Azza Negm

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Human Pathogenic Protozoa in Bivalves Collected
from Local Markets in Alexandria
By Azza Negm
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Introduction

• Bivalves (shell fish) commonly known as Om El
  Kholool and Gandofli are considered as a tasty
  delicacy and are usually eaten raw. They are
  however known as filter feeders concentrating
  pathogenic organisms from the surrounding water
  with the possibility of transmitting them to man.

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Aim of the Work

• To detect the possible presence of pathogenic
  protozoa in the bivalves collected from the local
  markets in Alexandria
• If organisms are detected then, their infectivity
  will be assessed by animal inoculation.

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Material and Methods

• The present study was conducted on two species of
  bivalves Donax trunculus (Om El Kholool) and
  Caelatura laronia (Gandofli)
• To identify the possible presence of pathogenic
  protozoa homogenization of the body of the
  shellfish was done and the sedimented homogenate
  was examined after staining with
• Ziehl Neelsen
• Modified Trichrome
• Positive samples were pooled together and were
  used to infect white mice by oral inoculation.

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• Experimental animals were grouped as follows
• Group I 10 animals infected with Gandofli
• GroupII10 animals infected with Om El Kholool
• Group III 5 animals served as non infected
  controls.
• Each animal was subjected to
• Stool examination
• Histopathological examination of the small
  intestine

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Conclusion

• Om el Kholool and Gandofli are potential sources
  of some protozoal organisms which can easily be
  transmitted to man especially that these
  shellfish are usually eaten raw.

• These protozoa Microsporidia, Cryptosporidia and
  Cyclospora are highly pathogenic especially in
  immunosupressed patients.Therefore as a
  precaution these shellfish should be properly
  cooked before being eaten.

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Detection of Trichomoniasis in Vaginal Specimens
by Both Conventional and Modern Molecular Tools
By Azza Y.Negm and Desouky Abd el Haleem
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Introduction

• Trichomoniasis has been associated with
  vaginitis, cervicitis,urethritis and pelvic
  inflammatory disease. In pregnancy it has been
  associated with premature rupture of membranes,
  premature delivery and low birthweight in
  neonates.It also increases the risk of HIV
  acquisition and transmission in women and was
  considered a predisposing factor for cervical
  cancer.

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• In men although trichomoniasis is usually
  asymptomatic yet it may lead to urethritis and
  infertility
• Symptoms and signs of trichomoniasis are not
  adequatly sensitive or specific for diagnosis
  besides being asymptomatic in up to 50 of
  infected individuals. So diagnostic tests are
  usually required to confirm the presence of
  organisms.

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Aim of the Work
To compare different techniques in diagnosis of
Trichomonas vaginalis in females
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Material and Methods

• The present study was conducted on 23 symptomatic
  females. Their age ranged between 18 and 45
  years.
• From each female vaginal secretions were
  collected on sterile cotton swabs and examined as
  follows
• Fresh wet mount
• Culture on Diamonds s medium
• Acridine orange stain
• PCR

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Results
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Analysis of PCR products by 2 agarose gel
electrophoresis
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Diagnosis of T. Vaginalis infection by different
techniques
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Conclusion

• Direct Microscopy is a practical widely used
  economical method, yet its sensitivity is low so
  all the suspected samples which are found
  negative by this method should be repeated
  primarily by culture followed by PCR

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• Culture of T.vaginalis is a relatively
  inexpensive option with high sensitivity but it
  requires specific culture media and an incubation
  period which may reach up to seven days so there
  is a delay in treatment
• PCR is easy to perform, highly sensitive and
  specific yet it is expensive and cannot be used
  for routine purposes. However it can be of great
  value in undiagnosed patients.

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Vaccination Against Congenital Toxoplasmosis
By Safeya M. Ali, Sonia R. Allam, Azza Y. Negm
and Lobna A. El Zawawy
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Introduction

• Infection of a woman with T. gondii for the first
  time during pregnancy may induce her protection
  but not that of her foetus.
• Experimentally it was proven that immunization by
  T. gondii tissue cysts induces immunity against
  vertical transmission in mice. However, such a
  procedure is relatively dangerous and the search
  for a safer vaccine is required.

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Aim of the Work

• An attempt to convey immunity to new born of
  experimentally infected pregnant mice by using
  irradiated live T. gondii cyst vaccine alone, or
  by the addition of IL-2 as an adjuvant as
  compared to the live cyst vaccine alone or with
  an adjuvant.

