الفهرس 
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Abstract
Pulmonary mycosis (PM) is an infectious process in the lungs
caused by one or more opportunistic fungi. Fungal colonization and
infection occur following the inhalation of spores. Case mortality
associated with PM can be as high as 75% in immunodeficient persons.
Fungal infections in lung often pose a great diagnostic challenge due to
the absence of characteristic pathognomonic and radiological features.
In the last decade, mycosis became a significant healthcare concern
due to the rampant use of broad- spectrum antimicrobials, prolong use of
immunosuppressive drugs, increased cases of critically ill, and
immunodeficient persons such as HIV/AIDS.
Fungi produce mycotoxins as nonvolatile secondary metabolites
and volatile organic compounds as byproducts of metabolism, The
volatile organic compounds are potential asthma triggers, pose a serious
health hazard for humans and animals. Presence of allergenic and
toxigenic fungi has previously been reported in damp homes and offices.
The aim of the present study was to: isolate and identify fungal
isolates from the respiratory tract of COPD patients, investigate the
predisposing factors, clinical characteristics, microbiology and suggest
the most effective antifungal agents against the recovered isolates. Also
the present study aimed at confirming the severity of fungal infection
through a histopathological study and investigating the mycotoxigenic
capacity by the isolated fungal species from the post mortem lung
samples in albino mice.
Summary
133
The study is a laboratory-based study conducted between October
2017 to September 2020 at Ain Shams University Hospitals (Ain-Shams
Specialized Hospital, Al-Demrdash hospital) located in Cairo and
Zagazaig University Hospitals located in Al Sharkia governorate. This is
a prospective cross-sectional analytical study. All the procedures used in
this study were approved by the research ethics committee of faculty of
Science, Ain Shams University, Egypt.
This study was carried out on Two hundred and fifty Egyptian
(250) patients. Their ages were more than 20 years old, they were
admitted to the emergency room, hospital wards, intensive care units
(ICU), with chronic obstructive pulmonary disease (COPD), and have
history, examination and investigations suggestive of fungal infection.
Their microbiology and pathology laboratories records were reviewed
daily. Clinical samples composed of sputum, Bronchoalveolar lavage
(BAL) and endotracheal tube (ETT).
The following data were collected from patient’s medical files:
Causes of admission, duration of mechanical ventilation, length of ICU
stay, underlying disease, and recovery of another respiratory pathogen.
The distribution of fungal isolates on the basis of some risk factors
showed that the highest percentage (36.6%) was for diabetes mellitus.
The second most common risk factor identified in this study was smoking
(25.9%). Prolonged antibiotic usage was found to be an important risk
factor (11.8 % of the patients). Prolonged corticosteroid antibiotic usage
also a risk factor for pulmonary fungal infection (11.8 % of the patients).
HIV infection and chemotherapy administration were also risk factors for
developing respiratory fungal disease, with percentage of 7.5% and 5.4%
respectively.
Summary
134
Sputum samples were collected in 150 patients by instructing them
to cough and expectorate about 5–10 ml in a sterile container usually
early in the morning.
Bronchoalveolar lavage (BAL): BAL & endotracheal tube ETT
samples were collected only from 100 patients who were mechanically
ventilated, with the help of a white light flexible bronchoscope attached
to a light source and digital camera.
All samples were labeled with individual identity numbers and
were opened aseptically at the time of analysis. For recovery of fungal
pathogens, the specimens were cultured onto Sabouraud dextrose agar
(Himedia-India) plates with 0.05 mg/ml of chloramphenicol disc and
other media. Plates were incubated at 28±2°C for 1-4 weeks and were
examined twice weekly.
Macro and Micro-Morphological as well as molecular
identification for the fungal species were performed for the fungal
isolates.
Out of the 250 patients included in this study, only 93 cases
(37.20%) were identified as having respiratory fungal infections.
In this study, most of the fungal agents were of the filamentous
type (70.97%), whereas Candida spp. comprised 29.03% of the causative
agents. A. fumigatus and A. flavus were the most prevalent mould fungal
pathogens (18.28% for each).
Also, molecular identification of the most potent producers of
mycotoxins, fungal pathogens was conducted. Agarose gel
electrophoresis was performed for the PCR amplified DNA product
obtained from the fungal isolates. Phylogenetic tree based on 18S rRNA
sequences depicting the relationship between the isolated strains was also
performed.
Summary
135
Susceptibility testing of pathogenic fungi to different antifungal
drugs was conducted.
The antifungal testing revealed that Ketoconazole exhibited the
highest effect against Candida albicans.
Itraconazole exhibited the highest effect against Candida
parapsilosis.
Amphotricin B and Itraconazole exhibited the most effect against
Candida tropicalis.
Caspofungin and Itraconazole exhibited the most effect against
Candida krusei.
Ketoconazole exhibited the highest effect against A. niger, A. flavus
and A. fumigatus.
Caspofungin and Flucytosine exhibited the highest effect against A.
cristatus.
Ketoconazole exhibited the highest effect against Pencillium spp.
Ketoconazole had the highest antifungal action against the
commonest fungal pathogens; Aspergillus spp., Pencillium spp. and
Candida albicans.
Four groups of isolates (Aspergillus flavus, Aspergillus fumigatus,
Aspergillus niger, and Penicillium sp.) were examined for the presence of
6 mycotoxins (AFB1, ochratoxin A, citrinin, patulin, cyclopiazonic acid,
kojic acid).
This study proved that human respiratory system is susceptible to
AFB1 and accumulation from A. flavus strains, and the highest mean yield
of AFB1 (93.7+0.51 μg/L) was detected in A. flavus isolate number 59 (A.
flavus Ali 1), at mean dry weight of 1.03+ 0.01 g/L.
