Comparative Study of Thick Smear, Thin Smear, QBC and Antigen Card Test in Diagnosis of Malaria

Abstract

Rapid diagnosis is pre-requisite for institution of effective treatment and reducing mortality and morbidity of malaria. The study was taken up to compare the efficiency of various methods available, ie thick and thin smear, Quantitative Buffy Coat (QBC) and Antigen card test (Parascreen). In this present study, thick smear was compared with thin smear, QBC and Antigen card test for the diagnosis of malaria. Malaria was diagnosed in 112, 81, 98 and 110 patient by thick smear, thin smear, QBC and antigen card test, respectively. In antigen card test, the sensitivity 98.2%, specificity 100%, Positive predictive value 100% and Negative predictive value 99.3% were observed. Although the antigen card test is superior than thin smear and QBC. Antigen card test has its advantages in terms of speed, sensitivity and specificity especially in an endemic area. Therefore, the authors have recommended antigen card test which was simple, reliable and effective for the diagnosis of malaria in remote and rural areas in Saudi Arabia.

Headnote

Abstract: Rapid diagnosis is pre-requisite for institution of effective treatment and reducing mortality and morbidity of malaria. The study was taken up to compare the efficiency of various methods available, i.e. thick and thin smear, Quantitative Buffy Coat (QBC) and Antigen card test (Parascreen). In the present study, thick smear was compared with thin smear, QBC and Antigen card test for the diagnosis of malaria. A total of 406 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smear were prepared and stained with Leishman's stain, taking thick smear as a gold standard. QBC and Antigen detection were done using commercially available kits. Malaria was diagnosed in 112, 81, 98 and 110 patient by thick smear, thin smear, QBC and antigen card test respectively. In antigen card test the sensitivity 98.2%, specificity 100%, Positive predictive value (PPV) 100% and Negative predictive value (NPV) 99.3% were observed. Although the antigen card test is superior than thin smear and QBC. Antigen card test has its advantages in terms of speed, sensitivity and specificity especially in an endemic area. Therefore we recommended antigen card test which was simple, reliable and effective for the diagnosis of malaria in remote and rural areas of our country.

Keywords: Malaria, thick smear, thin smear, QBC, Antigen card test.

1. Introduction:

Malaria presents a diagnostic challenge to the medical community worldwide. Its occurrence is noted in more than 90 countries. Malaria remains the most important parasite disease globally causing over 170 million cases usually of who over a million die in every year [1]. It is a serious, some time fatal, parasitic disease posing a major public health problem in India [2]. There is problem of diagnosis and control of malaria but the treatment has to be started immediately in order to avoid complications. The keen interest in this field will surely leads to early detection improvement in treatment regimen and implement better techniques of prevention [3].

Malaria is a mosquito- borne infectious disease of human and other animals caused by sporozoites of genus Plasmodium. It begins with a bite from an infected female Anopheles mosquito, which introduces the sporozoites through saliva into the circulatory system. In the blood, the sporozoites travel to the liver to mature and reproduce. Malaria causes symptoms that typically include fever and headache, which in severe cases can progress to coma and death. The disease is widespread in tropical and subtropical regions in a broad band around the equator, including which of sub-Saharan Africa, Asia and Americans [4].

Microscopic Examination of the peripheral blood smears (PBS) are the commonly employed method for diagnosis of malaria. Since its introduction 1903 thick PBS allows identification of the plasmodial parasite and its stages, the technique is labor intensive, time consuming and its interpretation at low level of parasitemia request considerable experienced microscopist for accurate identification [5].

In recent years newer, simple and rapid techniques like QBC (Quantitative Buffy Coat), antigen and enzyme detection have been developed to replace the conventional microscopic methods. The antigen detected is histidine rich protein-2 (HRP-2) and enzyme detected is plasmodium lactate dehydrogenase (pLDH) and Pan- specific aldolase all these techniques vary in there sensitivity, specificity, positive predictive value and negative predictive values [6]. Keeping in mind the seriousness of the condition and the current availability of diagnostic facilities across India we decided to conduct a comparative study of the thick smear with thin smear, QBC and antigen card test (parascreen).

