|Year : 2015 | Volume
| Issue : 1 | Page : 63-67
Usefulness of a centrifuged buffy coat smear examination for diagnosis of malaria
S Mohanty, R Sharma, M Deb
Department of Microbiology , VMMC and Safdarjung Hospital, New Delhi, India
|Date of Submission||15-Sep-2013|
|Date of Acceptance||02-Dec-2013|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology , VMMC and Safdarjung Hospital, New Delhi
Source of Support: None, Conflict of Interest: None
Purpose: Malaria continues to be a global public health challenge. Microscopic examination of peripheral blood smear (PBS) is the standard method for malaria diagnosis, which is easily available and has low cost but its reliability is questionable at low level of parasitaemia. The present study was undertaken to assess the usefulness of a modified centrifuged buffy coat smear (CBCS) technique for diagnosis of malaria and to compare it with conventional PBS examination and antigen detection test. Materials and Methods: The study was carried out over a 6-month period from July to December 2011. Blood samples (2-3 ml per patient) collected in EDTAvials from patients with a clinical suspicion of malaria were subjected to all three tests, that is PBS, CBCS and antigen test and results were compared with antigen test as the gold standard. Result: Of 1655 samples received, 394 (23.8%) samples were positive for infection with malaria parasites. All the three tests detected malaria infection equally in 279 samples, and gave varied results in the remaining 115 samples. Addition of centrifugation (i.e. CBCS) to the conventional method of PBS enabled detection of 80 more cases of plasmodia infection, especially (43, 53.7%) at low levels of parasitaemia (<200 parasites/μl). While both PBS and CBCS had excellent specificity (99.7% and 99.2%, respectively), PBS examination had low sensitivity (72.9%) in detecting malaria parasites in comparison to CBCS. The sensitivity of CBCS in detecting malaria parasites was 91.9%. Conclusion: The development of easy, rapid and accurate tests for the reliable detection of plasmodia infection is highly desirable. The CBCS technique fulfils most of these criteria and may be adopted for rapid and reliable diagnosis of malaria in resource-limited settings.
Keywords: Buffy coat, malaria, peripheral blood smear, rapid diagnostic test
|How to cite this article:|
Mohanty S, Sharma R, Deb M. Usefulness of a centrifuged buffy coat smear examination for diagnosis of malaria. Indian J Med Microbiol 2015;33:63-7
|How to cite this URL:|
Mohanty S, Sharma R, Deb M. Usefulness of a centrifuged buffy coat smear examination for diagnosis of malaria. Indian J Med Microbiol [serial online] 2015 [cited 2020 Feb 29];33:63-7. Available from: http://www.ijmm.org/text.asp?2015/33/1/63/148380
| ~ Introduction|| |
Malaria continues to be a global public health challenge with more than 200 million cases and over 660,000 deaths annually, especially in the tropical and sub-tropical countries.  Even in developed countries where the disease is not endemic, increasing immigration and worldwide travel to endemic regions have led to an increased incidence of imported malaria. In India, malaria is endemic throughout the country and is a major public health problem accounting for 1-2 million cases and 1100 deaths per year.  However, according to recently published studies, the burden of malaria appears to be much higher than the previously reported figures (i.e. a 9- to 50-fold underestimation of malaria-related cases and ~13-fold under-estimation of malaria-related mortality, respectively). , Some of the other notable epidemiological features of malaria in India include the increasing proportion of Plasmodium falciparum from 10% in the 1970s to around 50% in 2010, emerging chloroquine resistance, complications due to hitherto considered benign species P. vivax, and the impending threat of the fifth malaria species, P. knowlesi, reported from nearby countries especially Malaysia and now increasingly from Europe as well. 
The earliest symptoms of malaria are very non-specific and variable, such as fever, headache, body ache, malaise, fatigue and abdominal discomfort, and hence difficult to diagnose clinically. In such a scenario, control and eradication of malaria have become very challenging issues and requires prompt treatment to save the patient's life, which in turn requires a rapid and accurate diagnosis.
An optimal diagnosis of malaria requires the detection and identification of each Plasmodium species present in the patient's blood and especially in cases of P. falciparum infection, an evaluation of the level of parasitaemia. Microscopic examination of peripheral blood smears (PBSs) as stained thick and/or thin blood smears is the standard method for malaria diagnosis, which is easily available and has low cost but its reliability is questionable at low level of parasitaemia. Consequently, several other approaches have been developed in recent times to enable early and reliable diagnosis of malaria in clinical settings like the quantitative buffy coat (QBC) assay and the rapid diagnostic tests (RDTs) based on detecting parasitic antigens such as the histidine-rich protein-2 (HRP-2), Plasmodium lactate dehydrogenase (pLDH) and specific aldolase. , Other newer techniques include the detection of parasite-specific nucleic acid sequences in the sample by polymerase chain reaction (PCR) or by using specific complimentary biotinylated probes. , Each of the techniques have their own advantages and disadvantages.