Summary
136
Ochratoxin A was extracted from A. fumigatus, A. niger, and
Penicillium sp. at 214 nm. The mean highest concentration of Ochratoxin
A (28.7 + 0.35 μg/L) was extracted from isolate number 33 (A. fumigatus
Ali 2), with dry weight of 0.83 + 0.01 g/L. While, the isolate number 90
of A. niger was produced the highest concentration of Ochratoxin A (67.9
+ 0.3 μg/L), with dry weight of 1.33 + 0.46 g/L. The isolate number 23 of
Penicillium sp. (Penicillium chrysogenum Ali8) was produced the highest
concentration of Ochratoxin A (54+1.0 μg/L) with dry weight of
0.82+0.03 g/L.
Citrinin was extracted from Penicillium sp. at 366 nm. The mean
highest concentration (45+1.0 μg/L) was extracted from isolate number
73 (Penicillium crustosum Ali 3) with dry weight of 1.0+ 0.03 g/L.
Patulin was extracted from Penicillium sp. at 540 nm. The mean
highest concentration extracted from Penicillium sp was 63.3 + 1.5 μg/L,
it was produced at mean dry weight of 1 + 0.03 g/L.
Cyclopiazonic acid was extracted from A. flavus, A. fumigatus, and
Penicillium sp. at 550 nm. The mean highest concentration extracted from
A. flavus was 48.3 +0.58 μg/L, it was produced from the isolate number
59 (A. flavus Ali 1) at mean dry weight of 1.03+ 0.01 g/L. The mean
highest concentration extracted from A. fumigatus was 38+1.0 μg/L, it
was produced from the isolate number 33 (A. fumigatus Ali 2) at mean
dry weight of 0.83 + 0.01g/L. The mean highest concentration extracted
from Penicillium sp was 45.7+2.5 μg/L, it was produced from the isolate
number 23 (Penicillium chrysogenumAli8) with fungal dry weight of
0.82+ 0.03g/L.
Kojic acid was extracted from A. flavus, A. niger, and Penicillium
sp. at 540 nm. The mean highest concentration of Kojic acid extracted
from A. flavus was 496.3 + 2.5 μg/L, it was produced from the isolate
number 59 (A. flavus Ali 1) with mycelial dry weight of 1.03+ 0.01 g/L.
Summary
137
The mean highest concentration extracted from A. niger was 95.3+3.2
μg/L, it was produced from the isolate number 90 (A. nigerAli 4) with
fungal dry weight of 1.33 + 0.46 g/L. The mean highest concentration
extracted from Penicillium sp was 164.3 + 4.04 μg/L, it was produced
from the isolate number 23 (Penicillium chrysogenumAli8) with fungal
dry weight of 0.82+ 0.03g/L.
In histopathological examination, 35 male albino mice Balb/c
weighing about 18-25 g were obtained from the animal house unit of
Suez Canal University. The rooms and cages used for housing the
animals were thoroughly cleaned time to time. Mice were reared under
strict hygienic conditions during and before infecting them. All mice
were adjudged to be healthy. Mice were provided with food and water ad
libitum. Mice were observed daily for any morbidity or mortality.
Another mice model was examined for the effect of the extracted
mycotoxins (Ochratoxin A, AFB1, citrinin, patulin, CPA& Kojic acid)
after being immunosuppressed. Histopathological examination was
completed as with fungal spores.
Lung tissue changes in abino mice were evident in the form of high
density fungal conidia and proliferating hyphae, surrounded by necrotic
lung tissue and damaged alveoli. As regards the mycotoxins effects, there
were inflammatory cells infiltration in the interstitial space, interalveolar
septae, alveolar space, intrabronchial and intravascular as well as dilated
congested blood vessels, thickened alveolar wall and thickened bronchial
wall.
Data were analyzed using the SPSS statistical software (IBM
Corp., Armonk, NY, USA). Numerical data were expressed as mean+SD,
while qualitative data were presented as count (%), the appropriate tests
were used and statistical significance was considered at p<0.05.
Conclusion
138
Conclusion
A high prevalence of pulmonary fungal pathogens was found in
patients with COPD in this study. Of this, A. flavus and A. fumigatus were
the predominant isolates. Pulmonary fungal infections were more
prevalent at the older age group and males showed slightly higher
contribution.
Some risk factors were found to be related to the prevalence of
pulmonary fungal infections, they were mainly contributing in creation of
an immunosuppression state. In order of the frequency, they were
diabetes mellitus, smoking, prolonged antibiotic use, prolonged
corticosteroid use, HIV and chemotherapy.
Ketoconazole had the highest antifungal action against the
commonest fungal pathogens; Aspergillus spp., Pencillium spp. and
Candida albicans.
Human respiratory system is susceptible to AFB1, Ochratoxin A,
Citrinin, Patulin, CPA and Kojic acid accumulation from colonized
fungal strains, which predispose to several health hazards.
The inflammatory and destructive effects of the fungal infecting
pathogens as well as their mycotoxins were proved in the
histopathological examination.
Recommendations
139
Recommendations
We recommend the followings:
Further studies on larger number of patients are recommended.
Being life threatening, every effort should be prompted for the early
detection and elimination of fungal infections.
Attention should be paid to pick up cases with fungal infections
superimposed on the underlying chronic lung diseases, not to add
extramorbidity.
Special care should be provided to COPD patients, with more
focused consideration to the older age groups and patients with risk
factors.
Efforts should be subjected to introduce more save derivatives of the
drug ketoconazole, as it proved the highest efficacy.