Development of an Automated Malaria Discriminant Factor Using VCS Technology

Transcription

1 Microbiology and Infectious Disease / MALARIA DETECTION USING VCS TECHNOLOGY Development of an Automated Malaria Discriminant Factor Using VCS Technology Carol Briggs, FIBMS, 1 Anabela Da Costa, Nat Dip Med Tech, 2 Lyn Freeman, Nat Dip Med Tech, 3 Ilse Aucamp, Nat Dip Med Tech, 4 Busisiwe Ngubeni, Nat Dip Med Tech, 5 and Samuel J. Machin, FRCPath 1 Key Words: Malaria; Automated screening; Monocytes; VCS technology DOI: /0PL3C674M39D6GEN Abstract Malaria diagnosis presents a challenge to all laboratories. There is a need for rapid, sensitive, and cost-effective screening on all samples, particularly in areas where malaria is endemic. Response to malaria infection involves an increased monocyte count and production of large activated monocytes. These changes can be detected by volume, conductivity, and scatter (VCS) technology on certain automated blood cell counters (Beckman Coulter, Miami, FL). The SD of the volume of lymphocytes and monocytes demonstrates a significant difference from normal when malaria is present. By using a calculation derived from the SD volume of the lymphocytes and monocytes, herein termed the malaria factor, sensitivity of 98% and specificity of 94% were demonstrated for the detection of malaria. Based on this derived discriminant, VCS technology should become a useful tool in the detection of malaria. A flag to indicate the potential presence of malaria could then be generated by the instrument if the user or manufacturer chose to do so. The rapid diagnosis of malaria is essential in endemic areas and now in Western countries owing to increasing international travel, although diagnostic skills and facilities to detect malaria infection vary tremendously worldwide. 1 An ideal screening method for the diagnosis of malaria would be based on the automated CBC count results, but the data would have to demonstrate good sensitivity and specificity. Malaria has been shown to result in the activation of circulating blood monocytes with an increase in their size and number. 2 Mononuclear phagocytic cells recognize and ingest infected peripheral RBCs. Hemozoin, a crystalline brown pigment, is produced when malarial parasites detoxify free heme liberated during hemoglobin digestion. 3 Hemozoin is phagocytosed by neutrophils 4 and monocytes. 4,5 These monocytes have recently been analyzed to allow the identification of malaria infection by automated methods. Several groups have reported using depolarized laser light for the detection of malaria More recently, a preliminary study was published in which the Coulter Gen.S hematology analyzer and VCS (volume, conductivity, and scatter) technology (Beckman Coulter, Miami, FL) were used for the detection of malaria. 11 Differences in the SD volumes of the lymphocyte and monocyte populations (so-called research population data) were used to differentiate between malaria-positive and malaria-negative samples. A calculated discriminant factor then was defined. We conducted a large joint study in South Africa and London, England, using VCS technology to validate the sensitivity and specificity for the detection of malaria using a defined discriminant (SD volume of lymphocytes SD volume of monocytes/100) to determine whether a further adaptation of the discriminant is needed using additional parameters from Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN 691

2 Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY the CBC count analysis. Samples included in the study were from adult patients. Samples from healthy people constituted the control group. In addition to malaria-positive samples, samples in which malaria studies were requested but had negative results and samples known to be infected with HIV were also studied. HIV is a common disease in malaria-endemic areas and may also cause changes in the lymphocyte VCS population data. 12 Therefore, HIV infection may limit the sensitivity and specificity of malaria detection when a calculation including the lymphocyte SD volume is used. This derived discriminant factor would be compared with standard microscopic and routine immunologic diagnostic techniques. Materials and Methods Patients Peripheral blood samples collected into K 3 EDTA tubes (Becton Dickinson, Franklin Lakes, NJ) were analyzed at 5 laboratories. All samples were analyzed within 8 hours after collection. We randomly selected 1,079 samples from apparently healthy adults in whom all CBC count parameters were