Prepared By: Ken Robin A. Canada, RMT, Mls Ascpi

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Prepared by: Ken Robin A.

Canada, RMT, MLS ASCPi


 Coccidioides is a dimorphic fungus.

 In soil, Coccidioides grows as a mold (mycelium) with branching septate hyphae.

During the rainy season, the mycelia grow rapidly but they are also the least
infectious form of the organism.

 Arthrospores measure 3-5 µm and are extremely hardy, withstanding extreme heat,

desiccation, and changes in soil salinity and remaining viable in the soil for months to
years.
 Upon inhalation into lungs or on rare occasion after percutaneous implantation into
tissue, each arthroconidium transforms into a new multinucleated, spherical
structure called the spherule.
 As it grows, the spherule begins to divide internally. Spherules vary from 60 to over
100 um in diameter, while endospores remain 2 to 5 um in size.
 Mature spherules may contain 800 to 1,000 endospores.
 In its parasitic phase, each endospore grows into a new spherule.
 Coccidioides spp. are found in the hot, dry regions of the southwestern United
States, where winters are relatively mild and the soil is alkaline.
 Coccidioides spp. are most highly concentrated in the San Joaquin Valley of CA and
in south-central AZ.
 Historically, people at greatest risk for contact include farmers, construction
workers, and archaeologists.
 The spectrum of illness due to Coccidioides spp. is very broad.

 About 60% of clinical infections occur with few or no respiratory symptoms.

 The 40% of patients that are symptomatic may present with an acute or subacute
spectrum of illness, ranging from “flu-like” to progressive pneumonia.
 The incubation period of coccidioidomycosis usually begin within 7 to 21 days of
inhalation of arthroconidia.
 Symptomatic patients complain of fever, cough, chest discomfort, malaise, and
fatigue (generally last less than 3 weeks)
 Transient skin manifestations, including rash and erythema nodosum, may be seen in
10% to 50% of patients.
 Patients of black or Asian (especially Filipino) ethnic backgrounds, pregnant women
in the third trimester, and any immunocompromised patients appear to be at
significant risk for disseminated coccidioidomycosis.
 Dissemination may occur months to several years after the primary infection.
 Dissemination of spherules and endospores via the lymphatics and the bloodstream
may occur to any organ system, but skin, lymph nodes, and the skeletal system are
primarily involved.
 Hematology.
 Elevated erythrocyte sedimentation rate and eosinophilia may be seen in
coccidioidomycosis. Eosinophilia especially should heighten suspicion.

 Direct detection.
 (i) Microscopy
 This includes a mixed acute reaction consisting of an influx of polymorphonuclear
neutrophils and, to a far lesser degree, granulomatous cells.
 Tissue eosinophilia may be present and may be seen surrounding the offending organism
(Splendore-Hoeppli phenomenon).
Granuloma Formation Splendore-Hoeppli phenomenon
 Coccidioidomycosis can be diagnosed microscopically by visualization of endospore-
containing spherules in infected material.

 Potassium hydroxide (KOH) wet mounts are useful and readily available for
microscopic evaluations
 Calcofluor white (CFW) fluorescent stain

 Grocott-methenamine silver stain is most sensitive in detecting fungi in


histopathological preparations.

 The presence of endospore-containing spherules is diagnostic of coccidioidomycosis.


KOH Calcofluor White Grocott-methenamine
silver stain
 (ii) Molecular detection

 Nucleic acid amplification tests using PCR have been described by a number of
noncommercial laboratories and found to be both sensitive and specific.

 Confirmation of an isolate as Coccidioides spp. is best achieved using a


molecular genus-specific genetic probe
 Culture

 Coccidioides spp. can be grown on most fungal


media (Sabouraud medium, inhibitory mold agar,
and medium containing cycloheximide) and
bacterial media (sheep blood and chocolate agar
media as well as selective yeast extract medium
used for Legionella)

 Growth is usually recognizable within 4 to 5 days


on most media.
 There are four tests for diagnosis:
1. Complement-Fixation (C-F) is a PROGNOSTIC test. If the titer keeps
rising, then the patient is responding poorly and the course may be
fatal. If the C-F titer is dropping then the prognosis for that patient is
favorable. A titer of greater than 1:128 usually indicates extensive
dissemination.
2. Slide agglutination
3. Immunodiffusion methods are available for detection of IgM and IgG as
well, but they require longer incubation periods (up to 4 days) to rule
out negatives.
4. EIAC-F antibody is slow to rise and develop in about 1 month. This test
is excellent for coccidioidomycosis because it is quantitative.
 In most affected persons, however, illness is self-limited, spherule proliferation is
arrested, and markers of cellular immunity against antigens of Coccidioides spp.
become evident
 Recovery from an initial infection nearly always produces long-lived and complete
immunity to illness.
 The most protective coccidioidal vaccines to date have been spherule-derived or
from recombinant proteins that are abundantly, though not necessarily exclusively,
represented in the spherule phase
1. Strains and culture conditions.
2. Protein extraction
3. Two-dimensional differential in-gel electrophoresis.
4. MS/MS and bioinformatics.
5. Cloning and protein expression
6. ELISA and source of serum
7. Protection studies
8. Analysis and expression of an abundant spherule protein.
 Coccidioides posadasii strain Silveira stock cultures were maintained on 2XGYE agar
plates at room temperature.

