Curve Exam 2 Total points: 64

1
APRIL 1st, Second Exam CBIO4500/6500
Grade Distribution (67 students) A 16 A- 8 B
8 B 7 B- 10 C 2 C 5 C- 5 D 5 F
1 Class average 53.
Curve Exam 2 (Total points 64) A gt
60 100-94 A- 57.5-59.5 93-90 B 57-55 89-86
B 54.5-52 85-81 B- 51.5-48.5 80-76 C 48-45 7
5-70 C 44.5-42 69-66 C- 41.5-39 65-61 D 38.5-
32.5 60-51 F lt 32 50-0
2
AIDS and Opportunistic infections Medical
Parasitology CBIO4500 Cellular Biology,
UGA April 10, 2008 Silvia N. J. Moreno
3
Opportunistic Infections

• AIDS is the most severe acceleration of
  infection with HIV. HIV is a retrovirus that
  primarily infects CD4 T cells, macrophages and
  dendritic cells.
• When CD4 T cells are fewer than 200 CD4 T
  cells per microliter (µL) of blood, cellular
  immunity is lost and this is when AIDS is
  diagnosed (in the US). The sequential decline and
  ablation of cell-mediated immunity results in the
  developen t of opportunistic diseases.
• In the absence of antiretroviral therapy, the
  median time of progression from HIV infection to
  AIDS is nine to ten years, and the median
  survival time after developing AIDS is only 9.2
  months. These include factors that influence the
  body's ability to defend against HIV such as the
  infected person's general immune function. The
  use of highly active antiretroviral therapy
  (HAART) prolongs both the median time of
  progression to AIDS and the median survival time.

4
Opportunistic Infections
Most AIDS-defining conditions are opportunistic
infections, which rarely cause harm in healthy
individuals. In people with AIDS, these
infections are often severe

• Bacteria
• Mycobacterium Avium Complex
• Mycobacterium Tuberculosis
• Viruses
• Varicella-Zoster Virus
• Herpes Simplex Virus
• Cytomegalovirus
• Protozoa
• Coccidiosis (Cryptosporidiosis, Cyclosporiasis,
  and Isosporiasis)
• Toxoplasmosis
• Leishmaniasis (not in the U.S., but in Southern
  Europe and in many other parts of the world)
• Chagas
• Malaria

• Fungi
• Pneumocystis carinii Pneumonia
• Candidiasis
• Aspergillosis
• Cryptococcosis
• Histoplasmosis
• Coccidioidomycosis
• Microsporidiosis

5
Cyclospora cayetanensis

• Infection with cyclospora spp. is widely
  distributed probably cosmopolitan.
• Countries initially identified as having endemic
  cyclosporiasis Haiti, Guatemala, Peru and Nepal
• Infection most common in HIV/AIDS patients.
• Also important cyclosporosis in travelers
  infections acquired in Latin America, India or SE
  Asia
• Large, multi-state food-borne outbreaks of
  Cyclospora infection in the USA and Canada during
  the 1990s drew attention to this parasite.
• Associated with imported food items, specially
  raspberries and green leafy vegetables such as
  basil and mesclun lettuce.
• In 1996, fresh raspberries from Guatemala
  contaminated with a parasite called cyclospora
  cayetanensis sickened 1,465 people in 20 states,
  the District of Columbia and two U.S.
  territories.

