Introduction

Visual inspection of blood smears by light microscopy is the method available for quantifying malaria parasite density ,doing so by comparing the ratio of counted parasites within a given number of microscopic fields against either counted white blood cells (WBC) or counted red blood cells (RBC) within these same fields and then multiplying the ratio by either the measured or estimated density of WBCs or RBCs in the patients’ blood (O’meara et al, 2005)

The different methods for calculating parasites density described in literature can be as follows.

Expressing parasiteamia in a thick film as +,++, +++ based on the scheme

1-10 parasite/100 high powerfields(hpf) ———–+

11-100 parasites/100 high power fields (hpf) ————– ++

1- 10 parasites /every hpf            ———————- +++

>10 parasites/every hpf ———————- ++++

Counting parasites /WBC in a thick film until the high power field (hpf) with the 200^th WBC then multiplying with a ratio of the total WBC count /uL. (cheesbrough,2006)(O’meara et al ,2005) (Kakkalaya, 2004).

Counting parasitized RBCs in a thin film in 100 hpfs or until 1000^th RBC and multiplying with a ratio of total RBC count /uL. (O’meara et al ,2006) (cheesbrough,2006 )(Kakkilaya, 2004).

Examining 100hpf and if they all contain at least one parasite, then counting the number/ field in 10-100 fields depending on density and multiplying with aratio. A correction factor 2(-in(1-P) is applied . (Petersen et al 1996)

Finding the average number of parasites per high power field (hpf) after counting  the parasites in 10-50 hpfs, then multiplying by 500.

This last method proposed by greenwood and Armstrong was found by them to be more accurate and quicker than counting parasitesagainst white blood cells (WBCs) as described above. Using the count of parasitized RBC gave the most accurate assessments, however it is more time consuming and required much technical expertise.(Cheesebrough,2006)(O’meara 2005)(Greenwood and Armstrong ,1991).

The thick film is preferred to the thin film in estimating parasite density it is 30 times more sensitive than the thin film and has been known to detect as low 5 parasites/uL .(Cheesebrough,2006) . the rapid diagnostic tests including parasight , malaquick, ICT malaria pf are quick and single to carry out with no technical apparatus. The sensitivity is 90-95 % when parasiteamia is more than 100 parasite/UL, thus low parasiteamia is often missed . So a thick film is a more sensitive assay   than rapid diagnostic tests

Method which can be used to concentrate the parasites in a sample thus increasing malaria parasite density include

Centrifuging EDTA –anticoagulated venous blood in a capillary and using the blood just below the buffy coat layer. (quarantine buffy coat method). This gives excellent concentration especially of plasmodium vivax.(cheesbrough, 2006)

Using a magnetic field to concentrate malaria parasites. This is based on the paramagnetic property of haemozoin , a method called magnetic deposition microscopy .(Zimmerman et al 2006)

Magnetic deposition microscopy also concentrate gametocytes (Karl et al ,2008)

Density gradient centrifugation and removal of WBCs using cellulose powder column or filtration unit, thereby concentrating parasitized RBCs (Trang et al 2004)

Studies on malaria microscopy has often documented the frequency of false positive or negative results which increase with lower parasite density .(O’meara et al, 2005) (Mackenzie et al , 2003)(durrheim et al, 1997)(Beljaer et al, 1985)

These findings make the concentration of parasites very desirable when initial count shows a low density . however identical  consistent techniques may be more important than increasing the number of fields  read when precision among readers is desired , moreso , sequential samples taken from each patient in the course of a study stand to potentially improve the sensitivity and accuracy of estimates (O’meara et al, 2005)(O’meara et al 2006)

It is possible that total parasite quantity present  largely determines pathological and clinical manisfestation yet peripheral measurement of parasites density a subset and not total parasite load (Delley et al 2000)(Armstrong –schelenberg et al, 1994). During the course of a single infection , parasites density has been noted to fluctuate daily or even hourly in response to fever, immune mechanisms ,sequestration ,intra-host  competition , antigen swithching and other events. This  can lead to up to 20% or more under  estimation of parasite prevalence. (O’meara et al , 2007)(Delley et al,2000)(Genton et al , 1995)(Kibikamusoki,1967).

Malaria parasite density studies in Mali , determined  from peripheral blood (finger prick), varied during the day with a consistently higher parasite density measured at 13.00hours(12.00-13.00hours)(Delley et al 2000). A previous study had suggested an “internal check”  which resulted in synchronised explosion of schizonts around noon (Hawkings, 1990)

Above observations suggest that consistency of techniquesand time of blood collections around noon should give a fairly high and reliable estimation of malaria parasite burden . also studies which derive a correction factor for sequestered parasites could be added to give a fuller picture of malaria parasitaemia in an individual (Gravenor,  henbroek & kwiatkwoski,1998)(David et al ,1983)

Relating malaria parasite density to fever onset appears to be a major clinical implication of studies on malaria parasite density.

Malaria parasite density thresholds have been proposed for the development of fever due to malaria and irt appoearded to vary from study to study (Rogier et al ,1996)(Velema et al 1991)(bauden et al 1986)(Troupe et al 1985)(miller,1985).

