using aseptic technique, make a single-line streak inoculation of each isolate on the surface of an agar plate so as to divide the plate in half
• Incubate the plates in an inverted position for 3 to 5 days at 25°C.
• Following incubation, on the bottom of each plate draw four lines perpendicular to the growth of the antibiotic-producing isolate
• Aseptically make a single-line streak inoculation of each of the four test cultures following the inoculation template on each plate. Start close to, but not touching, the growth of the antibiotic-producing isolate and streak toward the edge of the plate.
• Incubate the plates in an inverted position for 24 hours at 37°C.
• Examine all plates for inhibition of test organisms, and record your observations in the Lab Report.

Observations and Results interpretations:

• I: Isolation of Antibiotic-producing micro-organisms.
  -Number of colonies showing zone of inhibition in different serial dilutions were noted and were further cultured to obtain pure cultures.
• II: Determination of anti-microbial spectrum of isolates.
  • Draw a representation of the observed antibiotic activity against the test organisms.
  • Based on your observations, record in the chart the presence (+) or absence (−) of antibiotic activity against each of the test organisms and the spectrum of antimicrobial activity (broad or narrow).

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Principle:

• Members of the anaerobic genera Clostridium and Desulfotomaculum and the aerobic genus Bacillus are examples of organisms that have the capacity to exist either as metabolically active vegetative cells or as highly resistant, metabolically inactive cell types called spores. When environmental conditions become unfavorable for continuing vegetative cellular activities, particularly with the exhaustion of a nutritional carbon source, these cells have the capacity to undergo sporogenesis and give rise to a new intracellular structure called the endospore, which is surrounded by impervious layers called spore coats. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called a free spore. Because of the chemical composition of spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation, desiccation, and chemical agents, as well as to the commonly employed microbiological stains. With the return of favorable environmental conditions, the free spore may revert to a metabolically active
  and less resistant vegetative cell through germination. It should be emphasized that sporogenesis and germination are not means of reproduction but merely mechanisms that ensure cell survival under all environmental conditions.

In practice, the spore stain uses two different stains and decolorizing agents:

1. Primary Stain (Malachite Green):

• Unlike most vegetative cell types that stain by common procedures, the free spore, because of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green.

2. Decolorizing Agent (Water):

• Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only the excess primary stain. The spore remains green. On the other hand, the stain does not demonstrate a strong affinity for vegetative cell components; the water removes it, and these cells will be colorless.

3. Counter stain (Safranin):

• This contrasting red stain is used as the second reagent to color the decolorized vegetative cells, which will absorb the counterstain and appear red. The spores retain the green of the primary stain.

Requirements

• i). Bacterial Culture: 48-72 hrs nutrient agar slant culture of Bacillus cereus and thioglycollate culture of Clostridium sporogenes.
• ii. Reagents: Malachite green and safranin.
• iii. Equipment: Microincinerator or Bunsen burnerhot plate, staining tray, inoculating loop, glass slides, bibulous paper, lens paper, and microscope.

Procedure for Spore staining:

1. Obtain two clean glass slides.
2. Make individual smears in the usual manner using aseptic technique.
3. Allow smear to air-dry, and heat fix in the usual manner.
4. Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. Note: Do not allow stain to evaporate; replenish stain as needed. Prevent the stain from boiling by adjusting the hot
   plate temperature.
5. Remove slides from hot plate, cool, and wash under running tap water.
6. Counterstain with safranin for 30 seconds.
7. Wash with tap water.
8. Blot dry with bibulous paper and examine under oil immersion.
9. In the chart provided in the Lab Report, complete the following:
   • Draw a representative microscopic field of each preparation.
   • Describe the location of the endospore within the vegetative cell as central, sub-terminal, or terminal on each preparation.
   • Indicate the color of the spore and vegetative cell on each preparation

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Central nervous system infections including Meningitis

• The first step in the diagnosis of a patient with suspected CNS infection is a lumbar puncture (spinal tap).

Specimen: Cerebrospinal fluid (CSF)

Collection and Transport of CSF:

• Aseptically CSF is collected.
• A needle is inserted into the subarachnoid space (lumbar puncture), at the lumbar spine region between L3, L4, or L5.
• In the sterile collection tubes, three or four tubes of CSF should be collected. It should not contain additives.
• Tube 1 is used for:
  • chemistry studies
  • glucose and protein count
  • immunology studies
• Tube 2 is used for culture.
• Tubes 3 and 4 are used for cell count and differential count.
• The amount of volume to be collected depends on the volume available in the patient which may differ between the adults and the neonates.
• When the needle first punctures the subarachnoid space, the opening pressure of the CSF is observed.
• In the high opening pressure, CSF should be collected slowly to prevent the collection of a larger volume of fluid.
• For the detection of mycobacteria and fungi, a minimum of 5 to 10 mL is recommended.
• Centrifugation and subsequent culture are done.
• The false-negative result may be seen if the sample is inadequate.
• CSF should be sent to the laboratory as soon as possible.
• In the case of delay after an hour or longer, agents such as Streptococcus pneumoniae, may not be detectable.
• CSF should not be refrigerated for microbiological studies.
• In the case of delay, it should be left at room temperature or incubated at the 35°C.
• For the viral study, CSF may be refrigerated, for as long as 23 hours after collection or frozen at −70°C.
• For hematology studies, CSF specimens can be refrigerated,
• For chemistry and serology, CSF can be frozen (−20° C).

Initial processing of CSF:

• All the CSF specimens for the bacterial, fungal, or parasitic studies should be centrifuged.
• Volume greater than 1 ml should be used.
• Centrifugation should be done at 1500× g for 15 minutes.
• Suspected specimens for cryptococci or mycobacteria should be handled carefully.
• When CSF fewer than 1 mL is available, Gram stain should be done and plated directly to the blood and chocolate agar plates.
• The supernatant is removed to a sterile tube, leaving approximately 0.5 mL of fluid.
• For visual examination and culture, the remaining fluid is used to suspend the sediment.
• The supernatant can be used:
• To test the presence of antigens
• rapid diagnostic test (vertical flow immunochromatography)
• for meningitidis
• For chemistry evaluations (e.g., protein, glucose, lactate, C-reactive protein).

Laboratory diagnosis:

• Communication between the physician and the microbiology laboratory is essential for the proper diagnosis and treatment of the patient.
• The diagnosis of acute bacterial meningitis can be excluded in patients with normal fluid parameters in almost all cases.
• Similar criteria have been used to exclude the performance of smear and culture for tuberculosis, as well as syphilis serology, on CSF specimens.

1. Visual Detection of Etiologic Agents in CSF:

• CSF sediment is examined for the presence of cells and organisms.

i) Stained Smear of Sediment:

• Gram staining should be performed on all the CSF sediments.
• The use of contaminated slides may give false-positive smears.
• The sediment should be thoroughly mixed and a heaped drop should be placed in the slide.
• The slide should be sterile or alcohol-cleaned.
• The sediment should never be spread out on the slide surface.
• It is because of the difficulty to find small numbers of microorganisms.
• The drop of sediment is allowed to air dry.
• Then it is heated or methanol fixed.
• Then it is stained by either Gram or acridine orange.
• A faster examination of the slide under high-power magnification (400×) can be done by the acridine orange fluorochrome stain.
• The brightly fluorescing bacteria can be visualized easily.
• Confirmation of the presence and the morphology of the organism can be done, using the Gram stain (directly over the acridine orange.
• The use of a cytospin centrifuge is an excellent alternative method for the preparation of slides for staining.
• It concentrates cellular material and bacterial cells up to 1000-fold.
• Centrifugation is done then the CSF is concentrated onto a circular area of a microscopic slide.
• It is then fixed, stained, and examined.
• Reporting should be done for the presence or absence of bacteria, inflammatory cells, and erythrocytes.

ii) Wet Preparation:

• Amoebas are best observed by this method.
• Sediment can be examined as wet preparation under phase-contrast microscopy.
• The light microscope can be used as an alternative, by slightly closing the condenser.
• Amoebas must be distinguished from motile macrophages, which occasionally occur in CSF.
• A trichrome stain can be used in the differentiation of amoebas from somatic cells.
• On the lawn of Klebsiella pneumoniae or Escherichia coli, the pathogenic amoebas can be cultured. Lawn.

iii) India Ink Stain:

• Cryptococcus neoformans consists of the large polysaccharide capsule which could be visualized by the India ink stain.
• For capsular antigen, latex agglutination testing is more sensitive and extremely specific.
• Antigen test is recommended than the India ink stain.
• Culture is essential in case of the AIDS patients because detectable capsules of neoformans may be absent.
• A drop of CSF sediment is mixed with one-third volume of India ink, for the India ink preparation.
• By the addition of 0.05 mL thimerosal, India ink can be protected from contamination.
• Smooth suspension is made by mixing the CSF and ink.
• Then a coverslip is applied to the drop.
• Then it is examined under high-power magnification (400×) for characteristic encapsulated yeast cells.
• Examination can be done under oil immersion.
• White blood cells must not be confused with yeasts.
• The presence of encapsulated buds, smaller than the mother cell, is diagnostic.

2. Direct Detection of Etiologic Agents:

Antigen detection:

• For the rapid detection of antigen in the CSF, commercial reagents and kits are available.
• By latex agglutination, rapid antigen detection can be done from CSF.
• An antibody-coated particle binds to a specific antigen which results in macroscopically visible agglutination.
• The soluble capsular polysaccharide, including the group B streptococcal polysaccharide, is well suited to serve as bridging antigens.
• Polyclonal or monoclonal antibody or an antigen from an infectious agent is present in the agglutination assay.
• Different commercial systems have been developed.
• Soluble antigens may concentrate in the urine from Streptococcus agalactiae and Haemophilus influenza.
• For the performance of antigen detection test systems, the manufacturers’ directions must be followed
• Some systems may also require the pretreatment of samples which is usually for 5 minutes.
• The pretreatment, called rapid extraction of antigen procedure (REAP), is recommended for laboratories that use commercial body fluid antigen detection kits.
• Only a limited number of clinically useful situations warrant bacterial antigen testing (BAT).
• Practice guidelines for the diagnosis and management of bacterial meningitis do not recommend routine use of BAT.