6
Cyclospora cayetanesis life cycle
In freshly passed in stools, the oocyst is not
infective (direct fecal-oral transmission cannot
occur this differentiates from Cryptosporidium).
In the environment, sporulation occurs after
days or weeks at temperatures between 22C to
32C, resulting in division of the sporont into
two sporocysts, each containing two elongate
sporozoites. Fresh produce and water can serve
as vehicles for transmission. The oocysts excyst
in the gastrointestinal tract, freeing the
sporozoites which invade the epithelial cells of
the small intestine. Inside the cells they
undergo asexual multiplication and they form two
types of meronts. First generation meronts w/
8-12 merozoites and 2nd generation meronts w/ 4
merozoites which penetrates new cells to form
gametes. Some of these gametes enlarge to form
the macro- and microgametes. Fertilization
produces the an immature oocyst that is passed
with stool. The potential mechanisms of
contamination of food and water are still under
investigation.
7
Sporulation of Cyclospora oocysts.
The sequence shows, as observed by DIC microscopy
of wet mounts an oocyst passed in fresh stool
(Day 0) sporulated oocysts at days 5 (Day 5) and
10 (Day 10), which both contain 2 sporocysts and
a ruptured oocyst (Rupture), with a sporocyst
still inside the oocyst and the other sporocyst
just outside. The coiled sporozoites are barely
visible inside the sporocysts.
8
Cyclospora oocysts
Four Cyclospora oocysts from fresh stool fixed in
10 formalin and stained with safranin, showing
the uniform staining of oocysts by this method
9
RELATIVE SIZES
Giardia lamblia cyst (length from 8 to 19 ? and
averages 11-12 ?), a Cyclospora cayetanensis
oocyst (8-10 ?), and a Cryptosporidium parvum
oocyst (4.5 ? 5 ?). The virus is not drawn to
scale. The Cyclospora oocyst shown is fully
sporulated it has 2 internal sporocysts, each
with 2 sporozoites. Whereas oocysts of
Cryptosporidium, are fully sporulated and
infectious when excreted, Cyclospora oocysts
sporulate in the environment, days to weeks after
excretion. Giardia, which is not a coccidian
parasite, does not have sporocysts or
sporozoites. (Figure courtesy of Dennis D.
Juranek.)
10
Oocyst comparison between cryptosporidium,
cyclospora and isospora
Three coccidian parasites that most commonly
infect humans, seen in acid-fast stained smears
(A, C, and F), bright-field differential
interference contrast (B, D, and G) and UV
fluorescence (E and H).
Isospora
Cyclospora
Cryptosporidium
11
Coccidian parasites from stool specimens
Acid -fast
Safranine
Cryptosporidium parvum (A), Cyclospora
cayetanensis (3 unstained oocysts and 1 oocyst
that stained acid fast B), and Isospora belli
(1 oocyst that stained acid fast C, bottom and
1 nonacid-fast oocyst that took up the
counterstain C, top). Note differences in size,
which highlights the importance of carefully
measuring what is found. Note also that the walls
of the unstained Cyclospora oocysts on the top
and lower right in B have a hyaline or
crumpled-cellophane appearance. The lower
photographs show the safranin-stained
preparations. Cyclospora oocysts stain more
uniformly with this technique (E) than with
acid-fast staining (B). (Figure courtesy of
Michael J. Arrowood.)
12
Pathology

• Cyclospora infects enterocytes of the small
  bowel where various stages, sexual and asexual
  stages have been observed. Villous blunting, mild
  crypt hyperplasia and variable increased chronic
  inflammatory cells in the lamina propria.
• Illness varies with age and condition of the
  host and the infectious dose is unknown. The main
  symptom is watery diarrhea, loss of appetite,
  weight loss, abdominal bloating and cramping,
  nausea, fatigue and low grade fever.
• Incubation period averages one week and illness
  lasts 6 weeks.
• In the immunocompromised patient, severe
  diarrhea can last up to 4 months or longer even
  if treated thus producing a disease syndrome that
  is debilitating and life threatening
• Extra-intestinal infection appears to be more
  common in AIDS patients

Duodenal biopsy showing immature schizonts (broad
arrow) and merozoites (arrow) in a
parasitophorous vacuole of C. cayetanesis in
surface enterocytes. (HE STAIN)
13
Diagnosis and treatment

• Detection of oocysts in stool sample by
  microscopy
• Recovery of oocysts in intestinal fluid or small
  bowel biopsy specimens
• Demonstration of oocyst sporulation
• PCR amplification of Cyclospora DNA