Important to note that the fact that high parasite levels >100000/uL can exist in persons with no symptoms at al , this is apparently a result of premonition. (Hogh,1996)(Hogh et al, 1983). Parasite density needed to trigger fever strongly varies from person to person, reflecting the possible role of immune factors on fever threshold . ( Delley et al 2000)(Roger et al 1996)

In a particular study maximum risk of fever occurred with parasite density >1000/uL especially >10,000/uL . the fact tended to occur 24-48hrs after peak of parasite density at which time measured parasiteamia may not be at peak anymore (Delley et al, 2006) . in the rainy season in Mali, the best prediction of fevers were in under age (<4yrs), enlarged spleen and high parasite density .(Bouvier et al, 1997)

An age dependent threshold effect of parasite density on occurrence of fever was described the threshold varied from a maximum of 2.45 trophozoites /leucocytes at 1 year of age

A minimum of 0.5 trophozoites /leucocytes  at 60 years of age

When an individual parasite density crossed the threshold level  corresponding to his/her age , the risk of fever multiplied by 44. (Rogier ,commanges and Trape , 1996)

The density found in children is explained to be higher than in adults in areas of high transmission and so is the risk of fever . the number of children <10yrs with parasite density >  100.000/uL increased progressively with axillary temperature (Ejezie and Ezedinachi ,1992) a regression model has been proposed describing the probability of being sick from malaria in relation to body temperature and parasite density it appears that the ability to control the disease and parasite density develops earlier than ability to prevent parasite infection (Hugh, 1996)

Several studies have estimated parasite density in the two most vulnerable subsets of human population –children and pregnant women . estimation of mean parasite density was more accurate when the individuals total WBC count was used instead of an assumed value which can lead to overestimation of parasite burden (Jeremiah and uko 2007)

Symptomatic  children in a hyperendemic  area of Nigeria (Ekpoma ,edo state ) showed a mean parasite density of 4707.6 + 995.53uL –showing moderately high parasiteamia (Nmorsi et al, 2008) . parasite density was significantly higher in children with splenomegaly than in those with non palpable spleen ,(Adeyemo et al, 1999) . it is also significantly lower in stable children with sickle cell trait and sickle cell disease  (Awoha- efebo et al, 2004)

In healthy pregnant women mean parasite density was lower than in children with a range of 600-800 parasite /uL. (Adefoye et al 2007)

References

1. Adefioye O.A, Adeyebi O.A, Hassan W.O, Oyeniran O.A.,(2007): Prevalence of malaria Parasite infection  among pregnant women in Osogbo southwest Nigeria. Am.-Eur.   J.  Sci. Res.  2(1): 43 – 45.
2. Adeyemo, A. A., Olumese, P. E., Amodu, O. K. and Gbadagesin, R. A.,(1999): Correlates of hepatomegaly and splenomegaly among healthy school children in a malaria endemic village. Nig. J. Paediatr. 26(1): 1 – 3.
3. Awotua-Efebo O, Alikor E.A.D, Nkanginieme K.E.O., 2004. Parasite density and splenic status by ultrasonography in stable sickle-cell anaemia (HbSS) children. Nig J. Med. 13(1), Jan-March .
4. Baudon D, Gazin P, Sanou J.M., 1986. Morbidite palustre en milieu rural au Burkina Faso ; etude de 526 cas febriles. Med. Dáf. Noire. 33:767-776.
5. Beljaer A.E, Brohult J.A, Sharma G.K, Haque M.A, Samantaray K.G., 1985. Studies on the detection of malaria at primary health centres. Part 1. Reliability of parasitological diagnosis by decentralised laboratories. Indian J. Malariol. 22:85-103.
6. Cheesbrough M, 2006, District Laboratory Practice In Tropical Countries Part 1, Cambridge University Press, U.K pp: 239-258
7. David, P. H., Hommel, M., Miller, L. H., Udeinya, I. J. and Oligino, L. D. (1983) Parasite sequestration in Plasmodium falciparum malaria :spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc. Natl. Acad. Sci. USA.  80: 5075 – 5079.
8. Delley, V., Bouvier, P. and Breslow, N. (2000): What does a single determination of malaria parasite density mean? A longitudinal survey in Mali. Trop. Med. Int. Health 5(6): 404 – 412.
9. Durrheim, D. N., Becker, P. J., Billinghurst, K. and Brink, A. (1997): Diagnostic disagreement the lesson learned from malaria diagnosis in Mpumalanga. S. Afr. Med. J. 87: 609-611
10. Ejezie, G. C. and Ezedinachi, E. N. (1992): Malaria parasite density and body temperature in children under 10 years of age in calabar, Nigeria; Trop. Geog. Med., 44(1-2) : 97-101.
11. Genton, B., Alyaman, F. and Alpers, M. P. (1995): Assessment of malaria morbidity in vaccine efficacy trials. Lancet 345: 804 – 864.
12. Genton, B., Smith, T. and Baea, K. (1994): Malaria: how useful are clinical criteria for improving the diagnosis in a highly endemic area? Trans. R. Soc. Trop. Med. Hyg. 88: 537 – 541.
13. Gravenor, M. B., Hensbroek, M. B. V. and Kwiatkowski, D. (1998): Estimating sequestered parasite population dynamics in cerebral malaria. Proc. Natl. Acad. Sci. USA. 95(13): 7620 – 7624.
14. Greenwood, B. M. and Armstrong, J. R. M. (1991): Comparison of two simple methods for determining malaria parasite density. Trans. R. Soc. Trop. Med. Hyg. 85: 186 -188.
15. Hawkings, F. (1970): The clock of the malaria parasite. Sci. Am. .222:123 – 131.