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Material and Methods

• Strains
• Virulent RH strain of T gondii was maintained by
  serial IP passages of tachyzoites in mice. These
  were used for challenge of pregnant female mice.
• Avirulent KSU strain of T. gondii used for
  production of vaccines

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• Vaccines
• Irradiated Cyst vaccine (ICV) Gamma rays applied
  to the live vaccine
• Irradiated Cyst vaccine IL-2 (ICV-IL-2).
• Live Cyst vaccine (LCV Prepared from brains of
  three months old infected mice with KSU strain
• Live Cyst vaccine IL-2 (LCV IL-2)
• Vaccines were administered orally before mating
  in a dose of 5 cysts per mouse.
• IL-2 was given IP in a dose of 0.2ml

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• 3. Grouping of Pregnant Female Mice
• Control Groups
• Normal non infected
• Non immunized infected
• Immunized non infected
• Experimental Groups Immunized Infected
• Subgroup 1 (S1) received ICV
• Subgroup 2 (S2) received ICV IL-2
• Subgroup 3 (S3) received LCV
• Subgroup 4 (S4) received LCV IL-2

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• All pregnant females were sacrificed near full
  term (18-21 days).
• Blood samples were collected and infection if
  present was proved by ELISA.
• Tissues were also examined by histopathological
  sections.
• Pups were removed by CS.
• Cord and Placental blood films were stained with
  Giemsa.
• Tissues from each pup were homogenized and
  injected IP into normal mice which were later
  examined for the presence of tachyzoites.in their
  peritoneal exudates.

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Results Conclusion

• Pre-immunization with the current vaccines
  offered significant protection of both dams and
  pups.
• The highest level of protection was noticed in
  mice which received

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• LCV-IL-2, followed by ICV-IL-2, then LCV and the
  least protection was elicited in dams immunized
  with ICV alone.
• Thus there is a possibility of applying such
  vaccines not only in mice but also in other
  mammalian hosts including human.

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DETECTION OF COPROANTIGEN IN EARLY TRICHINELLOSIS
By Boulos L.M., Ibrahim I.R., Negm A.Y., And
Aly S.M.
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Introduction

• Prompt diagnosis of early trichinosis not only
  relieves patients of unpleasant symptoms and
  serious complications, but can also avoid
  unnecessary and costly evaluations. Moreover,
  misdiagnosis of such early cases would be greatly
  minimized.

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• Nowadays, coproantigen detection has proved to
  be useful in diagnosis of many parasitic
  infections such as G.lamblia, E.histolytica,
  E.multilocularis, C.parvum and many other
  parasites. However its use in diagnosis of
  T.spiralis infection has not yet been assessed.

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Aim of the work

• Study the possibility of early diagnosis of
  T.spiralis by detection of coproantigen in stool
  using both the ELISA, as well as the Co-A test.

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Material and Methods
Parasite
The strain of T.spiralis was obtained from
infected pigs meat from the main slaughter house
in Alexandria.
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Exp. mice
CoproAg detection
Ag preparation
Adult crude Ag
Larval crude Ag
20 mice 300 L/m
20 mice 300 L/m
30 mice 500 L/m
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Preparation of hyperimmune sera (HIS)
HIS raised against T.spiralis Ags were prepared
in both rabbits and guinea pigs.
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Preparation of stool samples for Coproantigen
detection
Stoolanalysis
Preparation of faecal supernatant fractions 2000
g/30 min
Stoolcollection
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Modified double sandwich ELISA
Coproantigen detection
Co-agglutination test
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Detection of T.spirals antigen in stool of mice
at different durations using ELISA test
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Detection of T.spirals antigen in stool of mice
at different durations using CoA test
- Negative reaction Very weak ve Weak
ve Moderately ve High ve
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ve reaction
-ve reaction
Co-A reaction
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Advantages
Simple, rapid
Early detection
Non invasive
Past and present infection
Monitoring of parasitic development
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Conclusion
The current experimental study which was
carried out primarily as a model for human
trichinosis, indicated that coproantigen
detection was possible, even as early as the
first day post infection.
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The use of hyperimmune sera raised against
larval antigen is preferred since the latter is
more easily prepared in terms of amount and
cleanliness.
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The detection of coproantigens has showed a
great potential benefit for clinical and
epidemiological use and could play an important
part in the surveillance and control of
trichinosis in both developed and developing
countries.
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Coproantigen assay should be applied in
communities dealing with and suffering from
enteric diseases especially among pig raising
farmers and/or garbage collectors to depict the
actual incidence of trichinosis.
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Experimental Conversion of Virulent RH Toxoplasma
gondii Tachyzoites in Vitro
By N.A. Hammouda, I.R. Ibrahim, E.D. Elkerdany,
A.Y.Negm and S.R. Allam
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Introduction

