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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 2  |  Page : 200-202
A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria


Department of Microbiology, Jawaharlal Institute of Postgraduate, Medical Education and Research, Pondicherry - 605 006, India

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   Abstract 

Rapid diagnosis is prerequisite for effective treatment and reducing mortality and morbidity of malaria. This study was taken up to compare the efficacy of various methods available, i.e., thick and thin smear, quantitative buffy coat (QBC), plasmodium lactate dehydrogenase and aldolase in blood of patient. A total of 411 samples were collected from patients presenting with classic symptoms of malaria. For traditional microscopy; thick and thin smears were prepared and stained with Leishman's stain, taking thick smear as gold standard, thin smear had a sensitivity and specificity of 54.8% and 100%, respectively. QBC and antigen detection was done using commercially available kits; out of 411 samples, QBC and Malariagen were positive in 66 and 62 cases, with a sensitivity of 78% and 75%, respectively. Leishman's thick smear, although cost effective, is difficult to interpret for inexperienced microscopists; so if facilities are available, QBC should be used for routine diagnosis. In places where facilities are not available, rapid, simple and easy to interpret antigen detection test can be used despite low sensitivity.

Keywords: Malaria, diagnosis, QBC, fluorochrome dyes, antigen detection

How to cite this article:
Parija S C, Dhodapkar R, Elangovan S, Chaya D R. A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria. Indian J Pathol Microbiol 2009;52:200-2

How to cite this URL:
Parija S C, Dhodapkar R, Elangovan S, Chaya D R. A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Nov 19];52:200-2. Available from: http://www.ijpmonline.org/text.asp?2009/52/2/200/48917



   Introduction Top


Malaria presents a diagnostic challenge to the medical community worldwide. Its occurrence is noted in more than 90 countries. It is estimated that there are more than 50 million cases and 1.1-2.2 million deaths due to malaria every year. [1] In India, in the year 2005, there were approximately 1.8 million cases of malaria reported of which 44.5% were caused by Plasmodium falciparum. [2] Due to the serious nature of P. falciparum infections, prompt and accurate diagnosis of the condition is essential for effective management. The diagnostic modalities which are available for malaria range from conventional thick and thin smear to rapid modalities like fluorescent staining (QBC) and antigen detection tests detecting parasitic antigens like histidine-rich protein-2 (HRP 2), plasmodium lactate dehydrogenase (pLDH) and pan-specific aldolase. All these techniques vary in there sensitivity, specificity, positive and negative predictive values. 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 commonly employed diagnostic techniques in diagnosis of malaria, i.e., thick and thin smear, QBC and antigen detection using pLDH and aldolase.


   Materials and Methods Top


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.

Sample collection

Oral consent was taken from the patients prior to the collection of specimens. Approximately 5ml of venous blood was collected from each patient during the peak of fever and transported to the laboratory.

Thick and thin blood smears

Thick and thin blood smears were prepared as per the standard method described elsewhere. [3] The smears were stained with Leishman's stain. The slides were examined by an experienced microscopist, and the average time spent on each slide varied depending on parasite density. 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. [3]

Quantitative buffy coat technique (QBC)

Quantitative buffy coat technique (BD Diagnostics) was employed for the detection of malarial parasites in blood. Specially designed microhematocrit tubes coated with acridine orange were provided by the manufacturer. Approximately, 55-60µl 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's instructions. 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. [4]

Antigen detection using pLDH and aldolase

Commercially available antigen detection kit detecting plasmodium LDH and aldolase (Malariagen Pf/Pv Antigen Rapid test, Aspen Laboratories, India) were used. The test was conducted using anticoagulated venous blood; the sample was added to the test strip using a calibrated dropper provided with the kit, and the strip was placed in a microwell containing buffer. The result was read after 15 min as per the manufacturer's instructions. It was interpreted as positive for P. falciparum if T1 and T2 bands were seen along with control (C) band; if only T2 and C were seen, it was interpreted as positive for Plasmodium vivax.


   Results Top


A total of 411 samples were observed over the period of study. 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 were positive by the antigen detection test [Table 1]. 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. vivax detected by thin smear and antigen detection.


   Discussion Top


Rapid detection and effective treatment is a pre-requisite for reducing the morbidity and mortality due to malaria. Leishman's or Giemsa stained thick smears are considered to be the 'Gold standard' in diagnosis. However, the interpretation of thick smears is laborious and results depend on the quality of microscope, staining, technique with which blood film is prepared and also the concentration and motivation of microscopist. [5],[6] Newer techniques like QBC and Antigen detection assays are rapid, simple and easy to interpret.