Can be successfully treated with trimethoprim
(TMP)-sulfamethoxasole Trimethoprim is
5-(3,4,5-trimethoxyphenyl)methyl
-2,4-pyrimidinediamine. Sulfamethoxazole is
4-amino- N -(5-methyl-3-isoxazolyl)
benzenesulfonamide. The structural formula is
14
CYCLOSPOROSIS and RASPBERRIES
Multiple foodborne outbreaks, thousands in US and
Canada since 1990 Before 1996 mostly overseas
and 3 small US outbreaks May 1996 55 events
(all had raspberries served) of outbreaks in US
and Canada 1465 cases, 978 confirmed. Spring
1997 41 events, 1012 cases. Again the only
common food consumed in all events was
raspberries from Guatemala May 1998 Ontario,
Canada, 315 cases
ATLANTA-Guatemalan raspberries have been
fingered as the delectable vehicle of this
spring?s outbreaks of Cyclospora cayetanensis.
And while the epidemiologic links to the caviar
of fruit accumulated, a cousin berry was
exonerated. Despite much scuttlebutt, the
strawberry got a clean bill of health.
15
EPIDEMIOLOGY

• Food-borne pathogen
• Not sure if water could also have a role in
  transmission
• Infected humans are the only known sources of
  oocysts
• Not other reservoir host identified
• No methods available to grow them in the lab
• How the environment (water and food) gets
  contaminated with oocysts?
• Marked seasonality of cyclospora temp,
  humidity, may facilitate sporulation

16
Isospora belli

• Apicomplexan parasite. Coccida group. Isospora
  genus.
• Monoxenous life cycle.
• Coccida parasites are identified based on the
  structure of the sporulated oocyst.
• About 248 species described before 1986. I.
  belli, is a true member of the genus Isospora.
• The least common of the three intestinal
  coccidia that infect humans (Cryptosporidium,
  isospora and cyclospora)
• Can cause severe disease with fever, malaise,
  persistent diarrhea and even death in AIDS
  patients
• Also cause disease in pigs
• Infects the epithelial cells of the small
  intestine.

17
Isospora belli life cycle
Infection occurs by ingestion of
sporocysts-containing oocysts the sporocysts
excyst in the small intestine and release their
sporozoites, which invade the epithelial cells
and initiate schizogony. Upon rupture of the
schizonts, the merozoites are released, invade
new epithelial cells, and continue the cycle of
asexual multiplication . Trophozoites develop
into schizonts which contain multiple merozoites.
After a minimum of one week, the sexual stage
begins with the development of male and female
gametocytes . Fertilization produces oocysts that
are excreted in the stool.
18
Oocysts of Isospora belli
C
A
B
At time of excretion, the immature oocyst
contains usually one sporoblast (more rarely two)
(A and B) . During maturation the sporoblast
divides in two (two sporoblasts) and secrete a
cyst wall, thus becoming sporocysts which divide
twice to produce four sporozoites each (C).
Images contributed by Georgia Division of Public
Health. The oocysts are large (25 to 30 µm) and
have a typical ellipsoidal shape.
Sporogony it is dependent on moisture,
temperature and adequate oxygen. Optimun
temperature is at 30-37 C.
19
Isosporosis in AIDS patients

• Intestinal infections
• Diarrhea leads to dehydration requiring
  hospitalization
• Fever and weight loss are also common findings.
• Prevalence higher in foreign patients,
  specially from El Salvador
• or Mexico.
• Extraintestinal infections
• Two reports of disseminated Isosporiasis have
  been published. In one of them microscopic
  finding with I. belli were observed in the lymph
  nodes and walls of both the small and large
  intestines (Post-mortem).
• Second case showed severe villous atrophy and
  meront, gamonts, and oocysts of I. belli within
  enterocytes. Massive infection in the lymph nodes
  in association with plasmacytosis and some
  eosinophils .

Small Bowel Biopsy (Isospora belli arrows)
20
LEISHMANIASIS AND HIV
21
Global distribution of reported cases of
leishmaniasis and Leishmania/HIV co-infection,
1990-1998
Leishmania and HIV co-infections have been
reported in 35 out of the 88 countries in which
leishmaniasis is endemic and are a concern in
Africa, Brazil and the Indian continent where
both diseases overlap geographically
22
Three syndromes associated with Leishmania
infection in humans

• Visceral leishmaniasis (VL), (kala azar) is the
  most severe form of the disease. Mortality rate
  of almost 100. It involves the internal organs.
  After treatment and recovery, patients may
  develop chronic cutaneous leishmaniasis that
  requires long and expensive treatment. Leishmania
  donovani, Leishmania infantum, Leishmania chagasi