• Toxoplasmosis is an important protozoal disease
  of humans and domestic animals. In the
  intermediate host, as man Toxoplasma gondii
  exists in two forms rapidly dividing
  tachyzoites, which are thought to be responsible
  for the acute infection, and bradyzoites which
  are believed to persist for the life span of the
  host.

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• However, these dormant stages are able to
  reconvert into the virulent tachyzoites in
  immuno-compromised patients. The factors that
  influence interconversion of stages are still
  unknown .

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Aim of the Work

• Our aim was to convert tachyzoites to bradyzoites
  in culture simply by changing the pH of the
  culture medium. Changes in the morphology, DNA
  content and cell cycle phases during
  transformation were assessed.

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Material and Methods
Conversion of Tachyzoites

• RH tachyzoites, were harvested from previously
  infected mice and washed in culture medium.
• Macrophages were prepared from mice then
  collected in culture medium (RPMI 1640) 10
  foetal calf serum, L-glutamine, penicillin and
  streptomycin.

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• Tachyzoites were then placed together with
  macrophages in a modified medium with pH adjusted
  to 8 using sodium hydroxide.
• As a control organisms were cultured in the
  standard (RPMI 1640) 5 foetal calf serum pH
  7.4.

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Experiment

• Culture tubes were examined with their
  corresponding controls at
• Day 0 group 1
• Day 2 group 2
• Day 4 group 3

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• The following procedures were conducted-
• A . Giemsa and Feulgen Stains
• Giemsa stain
• Light microscopy
• Computerized image analyzer at 560 mm.
• Feulgen stain
• To determine the relative DNA content for each
  individual parasite.
• Tracing the cell cycle phase for each group.
• Organisms of group 2 and group 3 were compared
  with group1 as well as with controls and any
  morphological changes were noted

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• B. Assessment of infectivity after in vitro
  incubation
• Organisms from different groups were collected,
  washed, counted and inoculated into mice.
• Two to three months later the brains of infected
  mice were homogenized and examined for Toxoplasma
  cysts

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Results
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Cell cycle phases of the studied groups
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Conclusion

• It was possible to transform tachyzoites to a
  slowly proliferating stage simply by changing the
  pH of the culture medium. This observation will
  facilitate the study of the differences between
  the stages, and help in understanding more of the
  mechanism of the disease thus allowing better
  control of toxoplasmic reactivation.

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• Attenuation of toxoplasmic organisms by this
  simple method, combined with assessment by
  morphometry, DNA and cell cycle phase
  measurements and evaluation of their infectivity
  may help in the development of vaccines against
  Toxoplasma and other parasites.

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A New Approach in Cultivation of Leishmania major
Using Human Urine
By Mona M. El Temsahy and Azza Y. Negm
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Introduction

• A number of culture media, especially liquid
  media designed for the bulk cultivation of
  Leishmania contain either fetal calf serum (FCS)
  or a blood lysate as one of their essential
  ingredients.

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• Serum is a complex, highly variable and difficult
  to characterise reagent, beside being expensive.
• Using blood lysate in culture media, makes the
  medium translucent, and preparation of a sterile
  blood lysate is quite difficult. A cheap easily
  available, serum-free but good medium has been a
  goal for researchers.

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Aim of the Work

• The aim of the present work was to investigate
  the possibility of using human urine as an
  alternative ingredient for cultivating and
  isolating Leishmania major.

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Material and Methods
Parasite
L. major MHOM/SN/OO/DK1

• I- In vitro Leishmania culture
• Promastigotes grown on Tanabes medium were
  collected, washed and counted to reach a final
  concentration of 106 parasites/ml.

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RPMI 1640 5 urine
RPMI 1640 10 FCS
RPMI 1640

• Parasites were counted on alternate days starting
  from day two to day 14 to estimate the number of
  parasites/ml.

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II- Isolation of Leishmania

• Amastigotes were withdrawn from the footpads of
  infected Swiss albino mice and inoculated into
  the urine supplemented medium to determine the
  possibility of isolation on such medium.