2. Materials and Methods:

This prospective study was conducted in the department of pathology, Father Muller Medical College Hospital, Mangalore, Karnataka, India. The study was conducted from May 2011 to August 2012. This study was done in 406 cases of patients presenting pyrexia with chills, rigor and other suggestive symptoms of malaria. They were admitted in wards or attending the outpatient department of Father Muller Medical college Hospital, Mangalore, Karnataka, India.

2.1 Sample Collection:

Oral consent was taken from the patients prior to the collection of specimens. In this study 5ml of EDTA anticoagulated blood was taken and smear were prepared within an hour of collection of blood. All the sample was collected in Vacuntainer. The age group of patients varied from 4 to 80 years.

2.2 Thick and Thin Blood Smears:

Thick and thin blood smears were prepared as per the standard method. The smears were stained with Leishman's stain [7].

2.3 Quantitative Buffy Coat Technique (QBC):

The QBC is a high precision glass haematocrit tube, pre coated internally with acridine orange stain. This tube was filled with 55 - 60 ml of EDTA blood. A clear plastic closer was applied. A cylindrical float, designed to be suspended in a packed red cell was inserted. This tube was centrifuged at 12000 rpm for 5 minutes. This tube is examined under light microscope with standard white light, equipped with accessory illuminated microscope objective and fluorescence. Approximately 10-20 fields were examined over 2-5 minutes.

The principle of QBC technique is based on the fact that on centrifugation at high speed. The whole blood separates into plasma, buffy coat and packed red cell layer. The component of buffy coat separated according to their densities, forming discreat bands. The topmost area of the red cell band is enlarged 10 times more than normal, this area has red cells with parasites. Due to the high buoyancy of the infected cells they appear in the upper most part of the column. Due to acridine orange the malarial parasite are seen as fluorescent bodies standing at different level of the sedimentation column depending on the stage and species of the parasite [8, 9].

2.4 Antigen Detection using Parascreen:

Commercially available antigen detection kit Parascreen (Zephyr Biomedical Systems, lot: 101051) that detects the Histidine-rich protein 2 antigen (HRP II) of P.falciparum and the lactate dehydrogenase of Plasmodium was used.

The strips coated with anti HRP II antibody were used to detect the presence of the HRP II antigen by immuno-chromatography.

The test was done using EDTA anticoagulated blood according to standard operating procedure (SOP) given by manufactures. Interpretation of the test result was done as below:

1. When only one pink- purple band appears in the control window marked as "C" the test considered to be negative.

2. When one control band and another one bands appear only at region of "Pf" the test considered to be positive for P.falciparum.

3. When one control band and one pink purple band appear only at region "Pan" the test considered to be positive for other species (non falciparum).

4. When one control band and another two bands appear at regions "Pf" and "Pan" the test considered to be positive for P. falciparum or mixed infection.

5. When no bands appear on device the test should be considered invalid [10].

3. Results:

A total of 406 samples were evaluated by thick and thin Leishman stained peripheral blood smear, QBC technique and antigen card test. The blood film result indicated that malarial parasite was detected in 112 (27.58%) cases by thick blood smear and in 81(19.95%) cases by thin peripheral blood smear.QBC technique detected 98(24.13%) cases of malaria and antigen card test detect 110 (27%) cases of malaria (Table 1). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of antigen card test with respect to Leishman stain thick smear was 98.2%, 100%, 100% and 99.3%, respectively. The sensitivity of thin smear was low 72% but its specificity was 98%. The sensitivity and specificity of QBC were 87.5% and 99.0%, respectively with respect to Leishman stained thick smear (Table 2).