Bacteria involved in meningitis:

Cryptococcus neoformans:

• For the detection of polysaccharide capsular antigen of Cryptococcus neoformans, the reagents are available commercially.
• When the positive result for cryptococcal antigen is obtained in CSF specimens, a second latex agglutination test for rheumatoid factor should be done.
• Both latex agglutination assays (numerous commercial manufacturers) and enzyme immunoassays are available for the detection of Cryptococcus antigen.
• The false-negative reaction may be seen in the undiluted specimens which contain large amounts of capsular antigens.
• False-negative reaction is caused by a prozone phenomenon.
• Patients with AIDS may have an antigen titer over 100,000.
• It requires many dilutions to reach an endpoint.
• Parasites and Viruses are also involved in meningitis

  Conditions for the culture of free-living amoebae and viral agents should be maintained to detect viruses and parasites.

3. Molecular methods:

• PCR (Polymerase Chain Reaction )
• Real-time PCR

4. Other Tests

• the Limulus lysate test
• CSF lactate determinations,
• C-reactive protein
• mass spectrometry
• gas-liquid chromatography

5. Culture:

• Routine bacteriologic media: chocolate agar plate, 5% sheep blood agar plate, and an enrichment broth, usually thioglycolate without indicator.
• Blood agar plates help in the recognization of pneumoniae.
• For the isolation of influenzae and N.meningitidis, a chocolate agar plate is used.
• Plates should be incubated at 37° C in 5% to 10% carbon dioxide (CO2) for at least 72 hours.
• Candle jar can be used, if a CO2 incubator is not available.
• The broth should be incubated in the air at 37° C for at least 5-10 days.
• Anaerobic blood agar plate may also be inoculated, when Gram stain shows the morphologically resembling anaerobic bacteria.
• If a brain abscess is suspected then also anaerobic blood agar plate is used.
• For CSF fungal cultures, two drops of the well-mixed sediment should be inoculated onto:
• Sabouraud dextrose agar
• other non-blood containing medium
• brain-heart infusion with 5% sheep blood.
• Incubation of Fungal media should be done at 30° C for 4 weeks.
• If possible, two sets of media should be inoculated.
• One set should be incubated at 30° C and the other at 35° C.

Specimen: Brain Abscess/Biopsies samples

Collection, Transport, and Processing of brain absscess and biopsies

• Under anaerobic conditions, biopsy specimens or aspirates from brain abscesses should be submitted.
• Devices are commercially available too for transportation.
• Swabs are not considered an optimum specimen.
• If swabs are used to collect abscess material, during transportation, they should be maintained in an anaerobic environment.
• Before plating and smear preparation, biopsy specimens should be homogenized in sterile saline.
• processing should be kept to a minimum to reduce oxygenation.
• Inoculation should be done onto 5% sheep blood and chocolate agar plates, for the abscess and biopsy specimens.
• Incubation should be done in 5% to 10% CO2 for 72 hours at 35° C.
• In addition, an anaerobic agar plate and broth with an anaerobic indicator, vitamin K, and hemin should be inoculated and incubated in an anaerobic environment at 35° C.
• Incubation of the anaerobic culture plate is done at a minimum of 72 hours.
• It is examined after 48 hours of incubation.
• Anaerobic broths should be incubated for a minimum of 5 days.
• When fungi are suspected, fungal media, such as brain-heart infusion with blood and antibiotics or inhibitory mold agar, should be inoculated.

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• Francisella tularensis results tularaemia in man and certain small mammals, such as rabbits, hares, beavers and several rodent species.
• Tularaemia was originally described in Tulare county, California.
• It can be transmitted by direct contact, by biting flies, mosquitoes and ticks, by contaminated water or meat or aerosols.
• Francisella tularensis is also know as Pasteurella tularensis or Brucella tulerensis)

Morphology of Francisella tularensis:

• It is a very small, nonmotile, nonsporing, capsulate, gram-negative coccobacillus, about 0.3 to 0.7 μm × 0.2 μm in size.
• In culture it tends to be pleomorphic to and larger, even filamentous, forms are present.
• It stains poorly with methylene blue but dilute carbol fuchsin (10%) produces characteristic bipolar staining.

Cultural Characteristics of Francisella tularensis:

• F. tularensis is strictly aerobic.
• It will not grow on ordinary nutrient media but grows well on blood agar containing 2.5 percent glucose and 0.1 percent cysteine hydrochloride.
• Minute droplet-like colonies develop in 72 hours.

Biochemical characteristics of Francisella tularensis:

• Under suitable conditions acid is formed from glucose and maltose. Indole and urease tests are negative.
• Two biovars are recognized. Strains of F. tularen­sis have been subdivided into biotypes based on their virulence and epidemiological behaviour.
• Highly virulent strains are found only in N. America, while strains of low virulence are seen in Europe and Asia also.

Pathogenesis of Francisella tularensis:

• The infection, which is a typical zoonosis, is mainly spread by insects or ticks among lagomorphs and rodents.
• It is transmitted to man through handling of infected animals, e.g. rabbits or hares tick, mosquito or fly bites, inhalation of contaminated dust, ingestion of contaminated water or meat.
• Laboratory workers are at higher risk while handling infected laboratory animals or cultures of the organism.
• Man to man transmission of infection apparently does not occur.
• In human beings, tularemia may present as a local ulceration with lymphadenitis, a typhoid like fever with glandular enlargement or an influenza like respiratory infection.
• The severity of disease is much greater with type A strains and case fatality rates may exceed 5 percent.
• Disease caused by type B strains is much less severe, with very low mortality.

Laboratory Diagnosis of Francisella tularensis :

• F. tularensis is extremely dangerous to handle in the laboratory and Category 3 containment is required for all manipulations and animal work.
• Diagnosis may be made by culture or by inoculation into guinea pigs or mice. A PCR has been described, but is not widely available.
• Serology is most likely to be positive after 3 weeks.
• Rising titres of agglutinins to F. tularensis or individual titres of 160 are diagnostic.
• Serum from cases of F. tularensis may cross-react with brucellosis and vice versa, usually to relatively low titre.
• An intradermal delayed hypersensitivity test has been used in the past but the antigen is not readily available.

Treatment of Francisella tularensis:

• Streptomycin or gentamicin are the antibiotics of choice in tularaemia and are usually curative.

Prophylaxis:

• A vaccine based on the live-attenuated LVS strain confers some protection.
• It can be administered by scarification to persons who are subject to high risk of infection.
• F. tularensis has been developed as a biological warfare agent and has potential application in bioterrorism.

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• The infection within the subarachnoid space or throughout the leptomeninges is called meningitis.
• Meningitis is divided into two major categories based on the host’s response to the invading microorganism. They are:
  • purulent meningitis
  • aseptic meningitis.

 1. Purulent meningitis

• A patient with purulent meningitis typically has a marked, acute inflammatory exudative cerebral spinal fluid containing large numbers of polymorphonuclear cells (PMNs).
• The underlying CNS tissue, in particular the ventricles, may be involved.
• Ventriculitis means the involvement of ventricles.
• The cause of these infections is bacterial organisms.

Pathogenesis of purulent meningitis:

• Within the Central Nervous System, the blood-brain barrier is the important host defense mechanism.
• This barrier involves the choroid plexus, arachnoid membrane, and the cerebral microvascular endothelium.
• Vascular endothelium has got the unique structural properties.
• There is the presence of continuous intercellular tight junctions.
• It minimizes the passage of infectious agents into the CSF and acts as a barrier.
• The vascular endothelium helps in regulating the transport of nutrients in and out of the CSF.
• It includes low-molecular-weight plasma proteins, glucose, and electrolytes.
• Different underlying conditions and the host’s age may be responsible for the development of infectious meningitis.
• The highest rate of infection of meningitis is in neonates.
• It is because of the:
  • the immature neonatal immune system
  • the increased permeability of the blood-brain barrier in newborns
• The presence of colonizing bacteria in the female vaginal tract
• The most common bacterial pathogens responsible for meningitis in newborns are:
  • Group B streptococci
  • Escherichia coli
  • Listeria monocytogenes
• Before the development of the vaccine i.e Hib vaccine, the common cause of meningitis is Haemophilus influenza type b.
• It occurred in children of 4 months to 5 years of age.
• There is a decline in the Hib disease because of this childhood immunization program.
• Neisseria meningitidis causes meningitis in young adults.

Two meningococcal vaccines (vaccines for N. meningitidis) are available:

• The meningococcal polysaccharide vaccine (MPSV4): for older than 55 years of age
• The meningococcal conjugate vaccine (MCV4): for adolescents.
• The cause of meningitis in young children and elderly people is Streptococcus pneumonia.
• This meningitis develops from bacteremia or infection of the sinuses or middle ear.

Two pneumococcal vaccines (vaccines for S. pneumoniae) are:

• The pneumococcal conjugate vaccine (PCV13):
• protects against infection from 13 different serotypes of pneumonia
• used for vaccination of children and adults.
• Pneumococcal polysaccharide vaccine (PPSV):
  • protects from 23 serotypes of pneumonia
  • recommended vaccine for adults 65 years of age and older
  • recommended vaccine for anyone over the age of 2 who has long-term health problems or is immunocompromised.
• The primary portal of entry for causative agents of meningitis is the respiratory tract.
• Predisposing factors of meningitis to the adults are usually the same factors that cause pneumonia or other respiratory tract colonization or infection.
• Increased risk in:
  • Alcoholism
  • Splenectomy
  • diabetes mellitus
  • prosthetic devices
  • immunosuppression
• Patients with prosthetic devices, particularly CNS and ventriculoperitoneal shunts, are at increased risk for developing meningitis.
• Host defense mechanisms must be overcome by the organism to reach the CNS (primarily by the blood-borne route).
• The pathogen should colonize and cross the host mucosal epithelium.
• Then it should enter and thrive within the bloodstream.
• Pathogen should be able to evade the host defenses at each level.
• By breaking the blood-brain barrier at the level of microvascular endothelium, helps the organism to enter the CNS.