In the present study, we compared different methods available for rapid diagnosis with Leishman-stained thick smears. The sensitivity of Leisman-stained thin smear was found to be lowest (54.8%); however, this method had a high specificity and positive predictive value (100%) [Table 1].

QBC, which utilizes the principle of acridine orange staining with differential centrifugation, had a sensitivity of 78.94%, specificity, PPV and NPV were found to be 98%, 90%, and 95%, respectively. Although the sensitivity of QBC has been reported to be as high as 99.7% by Benito et al , [7] relatively low sensitivity of QBC was observed in our study. 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. Despite this, the specificity of the test as yielded by this study was in concurrence with that of other observers. [8] Although the test is proclaimed to be highly sensitive and easy to use, adjustment to the technique was found to be difficult by the first-time users who were involved in the study. Also the detection of parasites other than P. falciparum was not very sensitive as the two cases of P. vivax detected by thin smear and Malarigen were found to be negative by QBC. There were six cases found to be positive by QBC, which were subsequently found to be negative for thick and thin smear and Malarigen test. This can be explained by the fact that certain artifacts in blood like Howell Jolly bodies or platelet fragments might resemble the ring forms of P. falciparum.

Malarigen for detection of malaria antigen had a sensitivity, specificity and PPV of 75%, 100% and 100%, respectively. This test was based on detection of pLDH and aldolase with the help of monoclonal antibodies. The values obtained for this kit-based procedure were much lower than those observed for other tests based on similar principle. [9],[10] This low sensitivity could be attributed to low parasitemia levels as observed by Iqbal et al . [11] who observed 75% sensitivity at parasitemia < 100/µl. The specificity was comparable to other observers using the tests based on similar principle. [9],[10] However, the test was found to be user friendly and interpretation was more objective as compared to smear and QBC.


   Conclusion Top


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. Therefore, we recommend QBC to be used in setups where appropriate facilities are available; however, in situations where adequate laboratory back up is not available, simpler and easy to use techniques like antigen detection can be employed despite having lower sensitivity.

 
   References Top

1.Shivlal, Dhillon GP, Aggarwal CS. Epidemiology and control of Malaria. Indian J Pediatr 1999;66:547-54.  Back to cited text no. 1    
2.WHO regional office for South East Asia. New Delhi. [updated on 2007 Feb 27]; [cited on 2007 May 20]. Available from: http://www.searo.who.int/en/Section10/Section21/Section340_4021.htm.  Back to cited text no. 2    
3.Parija SC. Textbook of medical parasitology. 3 rd ed. Chennai: AIPHD; 2006.  Back to cited text no. 3    
4.Baird JK, Purnomo, Jones TR. Diagnosis of malaria in the field by fluorescence microscopy of QBC capillary tubιs. Trans R Soc Trop Med Hyg 1992;86:3-5.  Back to cited text no. 4    
5.Dowling MA, Shute GT. A comparative study of thick and thin blood films in diagnosis of scanty malaria parasitemia. Bull World Health Organ 1966;34:249-67.  Back to cited text no. 5    
6.Payne D. Use and limitations of light microscopy for diagnosing malaria at primary health care level. Bull World Health Organ 1988;66:621-6.  Back to cited text no. 6    
7.Benito A, Roche J, Molina RA, Amela C, Altar J. Application and evaluation of QBC ® malaria diagnosis in a holoendemic area. Appl Parasitol 1994;35;266-72.  Back to cited text no. 7    
8.Bosch I, Bracho C, Perez HA. Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of Venezuela. Mem Inst Oswaldo Cruz 1996;91:83-6.  Back to cited text no. 8    
9.Iqbal J, Hira PR, Sher A, Al-Enezi AA. Diagnosis of imported malaria by Plasmodium lactate dehydrogenase (pLDH) and histidine-rich protein 2 (PfHRP-2)-based immunocapture assays. Am J Trop Med Hyg 2001;64:20-3.  Back to cited text no. 9    
10.Palmer CJ, Lindo JF, Klaskala WI, Quesada JA, Kaminsky R, Baum MK, et al . Evaluation of the OptiMAL test for rapid diagnosis plasmodium vivax and plasmodium falciparum malaria. J Clin Microbiol 1998;36:203-6.  Back to cited text no. 10    
11.Iqbal J, Sher A, Hira PR, Al-Owaish R. Comparison of the OptiMAL ® test with PCR for diagnosis of malaria in immigrants. J Clin Microbiol 1999;39:3644-6.  Back to cited text no. 11    

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Correspondence Address:
S C Parija
Department of Microbiology, Jawaharlal Institute of Postgraduate, Medical Education and Research, Pondicherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.48917

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    Tables

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