starting from basic to advances in hemiparasite infection

1. RECENT ADVANCES IN
DIAGNOSIS OF HEMOPARASITE
INFECTIONS
BY: DR. PRERNA CHOUDHARY

2. HEMOPARASITES
• PLASMODIUM
• BABESIA
• LEISHMANIA (VISCERAL)
• TRYPANOSOMA
• MICROFILARIA (Brugia, Mansonella, and Wuchereria)

3. INTRODUCTION
• Microscopy remains the cornerstone of diagnostic laboratory testing for
blood parasites
• It requires minimal amount of reagents and equipment, but this method
depends on accuracy of well-trained and experienced technologists
• Other techniques for detection is Rapid methods (antigen
detection)which is on point care and antibody detection which has its
own advantages and disadvantages
• Molecular method (PCR) is the newest technique which is mainly used in
reference labs and research purposes.

4. PLASMODIUM
CASE I
• A 11 year boy came to emergency dept. with history of fever, temp 40 °C,
abdominal tenderness, no other abnormalities.
Hb – 8.3mmol/L
platelets- 56 × 109/L
leukocyte count of 4.5 × 109cells/L, RF of lymphocyte seen
CRP – 127mg/L
lactate dehydrogenase level 278U/L

5. 1. SMEAR PREPARATION: (CONVENTIONAL)
• Thick smear- parasite screening,
thin smear- species identification
• Stained by- giemsa/Leishman at pH7.2
• 5 minutes spent in thick film is equivalent to 1
hour spent in thin film.
• In thick smear-
• In thin smear- no. of parasites in 1ul of blood =
red cell count in million/cumm x parasite
percentage

6. • Advantage- Sensitivity of thick blood film is about 50parasite/ul
less equipments required
mostly used in tertiary centres
• Drawback- 60min of preparation time,
labour intensive
interpretation of the result requires expertise
low sensitivity in low parasite level
• .
In- Plasmodium falciparum malaria- parasites are often missed, so requires repetitive testing as the parasites are
sequestered in tissue capillaries for part of the asexual cycle and, parasite may not always be present in the
peripheral blood. Thus a P. falciparum infection might be missed unless blood samples are repeated daily

10. 2. Fluorescent microscopy
• A) The quantitative buffy coat or QBC11
• Blood collected in capillary with anticoagulant filled upto 2/3rd and ends sealed
centrifuge at 1200 rpm for 5 minutes
• Dye: acridine orange dye
• Tube is viewed using special objective (paralens) attached to florescence
microscope.
• Staining parasite DNA-
• ADVANTAGE: Rapid alternative to blood smear
Sensitivity of 90% in acute infection
can detect 100 parasite/ul
Nuclei- bright green,
Cytoplasm- yellow orange

11. • DISADVANTAGE: Expensive apparatus
Expertise
False positive: Howell- Jolly bodies and reticulocytes
50% sensitivity in low parasitamia
species identification difficult
B) Kawamoto technique
DYE: acridine orange process + anticoagulant
An interference filter is put in pathway of transmitted light beam
Similar to QBC
• benzothiocarboxypurine (BCP)-the Fluorochrome to stain the nucleic acids of
any malaria parasites in the sample.
This method overcomes some of the problems inherent in some fluorescence systems, such as the need for rapid examination
to prevent fading or precipitation of the dye

13. • Optical imaging techniques Cho, S., Kim, S., Kim, Y., & Park, Y. (2012). Optical imaging techniques for the study of malaria)
A) In vitro optical imaging of infected
red blood cells (iRBC)
using exogenous labeling agents
B) In vitro optical imaging of
iRBC with intrinsic optical
signals
C) In vivo optical
imaging of malaria
infection

14. 3. Rapid Diagnostic tests (antigen test)
• PRINCIPLE:
Immunochromatography relies on the migration of liquid
across the surface of a nitrocellulose membrane.
Tests are based on the capture of parasite antigen from
peripheral blood using monoclonal antibodies prepared
against a malaria antigen target and conjugated to either a
liposome containing selenium dye or gold particles in a
mobile phase.
A second or third capture monoclonal antibody applied to
a strip of nitrocellulose acts as the immobile phase.
The migration of the antigen-antibody complex in the
mobile phase along the strip enables the labeled antigen
to be captured by the monoclonal antibody of the
immobile phase, thus producing a visible colored line.
labeled goat antimouse antibody controls the migration

