C.C. Onyenekwe, O.G. Arinola, S.C. Meludu, L.S. Salimonu, I.F. Adewale & A.K. Obisesan

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J Vect Borne Dis 41,  September & December 2004, pp 72–75Malaria parasitaemia and Plasmodium falciparum specific-IgGin maternal peripheral, placental and cord circulatonC.C. Onyenekwea, O.G. Arinolab, S.C. Meludua, L.S. Salimonub, I.F. Adewalec &A.K. ObisesancaDepartments of Immunology and Chemical Pathology, College of Health Sciences, Nnamdi Azikiwe  University,Nnewi Campus, PMB 5001 Nnewi, Anambra state; bDepartment of Chemical Pathology; cDepartment of Obstetricand Gynaecology, University College Hospital, Ibadan, PMB 5116 Ibadan, Oyo state, NigeriaKey words Malaria parasitaemia – PfIgG – Pf hrp-2 – placental and cord circulation – pregnant womenClincial cases of malaria infection during pregnancymay result in placental1-5 and/or congenital malaria in-fections.  Emerging evidence has also shown thatpregnant women could remain asymptomatic with ma-laria parasitaemia6.  Hence, this study was designed toassess the possible impact of asymptomatic maternalperipheral malaria parasitaemia and Plasmodium fal-ciparum specific-IgG during pregnancy on placentaland cord circulations.Twenty-one pregnant women were recruited for thisstudy and blood was collected from maternal periph-eral, placental and cord circulations after delivery forserological screening of P. falciparum histidine richprotein (hrp-2), detection of malaria parasites andmalaria specific antibody. Informed consent was ob-tained from the subjects and the study design receivedethical approval.The detection of P. falciparum hrp-2 antigen in bloodsamples from peripheral, placental and cord circula-tion of the pregnant women was carried out using pre-coated monoclonal anti-P. falciparum hrp-2 wells(Cellabs Pty, Australia).  The procedure was followedas described by the manufacturer.  In brief, bloodsampels collected from the pregnant women stored inethylene-diamine-tetra acetic acid (EDTA) tubes werefrozen and thawed before analysis.  Thawed wholeblood samples 100 ?l reference positive and negativecontrols (Cellabs Pty, Australia) were added to theappropriately labelled pre-coated wells and incubatedat room temperature for 60 min.  This allows the fixingof hrp-2 antigen in the blood or reference positivecontrol to the anti-hrp-2 coated wells.  The wellswere washed with 0.01 M phosphate buffered saline(PBS)-tween solution thrice.  Then 100 ?l of (1 : 200)diluted conjugate of enzyme labelled anti-human glob-ulin was added to all the wells and incubated for 60min at room temperature.  After washing as describedabove, 50 ?l of (1 : 20) diluted 3,3’, 5,5’-tetramethyl-benzidine (TMB) substrate chromogen was added tothe wells and placed in the dark room for 15 min forcolour development. The 50 ?l of HCl (2.5 M) wasadded to stop the reaction and the colour developedwas read using a Dynatech MR 250 microplate read-er at 450 nm.  The absorbance of this test is not pro-ONYENEKWE et al :  MALARIA IN PREGNANT WOMEN 73portional to the level of parasitaemia.  The cut-offpoint for samples positive for P. falciparum hrp-2 an-tigen was determined as mean absorbance (+3 stan-dard deviation) reference negative control.The P. falciparum specific-IgG concentration in sam-ples of the pregnant women was performed using pre-coated P. falciparum antigen wells (Cellabs Pty, Aus-tralia).  The procedure followed as described by themanufacturer.  In brief, 100 ?l of diluted (1 : 100)pregnant women sera, reference positive and negativesera were added to appropriately labelled P. falci-parum antigen coated wells and incubated in a humidchamber for 60 min at 37 oC.  The washing, additionof conjugate antibody and substrate chromogen andstopping of the reaction and reading of absorbance ofcolours developed and determination of cut-off pointwas as described for P. falciparum hrp-2 antigen.However, the absorbance of this test is proportionalto the concentration of the P. falciparum specific-IgGin the samples.The malaria parasite density was reported as numberof parasites per  microlitre of blood as previously de-scribed by Rooth7.  