A Study of Malaria Parasite Density in HIV-1 Positive Under-fives in Benin City, Nigeria

A Study of Malaria Parasite Density in HIV-1 Positive Under-fives in Benin City, Nigeria Abstract Background Human immunodeficiency virus (HIV) and malaria are leading causes of morbidity and mortality among under-fives in sub-Saharan Africa. HIV infection could affect development of antimalarial immunity by impaired parasite clearance with predisposition to higher malaria parasitaemia. Objective The objective of this study is to assess asymptomatic malaria parasite density (AMPD) in HIV-1-infected under-fives in a holoendemic zone. Methods HIV-1-positive and -negative children <5 years on follow-up care were recruited and AMPD and CD4 counts were determined. Results A total of 358 children were studied. Significantly higher malaria parasitaemia was found in HIV-infected individuals (118.7 vs. 87.3 parasite/μl, p = 0.021). Disparity in AMPD was most pronounced at infancy with similar distribution at all age brackets and consistently higher parasitaemia in the subjects. Conclusion Parasitaemia is higher in HIV-infected than uninfected children. The burden is highest at infancy. Acquisition of antimalarial immunity is similar in both groups. Parasitaemia is not significantly affected by clinical disease stage or worsening immunosuppression. asymptomatic, parasite density, under-fives, immunity INTRODUCTION Human immunodeficiency virus (HIV) and Falciparum malaria infection are leading causes of morbidity and mortality in sub-Saharan Africa [1]. The epidemiologic overlap has been the source of concern, as coinfection could further increase the health burden [2]. Falciparum malaria is a major public health problem, with one child dying every minute in Africa [3], especially among under-fives owing to naive immunity [4–6]. In endemic areas, the transfer of maternal antibodies, repeated early childhood exposure and clinical episodes generate and sustain partial antimalarial immunity, which ameliorates infection but does not prevent asymptomatic malaria parasitaemia [7], i.e. the presence of malaria parasites in the blood without symptoms [8]. CD4+ T-cells are essential for developing antimalarial immunity [9]; they help B cells produce antibodies and indirectly support the control of parasitaemia through cytokine production and macrophage activation [10]. In HIV-infected children, malaria-specific immunity development and parasite clearance are impaired, resulting in increased predisposition to parasitaemia [9, 11–13]. Asymptomatic malaria parasitaemia is a predictive parameter for transmission and assessment of immunity in exposed populations [8, 14, 15]. This study highlights antimalarial immunity in a holoendemic region where sustained exposure generates immunity in childhood. Withworth et al. [2] found twice as many parasitaemia episodes, increasing with worsening immunosuppression in HIV-infected adults with symptomatic malaria in a moderate endemic area. The findings were collaborated by French et al. [16]. Coinfected pregnant adults living in stable and unstable malaria regions also showed increased parasitaemia [17–21]. Studies of asymptomatic malaria in HIV-infected individuals are few and limited in children from holoendemic regions having almost half of global malaria cases [22]. This cross-sectional study examines asymptomatic parasitaemia in HIV-infected children <5 years and the role of CD4 T-cells on parasite density. METHODS The study was conducted at the paediatric HIV clinic of University of Benin Teaching Hospital (UBTH), between March and July 2012. Malaria transmission is holoendemic; intense and stable transmission occurs all year round with peaks during the rainy season [23]. The HIV diagnosis for afebrile children 6 weeks to <18 months was by HIV DNA polymerase chain reaction, and those ≥18 months had HIV rapid antibody serial testing algorithm II using standard protocols [24]. Controls were age- and sex-matched HIV-1-negative children attending clinics for non-chronic medical conditions. Children with history of malaria treatment in the preceding 2 weeks and those on chemoprophylaxis were excluded. Haemoglobin genotype was done to exclude sickle cell disease in subjects and controls. Information on co-trimoxazole (CTX) prophylaxis is according to the national guideline; house screening and use of insecticide-treated nets (ITNs) were noted. Ethical approval was obtained from UBTH Ethics and Research Committee (ADM/E.22 A/VOL, VII/390), while informed, written consent was from parent(s)/guardian(s) of subjects and controls. Pre-test/Post-test counselling was done before and following HIV testing. The clinical (World Health Organization, WHO) staging of HIV/AIDS of the subjects was noted. Blood samples were collected into ethylenediaminetetraacetic acid (EDTA) anticoagulated containers and concomitant samples for CD4 count. Thick and thin blood smears were stained with 2% Giemsa for 30 min and read by WHO-certified microscopists who were uninvolved in direct patient care and blinded to the clinical status of the study participants. Asexual malaria parasitaemia was recorded as present or absent. Parasites/μl were calculated from thick blood smears by counting asexual parasites per 200 leukocytes, assuming a leukocyte count of 8000/ml. A blood smear was considered negative on examination of 100 high-power fields without parasites. Thin smears were for determination of parasite species [24]. CD4 counts was analysed using Flow Cytometer Cyflow SL Green® (Partec). The CD4 counts were documented according to the Centres for Disease Control and Prevention immunologic classification [24]. HIV screening test was carried out for the controls using HIV rapid testing serial algorithm