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1. Opportunistic Respiratory Tract Infection
Tankeshwar Acharya

2. Learning Objectives
Describe the morphology, pathogenesis and
laboratory diagnosis of agents causing opportunistic
infections in immunocompromised host.
Morphology, life cycle, pathogenesis and laboratory
diagnosis of Pneumocystsis jirovecii.

3. Host Defense Virulence:
Organism
Disease equation
Number of organisms x Virulence = Disease
Host Defense
State of Health

4. Host defenses
Innate (non specific defenses)
Physical barrier
Inflammatory response and Phagocytosis
Proteins: Complements, lysozyme and interferons
Acquired (specific) immunity
Antibody mediated: for extracellular organisms
Cell Mediated: For intracellular organisms
“Failure of Host Defenses predisposes to infections”

5. Immunocompromised conditions…
Immunity is compromised due to one of the many
health reasons
Cancer: Receiving chemotherapy
HIV/AIDS
Chronic use of corticosteroids or other medications that
weaken the immune system
Organ or bone marrow transplant
Individuals under immunosuppressive drugs

6. Nature of opportunistic pathogen
Normal flora or saprophytes that are ubiquitous in
environment
Weak virulence: Low pathogenic potential
High susceptibility by the host defenses

7. Normal Flora
Our body contains approx 10 trillion
eukaryotic cells
but
>100 trillion microorganisms as a
normal flora.
Normal microflora: Organisms that
live on or in the body but do not
cause disease.

8. Normal flora vs Opportunist pathogen
Opportunistic: Those species of normal flora that can
cause disease under certain conditions:
1. Failure of Host’s normal defenses (i.e
Opportunistic infections in immuno-compromised
host)
2. Introduction of the organisms into unusual body sites,
e.g…..??
3. Disturbances in the normal flora: Broad spectrum
antibiotics
“Too much of good thing is a bad thing”

9. Opportunisitc fungal pathogen
The fungi most frequently isolated from
immunocompromised patients are
saprophytic (i.e. from the environment/Contaminants)
or endogenous (a commensal).
Difficult to determine clinical significance
Each isolate is a potential pathogen
Some opportunistic infections:
Aspergillosis
Candidiasis
Cryptococcosis
Pneumocystosis
Histoplasmosis

10. Aspergillus: Aspergillosis/Aspergilloma
 Widespread in the environment
 Most frequently encountered fungi in the clinical
laboratory e.g. culture of respiratory secretions, skin
scrapings
 Transmission - inhalation of airborne conidia
 Mainly pulmonary infections:
immunocompetent and immunocompromised.
 Macrophage and Neutrophils are mainstay of pulmonary
host defense mechanisms.
 Immunocompromised esp. those with neutropenia:
Hemoptysis and granulomas

11. Invasive Aspergillosis
Important cause of morbidity and mortality in
Immunocompromised hosts.
 Neutropenic patients with hematologic malignancies
 Advanced stage of HIV infections
 Children in chronic granulomatous diseases etc
 Individuals who are in immuno-suppressive drugs.

12. Laboratory Diagnosis`
 Specimen: Sputum, Bronchoalveolar lavage fluid (BAL)
or transbronchial biopsy
 Direct examination: 10% KOH Mount: demonstration of
hyaline septate hyphae of Aspergillus species.
 Fungal Culture: Sabouraud dextrose Agar (SDA).
 Should be examined daily during first week and twice a week
for further four weeks before being considered as sterile.
 Identification based on colony morphology.

13. Laboratory Diagnosis
Fig-2: Dichotomously branched,
septate hyphae
Fig-1: Colonies on Czapek dox agar

14. Laboratory diagnosis
 Immunodiagnosis:
 IgG (Aspergilloma),
 ABPA (IgE) by
ELISA, RIA, Latex agglutination etc.
 Molecular diagnosis: PCR
 Animal Pathogenicity

15. CANDIDIASIS (Candida albicans)
An endogenous organism, found in
40 to 80% of normal human beings
as commensal.
Oval yeast with a single bud
Infections occur when a patient
has some alteration in cellular
immunity, normal flora or normal
physiology.
The more debilitated the host,
the more invasive the disease.
Prolonged antibiotic or steroid
therapy
Invasive procedures e.g. surgery
/ indwelling catheters
Candida albicans: Gram Stain

16. Candidiasis
Most common fungal infection in HIV infected
individuals.
Oropharyngeal candidiasis
Oesophagitis
Candidemia
Pneumonia
Specimen:
Depends on disease presentation
Lab diagnosis:
Colony on SDA
Gram Stain
Germ tube test: Positive

17. Culture
Colony of Candida albicans in SBHI agar Plate:
CDC

18. Cryptococcosis
Cryptococcus neoformans
Ubiquitous in environment
abandoned buildings contaminated with pigeon
droppings
Causes serious opportunistic infections in
immunocompromised patients
Primarily infections of lungs which can disseminated via
hematogenous route causing meningoencephalitis
High morbidity and mortality
Most common life threatening fungal disease in AIDS
patient