Virulence factors of Streptococcus pneumoniae:

• IgA protease: It is secreted by the Streptococcus pneumoniae and meningitidis. It can destroy the host’s secretory IgA and helps in bacterial attachment to the epithelium.
• Capsule: It is antiphagocytic and helps to evade destruction by the host immune system.
• Pili
• polysaccharide capsules
• lipoteichoic acids
• Organisms can enter by
  • disrupting tight junctions of the blood-brain barrier
  • transport within circulating phagocytic cells
  • crossing the endothelial cell lining within endothelial cell vacuoles.
• Then multiplication occurs within the CSF.

Clinical Manifestation of purulent meningitis:

i). Acute meningitis

• Symptoms of acute meningitis include:
  • Fever
  • stiff neck
  • headache
  • nausea and vomiting
  • neurologic abnormalities
  • change in mental status.
  • Presence of large numbers of inflammatory cells (>1000/mm3), primarily polymorphonuclear cells (PMNs) in the CSF.
• In CSF there is:
  • decreased glucose level relative to the serum glucose level
  • an increase in protein concentration.
  • In Normal condition:
    • The normal CSF glucose level is 0.6 of the serum glucose level and ranges from 45 to 100 mg/dL
    • The CSF protein range in an adult is 15 to 50 mg/dL; newborn CSF protein ranges run as high as 170 mg/dL with an average of 90 mg/dL.
• The sequelae of acute bacterial meningitis in children are frequent and serious. It includes:

• • Seizures
  • cerebral edema
  • hydrocephalus
  • cerebral herniation
  • focal neurologic changes.
• In about 10% of children recovering from bacterial meningitis, permanent deafness can occur.

ii). Chronic Meningitis

• May occur in immunocompromised patients.
• Symptoms:
  • Fever
  • Headache
  • stiff neck
  • nausea and vomiting,
  • Lethargy
  • Confusion
  • mental deterioration.
• Symptoms may persist for a month or longer before treatment is sought.
• Manifestation in CSF:
  • an abnormal number of white blood cells (usually lymphocytic)
  • elevated protein
  • decrease in glucose content

The pathogenesis of chronic meningitis is similar to that of acute disease.

Etiologic agents of Chronic Meningitis:

• HIV cytomegalovirus
• Enterovirus
• HSV
• Mycobacterium tuberculosis
• Cryptococcus neoformans
• Coccidioides immitis
• Histoplasma capsulatum
• Blastomyces dermatitidis
• Candida
• Aspergillosis
• Mucormycosis
• Miscellaneous other fungi
• Nocardia
• Actinomyces
• Treponema pallidum
• Brucella
• Borrelia burgdorferi
• Sporothrix schenckii
• Rare parasites—Toxoplasma gondii, cysticercus, Paragonimus westermani, Trichinella spiralis, Schistosoma , Acanthamoeba

2. Aseptic meningitis:

• It is usually viral and characterized by an increase of lymphocytes and other mononuclear cells (pleocytosis) in the CSF
• Bacterial and fungal cultures are negative.
• It is usually self-limiting.
• Symptoms:
  • Fever
  • Headache
  • Stiff neck
  • nausea, and vomiting
• Increase of lymphocytes and other mononuclear cells in the CSF
• Normal glucose level
• Normal or slightly elevated protein CSF level.
• Aseptic meningitis can also be a symptom for syphilis and some other spirochete diseases (e.g., leptospirosis and Lyme borreliosis).
• Stiff neck and CSF pleocytosis may also be associated with other disease processes, such as malignancy.

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• Different infectious agents may get entrance to the eye either through the external or endogenous source.
• Superficial structures like the conjunctiva and the cornea are affected during external infections.
• Microorganisms present in the blood (e.g., endocarditis )may cause infection endogenously
• Endogeneous infection may be caused by the reactivation of latent viruses or parasites (e.g., cytomegalovirus or toxoplasmosis).

Different types of eye infections

1. Blepharitis:

• It is the inflammation of the margins(edges) of the eyelids; (eyelids, eye lashes, or associated pilosebaceous glands or meibomian glands).
• Symptoms include irritation, redness, burning sensation, and occasional itching. Condition is typically bilateral.
• Causative agents:
  • Bacteria: Staphylococcus aureus
  • Virus: Herpes simplex virus
  • Fungi; Malassezia furfur
  • Parasites: Phthirus pulis

2. Conjunctivitis:

• Inflammation (conjunctivitis) produces redness (pink eye), itching, and a discharge, which may be mucous or purulent.
• In this case, eyelids may stick together because of the exudation in bacterial infections which are thick, sticky, and encrusted.
• In patients having seasonal allergies, acute noninfectious inflammation may also occur.
• Conjunctivitis is highly contagious and can be transferred easily to the other eye or other individuals by contact (e.g., rubbing the infected eye and then the normal eye).
• Causative agents:
• Bacteria
  • Streptococcus pneumoniae
  • Haemophilus influenzae
  • Staphylococcus aureus
  • Haemophilus spp.
  • Pseudomonas aeruginosa
  • Chlamydia trachomatis
  • Neisseria gonorrhoeae
  • Streptococcus pyogenes
  • Moraxella spp.
  • Corynebacterium spp.

• Viruses
  • Adenoviruses
  • Herpes simplex (HSV)
  • Varicella-zoster
  • Epstein-Barr virus (EBV)
  • Influenza virus
  • Paramyxovirus
  • Rubella
  • HIV
  • Enterovirus
  • Coxsackie A

3.Keratitis:

• Keratitis, inflammation of the cornea, is a much more serious infection than conjunctivitis.
• Although there are no specific clinical signs to confirm infection, most patients complain of pain.
• Usually decrease in vision may occur, with or without discharge from the eye.
• Keratitis can result in scarring and blindness.
• Causative agents:
• Bacteria
  • S. aureus
  • S. pneumoniae,
  • Pseudomonas, aeruginosa
  • Moraxella lacunata
  • Bacillus spp.
• Virus
  • Herpes Simplex Virus
  • adenoviruses,
  • varicella-zoster

• Fungi
  • Fusarium solani,
  • Aspergillus spp.
  • Candida spp.
  • Acremonium,
  • Curvularia

• Parasites
  • Acanthamoeba spp
• A different non-infectious injury like trauma and ultraviolet radiation can cause keratitis.

4. Keratoconjunctivitis:

• It is an infection that involves both the conjunctiva and cornea.
• Ophthalmia neonatorum is acute conjunctivitis or keratoconjunctivitis of the newborn which is caused by either gonorrhoeae or C. trachomatis.
• Causative agents:
• Bacteria
• It includes the agents for keratitis/ conjunctivitis.
  • Streptococcus pneumoniae,
  • Haemophilus influenzae,
  • Staphylococcus aureus,
  • Haemophilus spp.
  • Pseudomonas aeruginosa
  • Chlamydia trachomatis,
  •  Neisseria gonorrhoeae,
  • Streptococcus pyogenes,
  • Moraxella spp.,
  • Corynebacterium spp.
  • Bacillus spp
• Virus
  • It includes the agents for keratitis/ conjunctivitis
  • Adenoviruses,
  • Herpes simplex (HSV),
  • Varicella-zoster.
  • Epstein-Barr virus (EBV)
  • Influenza virus,
  • Paramyxovirus,
  • Rubella,
  • HIV
  • Enterovirus,
  • Coxsackie A

• Fungi
• It includes the agents for Keratitis
  • Fusarium solani,
  • Aspergillus spp.,
  • Candida spp.,
  • Acremonium,
  • Curvularia
• Parasites
  • Toxoplasma gondii,
  • Toxocara

5. Chorioretinitis and uveitis:

• It is the inflammation of the retina and underlying choroid or the uvea.
• The infection can result in loss of vision.
• Causatiive agents:
•  Bacteria:
  • Mycobacterium tuberculosis
  • Treponema pallidum,
  • Borrelia burgdorferi
• Virus
  • Cytomegalovirus
  • HSV

• Fungi
  • Candida spp.

• Parasites
  • Toxoplasma gondii,
  • Toxocara
  • Treponema pallidum

6. Endophthalmitis

• It is the infection of the aqueous or vitreous humor.
• This infection is usually caused by bacteria or fungi. It is rare, develops suddenly, and progresses rapidly, often leading to blindness.
• During the movement of the eye, there is pain. Vision is decreased.
• Causative agent:
• Bacteria:
  • S. aureus,
  • S. epidermidis,
  • pneumoniae,
  • Streptococcal spp.
  • P. aeruginosa,
  • Gram-negative organisms,
  • Nocardia spp

• Virus
  • HSV
  • Varicella zoster

• Fungi
  • Candida spp.,
  • Aspergillus spp.,
  • Volutella spp.,
  • Acremonium spp

• Parasite
  • Toxocara
  • Onchocerca volvulus

7. Lacrimal infections, canaliculitis:

• It is a rare, chronic inflammation of the lacrimal canals in which the eyelid swells and there is a thick, mucopurulent discharge.
• Causative agent:
• Bacteria:
  • Actinomyces,
  • Propionibacterium
  • Propionicum

8. Dacryocystis:

• It is the inflammation of the lacrimal sac that is accompanied by pain, swelling, and tenderness of the soft tissue in the medial canthal region.
• Causative agents:
• Bacteria:
  • S. pneumoniae
  • S. aureus,
  • S. pyogenes,
  • Haemophilus influenza

• Fungi:
  • C. albicans,
  • Aspergillus spp.

