a comparative study of blood smear, qbc

8/4/2019 A Comparative Study of Blood Smear, QBC

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A comparative study of blood smear, QBC

and antigen detection for diagnosis of

malaria

SC Parija, Rahul Dhodapkar, Subashini

Elangovan, DR Chaya


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Department of Microbiology,

Jawaharlal Institute of Postgraduate, Medical Education

and Research, Pondicherry


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Introduction

Malaria presents a diagnostic challenge to the medicalcommunity worldwide.

Its occurrence is noted in more than 90 countries.

It is estimated that there are more than 50 million casesand 1.1-2.2 million deaths due to malaria every year.

In India, in the year 2005, there were approximately 1.8million cases of malaria reported of which 44.5% werecaused by Plasmodium falciparum.


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Due to the serious nature of P. falciparum infections,

prompt and accurate diagnosis of the condition is

essential for effective management.


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Malarial parasite

Life cycle

- In man (Schizogony)

- In female anopheles mosquito (sporogony)


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Clinical feature

i) Fever 3 distinct stages

The cold stage

The hot stage

The sweating stage


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Anaemia occurs due to

i) Destruction of parasitised and non- parasitised redblood cells

ii) Hypersplenismiii) Autoimmune lysis of coated infected and uninfectedRBC

iv) increased fragility of RBC

v) Decreased RBC production from bone- marrow

supression.

Splenomegaly


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Complications ofmalaria

More common due to P.falciparum (malignant malaria )

Cerebral malaria

Acute renal failure

Liver damage

Dehydration

Gastrointestinal symptoms

Blackwater fever

Hyperpyrexia

hypoglycemia


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Laboratory diagnosis of malaria

Peripheral blood smear-- Thick smear 40 times sensitive than thin smear

- can detect as few as 5 parasites/l

- Thin smear essential for species identification of malarialparasite

Time of collection of sample- late in febrile paroxysm

Staining JSB stain, fields or geimsa stain


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Rough estimate can be made by counting the number of

parasites observed per thick film fields

1+ = 1-10 parasites per 100 thick film fields

++ = 11- 100 parasites per 100 thick film fields+++ = 1-10 parasites per thick film field

++++ = more than 10 parasites per thick film field.


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QBC system

Malarial parasites are concentrated by centifuging the

blood in a special capillary tube.

- The tube is coated with acridine orange and an

anticoagulant

- When examined by fluorescence microscopy, the acridineorange stained malarial parasite flouresce green yellow

against a dark red back ground .


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Kawamoto AO interference filter

- Thick and thin films are stained by adding a drop of AO

stain

- The nuclei of the malarial parasite and white cellsfluoresce bright green and cytoplasm of parasite

fluoresces red.


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Diagnosis using rapid immunologic strip tests

- HRP 2 test ( Histidine rich protein 2) - produced byP.falciparum.

- released from the parasitised cell into circulation

includes i)ParaSight F test- sensitivity is reported as84.2 96.6%

ii) ICT Pf test

- pLDH test (OptiMAL test) enzyme pLDH is produce byall human malarial parasite species during their growth inred cell


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Materials and Methods

The study was conducted in Department of Microbiology,

JIPMER, Pondicherry, a tertiary care institute in South

India.

Specimens were collected from patients presenting

clinically with fever with chills and rigor and other

suggestive symptoms of malaria.


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Sample collection

Oral consent was taken from the patients prior to thecollection of specimens.

Approximately 5ml of venous blood was collected fromeach patient during the peak of fever and transported tothe laboratory.


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Thick and thin blood smears

Thick and thin blood smears were prepared as per the

standard method described elsewhere.

- The smears were stained with Leishman's stain.

- The slides were examined by an experiencedmicroscopist, and the average time spent on each slide

varied depending on parasite density.


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- Thick smears were reported negative after examination

of 200-300 oil immersion fields (oif) with no parasites

observed;

- a thin smear was given negative when no parasites were

observed in 200 oif.


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Quantitative buffy coat technique (QBC)

- Quantitative buffy coat technique (BD Diagnostics) wasemployed for the detection of malarial parasites in blood.

- Specially designed microhematocrit tubes coated withacridine orange were provided by the manufacturer.

