IJCRIMPH Articles:

Evaluation of Falcivax against Quantitative Buffy Coat (QBC) for the Diagnosis of Malaria
International Journal of Collaborative Research on Internal Medicine & Public Health, 2010 Vol. 2 No. 5 (Pages 132-140)
Author: Bimala Gurung (1), Indira Bairy (1), Jagadishchandra (1), Chetan Manohar (2)

(1) Department of Microbiology, Kasturba Medical College, Manipal University, India
(2) Department of Pathology, Kasturba Medical College, Manipal University, India


Abstract  |  Full Text  |  PDF

Paper review summary:
Paper submission: February 10, 2010
Revised paper submission: April 30, 2010
Paper acceptance: May 04, 2010
Paper publication: May 07, 2010

Introduction
Malaria is one of the oldest diseases of mankind caused by a single-cell Apicomplexa of the genus Plasmodium and transmitted by biological vectors of the genus Anopheles. According to the world malaria report released in 2006 by the World Health Organization, there were 247 million malaria cases, 3.3 billion people at risk, and 881,000 deaths from 109 countries. These deaths were primarily in Africa (91%) and in children under 5 years of age (85%). India had an estimated 1.52 million malaria cases reported in 2008 that account approximately 60% of cases in the WHO South-East Asia Region. The states inflicted are Uttar Pradesh, Bihar, Karnataka, Orissa, Rajasthan, Madhya Pradesh and Pondicherry ¹. Because of immigrant population and resistant to insecticides, this part of Karnataka is witnessing an increasing prevalence of malaria cases over a period of 5 years. In the year 2008 alone, a total of 62,864 cases of malaria and 29 malaria deaths were reported from Karnataka state ². The global impact of malaria has spurred interest in developing diagnostic strategies that will be effective not only in resource-limited areas, where malaria has a substantial burden on society, but also in developed countries, where expertise in malaria diagnosis is often lacking because they do not come across adequate cases of malaria and are not properly trained to report cases ^3,4. Endemic malaria, migration, and foreign travel all contribute to the malaria diagnostic problems faced by the laboratory that may not have appropriate microscopy expertise available. Changing patterns of accepted morphologies appearances of malaria species, possibly due to drug pressure, strain variation, approach to blood collection, and have created diagnostic problems that can’t be easily resolved merely by references to an atlas of parasitology ⁵. The accurate diagnosis of malaria infection is important in order to reduce severe complications and mortality.

Microscopic detection of appropriately stained blood smear for the diagnosis of malaria has been the standard diagnostic technique for identifying malaria infections for more than a century. The technique is accurate and reliable when performed by skilled microscopists using defined protocols ^6,7. The problem associated with implementing and sustaining a level of skilled microscopy appropriate for clinical diagnosis; particularly has promoted the development of Malaria Rapid Diagnostic Devices (MRDD) ^8,9. The current MRDD are based on antigen capture immunoassays methodologies using immunochromatographic strip (ICS) technology. Most of the ICS will contain monoclonal antibodies directed against antigens such as histidine rich protein (HRP-2) and Plasmodium lactate dehydrogenase (pLDH) immobilized on a nitrocellulose strip ¹⁰. The newer generations of MRDD are using more antigens like merozoite protein 2 and circumsporozoite proteins.  Further these antigens are obtained using recombinant techniques. This study was done for evaluation of Antigen detection (Falcivax) against detection of parasites by QBC for the diagnosis of malaria P. falciparum & P. vivax, in patients attending Kasturba Hospital, Manipal.

Materials & Methods
The present comparative study was done from February 2008 to July 2009.

Hundred symptomatic patients attending outpatient department of Kasturba Hospital meeting the specific inclusion criteria were enrolled for the study.

The inclusion criteria were:

1)      Symptoms of fever > 38^oC, or headache, or history of fever within the past 72 hrs

2)      Age  ≥15 years

The exclusion criteria were:

1)      Patients who had been on anti-malarial therapy

2)       Treated with anti-malaria therapy within last 2 weeks

Study group 1- 70 Patients suspected of Malaria and are positive for malarial parasites (P. falciparum or P. vivax) by QBC.

Study group 2- 30 Patients suspected of Malaria but negative for malarial parasites (P. falciparum or P. vivax) by QBC.