• Cutaneous leishmaniasis (CL) large numbers of
  skin ulcers on the exposed parts of the body.
  Often self-healing but it can create permanent
  scars. After recovery or successful treatment,
  induces immunity to re-infection by the species
  that caused the disease. Represent 50-75 of all
  new cases. Leishmania tropica, Leishmania major,
  Leishmania aethiopica

• Mucocutaneous leishmaniasis (MCL), lesions can
  lead to extensive and disfiguring destruction of
  mucous membranes of the nose, mouth and throat
  cavities. Leishmania braziliensis, Leishmania
  mexicana, Leishmania amazonensis

• Leishmaniasis could become diffused due to
  immunosuppresion Difficult to treat due to
  disseminated lesions that resemble leprosy and do
  not heal spontaneously. Related to a defective
  immune system and it is often characterized by
  relapses after treatment.

23
Leishmania/HIV co-infection characteristics
Important epidemiological changes Humans become
reservoirs Co-infected patients harbor a high
number of Leishmania in their blood. This
increases the risk of future epidemics.
Atypical manifestations of Leishmaniasis
Leishmania/HIV co-infections modify the
traditional patterns of zoonotic VL.

• VL is the clinical form most frequently
  associated with HIV/AIDS specially in
  south-western Europe (some CL have been
  reported)
• The geographical distribution of VL and AIDS
  changes are due to
• the spread of AIDS in suburban and rural areas
  of the world,
• the spread of VL from rural to suburban areas.
• In southwestern Europe up to 70 of all adult
  cases of VL are related to HIV/AIDS.
• Since the introduction of HAART, the incidence
  of VL and HIV patients has dropped in Europe,
  although relapses still occur.

24
TRANSMISSION
The diagram suggests a few possible paths of
leishmaniasis infection. Female sandflies,
vectors for parasites of the genus Leishmania,
disseminate to humans mainly from animals.
Infected dogs, both symptomatic and asymptomatic,
have traditionally been reported to be the
reservoir of this disease foxes, jackals,
wolves, raccoons, sloths, hyraxes, rats, and
other rodents have been reported as reservoirs.
In HIV-Leishmania coinfection, intravenous drug
users seem to be human reservoirs. Reports of
transmission of leishmaniasis via needle-sharing
are increasing.
25
Average number of AIDS cases and Leishmania/HIV
co-infection cases per year, reported to WHO,
south-western Europe, 1990- June 1998
26
Percentage distribution of reported cases of
Leishmania/HIV co-infection and AIDS,
according to probable mode of HIV transmission,
south-western Europe, 1997-1999 Mode of
probable transmission for Leishmania/HIV
co-infection is based on the HIV transmission for
1990-1998. Mode of probable transmission for
AIDS is based on 1998.
27
HIV modifies the clinical presentation of
leishmaniasis in the co-infected patient

• Major characteristics for HIV-associated
  leishmaniasis, all related to the immunologic
  impairment caused by the virus
• Co-infected individuals may develop VL though
  infected with a leishmania strain that only
  causes cutaneous leishmaniasis in the
  immunocompetent
• The leishmaniasis may be severe and unresponsive
  to treatment
• They may have amastigotes in tissues, such as the
  intestine, that are never found infected in the
  immunocompetent
• Co-infected individuals may have unusual high
  numbers of amastigotes in their
  reticulo-endothelial system
• A chronic and relapsing course, with each patient
  typically experiencing two or three relapses
  despite proper treatment

28
A TH1 response is required for parasite control
and healing

• Stimulation with different cytokines leads to the
  development of two types of T-cells specialized
  for different immune responses
• Th1 and Th2 strongly down regulate each other
• This polarization has important consequences for
  the downstream response and can spell life or
  death
• Non healing Leishmania infections are
  characterized by a strong TH2 response
• Healing infections are characterized by TH1
• The parasites seems to manipulate this balance in
  his favor, we dont understand yet how that is
  done