II for HIV diagnosis according to national guidelines [25, 26]. Statistical analysis Clinical and laboratory data management and analysis were by IBM SPSS statistic 20. The test of normality (Kolmogorov–Smirnov) showed that malaria parasite density for microscopy positive slides was not normally distributed, hence the use of non-parametric tests for analysis. Mann–Whitney U test was to compare the mean parasite densities between cohorts. While Kruskal–Wallis test was for comparison of more than two means, the level of significance was at p < 0.05 and confidence level of 95%. RESULTS General characteristics In all, 179 HIV-1 positive and 179 HIV-1 negative sex- and age-matched controls were studied. The subjects consisted of 96 (53.6%) males and 83 (46.4%) females at the ratio of 1.16:1. The age range of the study population was 2–59 months and their age distribution is shown in Table 1. Table 1 Age distribution of the subjects and controls Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Table 1 Age distribution of the subjects and controls Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Most of the subjects (76; 42.5%) were in WHO clinical Stage 1, 44 (24.6%) in Stage 2, 46 (25.7%) in Stage 3, while 13 (7.3%) were in Stage 4. Majority of the subjects [152 (84.9%)] were in immunologic Categories 1 and 2, while 27 (15.1%) were in Category 3. Plasmodium falciparum was the only parasite specie identified. In all, 61 (34.1%) of the subjects and 32 (17.9%) of the controls had positive parasite smears (χ2 = 13.166, p = 0.001). Asymptomatic mean parasite density The mean parasite density (for microscopy-positive slides only) in subjects of 118.7 ± 70.5 parasite/µl was significantly higher than 87.3 ± 36.9 noted in controls (Mann–Whitney U = 705.5, p = 0.029) (Table 2). Table 2 Mean parasite density by age (groups) in subjects and controls Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Kruskal–Wallis test subjects: χ2 = 3.255, p = 0.516. Controls: χ2 = 2.531, p = 0.639, df = 4. mpd, mean parasite density/µl; SD, standard deviation. Table 2 Mean parasite density by age (groups) in subjects and controls Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Kruskal–Wallis test subjects: χ2 = 3.255, p = 0.516. Controls: χ2 = 2.531, p = 0.639, df = 4. mpd, mean parasite density/µl; SD, standard deviation. Asymptomatic parasitaemia in subjects was higher than in controls of all age brackets. Subjects 2 to <12 months (infancy) had the highest density of parasites (148.8 parasites/μl), and 12 to < 24 months age bracket had the least (84.7 parasites/μl). The differences in parasitaemia by age brackets of subjects and controls were not statistically significant (Table 2). The changes in asymptomatic malaria parasite density (AMPD) by age groups of subjects and controls are shown in Fig. 1. Fig. 1. View largeDownload slide Line graph depicting the mean parasite densities by age groups of the subjects and controls. Fig. 1. View largeDownload slide Line graph depicting the mean parasite densities by age groups of the subjects and controls. Parasitaemia by gender The AMPD of male subjects was 118.6 ± 70.5 parasites/µl as against 119.0 ± 54.0 in female subjects, p = 0.84. For the controls, it was 95.5 ± 36.4 parasites/µl in males and 78.0 ± 36.4 in females, p = 0.84. Parasitaemia by WHO stage and immune category The AMPD was least (111.6 parasite/µl) among subjects in WHO Stage 1 and highest (131.2 parasite/µl) among those in WHO Stage 4. Parasitaemia increased with worsening WHO clinical staging; however, this relationship was not statistically significant (Kruskal–Wallis test, χ2 = 1.514, p = 0.679) (Table 3). Table 3 Mean parasite density by WHO clinical staging of subjects WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) Kruskal–Wallis test χ2= 1.514, p = 0.679, df = 3. Table 3 Mean parasite density by WHO clinical staging of subjects WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) Kruskal–Wallis test χ2= 1.514, p = 0.679, df = 3. The AMPD was lowest among subjects in immune Category 1 and highest in immune Category 2. Association between parasitaemia and immune category was not statistically significant. (Kruskal–Wallis test, χ2 = 0.970, p = 0.616) (Table 4). Table 4 Mean parasite density by immune category of subjects Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Kruskal–Wallis test χ2 =0.970, p = 0.616, df = 2. Table 4 Mean parasite density by immune category of subjects Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Kruskal–Wallis test χ2 =0.970, p = 0.616, df = 2. Parasitaemia with antiretroviral therapy (ART) use Subjects on ART had higher AMPD (123.4 ± 73.7) than those not on treatment (88.1 ± 32.1) although the relationship was not significant (Mann–Whitney U = 165.500, p = 0.320). Mean parasite densities were higher in subjects receiving Zidovudine, Nevirapine, Lamivudine, Efavirenz and Stavudine (Table 5). None of the subjects on Ritonavir/Lopinavir and Abacavir had any parasitaemia. Table 5 Mean parasite density by ART use ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 Table 5 Mean parasite density by ART use ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 Parasitaemia with duration of ART use The AMPD was highest (128.4 ± 77.8) among those that received ART for <6 months, while the least parasite density (116.0 ± 45.7) was in those on ART for 18 to < 24 months. The lowest AMPD (88.1 ± 32.1) was among those that are ART naive. This relationship between AMPD and duration of ART was not statistically significant (Table 6). Table 6 Mean parasite density by duration of ART use ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) Kruskal–Wallis test χ2 = 1.799, p = 0.876, df = 5. Table 6 Mean parasite density by duration of ART use ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) Kruskal–Wallis test χ2 = 1.799, p = 0.876, df = 5. Parasitaemia and CTX prophylaxis Up to 46 of 154 subjects on CTX prophylaxis had positive result while 15 of 25 not receiving prophylaxis had positive malaria parasitaemia, a 3-fold risk (χ2 = 8.692, p = 0.003). The AMPD among subjects on prophylaxis of 127.5 ± 75.0 parasites/µl was not significantly higher than 91.9 ± 47.1 among those not on CTX, Mann–Whitney U = 242.500, p = 0.086. Parasitaemia and the use of ITN and window net The AMPD of infected children sleeping under ITNs was not significantly higher than those not sleeping under ITN; also for Children living in screened houses, AMPD was not significantly different from those living in unscreened houses (Table 7). Table 7 Mean parasite density of subjects by use of ITN and window net n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) Table 7 Mean parasite density of subjects by use of ITN and window net n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) DISCUSSION This study examines AMPD in HIV-infected children <5 years living in the holoendemic malaria region of Southern Nigeria. The study reveals significantly higher parasitaemia among HIV-infected compared with uninfected children. Malaria parasitaemia in infected children was higher at every age group, and the disparity was highest at infancy. Age was not a significant predictor of parasitaemia. The parasite density increased albeit non-significantly with worsening clinical staging and immunosuppression. Increased parasitaemia among HIV-infected children could be owing to the immune deficiencies and dysfunction attributable to HIV infection. HIV-related immunodeficiency is the probable cause, as the parasite may thrive better in infected than uninfected group. Malaria parasitaemia influences development of partial immunity [27], protection against clinical disease and ameliorates new infections in persons living in endemic areas [2, 28]. HIV-infected children therefore are at higher risk of having malaria parasitaemia, making HIV infection a risk factor for enhanced asymptomatic malaria parasitaemia [13–15]. The asymptomatic parasitaemia of 118.7 parasites/µl amongst HIV-infected children is higher than 85.2 parasites/µl reported by Adetifa et al. [29] in HIV-positive children 1–5 years in Lagos, South-West Nigeria. The reason for the difference in parasite density is not readily apparent. The AMPD reported in this study was smaller than 1922 parasites/µl reported by Owusu-Agyei et al. in Ghana among HIV-positive children 1–2 years and 1903 among 3–4-year-olds [15]. The study in Ghana reported geometric mean, while arithmetic mean was quoted in our study; this could have accounted for the large difference in parasitaemia. The pattern of parasitaemia in infected and uninfected children showed striking similarities with increasing age (Fig. 1). The narrow spectrum of parasitaemia may be owing to asymptomatic infection and small parasite densities recorded. Symptomatic malaria occurs with higher malaria parasitaemia [7]. Infancy was the peak of parasitaemia in both groups followed by a nadir at 12 to 24 months, to the staggered increase at 48 to <59 months. It seems probable therefore that infected children develop antimalarial immunity timely as non-infected peers albeit with higher parasitaemia. This is an area needing further exploration. Asymptomatic parasitaemia is necessary for development of specific antimalarial immunity and maintenance of the transmission cycle [7, 13–15, 30]. It can therefore be speculated that HIV infection does not distort the acquisition of antimalarial immunity. The finding of the highest parasitaemia and the worst disparity at infancy is an area needing further exploration. Comparative studies on HIV-infected/uninfected pregnant women have consistently reported higher parasitaemia in the infected groups. This may be partly owing to transient loss of acquired antimalarial immunity, leading to higher malaria and placental parasitaemia at delivery [19, 20]. The insufficient or defective transfer of antimalarial immune factors transplacentally from HIV-infected mothers could predispose their infants to higher AMPD as seen in this study. That uninfected infants had lower parasitaemia underscores this proposition. Therefore, infancy remains the critical point of enhanced parasitaemia in infected children. Appropriate vector avoidance is therefore recommended for malaria control. Malaria parasitaemia with HIV clinical disease stage and worsening immunosuppression is one of the objectives of this study. While parasitaemia increased with clinical staging, there was no consistent trend with immune category and the relationship was not significant. Clinical disease stage and immune category do not affect antimalarial immunity development among children <5 years. This finding differs from some studies on clinical (symptomatic) malaria [9, 17]. Apart from CD4+ T-cells, there are other factors required for development and sustenance of antimalarial immunity. Malaria parasitaemia was uninfluenced by ART, and there was increased parasitaemia among those on Zidovudine, Nevirapine, Lamivudine, Efavirenz and Stavudine. The duration of ART did not significantly affect parasitaemia. The reason for the non-association may be from lack of antimalarial properties by most ARTs. Their use, however, is known to enhance immune reconstitution and subsequently improve parasite clearance with attendant lower parasitaemia [31]. This is an expected occurrence with longer duration of ART administration. Our findings may be partly owing to the few number of children on protease inhibitors (Pi) (1.1%), which has distinct antimalarial properties [32]. The least