Comparison of Quantitative Buffy Coat technique (QBC) with Giemsa-stained thick film (GTF) for diagnosis of malaria

Abstract

The renewed interest in the use of fluorescent microscopy for malaria diagnosis prompted the comparison of Quantitative Buffy Coat technique (QBC) with the old standard Giemsa-stained Thick blood Film (GTF) in Ikeja General Hospital, now Lagos State University Teaching Hospital, in Lagos. Blood samples were collected from 353 patients, each examined with the QBC and GTF techniques. Of these, 68 were positive with GTF, 70 with QBC giving a positive rate of 19.3% and 19.8% respectively. The malaria positive rate was calculated as 19.3% using GTF as the standard. In general, females recorded higher percentages (58.6% and 54.4%) than males (41.4% and 45.6%) among those positive with QBC and GTF respectively. The overall sensitivity rate for QBC was 55.9% and the specificity was 88.8%. The positive and negative predictive values of QBC compared to GTF were 54.3%, 89.4% respectively while the concordance of the two techniques was 82.4%. These values were lower than those reported for QBC in previous studies. The sensitivity of QBC reduced further (33.3%) with samples having low parasite density (< 1000 parasites/ul). QBC test was not able to accurately differentiate between different Plasmodium species but with the GTF, 86.7% of the infected individuals had Plasmodium falciparum, 7.5% had P. malariae and only 5.9% had mixed infections. In spite of the speed and simplicity of QBC technique, it cannot be considered an acceptable alternative to GTF under routine clinical laboratory situation. However, its speed and ease of use make it an important new tool for the diagnosis of malaria.

Introduction

Malaria is the most important parasitic disease in the tropics with almost 300 million clinical cases of malaria occurring worldwide each year and over one million people dying [1]. Global figures for deaths from malaria range from 1.5 to 2.7 million each year, most of whom are children under 5 years of age and pregnant women [2]. Malaria reduces economic productivity due to absenteeism from schools and places of work during severe attacks [3]. As a result, malaria-endemic countries are some of the world's most impoverished. The cost of malaria control and treatment drains African countries and slows the rate of economic growth; early detection and treatment of malaria is therefore paramount [3].

The accepted laboratory practice for the diagnosis of malaria is the preparation and microscopic examination of blood films stained with Giemsa, Wright's, or Field's stain [4]. However, in resource-poor areas, microscopic diagnosis has been shown to be insensitive and non-specific, especially when parasitaemias are low or mixed infections are present [5], [6]. In field conditions, sensitivities and specificities as low as 71–72% have been reported [7]. Other limitations include false negativity due to relatively small amount of blood examined or low parasitaemia, and false positivity due to debris [8].

In an attempt to enhance the detection of malaria parasites in blood films, alternative methods have been introduced. Certain fluorescent dyes have an affinity for the nucleic acid in the parasite nucleus and will attach to the nuclei. When excited by UV light at an appropriate wavelength, the nucleus will fluoresce strongly. Two fluorochromes have frequently been used for this purpose, acridine orange (AO) and benzothiocarboxypurine (BCP), which are both excited at 490 nm and exhibit apple green or yellow fluorescence. Rhodamine-123 is also useful for assessing the viable state of parasites, since its uptake relies on an intact, working parasitic membrane. Several methods have been published in which AO is used either as a direct-staining technique or combined with a concentration method such as a thick blood film [9]. The centrifugal quantitative buffy coat or QBC II (QBC) (Becton Dickinson, Franklin Lakes, N.J.) combines an AO-coated capillary tube and an internal float to separate layers of white blood cells (WBC) and platelets using centrifugation. Parasites concentrate below this layer of cells, appearing in the upper layer of RBC but also sometimes appearing within the layers of platelets and WBC. Parasites can be viewed through the capillary tube using a special long-focal-length objective (paralens) with a fluorescence microscope [10]. Other newly developed techniques for rapid diagnosis of malaria include the ParaSight F [11], the Malaquick tests [12], the OptiMal [13] and PCR-based diagnostic tests[14].

In a comparative study [15], 35% of the 200 blood samples examined were positive for malaria by QBC, 31% by thick smears, 25% by thin smears and only 17% by the conventional buffy coat technique. Rickson et al. [16] recorded a low sensitivity of 35% for QBC technique at low parasitaemia. In a report from Kenya in which QBC was compared with Giemsa-stained thick blood films (GTF), high sensitivity and specificity values were obtained (93.6% and 91.5% respectively), but with low parasitaemia, the sensitivity dropped to 41.7% [17]. Higher (> 90%) Positive Predictive Values (PPV), Negative Predictive Values (NPV) and concordance were recorded for QBC and GTF at two study sites in China [18], while lower values (< 70%) were reported in Nigeria [8]. The objective of this study, therefore, was to make a comparative study of the QBC and GTF techniques in order to assess the potential of QBC as a possible replacement for GTF in the laboratory diagnosis of malaria in Nigeria.