29
Pathogenesis and immunological aspects of the
HIV-leishmania coinfection

• In HIV-1-infected patients, larger viral load is
  associated with a higher prevalence of
  leishmaniasis and a lower CD4-T cell count is
  linked to visceral and disseminated forms of
  leishmaniasis
• Both Leishmania and HIV share the same target
  cells
• A Th1 response (IFN-? and IL-2) is present
  during the early stages of HIV infection, the
  progression to AIDS being associated with a
  switch to a Th2 response (IL4 and IL-10).
• Prolonged, Th-2-type activation and increased
  viral replication have been shown in
  co-infections (high levels of IL-4, IL-6 and
  IL-10). HIV may exacerbates the Th2-type
  responses of VL patients.
• In vitro experiments showed that L. infantum
  induces expression of latent HIV-1 by
  up-regulating specific receptors on the surface
  of CD4 lymphocytes and macrophages.
• Humans co-infected with HIV are able to mount a
  T-cell response against the parasite after
  treatment but this response is lost as the viral
  infection progresses leading to relapse
• More studies are necessary on the immunological
  status of co-infected individuals for
  understanding the synergistic effect of both
  infections.

30
TREATMENT

• Similar medications used for leishmanisis but
  modes of administration and dosages may vary.
• Antimonials First-line therapy. SbV,
  Pentavalent antimonials include sodium
  stibogluconate and meglumine antimoniate. Higher
  doses. Intravenously or intramuscularly. Adverse
  effects of SbV are frequent but usually mild.
• Amphotericin B. AMB deoxycholate (Fungizone) is
  a second-line therapy because of its toxic
  effects.
• Liposomal AMB (L-AMB) is less toxic than AMB. It
  has been effective in the primary treatment of VL
  in both immunocompetent and immunocompromised
  patients
• Pentamidine. Pentamidine is definitely a
  second-line treatment for VL it might be useful
  if previous therapeutic approaches with
  antimonials or AMB have failed.
• Treatment of relapses. Up to 50 of patients
  experience a relapse six months after treatment
  and 90 after one year. These recurrences may be
  treated again with SbV. Treatment with L-AMB has
  been highly effective without any associated
  toxic effects.

31
MICROSPORIDIA

• Obligate intracellular parasite
• Agent of economically important diseases
  (insects, small mammals)
• Emerged as opportunistic during AIDS epidemics.
  Aprox. 15 overall prevalence in AIDS patients
• Small resistant spore stage (1-3 ?m)
• Defined by polar tube (extrusion apparatus)
• Classified as a fungus by molecular analysis of
  rRNA

• The phylum Microspora consists of approximately
  144 genera and over 1200 species.
• Eukaryotic features they possess a typical
  eukaryotic nucleus, endomembrane system,
  cytoskeleton
• Prokaryotic features the translational
  apparatus, genome size and organization, lack of
  mitochondria, peroxisomes and typical Golgi
  membranes

32
MICROSPORIDIA LIFE CYCLE

• The infective form is the spore which survives
  for a long time in the environment.
• The spore extrudes its polar tube and injects
  the host cell.
• The spore injects the infective sporoplasm into
  the host cell through the polar tube
• Inside the cell, the sporoplasm undergoes
  multiplication by merogony or schizogony.
• This development can occur either in direct
  contact with the host cell cytoplasm (e.g., E.
  bieneusi) or inside a parasitophorous vacuole
  (e.g., E. intestinalis).
• During sporogony, organisms mature into spores
  and a thick wall is formed.
• When the spores increase in number and completely
  fill the host cell cytoplasm, the cell membrane
  is disrupted releasing them.
• These free mature spores can infect new cells.

33
The microsporidian spore.

• Size from 1 to 10 mm.
• The spore coat an electron dense exospore
  (Ex), an electron lucent endospore (En) and
  plasma membrane (Pm).
• The sporoplasm (Sp) contains a single nucleus
  (Nu)
• Posterior vacuole (PV).
• The polar filament is attached to the anterior
  end of the spore by an anchoring disc (AD). The
  number and arrangement of coils vary with the
  genus/species .
• The lamellar polaroplast (Pl) and vesicular
  polaroplast (VPl) surround the manubroid polar
  filament (M).