9. Dacryoadenitis

• It is an acute infection of the lacrimal gland.
• These infections are rare and can be accompanied by pain, redness, and swelling of the upper eyelid, conjunctiva discharge.
• Causative agents:
• Bacteria:
• S. pneumoniae,
• S. aureus
• S. pyogenes

Laboratory Diagnosis of eye infection:

Specimen Collection and Transport

• A sterile swab should be taken for sample collection.
• From the lower conjunctiva sac and inner canthus (angle) of the eye, purulent material is collected on the sterile swab.
• Both eyes need to be cultured separately.
• For the Chlamydial culture, a dry calcium alginate swab should be taken.
• Then it should be placed in a 2-SP (2-sucrose phosphate) transport medium.
• If for the detection, Direct Fluorescent antibody (DFA) are to be used, then in such case additional slide also should be prepared.
• In that slide, the swab should be rolled across its surface which needs to be fixed with methanol.
• In the case of keratitis, scrapings of the cornea should be taken with a heat-sterilized platinum spatula.
• Multiple inoculations with the spatula are made to blood agar, chocolate agar, an agar for the isolation of fungi, thioglycollate broth, and an anaerobic blood agar plate.
• Other special media may be used if indicated.
• Corneal specimens for the detection of HSV and adenovirus should be cultured. They should be placed in viral transport media.
• Recently, the collection of two corneal scrapes (one used for Gram stain and the other transported in brain heart infusion medium and used for culture) was determined to provide a simple method for diagnosis of bacterial keratitis.
• From the anterior and posterior chambers of the eye, wound abscesses, and wound dehiscence (splitting open) specimens are collected for the culture of endophthalmitis.
• Lid infection material is collected on a swab conventionally.
• Under anaerobic conditions, transportation of the material should be done from the lacrimal canal in the case of canaliculitis.
• Aspiration of fluid from the orbit is contraindicated in patients with orbital cellulitis.

Direct Visual Examination:

• The smear should be prepared and a Gram stain should be performed.
• If there are other appropriate microscopic techniques, it should be performed.
• In bacterial conjunctivitis, polymorphonuclear leukocytes predominate; in viral infection, the host cells are primarily lymphocytes and monocytes.
• For the detection of Chlamydia, elementary body or inclusions should be checked.
• For this, it should be stained immediately with a monoclonal antibody conjugated to fluorescein.
• Using histologic stains, basophilic intracytoplasmic inclusion bodies are seen in epithelial cells.
• To detect herpes group infection in the conjunctivitis specimens, a Tzanck smear can be made. It shows the multinucleated epithelial cells.
• For the rapid diagnosis of the virus infection, DFA stains available for both HSV and VZV
• For the keratitis, the examination can be done using:
  • Gram stain
  • Giemsa stain
  • periodic acid-Schiff (PAS)
  • methenamine silver stains.
• Motile trophozoites should be observed by using the direct wet preparation in case of Acanthamoeba or other amebae and a trichrome stain should be added to the regimen.
• Culture is the most sensitive detection method for the diagnosis.
• In the case of endophthalmitis, the specimen needs to be examined by:
  • Gram
  • Giemsa
  • Periodic Acid-schiff (PAS)
  • Methenamine silver stains.
• Centrifugation should be done if the specimen is fluid and is in large volume.

Culture for eye infection:

• The number of organisms recovered from culture is low due to the washing action of tears.
• If the specimen is not purulent, large inoculums in a variety of media should be used to find out the etiological agent.
• The best result can be obtained when the conjunctival scrapings are placed directly onto the media.
• At a minimum, blood and chocolate agar plates should be inoculated and incubated under increased carbon dioxide tension (5% to 10% CO2).
• Sample from Both eyes should be cultured.
• Potential pathogens also may be present in an eye without causing infection.
• If the organism is isolated from both the infected and non-infected eye, it may not responsible for causing infection.
• If an organism only grows in culture from an infected eye, it might be the causative agent.
• Loeffler’s medium can be used when Moraxella lacunata is suspected.
• In this case, the growth of the medium causes the proteolysis and pitting of the medium.
• Loeffler’s or cystine-tellurite medium should be used if diphtheritic conjunctivitis is suspected.
• For the isolation of the organism from the keratitis, endophthalmitis, and orbital cellulitis, a reduced anaerobic blood agar plate, a medium for the isolation of fungi, and a liquid medium such as thioglycolate broth should be used.
• A reduced anaerobic blood agar plate should be used for the more serious eye infections.
• Blood culture also should be done in severe infections.
• From the transport broth, Chlamydia and virus should be inoculated inappropriate media.
• Cycloheximide-treated McCoy cells should be used for the Chlamydia
• For viral isolation, human embryonic kidney, primary monkey kidney, and Hep-2 cell lines can be used.

Molecular diagnosis for eye infection:

• Enzyme-linked immunosorbent assay (ELISA) tests and DFA staining are available for the detection of Chlamydia trachomatis.
• An ELISA test of aqueous humor is available for the diagnosis of Toxocara
• Single and multiplex polymerase chain reaction (PCR) assays

The post Eye infection: types, causative agents, clinical symptoms and diagnosis appeared first on Online Biology Notes.

Resident Microbial Flora of Ear

• Pneumococci
• Streptococcus pneumonia
• Propionibacterium acnes
• Staphylococcus aureus,
• More frequently Enterobacteriaceae are encountered.
• Occasionally Pseudomonas aeruginosa is found.
• Candida (non-C. albicans) is also common.

1. External Ear Infections: Otitis externa

• Otitis externa is similar to skin and soft tissue infection.
• Two major types of external otitis exist:
  • Acute
  • chronic

a) Acute external otitis:

• It may be localized or diffuse.

i. Acute localized disease:

• It occurs in the form of a pustule or furuncle.
• It is typically is caused by Staphylococcus aureus.
• Group A Streptococci causes Erysipelas.
• The soft tissue of the ear and external ear canal may be involved.

ii. Acute diffuse otitis externa (swimmer’s ear):

• It is related to softening of tissue(maceration) of the ear.
• It results from swimming or hot, humid weather.
• Pseudomonas aeruginosa, play an important role.
• aeruginosa also causes external otitis which can be severe and hemorrhagic.
• Occasionally it is found to be related to hot tub use which is also difficult to treat.

b)Chronic otitis externa:

• In the patients with chronic, suppurative otitis and the persons having perforated eardrums, the irritation of drainage from the middle ear results in chronic otitis externa.
• Malignant otitis externa is a necrotizing infection.
• It spreads to adjacent areas of soft tissue, cartilage, and bone.
• It may result in a life-threatening condition when the infection spreads to the central nervous system or vascular channel.
• It is caused by P. aeruginosa and anaerobes.
• In the diabetic patient having blood vessel disease, Malignant otitis media is seen.
• Occasionally, external otitis can spread to the cartilage of the ear.

Different parts of the ears like an external auditory canal, the soft tissue of the ear, or the tympanic membrane may be infected with the virus.

• Though it’s not established suspicion is made to the influenza A virus.
• Within the soft tissue of the ear and the ear canal. VZV causes painful vesicles.
• Viral agents such as influenza are the bacterial agents typically associated with acute otitis media:
  • pneumonia
  • influenza
  • catarrhalis
• Mycoplasma pneumoniae is rarely associated with this condition.

2. Middle Ear Infections: Otitis media

• It is the most common infection in children.
• Causative agents are:
  • Pneumococci
  • Haemophilus influenza
  • Group A streptococci (Streptococcus pyogenes)
  • Moraxella catarrhalis
  •  Staphylococcus aureus,
  • gram-negative enteric bacilli
  • anaerobes
  • Respiratory syncytial virus (RSV)
  • Influenza virus
• Otitis media with effusion (fluid) is considered a chronic sequela of acute otitis media.
• From the patients with otitis media with effusion, Alloiococcus otitis has been isolated.
• Pathogens of Chronic otitis media:
  • Peptostreptococcus
  • Bacteroides fragilis group,
  • Prevotella melaninogenica
  • Porphyromonas
  • Prevotella
  • Fusobacterium nucleatum a less
• The complication of chronic otitis media is mastoiditis.

Pathogenesis of otitis media:

• External ear infection (otitis media) can be caused due to many reasons such as:
  • Local trauma
  • the presence of foreign bodies
  • excessive moisture can lead to otitis externa
• Infection can progress up to the external ear by the purulent drainage from the middle ear.
• Otitis media also can be developed by anatomic or physiologic abnormalities.
• Infection may occur when the fluid develops in the middle ear.
• The auditory tube plays a major role in the protection of the middle ear:
  • protects from the nasopharyngeal secretion.
  • drains the secretions which are produced in the middle ear into the nasopharynx
  • Ventilates the middle ear
  • Maintains the air pressure in the equilibrium state with the external ear canal
• When there occurs any malfunctioning of these conditions, the infection may occur.
• Example: There is inflammation and swelling of the auditory canal when there is a viral upper respiratory infection. It hampers the ventilating function in the middle ear. These alternations in the pressure allow the potentially pathogenic bacteria present in the nasopharynx to get the entrance into the middle ear.

Laboratory Diagnosis of otitis media:

Specimen Collection and Transport

• Generally, culture is not done for the diagnosis of middle ear infection or otitis media.
• Diagnosis of external otitis is done by culture.
• Using mild germicide such as 1:1000 aqueous solution of benzalkonium chloride, the external ear should be cleansed.
• It helps to reduce the number of contaminating skin flora.
• Materials are obtained by spontaneous perforation of the eardrum or by the needle aspiration of the middle ear fluid (tympanocentesis) using sterile equipment.
• Specimens from the mastoid are generally taken on swabs during surgery, although actual bone is preferred.
• Specimens should be transported anaerobically.

Direct Visual Examination:

• Aspiration is done from the middle ear or mastoid, the direct examination is done for bacteria and fungi.
• Fungal elements can be revealed from the calcofluor white or PAS stains.
• Bacterial, fungal, and several parasitic species can be stained efficiently by the use of Methenamine silver stains.