parasitaemia, however, was among those that are ART naive. Parasitaemia in this study was unaffected by window netting or use of ITNs. The lack of association between ITN use and malaria parasite density is surprising, as ITN is a known factor for its beneficial vector avoidance property [31]. Although the fraction of infected children using ITNs is small (11.2%), infrequent use maybe contributory. Infected children on CTX had significantly lower prevalence of malaria parasitaemia as has been reported by other researchers and collaborated by this study. However, the finding of non-statistical higher parasitaemia among infected children on CTX is worrisome. This may be part of the higher AMPD associated with HIV infection; however, evidence abounds that CTX reduces symptomatic malaria incidence by 76%. ART and ITNs substantially reduce malaria frequency in HIV-infected adults [31]. In conclusion, malaria parasitaemia is higher in asymptomatic HIV-infected compared with uninfected children <5 years. In all age brackets, higher parasitaemia was maintained and a similar pattern is observed in both groups. Acquisition of antimalarial immunity in both groups may be similar albeit with higher parasitaemia in HIV-infected children. ACKNOWLEDGEMENT To all the children that attend the Paediatric HIV clinic in UBTH Benin City and all the staff that made this work possible, thank you for your support and immense contribution. FUNDING The study is self-funded with technical support of University of Benin Teaching Hospital, Benin City, Edo State, Nigeria. References 1 Joint United Nations Programme on HIV/AIDS (UNAIDS)/World Health Organization (WHO) . AIDS epidemic update: 2009. UNAIDS. Geneva: UNAIDS/WHO, 2009 . 2 Whitworth J , Morgan D , Quigley M , et al. 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Google Scholar CrossRef Search ADS PubMed 13 World Health Organization . Malaria and HIV-1/AIDS interactions and implications: conclusions of a technical consultation convened by WHO, June 23–25, 2004. Report no. WHO/HIV/2004.08. Geneva: WHO, 2004 . 14 Alves FP , Durlacher RR , Menezes MJ , et al. High prevalence of asymptomatic plasmodium vivax and plasmodium falciparum infections in native Amazonian populations . Am J Trop Hyg 2002 ; 66 : 641 – 8 . Google Scholar CrossRef Search ADS 15 Owusu-Agyei S , Smith T , Beck H-P , et al. Molecular epidemiology of Plasmodium falciparum infections among asymptomatic inhabitants of a holoendemic malarious area in northern Ghana . Trop Med Int Health 2002 ; 5 : 421 – 8 . Google Scholar CrossRef Search ADS 16 French N , Nakiyingi J , Lugada E , et al. Increasing rates of malarial fever with deteriorating immune status in HIV-1-infected Ugandan adults . Aids 2001 ; 15 : 899 – 906 . 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Google Scholar CrossRef Search ADS PubMed 21 Grimwade K , French N , Mbatha D , et al. HIV infection as a cofactor for severe falciparum malaria in adults living in a region of unstable malaria transmission in South Africa . Aids 2004 ; 18 : 547 – 54 . Google Scholar CrossRef Search ADS PubMed 22 Hay SL , Guerra CA , Tatem AJ , et al. The global distribution and population at risk of malaria: past, present, and future . Lancet Infect Dis 2004 ; 4 : 327 – 36 . Google Scholar CrossRef Search ADS PubMed 23 Federal Ministry of Health . Malaria in Nigeria . Nig Bull Epidemiol 1991 : 2 – 19 . 24 World Health Organization . Basic Malaria Microscopy, Part 1, Learners’Guide . A WHO Teaching Material. Switzerland : WHO , 1991 , 67 – 8 . 25 The Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children. Guidelines for the use of antiretroviral agents in pediatric HIV infection, 1 – 219 . http://aidsinfo.nih.gov/ContentFiles/PediatricGuidelines.pdf. 26 National Guidelines for Paediatric HIV & AIDS Treatment and Care . Nigeria : Federal Ministry of Health , 2010 , 1 – 233 . 27 Staalsoe T , Hviid L. The role of variant-specific immunity in asymptomatic malaria infections: maintaining a fine balance . Parasitol Today 1998 ; 14 : 177 – 8 . Google Scholar CrossRef Search ADS PubMed 28 Farnert A , Arez AP , Correia AT , et al. Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction . Trans R Soc Trop Med Hyg 1999 ; 93 : 50 – 3 . Google Scholar CrossRef Search ADS PubMed 29 Adetifa IM , Akinsulie AO , Temiye EO , et al. Effect of antiretroviral therapy on asymptomatic malaria parasitaemia in HIV-1 infected children . Nig Postgrad Med J 2008 ; 15 : 141 – 5 . 30 Orogade AA , Ogala WN , Aikhionbare HA. Asymptomatic malaria parasitaemia—a suitable index for evaluation of malaria vector control measures . Nig J Paediatr 2002 ; 29 : 23 – 6 . Google Scholar CrossRef Search ADS 31 Mermin J , Ekwaru JP , Liechty AL , et al. Effect of co-trimoxazole prophylaxis, antiretroviral therapy, and insecticide-treated bednets on the frequency of malaria in HIV-1-infected adults in Uganda . Lancet 2006 ; 367 : 1256 – 61 . Google Scholar CrossRef Search ADS PubMed 32 Parikh S , Gut J , Istvan E , et al. Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors . Antimicrob Agents Chemother 2005 ; 49 : 2983 – 5 . Google Scholar CrossRef Search ADS PubMed © The Author [2017]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Tropical Pediatrics Oxford University Press

A Study of Malaria Parasite Density in HIV-1 Positive Under-fives in Benin City, Nigeria

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Abstract