34
Species of microsporidia that infect humans

• Enterocytozoon bieneusi (Ent. bieneusi),
  originally described in humans, is associated
  with malabsorption, diarrhea and cholangitis.
  Most common species found in HIV-infected
  patients with intestinal infection. Self-limited
  diarrhea in immunocompetent hosts.
• Encephalitozoon hellem (Enc. hellem)
  superficial keratoconjunctivitis, sinusitis,
  respiratory disease, prostatic abscesses and
  disseminated infection.
• Encephalitozoon cuniculi (Enc. cuniculi)
  hepatitis, encephalitis, and disseminated
  disease.
• Encephalitozoon (Septata) intestinalis
  diarrhea, disseminated infection, and superficial
  keratoconjunctivitis.
• Trachipleistophora species T. hominis
  keratoconjunctivitis, myositis, sinusitis,
  skeletal muscle, rhinitis T. antropophtera
  disseminated infection, encephalitis, myositis
• Pleistophora sp. only a few infections in
  humans reported. Skeletal muscle involvement
  myositis
• Brachiola sp. B. vesicularum myositis B.
  algeraea mosquito parasite. keratoconjunctivitis.

35
DIAGNOSIS

• Transmission electron microscopyConfirmation
  and species identification. E. cuniculi and E.
  hellem are similar even at the ultrastructural
  level. The polar tube structure is an important
  feature.
• Light microscopy Genus and species
  differentiation is uncertain
• Cytologic diagnosis and stool examinations.
• Fluorescent staining methods
• Histologic diagnosis
• Antigen-based methods antibodies to
  species-specific surface of the spores or polar
  tube proteins
• Serology carbon immunoassay (CIA) indirect
  immunofluorescence test (IFA) enzyme-linked
  immunosorbent assay (ELISA), counterimmunoelectrop
  horesis (CIE) and Western blot
• detect IgG and IgM antibodies to E. cuniculi and
  E. intestinalis.
• Molecular-based methods The complete sequence
  of the whole genome of E. cuniculi is known
• PCR
• In situ hybridization

Monoclonal antibody-based immunofluorescence
identification of Encephalitozoon hellem. Spores
are present in a bronchoalveolar lavage sample of
a 30 year-old AIDS patient from Georgia. Note
the bright fluorescent spores, which have
extruded their polar tubules.
Stool smear stained with Quick-Hot Gram
Chromotrope stain containing Enterocytozoon
bieneusi spores. Black arrows indicate E.
bieneusi spores with their belt-like stripe
accentuated. The red arrow indicates an
unidentified yeast. The yellow arrow indicates a
vacuolated spore.
36
TREATMENT

• For ocular microsporidiosis (E. hellem, E.
  cuniculi, Vittaforma corneae) oral albendazole
  plus topical fumagillin.
• Albendazole is the drug of choice to treat
  intestinal (E. bieneusi, E. intestinalis) and
  disseminated microsporidiosis (E. hellem, E.
  cuniculi, E. intestinalis, Pleistophora sp.,
  Trachipleistophora sp., Brachiola vesicularum).
• Fumagillin, highly effective topically to treat
  keratoconcjutivitis due to Encephalitozoon sp.
  Systemically is effective against E. bienusi but
  has toxicity
• TNP-470, an analogue of fumagilin that is less
  toxic is effective against several species of
  microsporidia.
• Metronidazole may also be a useful therapy
  especially in non-HIV infected patients.
• Antiretroviral therapy (HAART) increases the
  CD4 T cell levels and reduces the HIV burden and
  leads to a reduction in the prevalence of
  microsporidia.

37
SUMMARY

• Opportunistic infections
• Most of these infections are benign but
  persistent in immuno-competent hosts
• In cases of infections that provoke symptoms in
  immuno-competent hosts, HIV worsens the symptoms
• Protection against the disease is mediated by
  the cellular arm of the immune system
• There is a reduction in the incidence of all of
  the opportunistic infections due to HAART
• The main problem is that in many African
  countries anti-HIV treatment is not available
• Leishmaniasis is an interesting case since the
  parasite lives in the same host cells as the
  virus. The co-infection may induce an
  uncontrollable spread of the parasite and an
  increase in viral replication.