Culture and Nonculture Methods:

• Inoculation of the ear specimens should be done in blood agar, MacConkey agar, and Chocolate agar.
• On the specimens obtained by tympanocentesis or those obtained from patients with chronic otitis media or mastoiditis, anaerobic culture also should be done.
• To detect the common middle ear pathogens, conventional and real-time PCR can be done because, in only 20% to 30% of patients, cultures are positive.

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 Classification:

• Order: Rickettsiales
• Tribe: Rickettsiae
• Family: Rickettsiaceae
• Genera: Rickettsia, Orientia, Ehrlichia

Introduction:

• Rickettsiae are obligate, intracellular, small Gram-negative bacilli.
• It multiplies within the cytoplasm of eukaryotic cells.
• Size: 0.3×1-2 µm.
• Genome: 1-1.5 million base pairs
• Rickettsiae are primary pathogens of arthropods like:
  • Lice
  • Fleas
  • Ticks
  • Mites
• Transmitted to humans by these arthropod vectors.
• Rickettsiae were originally thought to be a virus because:
  • Have small size
  • Stain poorly with Gram stain
  • Grows only in the cytoplasm of eukaryotic cells
  • Obligate intracellular parasites
• Rickettsiae are bacteria because:
  • Have Gram-negative cell wall
  • Contain both DNA and RNA
  • Contain enzymes for Kreb cycle
  • Contain ribosomes for protein synthesis
  • Susceptible to antibiotics

Morphology of Rickettsiae:

• They are small Gram-negative coccobacilli.
• Size: 0.3-0.6 to 0.8-2 µm.
• Non-motile
• Non-capsulated
• Stains poorly with Gram stain
• Stains well with these stains:
• Deep red with Machiavello and Gimenez stain
• Bluish purple with Giemsa and Castaneda stain

Culture characteristics of Rickettsiae:

• Rickettsiae do not grow in cell-free media.
• Most Rickettsia grows in the cytoplasm inside the cell.
• Rickettsia causing spotted fever grows in the nucleus of the cell.
• Cell lines:
  • HeLa, Hep2, Detriot-6, mouse fibroblasts, and other continuous cell lines.
• Chick embryo:
  • Grows in the yolk sac of 5-6 days old chick embryo.

Human Infections Caused by Rickettsia:

Antigenic Structure of Rickettsia:

1. Group-specific antigen:

• It is the soluble antigen.
• It is present on the surface of the organisms.
• From the repeated washings and centrifugation, it can be extracted.

2. Species- or strain-specific antigen:

• It is present in the cell wall of the bacteria.

3. Alkali-stable polysaccharide antigen:

• It is a surface antigen.
• It is present in some species of Rickettsia and some strains of Proteus (Proteus OX19, OX2 and OXK).
• This sharing of antigen form the basis of the Weil-Felix test.

Pathogenesis of Rickettsia:

• Rickettsia has the capacity of multiplication inside the cell.
• The important virulent factor is adhesion.
• Adhesins are the outer membrane protein that facilitates the entry of the organism into the host cells.
• When they enter the cell, multiplication occurs and accumulates in large numbers.
• It then lyses the host cells.
• Rickettsia can cause rickettsemia when it multiplies after reaching the circulation.
• In the endothelial cells of small arterial capillary and venous vessels, Rickettsia is localized.
• Then the endothelial cellular hyperplasia occurs at those sites.
• It results in multiorgan vasculitis.
• It may lead to the thrombosis and development of small nodules.
• Gangrene may result in the extremities, ear lobes, nose, and genitalia. It is due to the thrombosis of supplying blood vessels.
• Vasculitis may lead to:
  • Increased vascular permeability with consequent edema
  • Loss of blood volume
  • Hypoalbuminemia
  • Reduced osmotic pressure
  • Hypotension

Typhus Fever Group:

• Epidemic or louse-borne typhus caused by Rickettsia prowazekii
• Relapsing louse-borne typhus or Brill-Zinsser disease caused by prowazekii.
• Endemic or flea-borne murine typhus caused by Rickettsia typhi.

1. Epidemic or Louse-borne Typhus:

• It is caused by Rickettsia prowazekii.
• It is transmitted by the human body louse Pediculus humanus corporis causing the acute febrile illness.
• It is named after the scientist Von Prowazek. He died of typhus fever while studying this disease.
• prowazekii is an invasive bacterium.
• It leads to vasculitis by multiplying in the endothelial cells of blood vessels.
• The average incubation period is 8 days whereas it may vary low as 2-3 days.
• Characteristics of epidemic typhus:
  • High fever
  • Severe headache
  • Chills
• On the 4^th or 5^th day, the petechial or macular rash appears
• The rashes first start to appear on the trunk.
• Without affecting the face, palms, and sole it then spreads to the extremities.
• In nearly, 40 % of patients rashes are seen.
• The patient may become stuporous and delirious if they are left untreated.
• In the disease process, the cloudy state of consciousness appears. The name typhus is derived from it. The meaning of tyhus is cloud or smoke.
• Complications:
  • Myocarditis
  • Central nervous system (CNS) dysfunction
  • Mortality rate is as high as 60% in old or immunocompromised persons.

2. Relapsing or Recrudescent Typhus:

• Example of a recrudescent case of typhus fever is Brill-Zinsser disease.
• This condition was seen in the patients who were cured of the disease or the patients who were treated with antibiotics.
• Even after the antibiotic treatment, the recurrence of typhus fever has re-emerged after many months, years and decades.
• It is because of the persistence of prowazekii in the body tissues which re-emerges later.
• Primary reservoir of epidemic typhus: Human
• If a person is suffering from typhus fever from brill-Zinsser disease, and when lice feed on it, it will be infected with prowazekii.
• Vector of epidemic typhus: Body louse ( humanus corporis )
• Pubic louse does not transmit it.
• Occasionally transmission may occur by head louse (humanus capitis )
• In the alimentary tract of louse prowazekii, lives and multiplies.
• Within the 3-5 days of infection, the bacteria gets excreted in feces.
• After the infection, lice die.
• During the blood meal when the rickettsia-harboring louse bites the human, infection is transmitted.
• During the feeding, lice defecate.
• The contaminated louse feces gets inoculated into the minute lesion of the bite wound when the host scratches on it.
• From there the bacteria reach the circulation, multiplies and cause rickettsemia.
• Infection may also be transmitted rarely through the conjunctiva or inhalation of aerosols of dry louse feces.

3. Endemic or Flea-borne Murine Typhus:

• It is caused by typhi.
• It has a short duration and the disease is milder than epidemic typhus.
• Incubation period: 7 to 14 days.
• Symptoms:
  • Fever
  • Headache
  • Malaise
  • Myalgia
• In about 50% of infected patients, rash develops on the 3^rd to 5^th day of infection.
• Rash appears on the chest and abdomen.
• It may spread to palms and soles.
• May last up to 3 weeks in the untreated course.
• Reservoirs: Rat ( Rattus rattus ), mice, and cat
• Humans are the accidental hosts.
• Vectors for transmission of disease: Rat flea (Xenopsylla cheopis ) or cat flea (Ctenocephalides felis).
• It is transmitted from rats to rat by the rat flea.
• It is transmitted to humans accidentally by the feces of infected fleas.
• When the fleas feed on the mice, cat, or natural host, it becomes infected.
• It then transmits the disease to humans by biting.
• At the site of the bite, inoculation occurs.
• Disease transmission also may occur by:
  • Cat flea felis
  • Inoculation or inhalation of aerosolized infectious specimens
  • Ingestion
  • Contaminated food with infected rat urine or flea feces

Spotted Fever Group:

1. Rocky Mountain spotted fever caused by Rickettsia rickettsiae.
2. Rickettsialpox caused by R. akari

• Boutonneuse fever caused by R. conori
• Kenyatick-bite fever,
• African tick typhus
• The Mediterranean spotted fever
• Indian tick typhus
• Marseilles fever

1. Rocky Mountain Spotted Fever:

• Incubation period: 7 days
• Symptoms:
  • Fever
  • Severe headache
  • Chills
  • Myalgia
  • Development of rash three or more days
  • At first, a rash develops on the wrist, ankles, palms, and soles.
  • It then spreads to the trunk.
  • In the early stage, the rash is maculopapular but in the later stage, it becomes petechial and hemorrhagic.
• Complications:
  • Respiratory failure
  • Encephalitis
  • Renal failure
  • Patient may die within 5 days of onset of symptoms.
• Ticks are the host, reservoir, and vector of rickettsiae.
• Vectors:
  • Woodtick (Dermacentor andersoni )
  • American dog tick (Dermacentor variabilis)
  • Lone star tick (Amblyomma Americana ).
  • When the tick bites the human, it gets transmitted by saliva.

2. Rickettsial Pox:

• It is caused by akari.
• It is a milder form of infection.
• Natural reservoir: Common home mouse ( Mus musculus )
• By the bite of mouse mite (Liponyssoides sanguineus ), R. akari Iis transmitted from mouse to mouse.
• Incubation period: 7 days
• Papule develops at the site of the bite which progresses to ulcer and leads to eschar formation.
• In 3-10 days fever, headache, malaise, and myalgia develop.
• After the emergence of fever, a popular vesicular rash appears un 3-4 days.
• Recovery starts after the illness of 10-14 days.
• Without treatment complete healing of rash takes 2-3 weeks.

What are the conditions/ characteristics that differentiate Rickettsial pox from other rickettsial infections?

