NEUROPARASITIC INFECTIONS basis, diagnosis and limitations

1. NEUROPARASITIC
INFECTIONS
basis, diagnosis and limitations
Dr.T.V.Rao MD

3. How Parasites Enter Blood Brain Barrier
• Some intracellular and
extracellular parasites can
traverse the BBB during the
course of infection and cause
neurological disturbances and/or
damage which are at times fatal.
The means by which parasites
cross the BBB and how the
immune system controls the
parasites within the brain are
still unclear.

4. Methods to Diagnose Infections
• Methods for the diagnosis of
infectious diseases have
stagnated in the last 20–30
years. Few major advances
in clinical diagnostic testing
have been made since the
introduction of PCR,
although new technologies
are being investigated.

5. Are we Practising Older Methods
• Many tests that form the
backbone of the
“modern” microbiology
laboratory are based on
very old and labour-
intensive technologies
such as microscopy for
malaria or many parsites

6. Parasitic Infections Migrating from Developing
Nations to Developed Nations
• Parasitic infections of the
CNS, previously restricted
mainly to people living in
developing countries, are
becoming increasingly
more prevalent
throughout the world.
With the advent of
increasing global travel,

7. Immune suppression changes the
Adoptability of Infections
•Potent
immunosuppression,
and HIV infection,
parasitic infections
will likely become
even more
commonplace.

8. Overall familiarity is Important to Evaluate the
Matters
• Basic familiarity with
common pathogens can
make diagnosis more
expeditious and efficient.
For the clinician
confronted with a patient
with suspected parasitic
infection, additional
assistance with
diagnostic evaluation

9. Beginning of Career to Learn and Practice of
parasitology at Mansa General hospital Zambia

10. Is it easy to Diagnose Parasitic Infections of
Nervous system ?
• Parasitic infection of the
nervous system can
produce a variety of
symptoms and signs.
Because symptoms of
infection are often mild
or nonspecific, diagnosis
can be difficult.

11. Be familiar with Epidemiology and
Radiology
• Familiarity with basic
epidemiological
characteristics and
distinguishing
radiographic findings can
increase the likelihood
of detection and proper
treatment of parasitic
infection of the nervous
system.

12. We are still using the Microscope as a
Traditional tool in Diagnosis
•The primary tests currently used to
diagnose many parasitic diseases have
changed little since the development of the
microscope in the 15th century by Antonie
van Leeuwenhoek. Furthermore, most of
the current tests cannot distinguish
between past, , latent, acute, and
reactivated infections.

13. Diagnostic Methods in Parasitology are
Complex
If we wish sensitivity and specificity
• The methods currently in use
range from rather simple, easily
managed and routine
techniques to the extremely
complex cutting edge
technologies of modern
molecular biology and high-
throughput miniaturised
methods usually done as part of
thesis and research work and
rarely for diagnostic work

14. Newer Serological Assays
• Firstly, a number of newer
serology-based assays that are
highly specific and sensitive
have emerged, such as the
Falcon assay screening test
ELISA (FAST-ELISA) , Dot-ELISA
rapid antigen detection
system (RDTS) , and luciferase
immunoprecipitation system
(LIPS).

15. Emerging Molecular methods
• Secondly, molecular-based
approaches such as loop-
mediated isothermal
amplification (LAMP) , real-
time polymerase chain
reaction and Luminex have
shown a high potential for
use in parasite diagnosis
with increased specificity
and sensitivity.

16. Parasites Infections of the Central Nervous System
•Toxoplasma gondii associated with congenital
defects and AIDS
•African trypanosomes African sleeping sickness
•Plasmodium falciparum cerebral malaria
•Endamoeba histolytic rare invasion of the brain
•Free-living amebae rare cases

17. MALARIA

18. Malaria Continues to be a Emergency in
many countries
• Malaria should be
considered a potential
medical emergency and
should be treated
accordingly. Delay in
diagnosis and treatment is a
leading cause of death in
malaria patients in Many
Countries

19. Peripheral Blood Smear a Great tool
• Clinicians seeing a malaria
patient may forget to consider
malaria among the potential
diagnoses and not order the
needed diagnostic tests.
Laboratories may lack
experience with malaria and fail
to detect parasites when
examining blood smears under
the microscope

20. Making a Smear is Most Important Part of
Microscopy

21. Microscopic Diagnosis
• Malaria parasites can be
identified by examining under
the microscope a drop of the
patient's blood, spread out as a
"blood smear" on a microscope
slide. Prior to examination, the
specimen is stained
(most often with the Giemsa
stain) to give the parasites a
distinctive appearance. This
technique remains the gold
standard for laboratory
confirmation of malaria.