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• Assessment of infectivity after repeated
  subculture on urine supplemented medium

Mice
inoculated with promastigotes repeatedly
subcultured on urine supplemented media
inoculated with promastigotes repeatedly
subcultured on ordinary media for the same
duration
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Results
I. Growth curves of Leishmania promastigotes
Mean number of promastigotes/ml culture media in
the three studied groups
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• II- Isolation
• Leishmania amastigotes withdrawn from footpads of
  infected mice could not be isolated on
  urine-supplemented media.
• III- Maintenance and infectivity
• Mice inoculated with promastigotes repeatedly
  subcultured on urine-supplemented media showed
  typical lesion two weeks post infection as
  compared to the control group.

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Conclusion

• Human urine is an inexpensive, readily available
  growth supplement for culture and maintenance of
  L.major without affecting the parasite
  infectivity.
• No sophisticated facilities are required for its
  preparation, making it ideal for researchers
  working in developing countries.

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• Since the medium is free of blood or its
  products, it is well suited for immunologic as
  well as biochemical work on the parasite.
• Urine supplemented media is not suitable for
  isolation of Leishmania.

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Morphology, Histochemistry and Infectivity of
Blastocystis hominis Cyst
By Iman F. Abou el Naga and Azza Y. Negm
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Introduction

• Blastocystis hominis is a protozoal parasite
  frequently found in human fecal samples.
• Cystic and trophic stages were reported, the
  latter has different morphological forms. The
  life cycle of this parasite has not been
  definitely established and in recent years
  several studies suspected that the cystic form is
  the infective stage of this protozoa.

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Aim of the Work

• The present work aimed at studying the morphology
  and histochemistry of the cystic stage using
  different staining techniques. The infectivity of
  this stage was also verified by experimentally
  infecting albino mice.

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Material and Methods

• One hundred and twenty diarrheic stool samples
  were collected from different departments of
  Alexandria University hospitals and examined as
  follows.
• Direct saline and iodine smears.
• Formol-ether concentration technique.
• Morphological study was undertaken for both
  trophic and cystic stages present in the positive
  stool samples using
• Iodine smear
• Sargeaunts stain.

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• In vitro encystations was done by keeping fresh
  stool containing trophozoites of Blastocystis in
  2.5 potassium dichromate at 4ºC for 2 weeks.
• The in vitro induced cysts were subjected to
• Morphological study
• Histochemical study
• Test of infectivity

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• I- Morphological study
• Light microscope
• Direct and iodine smears
• Sargeaunts stain
• Iron haematoxyline stain
• Transmission electron microscope
• II- Histochemical study
• Alcian blue used for study of carbohydrates.
• Sudan III used for study of lipids.

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III- Test of infectivity
10 mice were inoculated with 1 ? 104 cyst/mouse
1 week P.I
Stool analysis
2 weeks P.I
Mice were sacrificed and small and large
intestine were studied histopathologically
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Results

• Morphological study
• Light microscopic examination of the cysts found
  in stool and the in vitro induced cysts showed
  that both had the same morphology.

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Conclusion

• Infective Blastocystis cysts could be induced in
  vitro.
• The morphological similarity between these
  induced cysts and those recovered in stool
  samples facilitates further studies on this
  protozoa as regards the life cycle and the
  development of control strategies to prevent its
  transmission.

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Effect of Topical Agents on Cercariae of
Schistosoma mansoni
By Azza Y. Negm, Iman R. Ibrahim, Mona M. El
Temsahy and Mervat Z. El Azzouni
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Introduction

• In Egypt schistosomiasis is still considered a
  major health problem despite the massive efforts
  to eradicate the disease.

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• Skin is the commonly known route of entry for
  this parasite into humans as the cercariae can
  penetrate intact skin within minutes after water
  contact.
• Therefore, several topically applied substances
  have been screened for their ability to confer
  protection against cercarial penetration.

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Aim of the Work

• The present work aimed to study the effect of
  local application of DEET (N-N-Diethyl
  Toluamide), controlled release DEET and white
  ppt. ointment (Ammoniated Mercury) on cercariae
  of S.mansoni both in vitro and in vivo.

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Material and Methods

• Parasite
• S.mansoni cercariae shed from infected
  Biomphalaria alexandrina snails.
• Drugs used
• DEET (N-N-Diethyl Toluamide).
• Controlled release DEET.
• White precipitate ointment (Ammoniated Mercury).