Abstract Background Human immunodeficiency virus (HIV) and malaria are leading causes of morbidity and mortality among under-fives in sub-Saharan Africa. HIV infection could affect development of antimalarial immunity by impaired parasite clearance with predisposition to higher malaria parasitaemia. Objective The objective of this study is to assess asymptomatic malaria parasite density (AMPD) in HIV-1-infected under-fives in a holoendemic zone. Methods HIV-1-positive and -negative children <5 years on follow-up care were recruited and AMPD and CD4 counts were determined. Results A total of 358 children were studied. Significantly higher malaria parasitaemia was found in HIV-infected individuals (118.7 vs. 87.3 parasite/μl, p = 0.021). Disparity in AMPD was most pronounced at infancy with similar distribution at all age brackets and consistently higher parasitaemia in the subjects. Conclusion Parasitaemia is higher in HIV-infected than uninfected children. The burden is highest at infancy. Acquisition of antimalarial immunity is similar in both groups. Parasitaemia is not significantly affected by clinical disease stage or worsening immunosuppression. asymptomatic, parasite density, under-fives, immunity INTRODUCTION Human immunodeficiency virus (HIV) and Falciparum malaria infection are leading causes of morbidity and mortality in sub-Saharan Africa [1]. The epidemiologic overlap has been the source of concern, as coinfection could further increase the health burden [2]. Falciparum malaria is a major public health problem, with one child dying every minute in Africa [3], especially among under-fives owing to naive immunity [4–6]. In endemic areas, the transfer of maternal antibodies, repeated early childhood exposure and clinical episodes generate and sustain partial antimalarial immunity, which ameliorates infection but does not prevent asymptomatic malaria parasitaemia [7], i.e. the presence of malaria parasites in the blood without symptoms [8]. CD4+ T-cells are essential for developing antimalarial immunity [9]; they help B cells produce antibodies and indirectly support the control of parasitaemia through cytokine production and macrophage activation [10]. In HIV-infected children, malaria-specific immunity development and parasite clearance are impaired, resulting in increased predisposition to parasitaemia [9, 11–13]. Asymptomatic malaria parasitaemia is a predictive parameter for transmission and assessment of immunity in exposed populations [8, 14, 15]. This study highlights antimalarial immunity in a holoendemic region where sustained exposure generates immunity in childhood. Withworth et al. [2] found twice as many parasitaemia episodes, increasing with worsening immunosuppression in HIV-infected adults with symptomatic malaria in a moderate endemic area. The findings were collaborated by French et al. [16]. Coinfected pregnant adults living in stable and unstable malaria regions also showed increased parasitaemia [17–21]. Studies of asymptomatic malaria in HIV-infected individuals are few and limited in children from holoendemic regions having almost half of global malaria cases [22]. This cross-sectional study examines asymptomatic parasitaemia in HIV-infected children <5 years and the role of CD4 T-cells on parasite density. METHODS The study was conducted at the paediatric HIV clinic of University of Benin Teaching Hospital (UBTH), between March and July 2012. Malaria transmission is holoendemic; intense and stable transmission occurs all year round with peaks during the rainy season [23]. The HIV diagnosis for afebrile children 6 weeks to <18 months was by HIV DNA polymerase chain reaction, and those ≥18 months had HIV rapid antibody serial testing algorithm II using standard protocols [24]. Controls were age- and sex-matched HIV-1-negative children attending clinics for non-chronic medical conditions. Children with history of malaria treatment in the preceding 2 weeks and those on chemoprophylaxis were excluded. Haemoglobin genotype was done to exclude sickle cell disease in subjects and controls. Information on co-trimoxazole (CTX) prophylaxis is according to the national guideline; house screening and use of insecticide-treated nets (ITNs) were noted. Ethical approval was obtained from UBTH Ethics and Research Committee (ADM/E.22 A/VOL, VII/390), while informed, written consent was from parent(s)/guardian(s) of subjects and controls. Pre-test/Post-test counselling was done before and following HIV testing. The clinical (World Health Organization, WHO) staging of HIV/AIDS of the subjects was noted. Blood samples were collected into ethylenediaminetetraacetic acid (EDTA) anticoagulated containers and concomitant samples for CD4 count. Thick and thin blood smears were stained with 2% Giemsa for 30 min and read by WHO-certified microscopists who were uninvolved in direct patient care and blinded to the clinical status of the study participants. Asexual malaria parasitaemia was recorded as present or absent. Parasites/μl were calculated from thick blood smears by counting asexual parasites per 200 leukocytes, assuming a leukocyte count of 8000/ml. A blood smear was considered negative on examination of 100 high-power fields without parasites. Thin smears were for determination of parasite species [24]. CD4 counts was analysed using Flow Cytometer Cyflow SL Green® (Partec). The CD4 counts were documented according to the Centres for Disease Control and Prevention immunologic classification [24]. HIV screening test was carried out for the controls using HIV rapid testing serial algorithm II for HIV diagnosis according to national guidelines [25, 26]. Statistical analysis Clinical and laboratory data management and analysis were by IBM SPSS statistic 20. The test of normality (Kolmogorov–Smirnov) showed that malaria parasite density for microscopy positive slides was not normally distributed, hence the use of non-parametric tests for analysis. Mann–Whitney U test was to compare the mean parasite densities between cohorts. While Kruskal–Wallis test was for comparison of more than two means, the level of