16. Hogh, B. (1996): Clinical and Parasitological studies on immunity to Plasmodium falciparum malaria in children; J. Infect. Dis. Suppl; 102: 1 – 53.
17. Hogh, B., Marbiah, N. T., Peterson, E., Dolopaye, E., Wilcox, M., Bjokjman A., (1993): Classification of clinical falciparum malaria and its use for the evaluation of chemosuppression in children under six years of age in Liberia, West Africa. Trop. 54: 105 – 115.
18. Jeremiah, Z. A. and Uko, E. K. (2007): Comparative analysis of malaria parasite density using actual and assumed white blood cell counts. Ann. Trop. Paediatr. 27(1): 75 -79.
19. Kakkilaya, B. S. (2004): Evaluation of cases of malaria; The malaria site-http://www.malariasite.com/malaria/Evaluation.htm
20. Kakkillaya, B. S. (2006): Drug resistance. Malaria site. http://www.malariasite.com.
21. Kibikamusoke, J. W. (1967): The examination of multiple slides for the demonstration of malaria parasites. Am. J. Trop. Med. Hyg. 70: 46 – 49.
22. McKenzie F.E, Sirichaisinthop J, Miller R.S, Gasser R.A, Wongsrichanalai C., 2003. Dependence of malaria detection and species diagnosis by microscopy on parasite density. Am. J. Trop. Med. Hyg. 69: 372 – 376.
23. Miller, J. (1958): Observation on the natural history of malaria in the semi-resistant west Africa. Tran. R. Soc. Trop. Med. Hyg. 52: 152 – 168.
24. Montanari, R. M., Bangali, A. M., Talukder, K. R., Baqui, A., Mahswary, N. P., Gosh, A., Rahman, M. and Mahmoud, A. H. (2001): Three case definitions of malaria and their effect on diagnosis, treatment and surveillance in Cox’s bazaar district, Bangladesh. Bull World Health Organisation. 79: 648 – 656.
25. Nmorsi,  O. P. G., Ukwandu, N. C. O. and Egwunyenga, A. O. (2007): Antioxidant status of Nigerian children with  Plasmodium falciparum malaria .Afr. J. Microbiol Res ; 61 – 64.
26. O’Meara, W. P., Mckenzie, F. E., Magill, A. J., Forney, J. R., Permpanich, B., Lucas, C., Gasser, Jr R. A. and Wongsrichanalai, C. (2005): Sources of variability in determining malaria parasite density by  microscopy. Am J. Trop. Med. Hyg. 73(3): 593-598.
27. O’Meara, W. P., Remich, S. and Ogutu, B. (2006): Systematic comparison of two methods to measure parasite density from malaria blood smears; Parasitol. Res. 99(4): 500 – 504
28. O’Meara, W.P., Hall, B.F. and McKenzie F. E. (2007): Malaria vaccine efficacy: the difficulty of detecting and diagnosing malaria. Malar. J. 6: 36.
29. Peterson, E., Marbiah, N. T., New, L. and Gottschau, A. (1996): Comparison ot two methods for enumerating malaria parasites in thick blood films. Am. J. Trop. Med. Hyg. 55(5): 485 – 489.
30. Rogier, C., Commenges, D. and Trape, J. F. (1996): Evidence for an age-dependent pyrogenic threshold of Plasmodium falciparum parasitaemia in highly endemic populations; Am. J. Trop. Med. Hyg. 54(6): 613 – 619.
31. Schellenberg, J., Smith, T., Alonso, P. L. and Hayes, R. J. (1994): What is clinical malaria : finding case definitions for field research in highly endemic areas .Parasitol. Today 10: 349 – 442.
32. Trape, J. F., Peelman, P. and Morault-Peelman, B. (1985): Criteria for diagnosing clinical malaria among a semi-immune population exposed for intense and perennial transmission. Trans. R. Soc. Trop. Med. Hyg. 79: 435-442.
33. Velema, J. P., Alihonou, E. and Chippaux, J. P. (1999): Malaria morbidity and mortality in children under three years of age on the coast of Benin, west Africa. Trans. R. Soc. Trop. Med. Hyg. 85: 430 – 435.
34. Zimmerman, P., Thomson, J. M., Fujioka H, Collins W.E, Zborowski, M. (2006): Diagnosis of malaria by magnetic deposition microscopy. Am. J. Trop. Med. Hyg.74(4):568-572.