15. • Antigens against which kits are available are 2 groups:
Expressed in all malarial species
They confirm infection
They are:
• PMA (plasma adolase)
• pLDH (Parasite Lactate Dehydrogenase)
Specific for species
• HISTIDINE rich protein – 2(HRP-2)- synthesized
by asexual blood stages and young gametocytes
of P.falciparum and expressed in red cell surface.
specificity >90%
• pfLDH

16. • ‘TWO LINE TEST’- anti-pfHRP2 band with positive control for falciparum
• ‘THREE LINE TEST’- P.falciparum specific antibody band +PMA/pLDH + positive control( species
specific only for p.falciparum)
• Research is happening on ADOLASE - enzymes within the glycolytic pathway of the malaria parasite
have been recognized and considered as targets for rapid malaria diagnostic tests.(common to all)

17. ADVANTAGE:
• Sensitivity 95% when 100 parasites per ml
• RAPID, SENSITIVE, NO SPECIAL EQUIPMENT, NO TRAINED PROFFESSIONAL
• HRP-2 can be used even in pregnancy
DISADVANTAGE:
• false positive : cross-reaction with autoantibodies (RA)
• Antigens remains positive even after 28 days of treatment
• Cannot differentiate parasite
• Few parasite have deleted HRP GENES – false negative
Few commercial available device are:
OptiMAL, ICT, Para-HIT-f, ParaScreen, Biolone, Paracheck

18. 4. Serological tests(antibody test)
IFAT, ELISA
Based on detection of antibodies against asexual blood stage malarial
parasite.
It is time consuming, subjective but highly sensitive and specific
DONE ON:
• screening blood donors involved in cases of transfusion-induced malaria
when the donor's parasitemia may be below the detectable level of blood
film examination
• testing a patient with a febrile illness who is suspected of having malaria but
repeated blood smears are negative
• testing a patient who has been recently treated for malaria but diagnosis is
questioned

19. ELISA
PRINCIPLE:
• The DiaMed ELISA malaria antibody test is based on binding of anti-
Plasmodium antibodies present in a serum sample to antigens
immobilized on 96-well plates.
• The antigens are a mixture of a total extract of cultured P. falciparum
and recombinant P. vivax antigens (MSP1 and CSP).
• SENSIVITY 94% SPECIFICITY 98%
• The test does not distinguish between IgG and IgM, or between
antibodies to P. falciparum, P. vivax,P. ovale and
P. malariae

21. IFA
• PRINCIPLE: following infection with any Plasmodium species, specific
antibodies are produced within 2 wk of initial infection, and persist for
3- 6 months after parasite clearance.
• IFA uses specific antigen or crude antigen prepared on a slide, coated
and kept at -30°C until used, and quantifies both IgG and IgM
antibodies in patient serum samples.
Titers > 1 : 20 are usually deemed positive
< 1 : 20 unconfirmed
Titers > 1 : 200 can be classified as recent infection

22. • Species-specific testing is available
• ANTIGEN USED: meronts (on shizonts)
CIRCUM-SPOROZOITE (protein of surface of sprozoite)
• The patient's serum is exposed to the organisms; homologous antibody,
if present, attaches to the antigen, forming an antigen-antibody (Ag-Ab)
complex.
• Fluorescein-labeled antihuman antibody is then added, which attaches
to the patient's malaria-specific antibodies.(MSP ANTIBODY) When
examined with a fluorescence microscope, a positive reaction is when
the parasites fluoresce an apple green color.