- Approximately, 55-60l of blood was loaded into the

tubes and stopper and float were applied at either ends;the tubes were centrifuged at 12000RPM in a pre-programmed centrifuge as per the manufacturer'sinstructions


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The interpretation was done using a standard

microscope fitted with Para Lens ultraviolet microscope

adaptor and a 60 objective connected to fiber optic

ultraviolet light module. The parasites were seen in buffy

coat layer and the interface between RBC and WBC

regions.


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Antigen detection using pLDH and aldolase

- Commercially available antigen detection kit detectingplasmodium LDH and aldolase (Malariagen Pf/Pv

Antigen Rapid test, Aspen Laboratories, India) were

used.

- The test was conducted using anticoagulated venousblood; the sample was added to the test strip using a

calibrated dropper provided with the kit, and the stripwas placed in a microwell containing buffer.


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- The result was read after 15 min as per the

manufacturer's instructions.

- It was interpreted as positive forP. falciparum if T1 andT2 bands were seen along with control (C) band; if only

T2 and C were seen, it was interpreted as positive for

Plasmodium vivax.


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Results

- A total of 411 samples were observed over the period ofstudy.

- Of these a total of 82 samples were positive by thick

smear and 45 were positive by thin smear.

- QBC was positive in 66 samples and 62 samples werepositive by the antigen detection test .


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Sensitivity specificity Positive

predictive

value

Negative

predictive

value

Thin smear 54.8 100 100 89

QBC 78.94 98 90 95

Antigen

detection

75 100 100 94.2


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- Total incidence of malaria was found to be 19.95%

(82/411).

- Most of the cases were detected to be due to P.

falciparum ; there were only 2 cases due to P. vivaxdetected by thin smear and antigen detection.


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Discussion

- Rapid detection and effective treatment is a pre-requisitefor reducing the morbidity and mortality due to malaria.

- Leishman's or Giemsa stained thick smears areconsidered to be the 'Gold standard' in diagnosis.

- However, the interpretation of thick smears is laboriousand results depend on the quality of microscope,

staining, technique with which blood film is prepared andalso the concentration and motivation of microscopist.


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- Newer techniques like QBC and Antigen detectionassays are rapid, simple and easy to interpret.

- In the present study, they compared different methods

available for rapid diagnosis with Leishman-stained thicksmears.

- The sensitivity of Leisman-stained thin smear was foundto be lowest (54.8%); however, this method had a highspecificity and positive predictive value (100%) .


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- QBC, which utilizes the principle of acridine orangestaining with differential centrifugation, had a sensitivityof 78.94%, specificity, PPV and NPV were found to be98%, 90%, and 95%, respectively.

- Although the sensitivity of QBC has been reported to beas high as 99.7% by Benito et al, relatively lowsensitivity of QBC was observed in their study.

- One of the reasons for this could be that as the hospitalis present in an endemic region for malaria the levels ofparasitemia could have been low.


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- Despite this, the specificity of the test as yielded by thisstudy was in concurrence with that of other observers.

- Although the test is proclaimed to be highly sensitiveand easy to use, adjustment to the technique was foundto be difficult by the first-time users who were involved inthe study.

- Also the detection of parasites other than P. falciparumwas not very sensitive as the two cases ofP. vivaxdetected by thin smear and Malarigen were found to benegative by QBC


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- There were six cases found to be positive by QBC,which were subsequently found to be negative for thickand thin smear and Malarigen test.

- This can be explained by the fact that certain artifacts inblood like Howell Jolly bodies or platelet fragments mightresemble the ring forms ofP. falciparum.


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- Malarigen for detection of malaria antigen had asensitivity, specificity and PPV of 75%, 100% and 100%,respectively.

- This test was based on detection of pLDH and aldolasewith the help of monoclonal antibodies.

- The values obtained for this kit-based procedure weremuch lower than those observed for other tests based on

similar principle.


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- This low sensitivity could be attributed to low parasitemialevels as observed by Iqbal et al. who observed 75%sensitivity at parasitemia < 100/l.

- The specificity was comparable to other observers usingthe tests based on similar principle. However, the testwas found to be user friendly and interpretation wasmore objective as compared to smear and QBC.


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Conclusion

Since malaria is endemic in certain regions of India, weneed to employ more sensitive tests, which are alsorapid to detect low levels of parasitemia in population.

Therefore, they recommend QBC to be used in setupswhere appropriate facilities are available; however, insituations where adequate laboratory back up is notavailable, simpler and easy to use techniques likeantigen detection can be employed despite having lower

sensitivity.


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a comparative study of blood smear, qbc

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