significance was at p < 0.05 and confidence level of 95%. RESULTS General characteristics In all, 179 HIV-1 positive and 179 HIV-1 negative sex- and age-matched controls were studied. The subjects consisted of 96 (53.6%) males and 83 (46.4%) females at the ratio of 1.16:1. The age range of the study population was 2–59 months and their age distribution is shown in Table 1. Table 1 Age distribution of the subjects and controls Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Table 1 Age distribution of the subjects and controls Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Age group (Months) Subjects n (%) Controlsn (%) 2 to < 12 9 (5) 9 (5) 12 to < 24 13(7.3) 13(7.3) 24 to < 36 25(14) 25(14) 36 to < 48 35(19.5) 35(19.5) 48 to 59 97(54.2) 97(54.2) Most of the subjects (76; 42.5%) were in WHO clinical Stage 1, 44 (24.6%) in Stage 2, 46 (25.7%) in Stage 3, while 13 (7.3%) were in Stage 4. Majority of the subjects [152 (84.9%)] were in immunologic Categories 1 and 2, while 27 (15.1%) were in Category 3. Plasmodium falciparum was the only parasite specie identified. In all, 61 (34.1%) of the subjects and 32 (17.9%) of the controls had positive parasite smears (χ2 = 13.166, p = 0.001). Asymptomatic mean parasite density The mean parasite density (for microscopy-positive slides only) in subjects of 118.7 ± 70.5 parasite/µl was significantly higher than 87.3 ± 36.9 noted in controls (Mann–Whitney U = 705.5, p = 0.029) (Table 2). Table 2 Mean parasite density by age (groups) in subjects and controls Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Kruskal–Wallis test subjects: χ2 = 3.255, p = 0.516. Controls: χ2 = 2.531, p = 0.639, df = 4. mpd, mean parasite density/µl; SD, standard deviation. Table 2 Mean parasite density by age (groups) in subjects and controls Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Age group (months) Subject mpd (SD) Controls mpd (SD) Mann-Whitney U P 2 to  <12 148.8 (93.3) 90.0 1.000 0.380 12 to < 24 84.7 (55.0) 65.0 (5.0) 6.000 0.437 24 to < 36 127.9 (54.8) 96.7 (44.5) 21.500 0.187 36 to < 48 107.6 (42.4) 81.3 (27.7) 22.000 0.189 48 to < 59 122.2 (83.3) 89.5 (41.4) 173.000 0.346 118.7 (70.5) 87.3 (36.9) 705.500 0.029 Kruskal–Wallis test subjects: χ2 = 3.255, p = 0.516. Controls: χ2 = 2.531, p = 0.639, df = 4. mpd, mean parasite density/µl; SD, standard deviation. Asymptomatic parasitaemia in subjects was higher than in controls of all age brackets. Subjects 2 to <12 months (infancy) had the highest density of parasites (148.8 parasites/μl), and 12 to < 24 months age bracket had the least (84.7 parasites/μl). The differences in parasitaemia by age brackets of subjects and controls were not statistically significant (Table 2). The changes in asymptomatic malaria parasite density (AMPD) by age groups of subjects and controls are shown in Fig. 1. Fig. 1. View largeDownload slide Line graph depicting the mean parasite densities by age groups of the subjects and controls. Fig. 1. View largeDownload slide Line graph depicting the mean parasite densities by age groups of the subjects and controls. Parasitaemia by gender The AMPD of male subjects was 118.6 ± 70.5 parasites/µl as against 119.0 ± 54.0 in female subjects, p = 0.84. For the controls, it was 95.5 ± 36.4 parasites/µl in males and 78.0 ± 36.4 in females, p = 0.84. Parasitaemia by WHO stage and immune category The AMPD was least (111.6 parasite/µl) among subjects in WHO Stage 1 and highest (131.2 parasite/µl) among those in WHO Stage 4. Parasitaemia increased with worsening WHO clinical staging; however, this relationship was not statistically significant (Kruskal–Wallis test, χ2 = 1.514, p = 0.679) (Table 3). Table 3 Mean parasite density by WHO clinical staging of subjects WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) Kruskal–Wallis test χ2= 1.514, p = 0.679, df = 3. Table 3 Mean parasite density by WHO clinical staging of subjects WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) WHO stage Subjects mpd (±SD) 1 76 111.6 (70.9) 2 44 113.1 (66.6) 3 46 129.0 (71.7) 4 13 138.2 (92.2) Total 179 118.7 (70.5) Kruskal–Wallis test χ2= 1.514, p = 0.679, df = 3. The AMPD was lowest among subjects in immune Category 1 and highest in immune Category 2. Association between parasitaemia and immune category was not statistically significant. (Kruskal–Wallis test, χ2 = 0.970, p = 0.616) (Table 4). Table 4 Mean parasite density by immune category of subjects Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Kruskal–Wallis test χ2 =0.970, p = 0.616, df = 2. Table 4 Mean parasite density by immune category of subjects Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Immune category Subjects Mean parasite density (±SD) 1 80 100.8 (49.0) 2 72 131.7 (72.3) 3 27 128.8 (91.5) Total 179 118.7 (70.5) Kruskal–Wallis test χ2 =0.970, p = 0.616, df = 2. Parasitaemia with antiretroviral therapy (ART) use Subjects on ART had higher AMPD (123.4 ± 73.7) than those not on treatment (88.1 ± 32.1) although the relationship was not significant (Mann–Whitney U = 165.500, p = 0.320). Mean parasite densities were higher in subjects receiving Zidovudine, Nevirapine, Lamivudine, Efavirenz and Stavudine (Table 5). None of the subjects on Ritonavir/Lopinavir and Abacavir had any parasitaemia. Table 5 Mean parasite density by ART use ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 Table 5 Mean parasite density by ART use ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 ART n (%) mpd (±SD) Mann-Whitney U P Zidovudine  Yes 144 (80.4) 118.9 (69.4)  No 35 (19.6) 117.9 (78.8) 266.500 0.873 Nevirapine  Yes 148 (82.7) 123.6 (75.1)  No 31 (17.9) 94.0 (31.0) 223.000 0.533 Lamivudine  Yes 151 (84.4) 123.4 (73.7)  No 28 (15.6) 88.13 (32.1) 165.500 0.320 Efavirenz  Yes 6 (3.4) 183.0 (113.6)  No 173 (96.6) 115.4 (67.5) 