24. ADVANTAGE
• Considered gold standard for antibody testing
• Simple and highly sensitive
DISADVANTAGES
• Time consuming
• Cannot be automated which limits the number sera for study
• Influenced by technician training
• Florescent microscope required

25. • 5. Molecular diagnostic methods:
PCR Technique
• Enzymatic synthesis to amplify nucleotides in
vitro
• Primer targets- 18sSSU RNA , OR TARGETS
circumsporozoite surface protein/cytochrome
B gene
• Used as reference test for evaluating the
newer diagnostic tests

27. Advantage- genotypic variation
PCR inhibitors or DNA degradation can give false negative result
Most specific and sensitive in low parasitemia or mixed infection
Detects 0.02-1 parasites/ ul of blood
Species of malaria can be identified
DISADVANTAGE: longer time required
cannot be used to determine exactly how many copies have been
generated.
Other pcr used are-
Modified PCR- reverse transcription PCR, Nested PCR real-time PCR

28. Real-time PCR (qPCR)
• that can be used to quantify the number of copies of a target sequence
in a patient’s sample
• used to detect the mRNA product of DNA transcription
• By detecting fluorescence (SYBR Green®) in the exponential phase
• By using a known series of diluted standards
• It attaches to double-stranded DNA in the minor groove and fluoresces

30. LAMP TECHNIQUE
• Simple and inexpensive-
detects conserved 18S
ribosome RNA gene of
P.falciparum
• Cells visualized with
FITC- CONJUGATED anti-
DIG antibody using
florescence microscope
• Reagents require cold
storage

32. • Microarrays:

35. • FCM assay
• RBC is lysed before fixation
• Blood cells+lysing agent +fixative Rupture of RBCs releasing of
parasites, WBC are intact
• Staining used for parasite is Hoechst33258 + propidium deoxide
other dyes: SYBR green 1 & CD 235A
• 1 million events acquired

39. • Analysis of blood-stage development and determination of susceptibility to drugs by
flow cytometry are reproducible and rapid and detection of blood-stage parasites
appears to be sensitive and reproducible
• DNA-specific dyes are especially useful because the parasites multiply inside the
RBCs. As RBC does not contain DNA, DNA-specific fluorescence from infected RBC can
only be due to fluorescence of dyes bound to parasite DNA
• The total DNA content of a parasite is 100-200 times less than that of nucleated
blood cells. Therefore, the nucleated blood cells can easily be distinguished from
parasites on the basis of the difference in fluorescence intensity
• Consequently, infected cells can be discriminated from noninfected cells based on
their fluorescence intensity.
• The advantages of flow cytometry are the speed of measurement, the accuracy,
reproducibility, and the large number of parasites analyzed

40. AUTOMATED BLOOD CELL COUNTERS (ACC)
• Principle: detection of hemozoin, which is produced when the intra-erythrocytic malaria
parasite digest host haemoglobin and crystallize the released toxic heme into hemozoin in
the acidic food vacuole.
• Hemozoin within phagocytotes can be detected by depolarization of laser light, as cells pass
through a flowcytometer channel.
• Cell-Dyn 3500- detects malaria pigment (hemozoin) in monocytes, sensitivity 95%
specificity 88%
• BECKMAN coulter- VCS-to detect activated monocytes 98%sensitivity, 94%specificity
Advantage -useful for diagnosing clinically
unsuspected malaria.
Disadvantages- labour intensiveness, the need for trained
technicians, costly diagnostic equipment, and that false-
positives may occur with other bacterial or viral infections.
-

41. MASS SPECTROPHOTOMETRY
• A novel method for in vitro detection of malaria parasites, with a sensitivity of 10
parasites/μl of blood, has been reported recently.
• It comprises a protocol for cleanup of whole blood samples, followed by direct
ultraviolet laser desorption mass spectrometry (LDMS).
• In malaria, heme from hemozoin is the parasite-specific biomarker of interest.
• LDMS is rapid, high throughput, and automated.
• Compared with the microscopic method, which requires a skilled microscopist
LDMS can analyze a sample in < 1 min

42. • FISH-Fluorescence in situ hybridization is a cytogenetic technique used to
detect and localize the specific nucleic acid (DNA or RNA) sequences by
hybridizing with complementary sequences labeled with fluorescent probes
Blood+ anticoagulant + Alexa 488 green fluorescent dye to label plasmodium
specific probe+ counter stain
16S ribosomal RNA (rRNA) was used as a target for developing FISH assays
for diagnosing malaria for the following reasons:
(1) rRNA is highly abundant in the cell cytoplasm and therefore can be
visualized using sequence-specific fluorescence probes under a
microscope, without target sequence amplification
(2) As a consequence of variable nucleotide sequence conservation, it is
possible to find short nucleotide stretches that are unique to the genus,
species, sub-species or strain

43. FISH detects only live parasites with intact rRNA
The assays consist of six steps: smear preparation,
fixation, hybridization, washing, counterstaining and
viewing the processed smear under a fluorescence
microscope.