• Presence of eschar at the site of the bite
• Presence of vesicular pustular eruption

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• Proper diagnosis of the infections aids in the treatment procedure. So, the combination of clinical observations along with laboratory investigations is crucial for the diagnosis of the infection.
• Different diagnostics approach applied to fungal infections are:
  1. Clinical approach for diagnosis of fungal disease
  2. Conventional Methods for diagnosis of fungal disease
  3. Molecular Methods for diagnosis of fungal disease
  4. Recently developed new techniques for diagnosis of fungal disease
  5. Other Miscellaneous Methods

Clinical approaches for diagnosis of fungal disease:

• The characteristics of lesions produced by superficial and subcutaneous mycoses suggest their fungal etiology though it may resemble other diseases too.
• There is no particular sign and symptom in the case of systemic mycoses. The infection is similar to bacterial, viral, or parasitic disease.
• Modern imaging techniques aids in the early diagnosis of fungal disease.
• Based on it and clinical significance, the suspected disease can be further identified by laboratory investigations.

Conventional (microbiological) approaches for diagnosis of fungal disease: 

Based on Sites and Types of Specimen:

• For the proper diagnosis of fungal disease, the specimen type and the site of collection should be accurate.
• a) Superficial Mycoses:
  • The site should be cleaned using 70% alcohol. Before taking the specimen it should be allowed to evaporate.
  • The material should be collected at the folded square of paper because it:
  • Permits drying of the specimen
  • Reduces bacterial contamination
  • Stores for a long time without losing the viability of fungi
  • Dermatophytic lesions should be collected using the scalpel blade which is held at 90°C to the skin surface. From the edges of the active lesion, materials should be scrapped outward and collected.
  • In case of lesions on glabrous skin ( smooth or hairless), cellophane tape can be used.
  • Scalp specimen should be collected with blunt scalpel including hair-stubs, contents of plugged follicles, and scales.
  • Instead of cutting, hair should be plucked.
  • Ringworm infection of the scalp is detected by Wood’s lamp examination. The infection hair produces fluorescence.
  • To obtain a specimen for fungal culture, a hairbrush sampling technique can be used. In this technique, the scalp is brushed thoroughly which is then used for inoculation on culture media.
  • Patients should be off antifungal agents, one week before the collection of specimens in the case of onychomycosis ( fungal infection of nails ).
  • The nail should be disinfected with 70 % alcohol and clipped from the free edge.
  • Culture can also be done from the borings taken from the base of the nail.
  • Scrapings are preferred over the swab for the infection of the mucous membranes.
• b) Subcutaneous Mycoses:
  • For the microscopy and culture, scrapings taken from the superficial parts of the subcutaneous lesions may be satisfactory.
  • Contamination is common with bacteria and saprophytic fungi. The aspirated sample of pus and/or biopsy material is valuable.
  • If sporotrichosis is suspected then a biopsy may be avoided. It is because of the chances of spread of infection and the lesions will also be healed slowly.
• c) Systemic Mycoses:
  • Specimen: Biopsy, pus, feces, urine, sputum, spinal fluid, blood, scrapings, or swabs from the edge of lesions.
  • The urine taken from the catheter bag and twenty-four hours’ sputum is unsatisfactory. It is because the commensal yeast can multiply rapidly.

Collection and transport of different fungal specimens

• a) Respiratory specimens:
  • Specimens: sputum, tracheal secretions, bronchoalveolar lavage (BAL), lung biopsy
  • An early morning sputum sample is collected.
  • In case of non-productive cough, sputum may be induced
  • Bronchoscopy is also used for the examination of lesions and collection of specimens in respiratory mycoses.
  • For concentrating the specimens:
    • Add 0.5 gm of N-acetyl L-cysteine (NALC) in sodium citrate buffer which is prepared freshly.
    • Then vortex for 10-30 seconds
    • Then M/15 phosphate buffer with pH 7.0 is added. Its volume should be double the volume that is already present in the tube.
    • Then centrifuge at 1000 rpm for 15 minutes.
    • The supernatant is discarded and the sediment is used for smear preparation and in media inoculations.
  • Opportunistic pathogens like Candida can be present in the sputum as the oropharyngeal contaminant. The presence of Candida in the respiratory specimens is not clinically significant unless it is found in tissue. Bronchial brushing and lung biopsy may also be used.
• b) Cerebrospinal Fluid:
  • For the culture of CSF, it should be centrifuged and the sediment is inoculated in the agar.
  • Inhibitory agents should not be used in the media because, in normal conditions, CSF is always sterile.
  • CSF should not be processed immediately. It should be kept at room temperature or incubated at 30°C
• c) Blood Culture:
  • Biphasic Brain-Heart Infusion Agar Broth can be used.
  • Though most of the fungi can be detected within the first four days of incubation, an occasional isolate of Histoplasma capsulatum may require 10 to 14 days.
  • Blood culture media should be incubated at both the 25°C and 37°C temperature.
  • Subculture should be done at two days and seven days respectively.
  • After the seven days of subculture preliminary report and after 28 days of subculture final report is sent.
• d) Tissue, Bone Marrow, and Body Fluids:
  • From the pyogenic and the necrotic areas of the wounds, tissue specimens should be taken.
  • Povidone-iodine should not be used as the chances of isolation of fungi will be bleak. It may be applied after the collection
  • The tissue specimen should be minced before culture. Then it should be inoculated in appropriate culture media and incubated at 37°C for 4 weeks.
  • Bone marrow can be directly inoculated on media and incubated.
  • Before the culture, centrifugation should be done for the body fluid which is collected from a sterile site.
• e) Semen Culture:
  • Done in Histoplasmosis and Cryptococcosis when the disease is somewhat hidden and patient without any improvement has taken repeated courses of antitubercular treatment.
• f) Skin:
  • For dermatophyte infection, skin scrapping should be used.
• g) Nail:
  • The nail should be clipped from the discolored, dystrophic, or brittle parts.
  • Since, in the distal part of the nail, the fungus is non-viable, it fails to grow in culture.
  • Before the inoculation in the suitable culture media, nails should be cut into pieces.
  • It can be only visualized by microscopy.
• h) Hair:
  • Instead of cutting, hair needs to be plucked with forceps.
  • It is then kept in a sterile petri dish or paper envelope.
  • Since low temperature may be detrimental to the dermatophytes, it should not be refrigerated.
  • Dermatophytes are cultured in Sabouraud dextrose agar with chloramphenicol and cycloheximide.
  • Before reporting them as sterile, cultures should be incubated at 25°C, 30°C, and 37°C for a minimum period of four weeks.
• i) Urine culture:
  • Twenty-four hour’s urine is not useful for the fungal culture.
  • If a delay is anticipated, it should be refrigerated at 4°C. It can be kept for up to 12 hours.
  • Before the culture, the urine sample needs to be centrifuged and the sediment is inoculated.
  • Culture media should contain antibacterial antibiotics to prevent bacterial contamination and isolate the fungi in pure form.
• j) Vaginal Secretions:
  • Clinical features along with the direct smear of secretions help in the diagnosis of vaginal candidiasis.
  • About 20 % of healthy females have yeasts as the normal flora, so culture can be misleading too.
  • Cultures can help monitor the therapy and for the management of chronic recurring diseases.
• k) Stool culture:
  • Biopsy of tissue is done than the culture of stool specimens for the diagnosis of fungal infections in the gastrointestinal tract.
  • Since yeast colonizes as the commensals in 40% of healthy individuals and 75% of compromised patients, positive cultures may be misleading.
• l) Eye:
  • Corneal scrapings are taken in the case of keratomycosis.
  • Kimura’s spatula is used aseptically to take the sample from the base and margin of the ulcer.
  • 4% xylocaine is used as the local anesthetic.
  • In keratomycosis, aspiration of hypopyon is done using the sterile needle.
  • In the case of fungal endophthalmitis, the posterior chamber may also be aspirated.

Sample preparation for laboratory diagnosis of Fugal disease

a) Direct Microscopic examination:

• The direct demonstration of fungi in the clinical specimen is taken as the “gold mine”.
• Fungi can be observed directly in the clinical specimens by:
  • Wet Mounts
  • Histopathology
  • Frozen-Section Biopsy
  • Fluorescent-Antibody staining

i)  For Wet Mounts preparation:

• Slide and tube KOH Mounts are prepared.
• Sodium hydroxide may also be used as an alternative.
• After the partial digestion with 10-20% KOH, specimens can be examined in wet mounts.
• On the slide, specimens like hair, nail, skin are mounted in KOH under the coverslip.
• Materials are digested under 5-20 minutes depending on the thickness.
• Under the low flame, it can be lightly warmed but should not be overheated.
• DMSO can be supplement in KOH to increase clearing of the fungi in the skin scrapings.
• Calcofluor and Blankophor are also used to prepare wet mounts. It offers excellent visualization of the fungi.
• The fungal cell wall under ultraviolet illumination, fluoresce brightly under the fluorescence microscope.
• For the detection of Cryptococcus wet mounts of India ink and Nigrosin staining are used.
• For evaluation of the viability of fungi, neutral red staining can be used.
• For the demonstration of fungi like Malassezia , Candida spp. and dermatophytes, Vinyl Adhesive Tape (VAT) preparation is also used.

ii) For Histopathology:

• Demonstration of the fungi in the tissue sections aids in diagnosis.
• If histopathology shows neither the fungal elements nor the tissue reaction, the fungal isolate can be the contaminant.
• Histopathological examination of the biopsy and autopsy specimens is the best method for the diagnosis of mycotic infections.
• In the histopathology laboratory, H&E stain is a routine procedure.
• For demonstrating fungi in tissue-specific fungal stains, such as Periodic Acid-Schiff (PAS), Grocott-Gomori’s methenamine silver stain, and Gridley stains are widely used.
• For a demonstration of capsular material of Cryptococcus and endospores and sporangia of Rhinosporidium seeberi, Mayer’s mucicarmine can be used.
• To demonstrate acid mucin, Alcian blue staining may be done.
• Disadvantage of use of special stain:
• Mask the natural color of fungal elements making it difficult to decide if it is hyaline or naturally pigmented.

iii) Frozen-Section Biopsy:

• This modality is adopted for making an intra-operative diagnosis of suspected malignancy.
• In mucormycosis and fungal rhinosinusitis, good results have been obtained.
• It is a useful tool to guide the extent of surgical debridement and/or onset of antifungal therapy.
• Frozen sections describe the morphology and infectious process.
• The evaluation of the frozen section has an important function in surgical pathology for diagnosis in tissue while the patient is undergoing an operative procedure.

iv) Fluorescent-Antibody Staining:

• It is used for the detection of fungal antigen in clinical material such as pus, blood, CSF, tissue impression smears, and in paraffin sections of formalin-fixed tissue.
• For the sputum specimen, it is less satisfactory.
• Advantage: detects fungus when few organisms are present.

b) Fungal Culture:

• Commonly employed medium is Emmons’ modification of Sabouraud Dextrose Agar.
• To minimize bacterial contamination, gentamicin and chloramphenicol can be supplemented in the media.
• To inhibit the saprotrophic fungi, cycloheximide can be supplemented in the media.
• Some fungi like Cryptococcus, Talaromyces marneffei, Aspergillus, or Scytalidium are sensitive to cycloheximide. So, cycloheximide should not be used in this case.
• For the isolation of the particular pathogens, a special medial can be used.
• For isolation of neoformans, Birdseed agar, Niger seed agar, Sunflower seed agar can be used.
• neoformans develop brown-colored colonies.
• Candida , Aspergillus spp. , Rhizopus spp. grows within 24-72 hrs of incubation.
• For the dimorphic fungi, incubation should be done at:
• 37°C for the isolation of yeast.
• Room temperature for the isolation of molds.