22. Appearance of P.vivax and P. falciparum

23. Malaria Diagnosis is a Emergency

24. Clinical examination a collaborating point
•Cerebral malaria, with
abnormal behaviour,
impairment of
consciousness,
seizures, coma, or
other neurologic
abnormalities

25. Antigen Detection
• Various test kits are available to
detect antigens derived from
malaria parasites. Such
immunologic
("immunochromatographic") tests
most often use a dipstick or
cassette format, and provide
results in 2-15 minutes. These
"Rapid Diagnostic Tests" (RDTs)
offer a useful alternative to
microscopy in situations where
reliable microscopic diagnosis is
not available.

26. HRP2 levels predict likelihood of cerebral malaria in African
children
• . While only about 1% of Plasmodium falciparum infections
progress to cerebral malaria, mortality occurs in 10–20% of
affected patients. plasma concentrations of the Plasmodium
protein HRP2 (histidine rich protein 2) can predict the odds of
developing cerebral malaria in Malawian children. Their data
show that mean plasma HRP2 concentrations were significantly
higher in the children who developed cerebral malaria than the
ones with uncomplicated malaria.

27. QBC SYSTEM CANNOT DIFFERENTIATE
Species

28. No Single Test is Perfect
• Current evidence indicates that no single method for
the diagnosis of malaria is perfect nor can any one of
them be a stand-alone accurate and effective
diagnostic criterion . Accurate and effective malaria
diagnosis should thus involve a rational approach to
each patient with suspected malaria employing both
symptoms/signs-based and laboratory-based malaria
diagnostic methods.

29. Emerging methods at even Smaller Clinks
• The prioritizing of any of the
malaria diagnostic methods, at
all times, should be influenced
by various factors including
malaria endemicity, transmission
pattern, the urgency of the
diagnosis, the experience of the
health worker, effectiveness of
the health care system, and
available budget resources.

30. Rapid Diagnostic Tests
• RDTs do not require laboratory equipment and are all
based on the same principle and detect malaria
antigen in blood flowing along a membrane
containing specific anti- malaria antibodies . Most of
the available RDTs are P. falciparum protein specific
(either histidine rich protein II -HRP-II or lactase
dehydrogenase-LDH) while some RDTs detect P.
falciparum and other Plasmodium proteins such as
aldolase or pan-malaria pLDH.

31. Rapid diagnostic tests proving useful
• Several studies have
reported the
performance of RDTs to
be excellent. Inarguably,
RDTs are enhancing the
benefits of parasite-
based diagnosis of
malaria though not
without problems or
limitations

32. Molecular Malaria laboratory diagnostic tests
• Molecular malaria techniques
such as PCR on blood or, more
recently, even on saliva samples
devised in Zambia by
(Mharakurwa et al), the loop-
mediated isothermal
amplification (LAMP),
microarray, mass spectrometry
(MS), and flow cytometry (FCM)
assay techniques are all new
developments mainly utilized in
research settings than during
routine patient care.

33. Serology
• Serology detects antibodies
against malaria parasites,
using either indirect
immunofluorescence (IFA)
or enzyme-linked
immunosorbent assay
(ELISA). Serology does not
detect current infection but
rather measures past
exposure.

34. Rapid Tests
•RDTs currently available
in the market are quite
a few and include
brands such as O p t i M
A L , P a r a c h e c k , I
C T, p a r a - s i g h t - F,
parascreen, and SD
Bioline.

35. Artefacts

36. Free Living Amoebic
Infections
PARASITIC MENINGITIS

37. Free Living Amoeba
• Naegleria fowleri and
Acanthamoeba spp., are
commonly found in lakes,
swimming pools, tap water, and
heating and air conditioning units.
While only one species of
Naegleria, N. fowleri, is known to
infect humans An additional agent
of human disease, Balamuthia
mandrillaris, is a related free-living
amoeba that is morphologically
similar to Acanthamoeba in tissue
sections in light microscopy.