Approximately 2ml of blood was collected by venipuncture into vacutainer containing EDTA as anticoagulant from all patients in study group 1 & 2. Tests were performed following manufacturer’s instructions on 8 samples at a time using both Falcivax (Zephyr biomedicals) & Anti-Malaria profile (Euroimmun).

Smear status by QBC, clinical features and relevant laboratory data of each sample was noted down.

Statistical Analysis
Validity of tests was statistically analyzed in terms of sensitivity, specificity, positive and negative predictive values. Results were analyzed by Mc Nemar’s test by using SPSS computer package.

Results
A total of 100 patients enrolled in the present study were belonging to the age group of the 15 to 65 years. Out of total 100 patients, 76 were males and 24 females. Among the tested 70 were positive and 30 were negative by QBC for malaria. Out of 70, 32 (45.7%) were due to P. falciparum and 37 (52.9%) were due to P. vivax and one (1.4%) of had mixed infection with P. falciparum as well as P. vivax (Table 1).

Table 1: Results of QBC for diagnosis of malaria 

Grading of malarial parasites was done by plus method. Most of the patients with P. falciparum infection had a lower parasite load of 1+ (39.4%) where as in contrast; majority of patients with P. vivax had a higher parasite load of 4+ (34.21%) (Table 2).

Table 2: Results of QBC for estimating relative quantity of parasites for P. vivax and P. falciparum

The Falcivax test showed 63 samples positive out of 100 in which 35 (55.5%) were P. falciparum, 26 (41.3%) for P. vivax, 2 (3.2%) cases tested positive for both P. falciparum and P. vivax (Table 3).

Table 3: Results of Falcivax test for diagnosis of malaria

For QBC in the detection of malaria, Falcivax test showed sensitivity, specificity, positive and negative predictive values of 90.0%, 100.0%, 100.0% and 81.0% respectively. The P value (p=0.04) was statistically significant (Tabel 4).

Table 4:  Comparison of QBC and Falcivax test for detection of malaria

P=0.04 s

Sensitivity-90.0%, Specificity-100.0%, Positive predictive value-100.0%, Negative predictive value-81.0%

In comparison with the study control QBC, the sensitivity, specificity, positive and negative predictive values of Falcivax test in detection of P. vivax were 73.68%, 100.0%, 100.0% and 86.2% respectively. The P value (0.004) is statistically very significant. In present study, comparing the sensitivity, pecificity, positive and negative predictive values of Falcivax test in comparison with QBC in detection of P. falciparum were 100.0%, 97.01%, 94.02% and 100.0% respectively.  

Discussion
Malaria is still a major global health problem, killing more than one million people every year.  A key to effective management of malaria to reduce mortality and morbidity is accurate and prompt diagnosis. Since the introduction of the MRDD in early 1990s new rapid diagnostic techniques have been developed and evaluated widely in recent years, but the rapid introduction, withdrawal, and modification of commercially available products, variable quality control in manufacturing, and potential decrements in test performance related to the stability of stored test kits have rendered these reviews largely obsolete ^5,14,15. The World Health Organization (WHO) has recommended a minimal standard of 95% sensitivity for P. falciparum densities of 100/μl and a specificity of 95% ^16,17. The development of easy, rapid, and accurate tests for the detection of plasmodial infection is highly desirable.

In our study out of 100 patients, 70 were  positive and 30 were negative for malaria by QBC and 63 patients were tested for malaria by Falcivax test, of whom 35 (55.5%) were for P. falciparum followed by 26 (41.3%) for P. vivax. Two (3.2%) cases were tested for both P. falciparum and P. vivax. MRDD are all based on the same principle and detect malaria antigen in blood flowing along a membrane containing specific anti-malaria antibodies; they do not require laboratory equipment. In contrary QBC is although simple, reliable fluorescent staining of malaria parasites; it requires specialized instrumentation ¹⁹. Studies of MRDD have demonstrated widely varying sensitivity, ranging from poor to 100%. The sensitivity of QBC for detection of malaria parasites in infections with parasite levels of >100 parasites/μl (0.002% parasitemia) has been reported to range from 41 to 93% and the specificity for infections with P. falciparum is excellent (>93%) ^20,21. 