Techniques for detection of fungi from culture:

• • Scotch Tape preparation
  • Wet Mount preparation
  • Lactophenol cotton blue staining
  • Germ Tube production test

i. Scotch Tape preparation:

• Over the growth of filamentous fungi, the sticking surface of scotch tape is touched.
• Then it is placed on a glass slide and observed under the microscope.
• Characteristics shape and arrangement of spores, type of hyphae and conidia, etc can be observed.
• Advantages:
  • It can be prepared quickly and easily.
  • Slide can be preserved for a longer time.
  • Fungi can be seen with their own pigmentation.
• Disadvantages:
  • Sample won’t be adequate if it is not pressed firmly.

ii. Wet Mount preparation:

• It is done for the observation of spores that were not seen by scotch tape.
• Example: Microconidia of Histoplasma capsulatum can be observed by the wet mount method.
• Procedure:
  • Bent wire of 90° is used.
  • Small portion is cut at the intermediate point of the center and periphery of the isolated colony.
  • It should contain a small amount of the supporting agar.
  • Then a drop of KOH is added.
  • It is covered by a coverslip and observed under the microscope.
• Disadvantages:
  • If pressure is applied to the coverslip, the characteristic arrangement of spores will be disrupted. In such a condition, definitive identification of fungi can’t be done.

iii. Lactophenol cotton blue staining:

• Fungal cytoplasm can be stained against which the walls of hyphae can be readily seen.

iv. Germ Tube production test:

• It is used for the definitive identification of fungi within 3hrs.
• Example: Yeast, Candida albicans
• Germ tube is the elongated tube-like structure originating from yeast cells.
• Nucleus is absent.
• It has half the width and 3-4 times greater the length of the yeast cell.
• Procedure:
  • Suspend the inoculums from the isolated colony of yeast cells in 0.5 ml sheep or rabbit serum.
  • Incubate the tube at 37°C for 3-4 hrs.
  • Then take the suspension and observe under the microscope.

c) Serological test:

• Agglutination tests:
  • Whole-cell agglutination (WCA)
  • Latex particle agglutination (LPA)
• Passive haemagglutination (PHA)
• Immunodiffusion (ID)
• Counterimmunoelectrophoresis (CIE)
• Complement fixation (CF)
• Indirect fluorescent antibody (IFA)
• Enzyme-linked immunosorbent assay (ELISA)
• Radioimmunoassay:
  • Solid-phase
  • Competitive RIA
• Complement fixation test (CFT):
  • Coccidioidomycosis, Histoplasmosis, Blastomycosis
  • Presence of the polysaccharide capsular antigens of  Cryptococcus neoformans in the CSF can be detected by the latex agglutination test.

Skin Test for fungal disease:

• Individuals infected with the Histoplasma or Coccidioides develop a Delayed-Type-Hypersensitivity (DTH) reaction with in1-14 days and may persist for many years
• Appropriate fungal antigen is inoculated intradermally.
• Within 24-72 hours induration and erythema occur.
• Skin test is used for:
• Establishing the etiological diagnosis
• Conducting the epidemiological survey
• Immunological classification of the subjects like atopic and non-atopic groups
• Find out the immunological status of the patients as in the immunodeficiency diseases

Various skin tests and fungal antigens used:

Molecular approaches for diagnosis of fungal disease:

1. Hybridization methods
2. Amplification methods
   • Broad range PCR
   • Nested PCR
   • Multiplex PCR
   • Nucleic acid Sequence-based Amplificatio
   • Fluorescence Resonance Energy Transfer
   • TaqMan
   • Molecular Beacons

3. Sequencing-based methods
   • Sanger’s Sequencing
   • Pyrosequencin
   • Next-Generation Sequencing
   • Ultra-Deep Sequencing
   • DNA Bar Coding

Recently Developed new Techniques for diagnosis of fungal disease:

• AccuProbe
• PNA FISH
• MALDI-TOF Mass Spectrometry
• Quantamatrix Multiplexed Assay Platform

Miscellaneous Methods:

• Point of Care Testing
• T2Candida Panel
• Biosensors for Medical Mycology
• Epidemiological Markers of Fungi
• Quality Control in Medical Mycology

Epidemiological Markers Used in Fungal Infections:

• Phage typing
• Secreted lethal factor typing
• Serotyping
• Morpho typing
• Mating typing
• Resistotyping
• Biotyping
• Protein electrophoresis (Immunoblot)
• Isoenzyme typing
• Restriction fragment length polymorphism (RFLP)
• Karyotyping
• Nucleic acid probes

The post Different approaches for Fungal Disease Diagnosis: Clinical, conventional and molecular approaches appeared first on Online Biology Notes.

• Mucormycosis is an opportunistic fungal infection which is rare but serious infection.
• It is also called as black fungus infection which has been reported recently in the recovered patients of COVID-19.
• Mucormycosis is sometimes called as zygomycosis.
• It is caused by mucormycetes which is group of fungi.
• Several fungi belonging to the phylum Glomeromycota cause it. They are the saprophytic fungi and are present ubiquitously in the soil and environment.
• The first authentic human case of mucormycosis was reported in 1855. It was described by Kurchenmeister in a patient of neoplastic lung on the basis of histopathology.
• Pulmonary mucormycosis was described by Furbringer in 1876 for the first time.
• Mycology:
  • Mucormycetes is a group of lower fungi.
  • Their hyphae are generally non-septate, sparsely septate or pauci-septate.
  • Reproduction occurs asexually by sporangiospores and/or by means of conidial development.
  • Reproduction occurs sexually by the formation of zygospore.

Common causative agents of Mucormycosis:

• Rhizopus arrhizus ( Old name Rhizopus oryzae )
• Rhizopus microspores var. rhizopodiformis
• Mucor racemosus
• Rhizomucor pusillus
• Lichtheimia corymbifera ( Mycocladus corymbiferus or Absidia corymbifera )
• Apophysomyces elegans
• Cunninghamella bertholletiae
• Saksenaea vasiformis
• Cokeromyces recurvatus
• Syncephalastrum recemosum
• Absidia corymbifera

Modes of Transmission of Mucormycosis:

•  Mucormycosis is caused by:
  • Inhalation of spores present in air.
  • Ingestion from contaminated food
  • Inoculation into skin amd soft tissues by trauma Trauma inoculates in skin and soft tissue.
  • Person to person and between animals and persons transmission doesn’t occur.

Virulence factor of Rhizopus arrhizus

• Rhizopus arrhizus is the commonest encountered mucormycetes which has several virulence factors such as:
  • Angioinvasive nature
  • Growth at the body temperature or above
  • Destructive enzymes production
  • Dormant spores
  • Active ketone reductase system
  • Hydroxamate siderophores

Pathogenesis of Mucormycosis:

• Infection can occur by either inhalation, percutaneous inoculation or ingestion of fungal spore.
• Phagocytes, polymorphonuclear neutrophils and macrophages play a critical role in patients of mucormycosis.
• When the spores are inhaled into lungs then alveolar macrophages ingest it.
• After ingestion, macrophages inhibit the germination of spores but the activity to kill them is limited. So, they can escape out from the antifungal activity of the macrophages and germinate into mycelia form .
• Then the expected to work against the fungi are polymorphonuclear neutrophils and peripheral monocytes.
•  The leukocytopenic patients are susceptible to this disease because they lack the sufficient cell like these which perform the antifungal activity.
• There is also the risk of mucormycosis to the patients of ketoacidosis ( metabolic acidosis due to accumulation of ketone bodies in the blood).
• It may be due to the release of iron bound to the protein.
• The low serum pH due to the ketoacidosis decreases the phagocytic effect of macrophages, chemotactic and oxidative burst of neutrophils.