within reference ranges. The patient diagnostic group studied consisted of 275 adult samples in which workup for malaria was requested by clinicians. Of these samples, 147 were positive, and as far as we were able to confirm, none of these samples were from HIV+ patients. The malaria-negative samples were mostly from patients undergoing workup for pyrexia; many of these samples later were found to be positive for other viral or infectious illnesses. Some samples were from people returning to London from malaria-endemic regions. Samples from 51 adults positive for HIV but negative for malaria also were analyzed. Automated Technology All samples were analyzed on hematology instruments using VCS technology. Four laboratories used the Coulter LH 750 (Beckman Coulter) and 1 the Gen.S (Beckman Coulter). The WBC count is performed using the impedance method. After lysis of the RBCs, the reagent modifies the nucleated cells, shrinking the cytoplasm and, therefore, affecting the original WBC size. The histogram produced after analysis of the WBC count differentiates the WBCs from the nucleated RBCs, platelets, and debris. The debris may contain apoptotic cells, cell membranes, and, perhaps, infective organisms. This separation is achieved on the Gen.S using a fixed volume threshold at 35 fl and on the LH750 with a fixed threshold and a moving threshold producing a result called the corrected WBC count. WBC size distribution histograms are illustrated in Figure 1, which demonstrates a normal population of WBCs (Figure 1A) and one from a patient with malaria infection (Figure 1B) and a visible peak before the 35-fL threshold. For the WBC differential count, the analyzer makes 3 measurements as each cell passes through a flow cell, which is an electro-optical flow cytometer. Volume, conductivity, and laser light scatter are measured for each cell. The WBC volume is measured using impedance; the cells are in their near-native state, and this gives a good indication of the cell volume as it circulates in the blood. The conductivity is measured using a radiofrequency probe that determines the nuclear shape, lobularity, density, and nuclear/cytoplasmic ratio. Laser light technology analyzes the median light scatter of each cell to quantify the specific granularity of the cells. The instrument provides a 2-dimensional histogram of volume and scatter showing the 4 main populations of WBCs, and this is included on the printed report. Figure 2 demonstrates 2 examples of this histogram, 1 from a normal population of WBCs (Figure 2A) and 1 from a patient with malaria infection (Figure 2B) demonstrating the A B Figure 1 A normal size distribution WBC histogram (A) and one from a patient with an infection with 3.6% Plasmodium falciparum (B) demonstrating a peak (arrow) at the threshold of the histogram. The x-axis gives the cell size (fl) and the y-axis, the cell count. 692 Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN

3 Microbiology and Infectious Disease / ORIGINAL ARTICLE A B Monocytes Monocytes Lymphocytes Lymphocytes V C S NE Mean SD LY MO EO NE LY MO EO Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD V C S Figure 2 A normal VCS (volume, conductivity, and scatter) plot (A) and one from a patient with an infection with 3.6% Plasmodium falciparum (B) demonstrating heterogeneity of the volume of the lymphocytes and monocytes. EO, eosinophils; LY, lymphocytes; MO, monocytes; NE, neutrophils. volume heterogeneity (anisocytosis) of the lymphocytes and monocytes. This is due to the presence of large activated cells of the monocyte lineage or monocytes with histiocytic changes Image 1. The range for the SD lymphocyte volume in normal patient samples observed in this study was 11.4 to 19.7 and for the monocytes, 13.7 to Reference Methods parasites can be seen when the tube is examined under a UV light source. 13 Immunologic Methods Malaria Antigen Histodine-Rich Protein 2. These commercial kits are based on the capture of the parasite antigen, histodine-rich protein 2 (HRP-2). BINAX NOW (Portland, ME) and MAKROmed (Makro Medical, Johannesburg, South Blood Films Malaria was diagnosed as positive or negative by careful examination of thick and thin blood films. In most laboratories, thin films were stained with May-Grünwald-Giemsa and thick films with Field stain. One laboratory used Giemsa stain for thick and thin films. Parasite counts were performed on all samples positive for Plasmodium falciparum by noting the number of RBCs containing parasites seen in 10,000 RBCs (approximately 