Commercially available antigen detection Falcivax test used to detect (Pf. HRP-2) for P. falciparum and specific pLDH for P. vivax were used. pLDH is a soluble glycolytic enzyme expressed at high levels in asexual stages of malaria parasites ²². It has been found in all four human malaria species ^23,24. Iqbal et al in their study concluded that pLDH has 97% sensitivity when parasite levels is > 100/μl parasites but failed to detect when parasite load was >50/μl parasites, but microscopy was able to detect ²⁵. In our study, sensitivity was 73.68% with the parasite load of >3+ (11-100 parasites per QBC field) in 25 cases out of 37 for detection of P. vivax. Several workers have noted that during therapy the clearance of parasites from blood films and decreased pLDH levels parallel each other ^26-28. This advocates the possible use of tests measuring pLDH as valuable tools in monitoring anti-malarial therapy particularly in areas where other facilities not available. Parija et al have found the sensitivity of 70.0 % where as we observed 73.68% of Falcivax test ²⁹.

In the study comparing the QBC with the Falcivax test for the detection of P. falciparum, the sensitivity, specificity was 100% and 94.02.  Most products target a P. falciparum-specific protein like HRP 2 ¹⁷ and HRP-2 from sexual stages of P. falciparum is more readily detected than pLDH. HRP-2 antigen detection for detection of P. falciparum in blood samples have shown an overall average sensitivity of 77 to 98% when >100 parasites/μl (0.002% parasitemia), and specificity of 83 to 98% for P. falciparum compared with thick blood film microscopy. We observed the sensitivity of 100.0% which is in agreement with the result of Moody et al ⁵.

Two cases were negative   by QBC but    positive by Falcivax test. This could be explained by persistence of antigenemia beyond the clearance of parasitemia in certain cases which reduce the usefulness of the test response ⁵.

Among the eight cases which were negative by Falcivax positive by QBC, seven were P. vivax and one was P. falciparum. This can be explained by certain artifact seen in blood like Howell Jolley bodies that resemble the ring form of P. falciparum ²⁹ and polymorphism of targeted antigens ³⁰.

Conclusion
The study results suggest that MRDD for the detection of plasmodial antigens may develop as an important diagnostic tool and can prove to be a valuable adjunct to clinical assessment of the patient and QBC. These tests are rapid, simpler to perform and to interpret.

 The 100.0% sensitivity for identification of P. falciparum conveys that this test using HRP-2 (Falcivax test) can substitute for diagnosis of malaria under certain cases but P.vivax targeting pLDH antigen (Falcivax test) has shown a lower sensitivity of 73.68% and a higher specificity of 100.0%, thus may rule out false positive.

Thus QBC still continues to be a better option than MRDDs for the detection of Plasmodium infections in health care facilities with all expertise.  But the limitation of the test is its being poor in species identification ⁵.  If facilities are available combination of QBC with MRDDs help in rapid diagnosis of malaria and help in monitoring the treatment.

References
1.        WHO, World Malaria Report, 2008.

2.        Malariasite.com

3.        Bell D, Wongsrichanalai C, Barnwell JW. Ensuring quality and access for malaria diagnosis: how can it be achieved? Nat. Rev. Microbiol. 2006; 4:S7–S20.

4.        Reyburn H, Mbakilwa H, Mwangi R, Mwerinde O, Olomi R, Drakeley C, Whitty CJ. Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: randomized trial. BMJ 2007; 334:403.

5.        Moody A. Rapid diagnostic Tests for Malaria Parasites. Clinical Microbiology reviews 2002; 15:66-78.

6.        Bain BJ, Chiodini PL, England M, Bailey JW. The laboratory diagnosis of Malaria. The Malaria working party of the general hematology task force of the British committee for standards in hematology. Clinical Lab. Hematology 1997; 19:165-170.

7.        Warshurst DC, Williams. JE. Laboratory diagnosis of Malaria. J. Clin. Patholgy. ACP broadsheet no. 148. 1996; 49:533-538.

8.        World Health Organization. Malaria diagnosis memorandum from a WHO meeting. Bull. WHO 1988; 66:575-594.

9.        World Health Organization. A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO informal consultation on recent advances in diagnostic techniques and Vaccines for Malaria. Bull. WHO 1996; 74:47-54.

10.    Shiff CJ, Premji Z, Minjas N. The rapid manual Parasight-F test. A new diagnostic tool for Plasmodium falciparum infection. Trans. R.Soc.Trop. Med. Hyg. 1993; 87:646-648.