38. Diagnostic Findings
• In Naegleria infections, the
diagnosis can be made by
microscopic examination of
cerebrospinal fluid (CSF). A wet
mount may detect motile
trophozoites, and a Giemsa-stained
smear will show trophozoites with
typical morphology. Confocal
microscopy or cultivation of the
causal organism, and its
identification by direct
immunofluorescent antibody, may
also prove useful.

39. Naegleria fowleri/Primary Amoebic
Meningo encephalitis
• Early symptoms include severe,
throbbing headache, fever, nausea,
and vomiting.Most patients have a
history of swimming or bathing in
stagnant water.
• Meningismus is common, and
some patients present with
seizures or coma. Differentiation
between PAM and bacterial
meningitis can be difficult but is
crucial given the rapid progression
of N. fowlerii infection.

40. Wet mounts are beneficial
• Organisms are not visualized
with Gram’s stain because
amoebas are killed during
the fixation process.
• CSF wet mount should be
performed to look for
trophozoites. Giemsa
staining of CSF may also be
useful. In the past,

41. Acanthamoeba
• In Acanthamoeba infections,
the diagnosis can be made
from microscopic
examination of stained
smears of biopsy specimens
(brain tissue, skin, cornea)
or of corneal scrapings,
which may detect
trophozoites and cysts

42. Acanthamoeba histolytica and Balamuthia
mandrillaris/Granulomatous Amoebic
• CNS infection by A.
histolytica is uncommon
in immunocompetent
hosts. In contrast to A.
histolytica, B.
mandrillaris causes
infection
inimmunocompetent and
immunosuppressed hosts
with equal frequency

43. Corneal scrapings
•Definitive diagnosis
can be obtained by
demonstration of
trophozoites or cysts
of A. histolytica on
stained smears of
biopsy specimens or
corneal scrapings

44. Immunofluorescence studies
• Direct IFA tests can be
useful. Differentiation
between B. mandrillaris and
A. histolytica infection
requires
immunofluorescence
studies. Examination of
contact lenses from patients
with keratitis can reveal A.
histolytica

45. 1Trophozoite of N. fowleri in CSF, stained with haematoxylin and eosin
2Trophozoite of N. fowleri in CSF, stained with haematoxylin and eosin

46. 1Cyst of Acanthamoeba sp. from brain tissue, stained with haematoxylin and
eosin
2Trophozoites of Acanthamoeba sp. in a corneal scraping, stained with H&E.

47. Real-Time PCR
• A real-time PCR was
developed at CDC for
identification of
Acanthamoeba spp.,
Naegleria fowleri, and
Balamuthia mandrillaris in
clinical samples.1 This assay
uses distinct primers and
TaqMan probes for the
simultaneous identification
of these three parasites

48. Toxoplasmosis

49. Cerebral toxoplasmosis : Centre for Disease
Control (CDC) criteria for diagnosis
•Recent onset of focal neurological
abnormality consistent with intracranial
disease or reduced consciousness
•Evidence from brain imaging of a lesion (CT
or MRI)
•Positive serum antibody to T. gondii or
response to treatment

50. Diagnosis of toxoplasmosis
• Diagnosis of toxoplasmosis is rarely made through the
detection or recovery of organisms, but relies heavily
on serological procedures. Parasites can be detected
in biopsied specimens, buffy coat cells, or cerebral
spinal fluid. These materials can also be used to
inoculated mice or tissue culture cells. However,
detecting tachyzoites from these materials is difficult..
Therefore, serologic tests are recommended for
diagnosis

51. IgM and Toxoplasmosis
• Acute infections are
characterized by high IgM
titres and/or a significant
increase in total antibody
titre in a sample taken two
weeks later. The serology
may also correlate with the
acute stage symptoms in
some individuals.

52. Diagnostic tests for Toxoplasma
•Sabin-Feldman dye test (DT)
•Enzyme immunoassay for T. gondii specific IgM
(EIA)
•Immunsorbent agglutination assay (ISAGA)
•Enzyme immunoassay for IgG avidity
•Isolation and culture of parasite
•Direct detection by microscopy and PCR

53. Persons with ocular disease
•Eye disease (most
frequently retinochoroiditis)
from Toxoplasma infection
can result from congenital
infection or infection after
birth by any of the modes of
transmission discussed on
the epidemiology and risk
factors page.