40 monolayer cell fields of a standard microscope using a 100 objective). The number of parasitized cells seen is reported as a percentage. The presence or absence of malaria was confirmed by a fluorescent or an immunologic method, depending on local laboratory protocol. Fluorescent Method The quantitative buffy coat method (QBC) (Becton Dickinson) combines an acridine orange coated capillary tube and an internal float to separate layers of WBCs and platelets using centrifugation. Parasites concentrate below this layer of cells and appear in the upper layer of RBCs. The Image 1 Blood film showing a large activated cell of monocytic lineage with histiocytic changes (May-Grünwald- Giemsa, 1,000). Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN 693

4 Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY Africa) were used in this study. HRP-2 is a protein produced by P falciparum and expressed on the RBC membrane in infected peripheral blood. A panmalarial antigen expressed by P falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae also is included. Malaria Antigen Plasmodium Lactate Dehydrogenase. One laboratory used the OptiMal test (OptiMal, Flow, Portland, OR) for rapid diagnosis of malaria. Three mouse monoclonal antibodies to Plasmodium lactate dehydrogenase (pldh) are used in the test. pldh is an enzyme expressed at high levels in asexual stages of malaria parasites. It has been found to be present in all 4 malaria species. 14 Two of the monoclonal antibodies are pan-specific, recognizing all 4 species of malaria; the other antibody is specific for P falciparum LDH. Statistical Methods CBC count, differential, and the values for the SD of the volume of the lymphocytes and monocytes were recorded for all samples. The malaria factor was calculated as follows: Malaria Factor = (SD Volume of Lymphocytes SD Volume of Monocytes)/100 Receiver operating characteristic (ROC) analysis was performed on the malaria factor to determine whether a satisfactory cutoff value could be established for the detection of malaria in a blood sample. The results of the SD volume of the lymphocytes and monocytes, mean volume of monocytes, the malaria factor, platelet count, and eosinophil count were analyzed by using the Mann-Whitney U test 15 to determine any statistical differences between malaria-positive and malaria-negative samples, HIV+ samples, and the control group. Results Of the malaria-positive samples, 120 were due to P falciparum, 11 to P vivax, 7 to P ovale, 1 to P malariae, and 1 to mixed P falciparum and P vivax infection. Seven samples gave positive results by an immunochromatographic and/or a fluorescent method, but no parasites were seen on the blood film. Table 1 shows the results of the Mann-Whitney U test comparing all 3 groups malaria-positive, malaria-negative and HIV+ with the control group. A P value of less than.05 was considered statistically significant. All 3 groups of patient samples had highly significantly different values from normal for the variables SD volume of lymphocytes, SD and mean volumes of monocytes, and the malaria factor, but most important, the malaria-negative and HIV+ samples also were significantly different from the malaria-positive samples. The malaria-positive samples were significantly higher for these parameters than the malaria-negative and HIV+ samples. The malaria-negative and HIV+ groups did not have a significant difference in values for mean and SD volume of monocytes. For platelets, the malaria-positive and malaria-negative groups had lower counts than the control group; however, the Table 1 P Values for Comparisons of SD Volume of Monocytes and Lymphocytes, Mean Volume of Monocytes, Malaria Factor, and Platelet and Eosinophil Counts in Four Groups * Diagnosis Malaria-Positive Malaria-Negative HIV+ SD volume of monocytes Control group <.0001 <.0001 <.0001 Malaria-negative.347 SD volume of lymphocytes Control group <.0001 <.0001 <.0001 Malaria-positive <.0001 <.0001 <.0001 Malaria-negative <.0001 Mean volume of monocytes Control group <.0001 <.0001 <.0001 Malaria-negative.318 Malaria factor Control group <.0001 <.0001 <.0001 Malaria-negative <.0001 Platelet count Control group < Malaria-negative.705 Eosinophil count Control group <.0001 < Malaria-negative.003 * Samples from healthy people constituted the control group. Significance was calculated using the Mann-Whitney U test; a P value of less than.05 was considered statistically significant. 694 Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN

5 Microbiology and Infectious Disease / ORIGINAL ARTICLE platelet counts of the malaria-positive samples were significantly lower than those of the malaria-negative group. Malaria-positive samples also had a lower platelet count than the HIV+ group, but platelet counts on malaria-negative and HIV+ samples were not significantly different. Eosinophil counts on the malaria-positive samples were significantly lower than for all the other patient groups. The malaria factor was submitted to a ROC analysis. Figure 3 shows the ROC curve, cutoff value for the malaria factor, corresponding sensitivity and specificity, and the area under the curve. By using a malaria factor of greater than 3.7 as the cutoff criterion, 144 of 147 samples were identified correctly by the instruments as positive for malaria (range of parasitemia, 0.001%-38.9%). The sensitivity was 98% and the specificity, 94%. True-positive, true-negative, false-positive, and false-negative results are given in Table 2. These figures were used to calculate the positive predictive value (PPV) and negative predictive value (NPV) for this study. The PPV was 65% and the NPV, 99.7%. The 78 false-positive samples were from 1 control subject, 45 patients undergoing workup for malaria but with negative results, and 32 patients with HIV. The 3 false-negative results were from patients with P falciparum; the parasite counts were reported as 0.01%, 0.1%, and less than 0.5%. There was no relationship found between the percentage of parasitemia and the value of the malaria factor. The type of Plasmodium species present did not seem to affect the value of the malaria factor. The average malaria factor for P falciparum was 6.2; for P vivax, 5.93; and for P ovale, 5.7. All 7 samples with positive results by a fluorescent or an immunologic method but with no parasites detected on the blood film had a malaria factor of greater than 3.7 and, therefore, were classified as positive by the hematology instruments. However, only 1 sample demonstrated a typical peak at the WBC threshold monitor. If a malaria factor of greater than 3.7 is used in conjunction with an SD volume of monocytes of greater than 23.2, a mean volume of monocytes greater than 180, a platelet count less than /µl ( /L), an eosinophil count less than 0.15% (0.0015), and the presence of a peak in the WBC threshold monitor histogram, the number of false-positive samples is reduced to 61 from the original 78. These were 40 samples that were tested for malaria but were negative, 20 HIV+ samples, and 1 control sample. The sensitivity was still 98%, but the specificity improved to 95%. By using the following algorithm: Malaria factor 3.7, the absence of a WBC peak, platelet count > /µl ( /L), eosinophil percentage >0.15%, SD volume of monocytes <23.2, and mean volume of monocytes <180 the PPV becomes 70% and the NPV, 99.7%. Sensitivity (True Positives) Specificity (False Positives) Figure 3 Receiver operating characteristic curve of the malaria factor. The cutoff value for the malaria factor was 3.7; sensitivity, 98%; specificity, 94%; and area under the curve, Table 2 Numbers of TP, TN, FP, and FN Results for Detection of Malaria When a Cutoff of 3.7 Is Used for the Malaria Factor * Discussion No. of Cases TP 144 TN 1,180 FP 78 FN 3 FN, false negative; FP, false positive; TN, true negative; TP, true positive. * 1,405 total samples. The accepted worldwide gold standard used for the routine laboratory diagnosis of malaria is the microscopic examination of stained thick and thin blood films. This procedure is difficult, time-consuming, and, particularly at low levels of parasitemia, requires special expertise. 16 During the past decade, efforts have been made to replace the traditional blood film for the diagnosis of malaria. Polymerase chain reaction has been proven to be sensitive in the diagnosis of all 4 species of malaria. 17,18 However, it is expensive and impractical in the routine diagnosis of malaria. The QBC blood parasite detection method is used in some laboratories as a backup to blood films or as an initial screening technique; thick and thin films are examined only on QBC-positive samples. The disadvantages of this method are the high cost of the equipment and consumables. In addition, the fluorescent stain is nonspecific, Howell-Jolly bodies Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN 695