A previous study from India  had developed, standardised and reported on the feasibility of a modified centrifuged buffy coat smear (CBCS) examination for diagnosis of malaria in which the authors had used a wide bore 4 ml tube instead of a Wintrobe's tube to obtain a buffy coat. This new technique combined most of the advantages of the existing techniques.
The present study was undertaken to assess the usefulness of the CBCS technique for diagnosis of malaria in our setting in comparison to the PBS and antigen detection.
| ~ Materials and Methods|| |
The study was carried out over a 6-month period from July to December 2011 in the Department of Microbiology of a 1531-bedded tertiary care hospital in north India, which also caters to a large outpatient population. Blood samples collected in EDTA vials from patients presenting with various clinical manifestations (fever, headache, abdominal pain, altered sensorium, anaemia, bleeding manifestations, jaundice, splenomegaly, hepatomegaly, etc.) with a clinical suspicion of malaria were included in the study. A single sample (approximately 2-3 ml) was collected from each patient.
First, thick and thin smears were prepared as per the standard method, stained with Giemsa's stain for 40-45 min, and examined under oil immersion at ×1000 magnification.  Levels of parasitaemia (asexual stages/μl) were calculated, using the thick smears, by counting asexual parasites against a fixed number of leucocytes (usually 200) and assuming each patient had 8000 leucocytes/μl blood.  A thick smear was considered negative if no parasites were seen in 200 oil immersion fields. Thin smears were used to confirm the Plasmodium species present. Samples with pure gametocytaemia were included among the positive samples.
Second, CBCSs were prepared as described previously.  Briefly, this consisted of centrifuging the 2 ml EDTA blood in a wide bore 4 ml tube at 2000-3000 rpm for 15 min. The supernatant plasma was separated and layer of buffy coat and equal thickness of red blood cells (RBC) layer just below was picked up to prepare smears, which were stained by Giemsa's stain. The CBCS were examined for 200 oil immersion fields before being reported negative. Levels of parasitaemia in CBCS were calculated if PBS were negative.
Third, antigen detection was performed using a commercially available antigen detection kit (Advantage Malaria Card, J. Mitra and Co. Pvt. Ltd., New Delhi, India) detecting P. falciparum HRP-2 and P. vivax pLDH malaria antigen in human blood as per manufacturer's instructions. Briefly, this consisted of adding the sample (using a calibrated dropper provided with the kit) and the Assay Buffer to their corresponding wells on the test card and allowing the reaction to occur for 20 min. The appearance of three pink coloured lines one each in P.v. region (V), P.f. region (F) and control region (C) indicated that the sample was reactive for both P. vivax and P. falciparum (mixed infection). Appearance of two pink coloured lines one each at V and C region indicated that the sample was reactive for P. vivax only and appearance of two pink coloured lines one each at F and C region indicated that the sample was reactive for P. falciparum only.
For analysing the results, we used the antigen test as the gold standard since both the PBS and CBCS methods are direct detection methods and hence can be compared against another test utilising a different principle.
| ~ Results|| |
During the study period, a total of 1655 samples from an equal number of patients were received for testing for malaria parasites, of which 432 were from the outpatient department and 1223 were from the wards; 1117 samples were received from males and 538 from female patients. The three diagnostic modalities gave varied results as shown in [Table 1]. The total number of malaria positive cases was found to be 394 (23.8%, 394/1655) [Table 1]. Of these, 210 (53.3%) were P. vivax, 178 (45.1%) were P. falciparum and 6 (1.5%) were mixed infections due to both P. vivax and P. falciparum [Table 2].The highest number of cases were detected by the antigen test (384, 23.2%) followed by the CBCS method (363, 21.9%), and PBS (283, 17.1%). However, the antigen test failed to detect malaria infection in 10 patients of which 3 were diagnosed by both the PBS and CBCS and 7 were diagnosed exclusively by the CBCS method. In contrast, the antigen test detected malaria infection exclusively in 30 patients.
|Table 1: Result of samples in three different methods (PBS, CBCS and antigen) for detection of malaria|
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|Table 2: Species distribution of malaria parasites in different methods (n=394)|
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[Table 2] shows the species distribution of malaria parasites detected by the different methods. In PBS, out of 283 samples, 115 were positive for P. falciparum, 165 for P. vivax and 3 samples were positive for mixed infection. The CBCS showed 363 positive for malaria of which 161 were P. falciparum, 198 were P. vivax and 4 were mixed infection. The antigen test was positive in 384 cases with 174 being positive for P. falciparum, 204 for P. vivax and 6 for mixed infection.