44. • FISH assays have other advantages over microscopic examination of
Giemsa stained smears viz. (1) FISH detects specific 18S rRNA fragment
in live parasites whereas by microscopy, Giemsa stained live and dead
parasites are detected since parasite detection and speciation is based
on the morphology only. (2) Since rRNA is present in the cytoplasm of
the parasite, the whole cell fluoresces when viewed under the
fluorescence microscope. Ribosomal rRNA is a unique molecule with
highly conserved and variable regions. Thus, it is possible to design
genus, species and strain specific probes.
• Dias- no experience in fluorescent microscopy.

45. • BABESIA
Case 2
A 50yr old previously spelenectized pt. came to OPD with fever and
chills for 2 weaks, sweating, myalgias, fatigue. On blood examination
pt. had Neutropenia, thrombocytopenia, decresed Hb, haematocrit,
Haptoglobulin, increased bilirubin, reticulocyte count, lactate
dehydrogenase, urobilinogen, and urine hemoglobin The laboratory
picture of hemolytic anemia with raised liver enzymes (aspartate and
alanine transaminase, alkaline phosphatase),

46. • incubation – 1-4 wk following tick bite (Ixodid ticks)
6 wk following infected blood transfusion
• CF: Fever, chills, sweat, headache, vomiting, abd pain,diarrhea, joint pain
Babesia species
Small Babesia---B.Microti B.GIBSONI
-
Large Babesia --B.BovisB. Canis B.divergans

47. • A)CONVENTIONAL
Microscopy: Ring forms can be round, oval, pyriform or ameboid shape
existing in singles, pairs tetrads- “MALTESE-CROSS APPEARANCE”
• This ring form is similar to plasmodium falci schizont bit we can differentiate
by absence of hemozoin pigment and absence of sizont and gametocytes in
Babesia
• QBC: Not recommended as poses difficulty
differentiating Plasmodium and Babesia.
• CULTURE:
Time taking, not used for human

48. IFAT:
• Most common serology test- only for B.microti the test has been
standardised
• Detects specific antibodies – CDC proposed IgG antibody kit
Titre > 1:64 – seropositive, even after 8-12 months of infection
> 1:1024 – acute infection
• IgG cant differentiate b/w recent and past infection
• So, IgM does but there is many false positive result.

50. IFA was and then probed with 1:128 dilutions of the plasma samples at 37°C for 1h.
Slides of Babesia sp. obtained from Focus Diagnostics were blocked with Phosphate Buffered Saline
(PBS) containing 5% bovine serum albumin (BSA) and 5% goat serum for 30 min at room temperature
After three washings with PBS at 5 min intervals, slides were incubated with a mixture of 1:100 dilution of 4′, 6-
diamidino-2-phenylindole (DAPI) to stain parasite DNA observed by blue fluorescence and anti-human Alexa
488 conjugated goat antibodies
After 1h incubation at 37°C in dark, slides were washed three times with PBS at 5 min interval
Cover glasses were then mounted using 1:1 mixture of glycerol and PBS and sealed with nail enamel to prevent
evaporation and drying. Slides were examined by fluorescent microscope using oil emersion objective. Green
fluorescence indicated that patient plasma contains antibodies against B. microti

51. 1.A single negative IFA test result does not exclude the possibility
of Babesia
2.Other Babesia antibodies may cross-react and yield a false
positive Babesia IFA test.
3.Results should be interpreted in conjunction with other laboratory and
clinical findings.
• The B. microti IFAT has a reported sensitivity of 88^96% and speci¢city of
90^100%.
• ELISA:
• Babesia merozoite surface proteins (BMN1-17 and MN-10) and have
been employed in ELISA