43.500 0.147 Stavudine  Yes 12 (6.7) 139.0 (91.9)  No 167 (93.3) 118.1 (70.5) 50.000 0.715 ART  Yes 155 (86.6) 123.4 (73.7)  No 24 (13.4) 88.1 (32.1) 165.500 0.320 Parasitaemia with duration of ART use The AMPD was highest (128.4 ± 77.8) among those that received ART for <6 months, while the least parasite density (116.0 ± 45.7) was in those on ART for 18 to < 24 months. The lowest AMPD (88.1 ± 32.1) was among those that are ART naive. This relationship between AMPD and duration of ART was not statistically significant (Table 6). Table 6 Mean parasite density by duration of ART use ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) Kruskal–Wallis test χ2 = 1.799, p = 0.876, df = 5. Table 6 Mean parasite density by duration of ART use ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) ART duration (months) n Mean parasite density(±SD) Parasite/µl Nil 25  88.1 (32.1) 0 to < 6 22 128.4 (77.8) 6 to < 12 34 123.2 (82.7) 12 to < 18 9 124.6 (77.3) 18 to < 24 16 116.0 (45.7) ≥24 73 121.4 (76.9) Kruskal–Wallis test χ2 = 1.799, p = 0.876, df = 5. Parasitaemia and CTX prophylaxis Up to 46 of 154 subjects on CTX prophylaxis had positive result while 15 of 25 not receiving prophylaxis had positive malaria parasitaemia, a 3-fold risk (χ2 = 8.692, p = 0.003). The AMPD among subjects on prophylaxis of 127.5 ± 75.0 parasites/µl was not significantly higher than 91.9 ± 47.1 among those not on CTX, Mann–Whitney U = 242.500, p = 0.086. Parasitaemia and the use of ITN and window net The AMPD of infected children sleeping under ITNs was not significantly higher than those not sleeping under ITN; also for Children living in screened houses, AMPD was not significantly different from those living in unscreened houses (Table 7). Table 7 Mean parasite density of subjects by use of ITN and window net n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) Table 7 Mean parasite density of subjects by use of ITN and window net n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) n (%) Mpd (±SD) Mann-Whitney U Window netting  Yes 114(63.7) 129.4 (71.3) 302.500; p=0.099  No 65 (36.3) 96.8 (65.0) ITN use  Yes 20 (11.2) 161.5 (101.2) 20.500; p=0.137  No 159 (88.8) 112.3 (63.5) DISCUSSION This study examines AMPD in HIV-infected children <5 years living in the holoendemic malaria region of Southern Nigeria. The study reveals significantly higher parasitaemia among HIV-infected compared with uninfected children. Malaria parasitaemia in infected children was higher at every age group, and the disparity was highest at infancy. Age was not a significant predictor of parasitaemia. The parasite density increased albeit non-significantly with worsening clinical staging and immunosuppression. Increased parasitaemia among HIV-infected children could be owing to the immune deficiencies and dysfunction attributable to HIV infection. HIV-related immunodeficiency is the probable cause, as the parasite may thrive better in infected than uninfected group. Malaria parasitaemia influences development of partial immunity [27], protection against clinical disease and ameliorates new infections in persons living in endemic areas [2, 28]. HIV-infected children therefore are at higher risk of having malaria parasitaemia, making HIV infection a risk factor for enhanced asymptomatic malaria parasitaemia [13–15]. The asymptomatic parasitaemia of 118.7 parasites/µl amongst HIV-infected children is higher than 85.2 parasites/µl reported by Adetifa et al. [29] in HIV-positive children 1–5 years in Lagos, South-West Nigeria. The reason for the difference in parasite density is not readily apparent. The AMPD reported in this study was smaller than 1922 parasites/µl reported by Owusu-Agyei et al. in Ghana among HIV-positive children 1–2 years and 1903 among 3–4-year-olds [15]. The study in Ghana reported geometric mean, while arithmetic mean was quoted in our study; this could have accounted for the large difference in parasitaemia. The pattern of parasitaemia in infected and uninfected children showed striking similarities with increasing age (Fig. 1). The narrow spectrum of parasitaemia may be owing to asymptomatic infection and small parasite densities recorded. Symptomatic malaria occurs with higher malaria parasitaemia [7]. Infancy was the peak of parasitaemia in both groups followed by a nadir at 12 to 24 months, to the staggered increase at 48 to <59 months. It seems probable therefore that infected children develop antimalarial immunity timely as non-infected peers albeit with higher parasitaemia. This is an area needing further exploration. Asymptomatic parasitaemia is necessary for development of specific antimalarial immunity and maintenance of the transmission cycle [7, 13–15, 30]. It can therefore be speculated that HIV infection does not distort the acquisition of antimalarial immunity. The finding of the highest parasitaemia and the worst disparity at infancy is an area needing further exploration. Comparative studies on HIV-infected/uninfected pregnant women have consistently reported higher parasitaemia in the infected groups. This may be partly owing to transient loss of acquired antimalarial immunity, leading to higher malaria and placental parasitaemia at delivery [19, 20]. The insufficient or defective transfer of antimalarial immune factors transplacentally from HIV-infected mothers could predispose their infants to higher AMPD as seen in this study. That uninfected infants had lower parasitaemia underscores this proposition. Therefore, infancy remains the critical point of enhanced parasitaemia in infected children. Appropriate vector avoidance is therefore recommended for malaria control. Malaria parasitaemia with HIV clinical disease stage and worsening immunosuppression is one of the objectives of this