19. Lab diagnosis
Specimen:
Depends on site of infection
CSF, biopsy materials, blood, urine etc.
Serological test: demonstrations of cryptococcal
anigen as well as Ab
A cryptococcal antigen titre of greater than or equal to 1:8
is diagnostically significant.
Microscopy:
India Ink Preparation: Oval budding yeast cell of
C.neoformans with distinct halo (polysaccharide capsule)
is observed.
Gram reaction: Gram positive budding yeast cells
In India Ink Preparation:

20. Lab diagnosis contd…
Culture
Blood Agar, SDA, BHI Agar, Bird seed agar etc.
Colony: highly mucoid, cream to buff colored colony on SDA
Cryptococcus neoformans Colony in SDA

21. PJP or PCP (P. jiroveci)
Previously it was called Pneumocystis carinii
Thought to be a protozoan. Presently it is believed to be
a fungus.
But antifungal drugs are ineffective
P. jiroveci is the only Pneumocystis species that infects
humans,
Pneumocystis jiroveci is common in the environment and
does not cause illness in healthy people.

22. Features
 Organism of low virulence
 Asymptomatic infection quiet common
 Antibodies present by 5 years (US Study)
 Pneumocystis jiroveci was a relatively rare infection before
the AIDS epidemic
 An important cause of opportunistic respiratory tract
infections in immunocompromised patients, particularly AIDS
patients.
 In tissue it appears as cyst: That resemble the cyst of
Protozoa.

23. Similarities with protozoa
I. Susceptible to anti-protozoan agents
II. Does not grow in vitro in fungal culture media
III. Requires tissue culture/cell lines for its growth and
viability.
IV. Absence of ergosterol in cytoplasmic
membrane: insensitive to antifungal drugs

24. Considered as fungi because
 Pneumocystis takes fungal stain eg. Methenamine
silver stain
 Possess chitin in all stages of its life cycle
 Cyst wall ultrastructure similar to fungi
 The protein synthesis elongation factor (EF3) and
thymidylate synthase of Pneumocystis are more
homologous to those of ascomycetous fungi
 The ribosomal RNA studies reveal that 16S like
RNA of Pneumocystis shares substantial sequence
homology with various species of Ascomycota

25. Fig: Life Cycle of Pneumocysits Jiroveci

26. Pathogenesis
 Transmission occurs by inhalation.
 Escape the defense of upper respiratory tract
 Deposition in alveoli
 Trophozoites attach to alveolar epithelium (alveolar
type I epithelial cells) and proliferate
 Alveolar type II cell hypertrophy: Macrophage infiltrate
and filling of alveolar spaces with foamy eosinophilic
material and plasma cells.
 Plasma cell pneumonia
 Foci of necrosis and cellular debris in extrapulmonary
sites.

27. Lab diagnosis
Specimen: Lung tissue, fluid obtained by bronchoscopy,
bronchial lavage or open long biopsy.
Sputum: Not suitable
Delay in sample transport might result in false
negative result
Microscopy: visualization cysts or trophozoitic forms
after Giemsa or other tissue staining.
Does not grow in fungal culture Media/cell culture
Fluorescent- labeled immunocytochemistry: detects both
cystic and trophic forms by using monoclonal antibodies
Recent diagnostic methods
Detection of P. jiroveci DNA by PCR in BAL
Measurement of S-adenosylmethionine serum levels

28. Others opportunistic mycoses: Mucormycosis
Zygomycosis/Phycomycosis
Caused by saprophytic molds
( Mucor, Rhizopus, Absidia)
Ubiquitous in environment
Not dimorphic
Transmission by airborne asexual spores
Patients with diabetic ketoacidosis, burns, or lukemia are
particularly susceptible
Sample:
Biopsy specimen or based on sites of infection
Lab diagnosis:
Microscopy and Culture

29. Others:
Histoplasmosis:
Pulmonary compromise is more severe in
immunosuppressed individuals
Nocardiosis:
Nocardia asteroides is the most frequent species isolated
in severely immunocompromised patients
Pulmonary, brain, cutaneous or disseminated disease.

30. Others:
Tuberculosis: M. tuberculosis/MAC
Patients deficient in cellular immunity such as AIDS
patients, are at higher risk of disseminated, life-
threatening tuberculosis.
Mycobacterium avium-intracellularae complex (MAI,
MAC)
Most common in immunocompromised patients such as
those who have CD4 counts of less than 200/µl

31. Others..
Cryptosporiodiosis: Cryptosporidium parvum
Diarrhea is most severe in immunocompromised
patients e.g. AIDS Patient
Symptoms are self limited in immunocompetent
patients

32. Any Questions?