52. • MOLECULAR:
FISH:
• The FISH assay is based on two fundamental principles:
Hybridization directly on a thin blood smear with a fluorescein-
labeled Babesia specific probe
Identification of Babesia parasites on a blood smear by viewing with a
fluorescent microscope
is designed for qualitative detection of ribosomal RNA of Babesia parasites
directly in a blood smear. The test’s highest degree of specificity is
provided by nucleic acid probes, which bind to RNA sequences of
the Babesia. This test detects all species of Babesia

53. PCR:
• Diagnostic method of choice
• Target is detection of babesia srr is 18S rRNA
• Real time PCR – high sensitivity of detecting 20 genome copies/ul ~ 0.0001%
Agarose gel (2%) analysis of a PCR diagnostic test for detection of Babesia microti DNA.
PCR was performed using a nested protocol with primers BAB1 and BAB4 (first round)
and BAB2 and BAB3 (second round).
* Lane S: Molecular base pair standard (50-bp ladder). Black arrows show
the size of standard bands.
* Lane 1: First step amplification with primers BAB1 and BAB4 of the
nested PCR protocol for detection of B. microti in DNA extracted from
whole blood. The specimen, serologically positive for B. microti, was
submitted to the CDC by the American Red Cross. The red arrow shows
the single-step PCR diagnostic band for B. microti (size: 238 bp).
* Lane2: Nested PCR with primers BAB2 and BAB3 using as template the
product of the first step amplification. The blue arrow shows the nested
PCR diagnostic band for B. microti (size: 154 bp). Please note the
enhanced sensitivity of B. microti DNA detection with the nested reaction.

54. • The Babesia microti/duncani Polymerase Chain Reaction (PCR) screen
is an assay that detects Babesia DNA in whole blood and speciates
to B. microti and B. duncani. The combination of the following three
steps imparts a very high specificity and sensitivity to the test:
• Hybridization/Selection
• Amplification of Babesia-specific DNA
• Detection of Babesia-specific amplified DNA fragments

55. • LEISHMANIA
• A 41-year-old, HIV-positive man from Bangladesh presented to the HIV
clinic with fatigue and abdominal fullness, high fever. The laboratory
workup revealed mild pancytopenia and elevated liver function
enzymes. Computed tomography identified hepatosplenomegaly and
inguinal nodules. bone marrow (BM) biopsy was performed. The
peripheral blood (PB) smear showed rouleaux and monocytes with
intracellular microorganisms bearing prominent nuclei and adjacent rod-
shaped. The BM biopsy and aspirate smear were hypercellular, and also
demonstrated histiocytes with similar intracellular microorganisms.
There was no evidence of hemophagocytosis..

56. SMEAR: geimsa stain: LD
bodies(Amastigote)
CONVENTIONAL TESTS:
Splenic aspirate or liver biopsy.
Bone marrow aspiration of a patient with visceral leishmaniasis
showing Leishman-Donovan bodies being visualized as small,
ovoid structures with a rounded basophilic nucleus and an
elongated kinetoplast, within macrophages as well as
extracellularly

57. SEROLOGICAL TESTS:
IFAT
• If the anti- bodies persist in low litres it is an indication of a probable
relapse.
• Titres above 1:20 are significant and above 1:128 are diagnostic
• The test is based on detecting antibodies, which are demonstrated in the
very early stages of infection and are undetectable six to nine months
after cure.
• sensitive (96%) and specific (98%)

58. ELISA
• Recently, several recombinant antigens like rGBP from L. donovani, rORFF
from L. infantum, rgp63, rK9, rK26 and rK39 from L.chagasi have been
developed and tested.
• Of these, the rK39 antigen is found to be highly sensitive and predictive
of onset of disease manifestation in VL patients.
DAT
• The antigen is prepared from promastigotes of L. donovani and test can
be carried out on plasma, serum, blood spots and whole blood.
• Antibody titres of 1: 3200 are considered positive
Culture: Nicolle-NovyMacneal (NNN media) from clinical specimen

59. • CHROMATOGRAPHIC STRIP TEST
• A membrane strip which also contains a conjugate dye region is coated
with this protein. Through capillary action, the patient serum will react
with the dye and antigen to quickly indicate the presence of anti-rK39
immunoglobulin G (IgG) in a patient sample.