Risk factor for Mucormycosis

1. Diabetes patients with diabetes kitoacidosis:
   • The patients of diabetes are at risk of mucormycosis when they have got ketoacidosis.
   • It is because diabetic patient have the high glucose level in their blood and Rhizopus  can thrive in it too.
   • Dye to the presence of active ketone reductae system Rhizopus can survive in high glucose and acidotic conditions.
   • Due to the impaired glutathione pathway, these patients also have decreased phagocytic activity.
   • Though the exact phenomenon is unknown, it might be due to the metabolic abnormalities in combination with diabetic patients.
   • Similarly, the invivo growth of fungus is not supported solely by hyperglycemia o acidosis.
   • Acidosis without hyperglycemia has been reported with the invasive mucormycosis.
   • Rhizopus gets inhibited at the normal serum but the growth is stimulated in the patients of diabetic ketoacidosis.
   • It has been found that the patients on dialysis and iron overload, who are being treated with deferoxamine, an iron chelator are susceptible to mucormycosis.
   • It may be due to Mucorales which to obtain more iron use the siderophore as the chelator.
2. Other risk factors includes:
   • Neutropenia ( abnormal decrease of the neutrphil in circulating blood )
   • High-dose systemic steroids
   • Protein-calorie malnutrition
   • Solid organ and bone marrow transplantation
   • Immunodeficiency
   • Leukemia (blood cancer caused by increase in White blood cell )
   • Intravenous drug use

Histopathology of Mucormycosis:

• The primary histopathological features of mucormycosis are:
  • Angioinvasion: ( spread of tumor into a blood vessel, also called as vascular invasion)
  • Thrombosis: (formation of the fibrinous clot in any part of the circulatory system)
  • Ischemia: (deficiency of blood supply to an organ or tissue)
  • Tissue necrosis: (death of tissue in the living body)

Clinical features of Mucormycosis:

• It is the fatal disease which progresses rapidly due to its involvement in blood vessels and being angioinvasive in nature.It is of six types:
  • Rhinocerebral Mucormycosis
  • Pulmonary Mucormycosis
  • Cutaneous Mucormycosis
  • Gastrointestinal Mucormycosis
  • Isolated Renal Mucormycosis
  • Disseminated Mucormycosis

i) Rhinocerebral Mucormycosis:

• It is the most common and fulminating type of mucormycosis.
• If it is left untreated it may lead to fatal consequences within a week.
• It occurs when the inhaled spores germinates in the nasal passage then making spreading from the nasal mucosa to the turbinate bones, paranasal sinuses, orbit and palate.
• It then spreads upto the brain where its invades blood vessels massively causing the major infarct (area of necrosis due to deficient blood supply )
• The imaging techniques like CT or MRI shows the destruction of bones.
• Mostly it is caused by Rhizopus arrhizus and others etiological agents are also reported.
• Symptoms includes:
  • Facial pain
  • Head-ache
  • Lethargy
  • Loss of vision
  • Brownish, bloodstained nasal discharge
  • Black eschar on palate ( due to hemorrhage and tissue necrosis )
  • Fixed and dilated pupil
  • Global proptosis and ptosis
  • Dysfunction of cranial nerves ( especially 5^th and 7^th nerves )
  • Extensive and rapid destruction to the surrounding tissues.
  • Sometimes the infection may spread to other parts like lungs, gastrointestinal tract, skin.
  • Symptoms of orbital (related to orbit which is the socket in the skull where eyeball is present) mucormycosis:
  • Chemosis (oedema of the conjunctiva producing the pronounced ring around the cornea)
  • Periorbital cellulitis
  • Ophthalmoplegia ( partial or total paralysis of the muscles that moves the eye)
  • Proptosis ( forward displacement of any organ )
  • Ptosis ( drooping or falling or lowering out of eyelids or other organs )
  • Abrupt visual loss
  • Orbital pain
  • Facial hypoesthesia ( decreased sensitivity particularly of touch )
  • Infection may spread from orbit to brain which may lead to frontal lobe necrosis and abscesses formatin.
  • Invasion of the fungi in blood vessels causes necrotizing (causing the death of tissues ) vasculitis ( inflammation of blood vessels ) which results in the thrombosis (formation of fibronous clot in any part of circulatory system ) of vessel lumen.

ii) Pulmonary Mucormycosis :

• It occurs through the inhalation of the sporangiospores.
• The invasion of the blood vessel may lead to the destruction of lung parenchyma.
• From the onset of the illness to the terminal stage of illness it takes about 1 to 4 weeks.
• Symptoms includes:
  • Chest pain
  • Dyspnea
  • Hemoptysis
  • Infiltration can be seen in the chest x-ray which shows the progressive infectiom from an anatomical site to the multiple adjoining areas on the same lung.
• The pulmonary mucormycosis and its extension is best determined by the HRCT (High Resolution Computed Tomography) scan.
• When the patients have a reverse halo sign on CT of the chest, this entity is suspected.
• Rarely the members of the order Mucorales are involved in forming the fungus ball like that of aspergilloma.
• It has been reported that the Rhizopus caused the hypersensitivity pneumonitis in Scandinavian Sawmill workers (so-called Wood trimmer’s disease) and in farm workers.

iii) Cutaneous Mucormycosis:

• It occurs in the patients of severe burns which spread to the underying tissue.
• The severe underlying necrosis develops.
• In the diabetic patient cutaneous lesions may occur at the site of injection.
• It may occur when the contaminated surgical dressings or the splints are applied in the skin.
• Clinical manifestation may vary from the pustules or vesicles to wounds with wider areas of necrotic zones.
• Lesions in the early stages resembles ecthyma gangrenosum. In this condition, cotton like growth can be seen over the surface of the tissue. This clinical sign is called as “hairy pus”.
• Cutaneous mucormycosis can be primary infection or it can be secondary to the disseminated form.
• For the primary cutaneous mucormycosis, Saksenaea and Apophysomyces should be suspected as the causative agent. It occurs when there is contamination of the wound and traumatic injured areas with dust or soil. After few days blistering and necrotic lesions occurs.
• Apophysomyces tends to invade the vascular lumen causing thrombosis leading to ischemia and finally tissue necrosis.

iv) Gastrointestinal  Mucormycosis:

• Rarely occurring mucormycosis which is about 7 % of all the mucormyccosis.
• It most often involves in stomach.
• It occurs in patients with extreme malnutrition.
• Ingestion of foods like fermented milk, porridge, and alcohol made from corn and herbal products  contaminated with fungal spores causes gastrointestinal mucormycosis.
• Lesions occurs  in stomach which are followed by colon, ileum and esophagus.
• Invasive mucormycetes either colonize or invade gastric mucosa.
• Non-specific symptoms include:
  •  Abdominal pains
  • Diarrhea
  • Hematemesis
  • Melena
• Patients on dialysis who are undergoing treatment with desferrioxamine are also reported to develop mucormycosis.
• Causative agents of gastrointestinal mucormycosis:
  • Lichtheimia corymbifera
  • Basidiobolus ranarum

v) Isolated Renal Mucormycosis:

• It is one of the emerging clinical entity.
• It is an unusual cause of renal infarction, if not detected in time may be fatal.
• It infects the kidney.
• Symptoms includes:
  • Flank pain
  • Fever
  • Pyuria
  • Infarct of renal tissue leading hematuria
  • Initially patents are assumed to be infected with bacterial pyelonephritis and does not respond to the antibiotics.
  • Acute pyelonephritis occur in immunocompromised patients.

vi) Disseminated Mucormycosis:

• Portal of entry is skin which may be from trauma. It then invades blood vessels and disseminates.
• Dissemination may occur to different parts of body which affects lungs, kidney, gastrointestinal tract, heart and brain.
• Lungs is the most commonly involved site.
• Symptoms includes:
  • Pneumonia
  • Stroke
  • Subarachnoid hemorrhage
  • Brain abscess
  • Cellulitis
  • Gangrene of skin
  • Gastrointestinal bleeding
  • Peritonitis
  • Acute myocardial infarction
  • Hepatitis
• Disseminated Mucormycosis is caused by:
  • Apophysomyces
  • Saksenaea
  • Rhizopus
• Cerebral infection is distinct from the rhinocerebral mucormycosis because it leads to the focal neurological signs.
• Disseminated infections by Mucor species:
• Endophthalmitis
• Prosthetic mitral valve mucormycosis

Laboratory diagnosis of mucormycosis:

• Diagnosis is slightly difficult because of rapid and fulminant course of disease.
• Doubtful significance of isolates because they are usually encountered as laboratory contaminants.

Specimens:

• Nasal discharge
• Biopsy
• Sputum
• Bronchoalveolar lavage
• Necrotic lesions
• CSF

Direct Examinations: by Microscopy

• KOH Wet mount preparation of the nasal discharge or biopsy material shows  broad, non-septate, thick walled ribbon-like hyphae with wide-angle or right-angle branching at irregular intervals which are the characteristics of  hyphal elements of mucormycetes. It is  different  from other fungi. For example: Hyphae of Aspergillus, Fusarium and/or Scedosporium spp.have  slender hyphae with regular dichotomous branching and frequent septation.
• Microscopic morphology of Saksenaea vasiformis shows a typical trumpet-shaped sporangiophore.
• Calcofluor white staining
• Well stained in H & E but poorly stained with PAS
• Other staining: Gridley, Gram’s and GMS
• Immunochemical staining methods
• Frozen section evaluation while patient is undergoing operative procedure

Fungal Culture:

• Specimens should be directly inoculated in the culture media avoiding grinding. It is because the hyphal elements are prone to physical damage.
• Mucormycetes grows at conventional media like Sabouraud Dextrose Agar (SDA) with antibiotics at 25°C and 37°C.
• BHI broth can be used for biopsy material.
• Portion of tissue can be kept in water which is added with  malt yeast broth. Then molecular techniques can be done directly from the sample.
• Fibrous and cotton-candy growth in media. Also called as “lid-lifters”.
• Apophysomyces spp. , Saksenaea spp. and Mortierella wolfii under routine cultural conditions on SDA, CMA or PDA may produce only sterile hypahe without any spores.
• For the induction of sporulation various induction techniques are designed:
• Czapek Dox agar and slide culture in humid atmosphere
• Padhye and Ajello technique: use of saline agar (1%) supplemented with grass and hay
• Soil extract medium

Serological test:

• No routine serological test

Treatment of Mucormycosis:

• Rapid correction of underlying predisposing factor of the host like diabetic ketoacidosis
• Surgical debridement of necrotizing tissue
• Antifungal therapy
• Consideration of adjunctive therapy such as hyperbaric oxygen
• Drugs: Intravenous amphotericin B, Posaconazole and Isavuconazole

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