 For growth in the spherule phase, arthroconidia were harvested from 4-week-old
cultures. Inoculated into 1 liter modified Converse medium (7) supplemented with
0.05% NZ-Amine and cultures were incubated at 39°C, 8% CO2, and 160 rpm, for
96h.

 Mycelia for protein extraction were grown in liquid 2XGYE at 37°C and 180 rpm, for
48 h.
Spherules were harvested by centrifugation and the carbohydrate rich outer wall
material removed from the surface of the cell pellet.

 The cell pellet was washed twice with water, then frozen at 80°C.

 Mycelia were harvested by filtration, washed extensively with water, and frozen at
80°C.
 The protein concentration for each extract was determined using the 2-D Quant kit.

 Forty protein spots revealed as differentially expressed were manually excised at


the center of the visible spot from one of the gels after post staining of the
unlabeled protein with colloidal Coomassie stain.

 Proteins in each spot were digested in gel with trypsin.


 Protein identities based on peptide sequences deduced by the SEQUEST program
were verified.
 One protein, identified by SEQUEST as a peroxisomal membrane protein from
C. posadasii, and highly expressed in the spherule phase, was selected for further
study.

 This approach led to the identification of a protein, Pmp1.


 To assess whether Pmp1 was recognized by the immune system of naturally infected
patients, we assayed for anti-Pmp1 immunoglobulin G (IgG) antibody in several
patient populations.
 Ninety-six-well plates were coated with 10 ng rPmp1 in Dulbecco’s phosphate-
buffered saline (PBS) containing 0.2 M MgCl2.
 Enzyme-linked immunosorbent assays (ELISAs) were performed
1. Serum from individuals with complicated coccidioidal disease
2. Sera from patients with primary coccidioidal pneumonia
3. Serum samples from patients with no apparent coccidioidal disease were
deidentified samples selected randomly from the clinical chemistry laboratory at
a hospital in Tucson.
 A panel of serum from 20 patients with complicated disease showed a median titer
of 1:20,480. Only one of these patients showed an undetectable level (1:80) of
anti-Pmp1 antibody.

 In comparison, in a panel of serum from 18 patients with primary coccidioidal


pneumonia, half showed an undetectable level of antibody.

 Serum samples from 98 patients with no evidence of coccidioidal disease,59% had


undetectable levels of anti-Pmp1 antibody.
 Female, 6-week-old C57BL/6 mice were purchased from Harlan-Sprague-Dawley
(Indianapolis, IN) and maintained according to National Institutes of Health
guidelines.
 Groups of 10 mice were immunized subcutaneously with 1, 5, or 50 g rPmp1 with
monophosphoryl lipid A-stable emulsion (MPLSE) adjuvant on day 0 and day 14.
 Control mice received adjuvant only
 Four weeks after boosting (day 42), the mice were infected intraperitoneally
with 310 arthroconidia and sacrificed 2 weeks later.
 The right lung and spleen were quantitatively cultured and results were expressed
as log10 CFU per organ.
 Lungs of mice vaccinated with 1 or 5 g of
rPmp1 exhibited mean log fungal CFU per
organ approaching the lower limits of
detection.
 Less protection was observed using 50 g
rPmp1, but reduction in CFU for all doses was
significant compared to MPL-SE adjuvant
alone.

 Spleens of vaccinated mice also exhibited a


significant reduction in CFU compared to
controls when lower doses of antigen were
used, but protection was lost when the
immunizing dose was increased to 50 g
protein.
FIG. 4. Prolonged survival following immunization with Pmp1.
Groups of 10 mice were immunized intradermally with 1 ug rPmp1 or ISS adjuvant only
The groups were challenged on day 42 with 82 arthroconidia intranasally
Survival was assessed for 56 days. , Pmp1; ,ISS adjuvant alone.
 By homology, Pmp1 appears to be related to a thiol-specific antioxidant protein of
S. cerevisiae (Ahp1/TSA2/YLR109w) which is reported to be an alkyl hydroperoxide
reductase.
 Pmp1 shows homology to an Aspergillus fumigatus antigen, Asp f3, and related
fungal allergens.
 Of the 32 different proteins identified in the analysis of 40 spots, none met all three
postulated criteria for an optimally protective antigen:
1. abundance and preferential expression in the spherule phase,
2. motifs indicative of secreted or cell wall localization
3. Lack of similarity to host (self) antigens.

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