60. • MOLECULAR
PCR
• Recently, molecular probes, using kinetoplast DNA (kDaNA), ribosomal
RNA (rRNA), mini exon derived RNA (medRNA) and genomic repeats
have been evaluated.
• Different DNA sequences in the genome of leishmania like ITS region,
gp63 locus, telomeric sequences, sequence targets in rRNA genes such
as 18s rRNA and SSU-rRNA and both conserved and variable regions in
kinetoplast DNA (kDNA) minicircles are being used by various workers
• Drawback - this technique is not automated and is laborious, which
limits its use to a small number of samples.

61. • PCR-ELISA
• The kDNA PCR-ELISA is based on the capture of an amplified product
with a sense primer labelled with biotin at the 5′end in the polystyrene
plate coated with streptavidin, followed by hybridisation with a
fluorescein-labelled probe at the 5′ end and an antifluorescein-
peroxidase conjugate for detection
• DNA TARGET- kDNA from parasites
• DNA extracted BY- “QIAamp DNA mini” kit .
• Negative DNA extraction controls were performed for each experiment
by the addition of all reagents except the sample.
• The yield was determined by absorbance at 260 nm in a
spectrophotometer PCR-ELISA

62. TRYPANOSOMA
AMERICAN TRYPANOSOMIASIS (CHAGA’S DISEASE)
AFRICAN TRYPANOSOMIASIS(SLEEPING SICKNESS)

63. • IN HUMANS, T.cruzi is in 2 forms- amatigote
and trymastigote
A)CONVENTIONAL
• Giemsa stain,thick and thin smear
• Quantitative Buffy Coat (QBC®; Becton
Dickinson)

64. • B)SEROLOGY
• ICT Card test- cytoplasmic repetitive antigen (CRA) and flagellar
repetitive antigen (FRA) proteins from Trypanosoma cruzi
• ELISA
• C) molecular
• Pcr- TARGET- Highly repetitive nuclear & kinetoplast DNA (k DNA)
• Primer TCZ1-TCZ2 for nuclear repetitive 188bp sequence
• Primer S35-S36 against 330 bp kinetoplast minicircle
• PCR is useful in – Persons with borderline serology results Received
specific treatment Acute or congenital disease

65. MICROFILARIA

67. A)Conventional tests
DEMONSTRATIONG THE CIRCULATING MICROFILARIA
Microfilariae circulate in blood at night (nocturnal periodicity) so optimal
time for drawing blood is between 10pm to 4am
Blood collected in EDTA, GEIMSA, WRIGHT- GEIMSA STAIN,
AHAD proposed rapid method using Zeil-Neelson carbol fuschin followed
by Leishman’s stain.
Thick blood smear :for detection of microfilarie

68. CONCENTRATION OF BLOOD WITH
POLYCARBONATE MEMBRANE FILTER
It is a concentration technique used to trap
microfilariae on polycarbonate filter after red
blood cells are lysed. 1-2ml intravenous blood
filtered through 3um pore size membrane
filter and the filter paper is examined
directly under microscope.
Used when microfilarial load is low
DEC
For day time filarial parasite testing
QBC
Detection of microfilariae using
microhematocrit tube coated with acridine
orange

69. B) Antigen and antibody testing
IMMUNO CHROMATOGRAPHIC TEST:
• ANTIGEN TESTING:Most sensitive and convenient
Gold standard for W.bancrofti
2 types: POC (point of care) , ELISA
Poc-BinaxNOW (rapid)
Filarial card test(rapid)
Filarial strip test (2013)rapid
Og4c3 antigen

70. ELISA
Og4C3 antigen detection
• Antibodies detection: Wb123
BM14/Wb123
Multiplex bead assay
Biplex WD123/OV16 (rapid)
Wb- SXP-1 ELISA
• 3)DNA
• PCR – detection of filarial DNA in human blood
targets pWb12 for w.bancrofti
Shotgun metagenomics, which is applied to the direct sequencing of DNA
extracted from a sample without culture or target-specific amplification
or capture, has been widely used

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