Statistical anaylsis was performedon data using ANOVA, student t-test, likelihood ratioand p-value < 0.05 was considered significant.The result showed that 15 (71%) of the maternalperipheral, 18 (86%) of the placental and 9 (43%) ofthe cord samples were sero-reactive to anti-P. falci-parum hrp-2 antigen; while 6 (29%), 3 (24%) and 12(57%) of the maternal peripheral, placental and cordsamples were non-reactive  with anti-P. falciparumhrp-2 antigen respectively.  There was no strong likeli-hood that maternal peripheral malaria parasitaemia inasymptomatic pregnant women will result in parasi-taemia of the cord circulation (likelihood ratio: 0.704,p >0.1), or placental circulation (likelihood ratio:0.038, p > 0.1).  However, there was a strong likeli-hood that malaria parasitaemia of the placental circu-lation will result in malaria parasitaemia of the cordcirculation (likelihood ratio: 4.357, p<0.05) (Table 1).The mean (+ 2 SD) malaria parasites density ex-pressed as number of parasites per microlitre of bloodwas not significantly different amongst the differentcirculations (p>0.l) (Table 2). However, the mean(+ 2 SD) P. falciparum specific-IgG concentrationwas significantly different amongst the malaria parasi-taemia maternal peripheral, placental and cord circula-tions (p<0.01) (Table 2).Table 1. The likelihood ratio for malaria parasitaemiabetween the maternal peripheral, placental andcord circulations (n = 21)Circulations Likelihood ratio p-valueMaternal peripheral vs. cord 0.704 0.1 nsPlacental vs. cord 4.357 < 0.05Maternal peripheral vs. placental 0.038 0.1 nsn—Number of subjects; ns—Not significant.Table 2.  P. falciparum specific-IgG and malaria parasites density in maternal peripheral, placental andcord circulations with malaria parasitaemiaCirculations Maternal Placental Cord ANOVAperipheralPf-IgG 0.547 + 0.133 0.605 + 0.102 0.422 + 0.198 < 0.01(n =15) (n =18) (n = 9)MPD 483 + 274 356 + 230 386 + 273 > 0.1(n =15) (n =18) (n = 9)n — Number of subjects; MPD — Malaria parasite density.J  VECT  BORNE  DIS  41, SEPTEMBER & DECEMBER  2004 74The finding of the present study shows high incidenceof malaria parasitaemia in various circulations in as-ymptomatic individuals during pregnancy. This findingis consistent with our earlier report of high prevalenceof asymptomatic malaria parasitaemia amongst preg-nant women6.Similarly, the present study observed that malariaparasitaemia in the peripheral circulation of asymp-tomatic pregnant women might not necessarily result inplacental and or congenital malaria parasitaemia in allinstances. However, in most instances, malariaparasitaemia of the placental circulation resulted incongenital malaria parasitaemia of the neonates. Thus,over-reliance on maternal peripheral circulation for di-agnosing and excluding possible congenital malaria in-fections may conspicuously encourage high rates ofcongenital malaria infection of the neonates becauseclearance of the malaria parasites from the maternalperipheral circulation does not exclude congenital ma-laria. Hence, placental malaria parasitaemia should beprevented in pregnant women due to the risk associ-ated with congenital malaria infection.However, the most effective way of controlling oravoiding placental malaria parasitaemia is by prevent-ing exposure of pregnant women to infective bites ofmosquitoes or by administration of effective antimalar-ia drugs which helps to limit and/or clear the parasi-taemia in pregnant women. It may not be easy toidentify such asymptomatic pregnant women with ma-laria parasitaemia except by compulsory routine ante-natal malaria screening. This is because such patientsrarely present with signs and symptoms of malaria8.The lack of clinical evidence based on signs andsymptoms, excludes these subjects