4. Discussion:

Rapid detection and effective treatment is a pre requisite in reducing morbidity and mortality due to malaria. Peripheral blood smear study is simple, least expensive, labor intensive, time consuming and therefore delay in diagnosis. Leishman's or Giemsa stained thick smears are considered to be the 'Gold standard' in diagnosis. However, the accurate species differentiation and quantitation of parasitemia is possible only when performed by skilled microscopist [11, 12]. Apart from clinical and microscopic examination, many rapid diagnostic tests have been developed. Most frequently, they deploy a dipstick or test strip bearing monoclonal antibodies detected against the target parasite antigen [2]. Newer technique like QBC and Antigen card test are rapid, simple and easy to interpret.

In the present study, while comparing the available different methods of rapid detection of malarial parasites with gold standard Leishman stained thick smear, the sensitivity of thin smear found to be lowest (72.3%) which may be because of the undefined ring stage of the parasite. While Parija et al [13] reported a very low sensitivity of (54.8%) leishman stained thin smear. In our study we got a high specificity and positive predictive value (98% and 93%) respectively (Table 2). Leishman stained thin blood smear detects malarial parasite only when there are 50 parasite/ ml of blood [5]. Even though this method can be used for species identification of malarial parasite.

When we compared QBC with Leishman stained thick smear the sensitivity of QBC was found to be low 87.5% but specificity, PPV and NPV was 99.0 %, 97.0 %, 95.4 % respectively. Although the sensitivity of QBC has been reported to be high as 92.6% by Mendiratta et al and 100 % by Bhandari et al [14, 3]. Whereas in our study we observed low sensitivity of QBC. One of the reasons for this could be that as the hospital is present in an endemic region for malaria the levels of parasitemia could have been low. In another study Benito et al [15] revealed that QBC has a low sensitivity and greater rapidity as compared to Leishman stained thick smear. Similarly in the present study we also observed same findings with lower sensitivity and higher specificity. QBC has the advantage that screening is much faster. However, it requires a fluorescence microscope which is expensive and the QBC mounts cannot be preserved, unlike the Leishman stained smears. One more disadvantage of QBC technique is that a permanent record of test cannot be kept [16].

In the present study we observed 98.2 % of sensitivity, 100% of specificity and 100% PPV in Parascreen antigen card test comparing with the thick smear. Similarly some authors also observed same findings [17, 2]. While few authors [13, 18] observed a lower sensitivity of 75% by using other kit (malarigen) based on similar principle, whereas the specificity (100%) and PPV (100%) were in accordance with our study.

However, in the present study parascreen antigen card test was negative in two cases which were positive cases in Leishman's stained thick smear; (Table1) these were probably false negatives because of immuno-chromatography (ICT) is unable to detect HRP II below 100 parasites/µL of blood. One should bear in mind that HRP II has been shown to persist in blood for 7-14 days and up to 28 days following antimalarial therapy; hence it is also important to be familiar with the history of antimalarial treatment of the individual patient to rule out false positives [19, 20].

In this study we also compared thin smear with antigen card test, 81 cases were positive by both these methods, while thin smear missed to rule out malaria in 29 cases which was positive for malaria by antigen card test (Table1). Which is comparatively similar to the study done by Iqbal et al [21] in their study microscopy missed 47 cases but these 47 cases were positive by optiMAL antigen test. However, the test was found to be user friendly and interpretation was more objective as compared to smear and QBC.

There were some limitations in the present study, sample size was small and it was a hospital based study, so can't represent whole population. There is need to perform such studies on larger and community based population.

Conclusion:

Since malaria is endemic in certain regions of India, we need to employ more sensitive tests, which are also rapid to detect low levels of parasitemia in population. Where QBC method is useful in laboratories only for screening large number of samples and also where appropriate laboratory facilities are available. But some peripheral health care units there are non available of skilled persons and good laboratory facilities for blood film examination. Therefore we recommend antigen card test which was simple, reliable, rapid and effective for the diagnosis of malaria. Even though the test can be a promising alternative to microscopy in remote and rural areas of our country.

References

References

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