54. Persons with compromised immune
systems
•Persons with
compromised immune
systems may
experience severe
symptoms if they are
infected with
Toxoplasma while
immune suppressed.

55. Serology
•In the second situation, a second specimen
should be drawn and both specimens
submitted together to a reference lab which
employs a different IgM testing system for
confirmation. Prior to initiation of patient
management for acute toxoplasmosis, all
IgG/IgM positives should be submitted to a
reference lab for IgG avidity testing.

56. Diagnosis
• The diagnosis of toxoplasmosis is
typically made by serologic testing.
A test that measures
immunoglobulin G (IgG) is used to
determine if a person has been
infected. If it is necessary to try to
estimate the time of infection,
which is of particular importance
for pregnant women, a test which
measures immunoglobulin M (IgM)
is also used along with other tests
such as an avidity test.

57. Diagnosis by staining methods
• Diagnosis can be made by
direct observation of the
parasite in stained tissue
sections, cerebrospinal fluid
(CSF), or other biopsy
material. These techniques
are used less frequently
because of the difficulty of
obtaining these specimens.

58. :
Cysticercosis

59. Cysticercosis
• This infection is caused by pork
tapeworm larvae (see Tapeworm
Infection). It is the most common
parasitic infection in the Western
Hemisphere. After people eat food
contaminated with cysticercus
eggs, secretions in the stomach
cause the eggs to hatch into larvae.
The larvae enter the bloodstream
and are distributed to all parts of
the body, including the brain

60. MRI AND CT SCNNING CONTINUES TO BE MAIN IN
STAY IN DIAGNOSIS
•Magnetic resonance
imaging (MRI) or
computed tomography
(CT) can often show the
cysts. But blood tests and
a spinal tap (lumbar
puncture) to obtain a
sample of cerebrospinal
fluid are often needed to
confirm the diagnosis.

61. PRIMARY EXAMINATIONS
• Infection with adult T. solium
worms can usually be
diagnosed by microscopic
examination of stool samples
and identification of eggs
and/or proglottids. However, T.
solium eggs are present in ≤
50% of stool samples from
patients with cysticercosis.

62. CDC standarsises the Immunoblot Testing
• The CDC's immunoblot
assay (using a serum
specimen) is highly specific
and more sensitive than
other enzyme
immunoassays (particularly
when > 2 CNS lesions are
present; sensitivity is lower
when only a single cyst is
present).

63. Immunoblot assay
• CDC's immunoblot assay with
purified Taenia solium antigens
has been acknowledged by the
World Health Organization and
the Pan American Health
Organization as the
immunodiagnostic test of choice
for confirming a clinical and
radiologic presumptive diagnosis
of neurocysticercosis.

64. We mainly Dependent on
• There are two available
serologic tests to detect
cysticercosis, the
enzyme-linked
immunoelectrotransfer
blot or EITB, and
commercial enzyme-
linked immunoassays.

65. Antigen Detection
• Tests that detect circulating
cysticercal antigens in serum and
CSF have been developed and may
prove to be most useful to follow
response to therapy in in
subarachnoid and ventricular forms
of neurocysticercosis. Antigen
levels drop quickly in cured NCC
patients, so serum antigen
monitoring is useful for assessing
treatment and determining of
clinical cases.

66. Antigen Detection Methods lack sensitivity
•Antigen detection
testing is not as
sensitive as antibody
detection and should
not be used to
diagnose
neurocysticercosis

67. Molecular Detection
•PCR tests have been
developed to detect T.
solium DNA in CSF but
these are not widely
used for clinical
laboratory diagnosis of
neurocysticercosis.

