Lab. Investigation of MP

Sujan Chandra Mondal

Laboratory investigations of

Malarial Parasite

Dr. Md Moyez UddinDirector

Institute of Public Health


Tests for Malarial Parasites

Microscopic test1.Peripheral blood film study2.Quantitive Buffy Coat (QBC) test

Non microscopic testRapid Diagnostic Tests (RDTs) Para Sight F test OptiMal Assay The immunochromatographic test (ICT Malaria P. f. test) Polymerase Chain Reaction Detection of antibodies by Radio immuno assay, immunofluorescence or enzyme immuno assay


Procedure of Diagnosis

Collection of blood

Preparation

Stain-Giemsa stain (1:20 dilute) 30min

-Field’s stain 4-5 second ‘A’ Polychrome methyline blue ‘B’ Eosine

-Leishman stain


Preparation of PBF

Hold the third finger of the left hand and wipe its tip with spirit/Savlon swab; allow to dry

Prick the finger with disposable needle/lancet; allow the blood to ooze out

Step 1 Step 2


Preparation of PBF

Take a clean glass slide. Take 3 drops of blood 1 cm from the edge of the slide, take another drop of blood one cm from the first drop of blood

Take another clean slide with smooth edges and use it as a spreader...

Step 3 Step 4


Preparation of PBF

and make thick and thin smears. Allow it to dry

Prepared smear.(Slide number can be marked on the thin smear with a lead pencil.)

Step 5

Step 6


Thick film of MP Microscopic

view RBC disappeared

Streaks of blue cytoplasm with detached nuclear dot appearance like ‘coma’ or ‘exclamation mark’

WBC remain unchange


Thick film

Parasite density-Number of parasites counted in each

microscopic field

-density considered in thick film

20 or more /field : high density

2-19 / field : medium density

1 or less ; low density

-It is important to report parasite density

Advantage- Rapid detection of parasite (*)- Concentrating parasite 20 fold

than thin film - Guide to Rx for testing efficacy

of antimalarial drug

Disadvantage - species cannot detected

( *) 1 microscopic field of thick film equavalent to 50 microscopic field of thin film


Thin film

Certain rules before declared negative -upper & lower margin of the tail end of film should examine thoroughly because parasite mostly numerous here -minimum 100 field must examined -time taken for examintion at least 8 to 10 min.


Diagnostic points:- Red Cells are not

enlarged. Rings appear fine and

delicate and there may be several in one cell.

Some rings may have two chromatin dots.

Presence of marginal or accole forms.

It is unusual to see developing forms in peripheral blood films.

Gametocytes have a characteristic crescent shape appearance. However, they do not usually appear in the blood for the first four weeks of infection.

Maurer's dots may be present.


Diagnostic points:- Red cells containing

parasites are usually enlarged.

Schuffner's dots are frequently present in the red cells as shown above.

The mature ring forms tend to be large and coarse.

Developing forms are frequently present.


Diagnostic points :- Ring forms may have a squarish appearance. Band forms are a characteristic of this species. Mature schizonts may have a typical daisy head appearance with up to

ten merozoites. Red cells are not enlarged. Chromatin dot may be on the inner surface of the ring.


Diagnostic points :- Red cells enlarged. Comet forms

common (top right) Rings large and

coarse. Schuffner's dots,

when present, may be prominent.

Mature schizonts similar to those of P. malariae but larger and more coarse


P. vivex P.falciparum

P.malariae P.ovale


QBC testnew method

involves staining of the centrifuged and compressed red cell layer with acridine orange and its examination under UV light source.

key feature-centrifugation & concentration of the red blood cells in predictable area of the QBC tube, making detection easy and fast. Parasitized RBC less dense than normal ones & concentrate just below the leukocytes, at the top of the erythrocyte column. The float forcs all the surrounding red cells into the 40 micron space between its outside circumference and the inside of the tube. Since the parasites contain DNA which takes up the acridine orange stain, they appear as bright specks of light among the non-fluorescing red cells. Virtually all of the parasites found in the 60 microliter of blood can be visualized by rotating the tube under the microscope.


Fluorescense UV ray microscopy


Other Rapid test (RDT’s) of MP new method

OptiMal Assay result

OptiMal Assey Kit


Immunochromatographic (ICT) Tests for Malaria Antigens ( HRP-2, LDH, Aldolase)

based on the capture of the parasite antigens from the peripheral blood using either monoclonal or polyclonal antibodies against the parasite antigen targets.

can target the histidine-rich protein 2 of P. falciparum, a pan-malarial Plasmodium aldolase, and the parasite specific lactate dehydrogenase

. do not require a laboratory,

electricity, or any special equipment.


Comparison between peripheral smear and QBC test for detecting malaria

Peripheral smear

QBC

Method Cumbersome Easy

Time Longer, 60 - 120 minutes

Faster, 15 - 30 minutes

Sensitivity 5 parasites/µl in thick film and 200 /

µl in thin film

Claimed to be more sensitive, at least as good as a thick film

Specificity Gold standard ? False positives, artifacts may be reported as positive by not-so-well-trained technicians

Species identificatio

n

Accurate, gold standard

Difficult to impossible

Cost Inexpensive Costly equipment and consumables

Acceptability

100% Not so

Availability Everywhere Limited

Other -- Accidentally can detect filarial worms


Peripheral Smear Rapid Diagnostic Tests

Format Slides with blood smear Test strip

Equipment Microscope Kit only

Training Trained microscopist 'Anyone with a little training'

Test duration 20-60 minutes or more 5-30 minutes

Test result Direct visualization of the parasites

Color changes on antibody coated lines

Capability Detects and differentiates all

plasmodia at different stages

Detects malaria antigens (PfHRP2/ PMA/pLDH) from asexual and/or

sexual forms of the parasite

Detection threshold 5-10 parasites/µL of blood

1 00-500/µL for P. falciparum, higher for non-falciparum

Species differentiation

Possible Cannot differentiate among non-falciparum species; mixed infections of P. falciparum and non-falciparum

appear as P. falciparum

Quantification Possible Not possible

Differentiation between sexual and

asexual stages

Possible Not possible

Disadvantages Availability of equipment and skilled microscopists,

particularly at remote areas and odd hours

Unpredictable efficiency at low and very high parasitemia; cross

reactions among plasmodial species and with auto-antibodies; persistence

of antigens

Status Gold standard Not yet approved by the FDA

Lab. Investigation of MP

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