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I- In vitro study

• Light microscopy
• Motility
• Viability
• Scanning electron microscopy

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2- In vivo study

• Experimental animals
• Group I 10 mice sprayed with DEET (OFF) prior to
  infection.
• Group II 10 mice painted with controlled release
  DEET prior to infection.
• Group III 10 mice painted with white ppt.
  ointment prior to infection.

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• Infected control group
• Infected with S.mansoni only.
• Assessment of in vivo study
• Perfusion
• Histopathological study.

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Results
I- In vitro study

• Light microscopy
• Cercariae exposed to free DEET were motile for 20
  min. when stained with toluidine blue most of
  them took up the dye and appeared blue.

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• Cercariae exposed to controlled release DEET
  formulae. Mobility was observed for five min
  only. All cercariae appeared blue when stained.
• Control Cercariae were motile and remained
  unstained with toluidine blue stain.

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II- In vivo study
Mean Schistosomal Worm Load Recovered From
Different Experimental Groups As Compared To The
Control Group
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Mean Number Of S. mansoni Granulomas/LPF at 7
Weeks Post Schistosomal Infection In Different
Experimental Groups As Compared To The Control
Group
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Conclusion

• Controlled release formula of DEET is recommended
  for use as a safe, long acting cercaricidal
  agent.
• It can also be beneficial in reducing dermatitis
  associated with swimmers itch.

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Influence of Temperature and Salinity on the
Viability and Infectivity of Giardia lamblia and
Cryptosporidia parvum
By Salwa T. El Mansoury, Iman F. Abou El Naga,
Azza Y. Negm and Eglal E. Amer
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Introduction

• Giardia lamblia and Cryptosporidium parvum are
  worldwide intestinal protozoa. Several human
  outbreaks containing such protozoa have resulted
  from drinking polluted water.

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• The factors affecting the survival and
  infectivity of these protozoa in water are of
  considerable importance for assessing the risk
  potential of contracting the infection. Such
  factors include use of disinfectants,
  temperature, salinity, storage time pH etc.

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Aim of the Work

• To assess the effect of different degrees of
  temperature and salinity on the viability and
  infectivity of Giardia lamblia and
  Cryptosporidium parvum at different storage time.

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Material and Methods

• Giardia lamblia cysts and Cryptosporidium parvum
  oocysts were collected from diarrheic stool
  samples from the different departments of
  Alexandria University Hospital.
• 1- Stool Samples
• Identification
• Giardia lamblia was identified by direct saline,
  iodine smears and trichrome stain.
• Cryptosporidium parvum was identified by
  modified Z-N stain.

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• Preservation and Purification of Positive samples
  
• Giardia lamblia and Cryptosporidium parvum were
  filtered through two layers of gauze and stored
  in an equal amount of 2.5 potassium dichromate.
• Giardia lamblia was purified by repeated washing
  and sedimentation by 0.85 NaCl.
• Cryptosporidium parvum was separated by
  Sheathers's sugar floatation .

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• 2- Testing Viability
• I.Using fluorogenic dyes
• Fluorescence diacetate (FDA) and Propidium iodide
  (PI)
• Viable organisms fluoresced intensely green with
  FDA and dead organisms fluoresced bright orange
  with PI
• II. Oral Infection of Experimental Animals and
  their Stool Examination
• Rats were infected with Giardia lamblia.
• Mice were infected with Cryptosporidium parvum.

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• 3-For Testing the Effect of Temperature and
  Salinity
• Temperature used was 100 C, 4C and -4C for
  different durations of time.
• Salinity was examined at 10ppt, 30ppt and 50ppt.
• 4- Assessment of Viability of Protozoa after
  Exposure to the Different Factors
• Vital dyes
• Animal infectivity was assessed by stool
  examination and histopathological study of the
  ileum

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Conclusion

• The effect of temperature on the viability of
  Giardia lamblia cysts and Cryptosporidium parvum
  oocysts showed that temperature was only
  effective at 100C, while at 4C and -4C, the
  viability was preserved up to seven days.

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• Water used for drinking or preparing food, should
  be boiled for at least 1 min, or preserved at
  room temperature for at least 7 days to minimize
  the risk of contracting infection.
• Salinity at low concentration has an impact on
  protozoal viability only if they were exposed for
  long duration of time, whereas, high
  concentration salinity is the most influential
  factor. This demonstrates that sea food should
  not be eaten unless perfectly cooked also
  swimming in contaminated areas with such protozoa
  should be avoided.

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