from early drug in-tervention and thus exposes them to possible risk ofcongenital malaria infection of the neonates.It has been reported that malaria in pregnancy espe-cially during the last trimester may result in alterationof placental functionl,2 and could adversely affect thefeto-placental unit irrespective of the parity3,9-11.Hence, extent of damage to the placental integritymight be a strong indicator that determines the fre-quency of parasites transmission to cord circulations inan asymptomatic state as is likely in the present study.The P. falciparum specific-IgG concentration wassignificantly different amongst the different malariaparasitaemia circulations. The concentration was leastin malaria parasitaemia cord circulations. However, in-fection of cord circulations with malaria parasitesseemed to result in blood reduction in P. falciparumspecific-IgG concentration. This was clearly reflectedin this finding in which the concentration of the specificantibody was significantly higher in un-infected cordcirculations compared with infected circulations, bothcirculations having similar history of maternal peripheralmalaria parasitaemia. This will imply possible earlier ex-haustion of protective specific antibody during neona-tal life by neonates with congenital malaria infections.In conclusion, this study showed that malariaparasitaemia of the placental calculations has stronglikelihood to result in congenital malaria infection.Therefore, over-reliance on maternal peripheral circu-lation for high-risk congenital malaria infection shouldbe avoided.AcknowledgementWe are grateful to Mr. Inyang Ekanem and Mrs.Omoyeme Okiwelu, for their technical assistance inthe laboratory. We are also grateful to members ofstaff of the antenatal clinic. This study was partlysponsored by Stephen Oluwole Awokoya FoundationScience Education, Nigeria.References1. Brabin BJ. An analysis of malaria in pregnancy in Africa.WHO 1983; 61 : 1005–16.2. McGregor IA. Epidemiology, malaria and pregnancy. Am JTrop Med Hyg 1998; 33 : 517–25.3. Morgan HG.  Placental malaria and low birth weight neo-nates in urban Sierra-Leone. Annal Trop Med Parasitol1994; 88 (6) : 575–80.ONYENEKWE et al :  MALARIA IN PREGNANT WOMEN 754. Menedez C. Malaria during pregnancy: a priority area ofmalaria research and control. Parasitol Today 1995; 11 (5):178–83.5. Fischer PR. Congenital malaria: an Aftican survey. ClinPeadiatr Phil 1997; 36 (7) : 41–3.6. Onyenekwe CC, Arinola OG, Salimonu LS. Detection ofPlasmodium falciparum IgG and incidence of asymptom-atic malaria in pregnant women in Nigeria. Indian J Malar-iol 2002; 39 (1-2) : 39–42.7. Rooth I, Bjorkman A. Fever episodes in a holoendemicmalaria area of Tanzania: parasitological and clinical find-ings and diagnostic aspects related to malaria. Trans RSoc Trop Med Hyg 1992; 86 : 470–82.8. Malaria diagnosis: new perspectives. Report of a jointCorresponding author : Dr. Charles C. Onyenekwe, Deptt. of Immunology and Chemical Pathology, College of Health Sciences,Nnamdi Azikiwe  University, Nnewi Campus, PMB 5001 Nnewi, Anambra state, Nigeriae-mail : charleschinedum2002@yahoo.comWHO/USAID informal consultation, 25–27 October 1999.Geneva : World Health Organization 2000.9. Reinhardt MC, Ambrose-Thomas P, Cavallo-Serra R, Mey-lan C, Gautier R. Malaria at delivery in Abidjan. Helv Pae-diatr Acta 1978; 33 (Suppl): 65–84.10. McGregor AI, Wilson ME, Billewiez WZ. Malaria infectionof the placenta in the Gambia, West Africa: its incidenceand relationship to stillbirth, birth weight and placentalweight. Trans R Soc Trop Med Hyg 1983; 77 : 232–44.11. Warkinson M, Rushton DI, Lunn PG. Placental malariaand feto-placental function, low plasma oestradiol asso-ciated with malaria pigmentation of the placenta. Trans RSoc Trop Med Hyg 1985; 79 : 448–50.

Malaria parasitaemia and Plasmodium falciparum specific-IgG in maternal peripheral, placental and cord circulaton

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