68. Schistosomiasis (Bilharzia)
• EPIDEMIOLOGY—Schistosomiasis
occurs in up to 300 million people
worldwide each year and is caused
by five species of blood flukes
(digenetic trematodes):
Schistosoma mansoni,
S.haematobium, S. japonicum, S.
intercalculatum, and S. mekongi.62
CNS involvement has beenreported
with three of the five species: S.
mansoni, S. haematobium, S.
japonicum

69. Neurological involvement
• Neurological involvement usually
appears weeks ormonths after
initial infection when eggs migrate
through the vascular system to the
brain orspinal cord; symptoms may
result from mass effect of the egg
itself or from granulomaformation
around the egg. Because the
parasite likely enters the CNS via
Batson’s plexus,the spinal cord and
posterior fossa are the most
common sites of involvement

70. DIAGNOSIS
• Definitive diagnosis of CNS
schistosomiasis is obtained by
identification ofan egg in biopsy
tissue. Detection of schistosomal
eggs in stool or urine confirms
the diagnosisof
schistosomiasis.14 Stool
examination is more sensitive for
S. mansoni and S. japonicum,and
examination of urine is best for
S. hematobium.

71. Echinococcus
(Hydatid Disease)

72. Echinococcus (Hydatid Disease) CESTODES
• Hydatid infection often remains
undetected until cyst
enlargement produces
symptoms. The cyst can cause
more severe symptoms if it
ruptures or becomes super-
infected. Central nervous system
(CNS) involvement complicates 2
and 5% of infections with E.
granulosis and E. multilocularis,
respectively.1,3

73. DIAGNOSIS
• Diagnosis of E. granulosus infection
can be confirmed by serum indirect
hemagglutination (IHA), indirect
fluorescent antibody (IFA), or
enzyme-linked immunosorbent
assay (ELISA), with assay sensitivity
rates ranging from 50 to 60% in
patients with pulmonary cysts to
98% in patients with hepatic cysts.9
Serum assays to detect E.
multilocularis are more sensitive
than assays for E. granulosis and are
not cross-reactive

74. NEUROIMAGING
• ontrast-enhanced computerized
tomography (CT) of the brain is
usually sufficient for evaluation,
but magnetic resonance imaging
(MRI) is warranted if surgical
intervention is planned. CT
demonstrates cysts of various
sizes, sometimes in grapelike
clusters.13 Chronic disease may
develop a granulomatous
appearance

75. Serum indirect hemagglutination
(IHA),
• Diagnosis of E.granulosus infection
can be confirmed by serum indirect
hemagglutination (IHA),
indirectfluorescent antibody (IFA),
or enzyme-linked immunosorbent
assay (ELISA), with assaysensitivity
rates ranging from 50 to 60% in
patients with pulmonary cysts to
98% in patientswith hepatic cysts.9
Serum assays to detect E.
multilocularis are more sensitive
than assaysfor E. granulosis and are
not cross-reactive.

76. CDC Helps in Diagnosis of
Parasitic Infections

77. I wish many use this Facility
• The modern generation
of Microbiologists can
use digital imaging
technology of parasitic
infections with Web site
developed and
maintained by CDC's
Division of Parasitic
Diseases and Malaria
(DPDM)

78. CDC Helps with Tele diagnosis of Paasitic
Infection
• DPDx is a unique online
educational resource that
includes visual depictions of
parasite lifecycles, a
reference library of free
images of parasites, and
guidance on proper
laboratory techniques for
diagnostic parasitology. But
it is much more than a Web
site.

79. 1Encysted larvae of Trichinella sp. in muscle tissue,
2 Babesia and Falciparum

80. Diagnostic Assistance function
A Diagnostic Assistance
function, in which
laboratory and other health
professionals can ask
questions and/or send
digital images of specimens
for expedited review and
consultation with DPDx
staff. This assistance is free
of charge.

81. Why we Stand Today In Diagnosis
•In spite of many Advances in Medical profession
the Parasitology suffers much lacunae in
diagnosis for optimal treatment, great reason
being lack of human dedication in the matters
concerned, and lack of evaluation of skills in
matters of diagnostic talents in postgraduate
examinations, and above all non-availability of
advancing technologies

82. The Great Question hunts many of us,
If we are true to our Job
Are we really
diagnosing the
Parasitic Infections?

83. Say
OR NO
•

84. What Can be Done?
• Speciality of Parasitology
hangs between the
domains of Microbiology
and Pathologists. However
it needs more inputs and
coordination of Vetenary
sciences and Zoology
Professionals.