11.    White NJ. Malaria, In: Jauregg JW, Lebenserinnerungen L, Schonbauer (editor) Manson’s Tropical diseases. 21^st edition. U.S.A, Elsevier 2008: 1205-1295.

12.    Jauregg JW, Lebenserinnerinnerungen L, Schonbauer M. Jantsch (eds), Wein, Springer Verlag 1950; 157.

13.    White NJ, Berman JG. Malaria and Babesiosis: Disesases Caused by Red Cell Parasites In: Anthony WJ, Fauci S. (editors) Harrison's Principles of Internal Medicine. 16^th edition Baltimore, MD U.S.A, McGraw-Hill Professional 2007:1219-1233.

14.    Marx, A, Pewsner D, Egger M, Nuesch R, Bucher HC, Genton B, Hatz C, Juni P. Meta-analysis: accuracy of rapid tests for malaria. Annals of Internal Medicine 2005; 142(10):836-846.

15.    Murray, CK, Mody RM, Dooley DP, Hospenthal DR, Horvath LL, Moran KA, Muntz RW. The remote diagnosis of malaria using telemedicine or e-mailed images. Mil. Med 2006; 171:1167–1171.

16.    Bell D, Peeling RW. Evaluation of rapid diagnostic tests: malaria. Nat. Rev. Microbiol 2006; 4 (Suppl. 9):S34–S38.

17.    WHO, Western Pacific Region. Towards quality testing of malaria rapid diagnostic tests: evidence and methods. WHO, Western Pacific Region, Manila, Philippines. 2006.

18.    Pinto MJ, Rodrigues SR, Desouza R, Verenkar MP. Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria. Indian J Med Microbiol 2001; 19:219-21.

19.     Tangpukdee N, Duangdee C, Wilairatana P, Krudsood S. Malaria Diagnosis: A Brief review. Korean J Parasitol 2009; 47(2):93-102.

20.     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; 6:273-275.

21.     Wongsrichanalai C, Pornsilapatip J, Namsiriponpun V, Webster HK, Luccini A, et al. Acridine orange fluorescent microscopy and the detection of malaria in populations with low-density parasitemia. Am. J. Trop. Med. Hyg 1991. 44:17–20.

22.    Makler MT, Piper RC, Milhous W. Lactate dehydrogenase and diagnosis of malaria. Parasitol. Today 1998; 14:376-377.

23.    Piper R, Lebras J, Wentworth L, Hunt Cooke A, Houze S, Chiodini P, Makler M. A capture diagnostic assay for malaria using Plasmodium lactate dehydrogenase (pLDH). Am. J. Trop. Med. Hyg 1999; 60:109–118.

24.     Piper RC, Vanderjagt DL, Holbrook JJ, Makler M. Malaria lactate dehydrogenase: target for diagnosis and drug development. Ann. Trop. Med. Parasitol 1996; 90:433.

25.    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; 39:3644–3646.

26.    Moody A, Hunt-Cooke A, Gabbett E, Chiodini P. Performance of the OptiMAL® malaria antigen capture dipstick for malaria diagnosis and treatment monitoring at the Hospital for Tropical Diseases, London. Br. J. Haematol 2000; 109:891–894.

27.    Oduola AM, Omitowoju GO, Sowunmi A, Makler MT, Falade CO, Kyle DE, Fehintola FA, Ogundahunsi OA, Piper RC, Schuster BG, Milhous WK. Plasmodium falciparum: evaluation of lactate dehydrogenase in monitoring therapeutic response to standard anti-malarial drugs in Nigeria. Exp. Parasitol 1997; 87(3):283–289.

28.    Srinavasan S, Moody AH, Chiodini PL. Comparison of blood-film microscopy, the OptiMAL® dipstick, Rhodamine 123 and PCR for monitoring anti-malarial treatment. Ann. Trop. Med. Parasitol 2000; 94:227–232.

29.    Parija SC, Dhodapkar R, Elangovan S, Chaya DR. A comparative study of blood smear, QBC and antigen detection for diagnosis of malaria. Indian J Pathol Microbiol 2009;52:200-2

30.    Tanabe K. Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123. J. Protozool 1983; 30(4):707–710.