6 Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY will fluoresce with acridine orange, and the specificity for non P falciparum malaria is low owing to the denser late stages of parasites, which may be hidden in the mononuclear layer. Sensitivities of immunologic methods for detecting malaria remain a problem. In nonendemic areas for malaria, the parasitemia is often very low. The average parasitemia seen in patients attending the Hospital for Tropical Diseases, London, is between 0.1% and 0.001%. 19 These levels are at the lower limit of the capabilities of most devices involving capture of HRP-2 and pldh. These methods also are prohibitively expensive for some laboratories. We reevaluated a fully automated rapid method as a potential diagnostic tool for malaria using Coulter VCS technology on a large number of samples. This detection method depends on changes in the SD volumes of lymphocytes and monocytes in the presence of malaria. By using a calculation, a malaria factor is produced. The values for the SD volumes of lymphocytes and monocytes in blood anticoagulated in EDTA stored at room temperature were demonstrated previously to be stable for 11 hours. 11 There is little variation in the parameters between instruments or in samples from healthy people tested repeatedly during a 1-month period, with coefficients of variation of 4%. 11 By using a cutoff value for the malaria factor of greater than 3.7 as an indicator of malaria infection, the specificity was 94% and the sensitivity, 98%. These values are better than any previously reported using other automated hematology instrumentation and also are superior to some quoted sensitivities and specificities for fluorescent techniques and pldh capture methods Table 3 ). 7,9,11,20-26 False-positive results from this study were almost exclusively from samples from patients with infections other than malaria and patients with HIV. Some bacterial and viral infections, including HIV, seem to increase the SD volume of lymphocytes 12 and, therefore, may cause false-positive results for malaria when the malaria factor is used for diagnosis. When a malaria factor of greater than 3.7 is used in conjunction with the algorithm using the SD volume of monocytes, mean volume of monocytes, low platelet and eosinophil counts, and the presence of a peak in the WBC threshold monitor histogram to detect malaria, specificity is improved. Seven samples gave a positive result for malaria by an immunologic test but were negative on the blood film, and 3 of these also were positive by the QBC. All samples had a malaria factor greater than 3.7, indicating the presence of large activated cells of monocytoid lineage. Six of these samples did not demonstrate a visible peak at the threshold of the WBC size distribution curve, indicating that there probably were no malaria-infected erythrocytes or gametocytes present. A previous study using the Abbott Cell-Dyn 4000 (Santa Clara, CA) and laser light depolarization analysis found that cases of treated convalescent malaria with no residual parasitemia demonstrated abnormal depolarization patterns. 9 This is explained by the kinetics of hemozoin clearance. The removal of pigment-containing monocytes is slower (median, 216 hours) than parasitized erythrocytes (median, 72 hours). 27 In the same way that other authors have detected malaria using the presence of hemozoin in the monocytes, we observed that the histiocytic monocytes of infected patients were larger and, therefore, had increased volumes and SD volumes. These cells persist in the circulation after parasite clearance. HRP-2 also has been shown to persist after the symptoms of malaria subsided and parasites cleared from the blood. 28 The detection of unsuspected malaria is important. In endemic areas, there is limited access to clinical and laboratory expertise, and immunologic and fluorescent tests often are Table 3 Comparison of Results From the Present Study for Detecting Malaria With Other Automated Hematology Instruments or Tests Reported in the Literature * Instrument/Test Sensitivity (%) Specificity (%) Author CD3500, Abbott, Santa Clara, CA Hanscheid et al 7 CD3200, Abbott Scott et al 26 CD3700, Abbott Scott et al 26 CD3000, Abbott Grobusch et al 20 CD4000, Abbott Scott et al 9 Gen.S, Beckman Coulter, Miami, FL Fourcade et al 11 Gen.S/LH 750, Beckman Coulter Malaria factor, 98; algorithm, 98 Malaria factor, 94; algorithm, 95 Present study QBC (fluorescent), Becton Dickinson, 93 (Plasmodium falciparum, 93 Wongsrichanalai et al 21 Franklin Lakes, NJ 93 (Non P falciparum, 52 Gaye et al 22 ICT P.f/P.v (immunologic histodine-rich P falciparum, 96 P falciparum, 99 Mills et al 23 protein 2), Binax, Portland, ME Plasmodium vivax, 87 P vivax, 99 Farcas et al 24 OptiMAL (immunologic Plasmodium P falciparum, 94; P vivax, 88 P falciparum, 100; P vivax, 99 Palmer et al 25 lactate dehydrogenase), Flow, Portland, OR * The algorithm is as follows: malaria factor 3.7, the absence of a WBC peak, platelet count > /µl ( /L), eosinophil percentage > 0.15%, SD volume of monocytes <23.2, and mean volume of monocytes < Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN

7 Microbiology and Infectious Disease / ORIGINAL ARTICLE prohibitively expensive. In nonendemic countries, symptoms of fever are rarely caused by malaria, and it may not be considered in the differential diagnosis. Even when a malaria test is requested, there may be poor skills for the detection, identification, and quantification of malaria. A study in Canada found that community-based microscopic diagnosis provided incorrect species identification in 64% of cases. 29 In a published audit in the United Kingdom, 13 of 34 cases in which malaria parasites were not seen by the reference laboratory were reported as positive by the submitting laboratories. 30 A false diagnosis of malaria had been reported. High frequencies of technical errors such as wrong ph in staining or poor quality film also were noted. In the last 3 exercises from the United Kingdom National External Quality Assessment Scheme, 6 blood films were sent to all laboratories registered. From 1,389 results, 13 malaria infections were missed and 14 malaria infections reported when the blood film was negative. In 117 other cases, parasite infections, such as microfilaria or trypanosomes, also were reported falsely. 31 The present study demonstrated a fully automated rapid method for the potential detection of malaria infection in adults. The results of the malaria factor are available at the same time as part of the CBC count and at no extra cost because these data are reported automatically with the differential results. Further studies are needed to validate the usefulness of the malaria factor in pediatric patients. The VCS parameters may be different in children, and children may have changes different from those in adults in response to general infection. By using the previously described algorithm, a suspect malaria flag could be generated on the analyzer, allowing the detection of cases of unsuspected malaria and, therefore, early diagnosis of the disease with the potential of reducing the possibility of serious complications. The flag could be used as a screening device for people returning from malaria-endemic areas even if there are no symptoms of the disease. With an NPV of 99.7%, the absence of the flag would indicate the absence of malaria. Kain et al 29 reported that for cases of P falciparum malaria involving patients admitted to centers other than tropical disease units, the diagnosis of malaria was missed by the physician in 61% of cases. In addition, 16% of patients with a history of fever reported that they were examined by 3 or more physicians before malaria was suspected and the test requested. The suspect malaria flag should not be considered a replacement for the gold standard microscopic examination of the blood film but should alert laboratory staff to give particular attention to the examination of blood films when the instrument has reported the flag. A flag to indicate the potential presence of malaria parasites could be a valuable diagnostic method for the detection of malaria and may become a routine parameter in the diagnosis of the disease. From the 1 Department of Haematology, University College Hospital London, London, England; 2 Haematology, Doctors du Buisson, Bruinette Kramer, Pretoria, South Africa; 3 Haematology, 1 Military Hospital, Pretoria; 4 Haematology, Lancet Laboratories, Pretoria; and 5 National Health Laboratory Services, Johannesburg. Address reprint requests to C. Briggs: Dept of Haematology, University College Hospital London, 60 Whitfield St, London, W1T 4EU England. Beckman Coulter provided financial travel support to participants in the study to discuss and analyze the data. References 1. World Health Organization. New Perspectives: Malaria Diagnosis: Report of a Joint WHO/USAID Consultation, October Geneva, Switzerland: World Health Organization; Ho M, Webster HK. Immunology of human malaria: a cellular perspective. Parasite Immunol. 1989;11: Arnse P, Schwarzer E. Malaria pigment (haemozoin): a very active inert substance. Ann Trop Med Parasitol. 