study. While parasitaemia increased with clinical staging, there was no consistent trend with immune category and the relationship was not significant. Clinical disease stage and immune category do not affect antimalarial immunity development among children <5 years. This finding differs from some studies on clinical (symptomatic) malaria [9, 17]. Apart from CD4+ T-cells, there are other factors required for development and sustenance of antimalarial immunity. Malaria parasitaemia was uninfluenced by ART, and there was increased parasitaemia among those on Zidovudine, Nevirapine, Lamivudine, Efavirenz and Stavudine. The duration of ART did not significantly affect parasitaemia. The reason for the non-association may be from lack of antimalarial properties by most ARTs. Their use, however, is known to enhance immune reconstitution and subsequently improve parasite clearance with attendant lower parasitaemia [31]. This is an expected occurrence with longer duration of ART administration. Our findings may be partly owing to the few number of children on protease inhibitors (Pi) (1.1%), which has distinct antimalarial properties [32]. The least parasitaemia, however, was among those that are ART naive. Parasitaemia in this study was unaffected by window netting or use of ITNs. The lack of association between ITN use and malaria parasite density is surprising, as ITN is a known factor for its beneficial vector avoidance property [31]. Although the fraction of infected children using ITNs is small (11.2%), infrequent use maybe contributory. Infected children on CTX had significantly lower prevalence of malaria parasitaemia as has been reported by other researchers and collaborated by this study. However, the finding of non-statistical higher parasitaemia among infected children on CTX is worrisome. This may be part of the higher AMPD associated with HIV infection; however, evidence abounds that CTX reduces symptomatic malaria incidence by 76%. ART and ITNs substantially reduce malaria frequency in HIV-infected adults [31]. In conclusion, malaria parasitaemia is higher in asymptomatic HIV-infected compared with uninfected children <5 years. In all age brackets, higher parasitaemia was maintained and a similar pattern is observed in both groups. Acquisition of antimalarial immunity in both groups may be similar albeit with higher parasitaemia in HIV-infected children. ACKNOWLEDGEMENT To all the children that attend the Paediatric HIV clinic in UBTH Benin City and all the staff that made this work possible, thank you for your support and immense contribution. FUNDING The study is self-funded with technical support of University of Benin Teaching Hospital, Benin City, Edo State, Nigeria. References 1 Joint United Nations Programme on HIV/AIDS (UNAIDS)/World Health Organization (WHO) . AIDS epidemic update: 2009. UNAIDS. Geneva: UNAIDS/WHO, 2009 . 2 Whitworth J , Morgan D , Quigley M , et al. Effect of HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort study . Lancet 2000 ; 356 : 1051 – 6 . Google Scholar CrossRef Search ADS PubMed 3 Krogstad DJ. Malaria as a re-emerging disease . Epidemiol Rev 1996 ; 18 : 77 – 89 . Google Scholar CrossRef Search ADS PubMed 4 Samba E. Preface: the economic burden of malaria . Am J Trop Med Hyg 2001 ; 64 : ii . Google Scholar CrossRef Search ADS PubMed 5 Breman JG , Egan A , Keusch GT. The intolerable burden of malaria: a new look at the numbers . Am J Trop Med Hyg 2001 ; 64 (suppl): iv – vii . Google Scholar CrossRef Search ADS PubMed 6 Najera JA , Liese BH , Hammer J. Malaria In: Jamison DJ , Mosley WH , Measham AR , et al. (eds). Disease Control Priorities in Developing Countries . New York : Oxford University Press , 1993 , 281 – 302 . 7 Bruce-Chwatt LJ. Malaria in African infants and children in southern Nigeria . Ann Trop Med Parasitol 1952 ; 46 : 173 – 200 . 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Google Scholar CrossRef Search ADS PubMed 17 Patnaik P , Jere CS , Miller WC , et al. Effects of HIV-1 serostatus, HIV-1 RNA concentration, and CD4 cell count on the incidence of malaria infection in a cohort of adults in rural Malawi . J Infect Dis 2005 ; 192 : 984 – 91 . Google Scholar CrossRef Search ADS PubMed 18 Cohen C , Karstaedt A , Frean J , et al. Increased prevalence of severe malaria in HIV-infected adults in South Africa . Clin Infect Dis 2005 ; 41 : 1631 – 7 . Google Scholar CrossRef Search ADS PubMed 19 Steketee RW , Wirima JJ , Bloland PB , et al. Impairment of a pregnant woman's acquired ability to limit Plasmodium falciparum by infection with human immunodeficiency virus type-1 . Am J Trop Med Hyg 1996 ; 55 : 42 – 9 . Google Scholar CrossRef Search ADS PubMed 20 Verhoeff FH , Brabin BJ , Hart CA , et al. Increased prevalence of malaria in HIV-infected pregnant women and its implications for malaria control . Trop Med Int Health 1999 ; 4 : 5 – 12 . 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Asymptomatic malaria parasitaemia—a suitable index for evaluation of malaria vector control measures . Nig J Paediatr 2002 ; 29 : 23 – 6 . Google Scholar CrossRef Search ADS 31 Mermin J , Ekwaru JP , Liechty AL , et al. Effect of co-trimoxazole prophylaxis, antiretroviral therapy, and insecticide-treated bednets on the frequency of malaria in HIV-1-infected adults in Uganda . Lancet 2006 ; 367 : 1256 – 61 . Google Scholar CrossRef Search ADS PubMed 32 Parikh S , Gut J , Istvan E , et al. Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors . Antimicrob Agents Chemother 2005 ; 49 : 2983 – 5 . Google Scholar CrossRef Search ADS PubMed © The Author [2017]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Tropical PediatricsOxford University Press

Published: Aug 1, 2018

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