[Table 3] shows the demographic profile of the patients' positive for malaria infection as regards to their age, sex and admission status. Of the 394 malaria-positive patients, 232 (58.8%) were males and 162 (41.1%) were females. The youngest patient was a 9-month-old child and the eldest was a 72-year-old female. The age distribution of the 394 patients was as follows: 169 (42.8%) between 0 and 14 years, 222 (56.3%) between 14 and 60 years and 3 (0.7%) aged over 60 years. It can be observed from the table that a large proportion of malaria-positive patients (355/394, 90.1%) required admission in the hospital wards. This is also reinforced from the fact that more number of samples from the wards (355/1223, 29.0%) tested positive for malaria as compared with that from the outpatient department (39/432, 9.0%) [Table 3].
|Table 3: Demographic profile of malaria-positive patients (n =394) under study|
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[Table 4] shows the specificity, sensitivity, positive predictive value, negative predictive value and area under the curve (AUC) of PBS and CBCS in comparison to antigen test. It was observed that while both PBS and CBCS had excellent specificity, PBS had low sensitivity (72.9%) in detecting the malaria parasites as compared with CBCS (91.9%). The usefulness of CBCS in detecting malaria parasites was further revealed by a statistically significant difference (P < 0.001 by Chi-square test) in the AUC of PBS and CBCS.
|Table 4: Sensitivity, specificity and validity of PBS and CBCS in comparison to antigen test|
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[Table 5] shows the results of PBS, CBCS and antigen test according to the parasitaemia level in the microscopy-positive cases. All the three tests detected malaria infection equally when the sample had a high parasite count of >1000 parasites/μl. However, at lower levels of parasitaemia (<200 parasites/μl), the PBS failed to detect malaria infection as compared with the CBCS and antigen test in 43 and 39 samples, respectively. Similarly, at moderate levels of parasitaemia (200-1000 parasites/μl), the PBS failed to detect malaria infection as compared with the CBCS and antigen test in 13 and 11 samples, respectively. The results of CBCS and antigen test generally correlated well. Thus, the addition of centrifugation (i.e. CBCS) to the conventional method of PBS enabled detection of 80 more cases of plasmodia infection, which included 43 (53.75%) with low levels of parasitaemia, 13 (16.25%) with moderate levels and 24 (30.0%) with pure gametocytaemia.
|Table 5: Parasite density in specimens from patients with microscopy-confirmed malaria infections|
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| ~ Discussion|| |
The present study demonstrated the performance of a modified technique for diagnosis of malaria by incorporating a centrifugation-enhanced step into the conventional method of smear preparation and examination for malaria. This helps to concentrate the parasites, which are then easily visualised by microscopy.
The results of the study show that as compared with traditional PBS examination, the CBCS detected 80 more cases as malaria-positive, especially at low- and moderate-levels of parasitaemia, which also correlated with the antigen test to a great extent. The PBS failed to detect true malaria infection in 104 (6.2%) samples, which is not at all desirable in a malaria endemic country like India. The CBCS, in contrast, failed to detect malaria infection in only 31 (1.8%) patients. Similar results were obtained in the study by Akhtar et al., in which out of 120 patients, the CBCS detected 6 more cases (49%) as malaria positive as compared with the peripheral smear (44%). Similarly, in another study  where the authors used centrifugation-enhanced heparinised capillary tubes for smear preparation and examination found that, out of 100 patients, the modified centrifuged buffy coat detected 7 more samples as malaria-positive as compared with the conventional smear technique. The addition of centrifugation to the conventional smear technique improved its sensitivity from 86.79% to nearly 100%.  In yet another study from north India,  out of 50 patients clinically diagnosed as cases of cerebral malaria, only 28 patients (56%) were positive by Leishman stained blood smear examination for various stages of P. falciparum, whereas QBC and ParaSight-F (antigen) test were positive in 47 (94%) and 46 (92%) patients, respectively.
In the present study, we have used the antigen test as the gold standard. If, in contrast, we had used PBS as the reference standard, which is the conventional method, it would have rendered 104 samples detected as malaria-positive by the antigen test to be labelled as false-positive. This can act as a deterrent to malaria testing in field conditions where antigen detection systems are used as diagnostic tests for rapid diagnosis of malaria. However, the use of CBCS lead to the demonstration of the parasites in 74 (71.1%) of these 104 samples thereby providing excellent correlation between the antigen test and direct demonstration of the parasites. Still, 30 cases were detected by antigen testing alone, which were microscopy negative. It is, however, probable that most of these apparently false-negative cases by CBCS were true-positives, which were not detected by microscopy, particularly in case of P. falciparum malaria due to sequestration limiting the number of circulating parasites at the time of blood collection or due to the parasitaemia being below the detection limit of approximately 50 parasites/μl by microscopy. This is evident from the fact that CBCS as compared with the PBS enabled detection of 80 more cases of plasmodia infection particularly at low and moderate levels of parasitaemia.
Of the numerous methods available for malaria diagnosis, one of the quick and new methods using the principle of centrifugation is the QBC assay. QBC, which utilises high-speed centrifugation along with a larger volume of blood (55-65 μl), has a definite advantage in detecting Plasmodium infection in samples with low levels of parasitaemia (1 parasite/μl).  However, the drawbacks of QBC are that it is expensive, chances of leaking and breaking of blood-filled QBC tubes in the centrifuge and inability to keep a permanent record of the test. The modified CBCS, in contrast, is cheap and provides a permanent record of the smear.
It was concluded that CBCS is an easy, rapid and accurate technique and could be adopted for reliable diagnosis of malaria in resource-limited settings where RDT and QBC may prove to be costlier options.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]