1997;91: Amodu OK, Ademeyo AA, Olumese PE, et al. Interleucocyte malaria pigment in asymptomatic and uncomplicated malaria. East Afr Med J. 1997;74: Amodu OK, Ademeyo AA, Olumese PE, et al. Interleucocyte malaria pigment and clinical severity of malaria in children. Trans R Soc Trop Med Hyg. 1998;92: Mendelow BV, Lyons C, Nhlangothi P, et al. Automated malaria detection by depolarization of laser light. Br J Haematol. 1999;104: Hanscheid T, Melo-Christino J, Pinto BG. Automated detection of malaria pigment in white blood cells to diagnose malaria in Portugal. Am J Trop Med. 2001;64: Wever PC, Henskens YM, Kager PA, et al. Detection of imported malaria with the Cell-Dyn 4000 hematology analyser. J Clin Microbiol. 2002;40: Scott CS, van Zyl D, Ho E, et al. Automated detection of malaria-associated intraleucocytic haemozoin by Cell-Dyn CD4000 depolarization analysis. Clin Lab Haematol. 2003;25: Suh IB, Kim HJ, Kim JY, et al. Evaluation of the Abbott Cell- Dyn 4000 hematology analyser for detection and therapeutic monitoring of Plasmodium vivax in the Republic of Korea. Trop Med Int Health. 2003;8: Fourcade C, Casbas MJC, Belaouni H, et al. Automated detection of malaria by means of the haematology analyser Coulter GEN.S. Clin Lab Haematol. 2004;26: Simon R, Lima M, Arroyo JL, et al. Utility of the Beckman Coulter GEN.S investigation screen parameters in order to diagnostic the etiology of a lymphocytosis [abstract]. Blood. 2000;96:45b. Abstract Baird J, Purnomo K, Jones TR. Diagnosis of malaria in the field by fluorescence microscopy of QBC capillary tubes. Trans R Soc Trop Med Hyg. 1992;86: Piper R, Lebras J, Wentworth L, et al. A capture diagnostic assay for malaria using Plasmodium falciparum lactate dehydrogenase (pldh). Am J Trop Med Hyg. 1999;60: Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN 697

8 Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY 15. Hart A. Mann-Whitney test is not just a test of medians: differences in spread can be just as important. Br J Haematol. 2001;323: Warhurst DC, Williams JE. Laboratory diagnosis of malaria. J Clin Pathol. 1996;49: Snounou G, Viriyakosol S, Jarra W, et al. Identification of four human malaria species in field samples by the polymerase chain reaction and detection of high prevalence of mixed infections. Mol Biochem Parasitol. 1993;58: Kawamoto F, Miyake H, Kaneko O, et al. Sequence variation in the 18S rrna gene, a target for PCR-based malaria diagnosis in Plasmodium ovale from southern Vietnam. J Clin Microbiol. 1996;34: Moody A. Rapid diagnostic test for malarial parasites. Clin Microbiol Rev. 2002;15: Grobusch MP, Hansscheid T, Kramer B, et al. Sensitivity of hemozoin detection by automated flow cytometry in nonand semi-immune malaria patients. Cytometry B Clin Cytom. 2003;55B: Wongsrichanalai C, Pornsilapatip J, Namsiriponpun V, et al. Acridine orange fluorescent microscopy and the detection of malaria in populations with low-intensity parasitemia. Am J Trop Med Hyg. 1991;44: Gaye O, Diouf M, Diallo S. A comparison of thick films, QBC malaria, PCR and PATH falciparum malaria test strip in Plasmodium falciparum diagnosis. Parasite. 1999;6: Mills CD, Burgess DCH, Taylor HJ, et al. Evaluation of rapid and inexpensive dipstick assay for the diagnosis of Plasmodium falciparum malaria. Bull World Health Organ. 1999;77: Farcas GA, Zhong K, Lovegrove FE, et al. Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers. Am J Trop Med Hyg. 2003;69: Palmer CJ, Lindo JF, Klaskala WI, et al. Evaluation of the OptiMAL test for rapid diagnosis of Plasmodium vivax and Plasmodium falciparum malaria. J Clin Microbiol. 1998;36: Scott CS, van Zyl D, Ho E, et al. Automated detection of WBC intracellular malaria-associated pigment (hemozoin) with Abbott Cell-Dyn 3200 and Cell-Dyn 3700 analyzers: overview and results from the South African Institute for Medical Research (SAIMR) II evaluation. Lab Hematol. 2002;8: Day NP, Pham TD, Phan TL, et al. Clearance kinetics of parasites and pigment-containing leukocytes in severe malaria. Blood. 1996; Laferi H, Kandel K, Pichler H. False positive dipstick test for malaria [letter]. N Engl J Med. 1997;337: Kain KC, Harrington MA, Tennyson S, et al. Imported malaria: prospective analysis of problems in diagnosis and management. Clin Infect Dis. 1998;27: Milne LM, Kyi MS, Chiodini PL, et al. Accuracy of routine laboratory diagnosis of malaria in the United Kingdom. J Clin Pathol. 1994;47: UK NEQAS (H), United Kingdom National External Quality Assessment Scheme Surveys (October 2004, January 2005, April 2005) [survey results]. 698 Am J Clin Pathol 2006;126: DOI: /0PL3C674M39D6GEN