[Show abstract][Hide abstract] ABSTRACT: As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material.
[Show abstract][Hide abstract] ABSTRACT: Background
Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear.
We evaluated the effect of baseline S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥37.5°C) and parasitemia (≥2500 asexual parasites/µl). Secondary definitions of malaria using different parasite densities were also explored.
After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression (hazard ratios 0.71 and 0.44; P = 0.01 and 0.02; reference group: uninfected at baseline). Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode.
Co-infection with S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.
[Show abstract][Hide abstract] ABSTRACT: In malaria-naïve individuals, Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, individuals in endemic areas who are repeatedly infected are often asymptomatic and have low levels of iRBCs, even young children. We hypothesized that febrile malaria alters the immune system such that P. falciparum re-exposure results in reduced production of pro-inflammatory cytokines/chemokines and enhanced anti-parasite effector responses compared to responses induced before malaria. To test this hypothesis we used a systems biology approach to analyze PBMCs sampled from healthy children before the six-month malaria season and the same children seven days after treatment of their first febrile malaria episode of the ensuing season. PBMCs were stimulated with iRBC in vitro and various immune parameters were measured. Before the malaria season, children's immune cells responded to iRBCs by producing pro-inflammatory mediators such as IL-1β, IL-6 and IL-8. Following malaria there was a marked shift in the response to iRBCs with the same children's immune cells producing lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines (IL-10, TGF-β). In addition, molecules involved in phagocytosis and activation of adaptive immunity were upregulated after malaria as compared to before. This shift was accompanied by an increase in P. falciparum-specific CD4+Foxp3- T cells that co-produce IL-10, IFN-γ and TNF; however, after the subsequent six-month dry season, a period of markedly reduced malaria transmission, P. falciparum-inducible IL-10 production remained partially upregulated only in children with persistent asymptomatic infections. These findings suggest that in the face of P. falciparum re-exposure, children acquire exposure-dependent P. falciparum-specific immunoregulatory responses that dampen pathogenic inflammation while enhancing anti-parasite effector mechanisms. These data provide mechanistic insight into the observation that P. falciparum-infected children in endemic areas are often afebrile and tend to control parasite replication.
[Show abstract][Hide abstract] ABSTRACT: Background. Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear.Methods. We assessed pre-malaria season PfRH5-specific IgG levels in 357 Malian children and adults who were uninfected with Plasmodium. Subsequent P. falciparum infections were detected by PCR every 2 weeks and malaria episodes by weekly physical examination and self-referral for 7 months. The primary outcome was time between the first P. falciparum infection and the first febrile malaria episode. PfRH5-specific IgG was assayed for parasite growth-inhibitory activity.Results. The presence of PfRH5-specific IgG at enrollment was associated with a longer time between the first blood-stage infection and the first malaria episode (PfRH5-seropositive median: 71 days, PfRH5-seronegative median: 18 days; P = .001). This association remained significant after adjustment for age and other factors associated with malaria risk/exposure (HR, .62; P = .02). Concentrated PfRH5-specific IgG purified from Malians inhibited P. falciparum growth in vitro.Conclusions. Naturally acquired PfRH5-specific IgG inhibits parasite growth in vitro and predicts protection from malaria. These findings strongly support efforts to develop PfRH5 as an urgently needed blood-stage malaria vaccine.
The Journal of Infectious Diseases 10/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. In experimental models of human and mouse malaria, sterilizing liver stage immunity that blocks progression of Plasmodium infection to the symptomatic blood stage can be readily demonstrated. However, it remains unclear whether individuals in malaria-endemic areas acquire such immunity. Methods. In Mali, 251 healthy children and adults aged 4 to 25 years who were free of blood-stage Plasmodium infection by PCR were enrolled in a longitudinal study just prior to an intense six-month malaria season. Subsequent clinical malaria episodes were detected by weekly active surveillance and self-referral. Asymptomatic P. falciparum infections were detected by blood-smear microscopy and PCR analysis of dried blood spots which had been collected every two weeks for seven months. Results. As expected, the risk of clinical malaria decreased with increasing age (log-rank test, P=.0038). However, analysis of PCR data showed no age-related differences in P. falciparum infection risk (log-rank test, P=.37). Conclusions. Despite years of exposure to intense P. falciparum transmission, there is no evidence of acquired, sterile immunity to P. falciparum infection in this population, even as clinical immunity to blood-stage malaria is clearly acquired. Understanding why repeated P. falciparum infections do not induce sterile protection may lead to insights for developing vaccines that target the liver stage in malaria-endemic populations.
[Show abstract][Hide abstract] ABSTRACT: Heterozygous states of hemoglobin (Hb) A and HbS (HbAS, sickle-cell trait) or HbC (HbAC) protect against Plasmodium falciparum malaria by unclear mechanisms. Several studies suggest that HbAS and HbAC accelerate the acquisition of immunity to malaria, possibly by enhancing P. falciparum-specific antibody responses.
We used a protein microarray representing 491 P. falciparum proteins expressed during exoerythrocytic and erythrocytic stages of the life cycle to test the hypothesis that HbAS and HbAC enhance the P. falciparum-specific IgG response compared with normal HbAA. Plasma samples were collected from Malian children aged 2-10 years before and after a 6-month malaria season and were probed against the microarray. Immunoglobulin G (IgG) profiles of children with HbAA (n = 106), HbAS (n = 15), and HbAC (n = 20) were compared.
Although the magnitude and breadth of P. falciparum-specific IgG responses increased with age and from before to after the malaria season in each antigen category, Hb type did not independently predict significant differences in P. falciparum-specific IgG profiles.
These data do not support the hypothesis that HbAS and HbAC protect against malaria by enhancing P. falciparum-specific antibody responses. It remains possible that HbAS and HbAC protect against malaria by enhancing antibody responses to antigens not studied here or through other immune mechanisms.
The Journal of Infectious Diseases 12/2011; 204(11):1750-61. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In 2003, Mali introduced intermittent preventive therapy in pregnancy (ITPp) with sulfadoxine-pyrimethamine (SP) for the control of malaria in pregnancy, consisting of 2 doses of SP given in the 2nd and 3rd trimester. This widely used regimen, although very effective, leaves many women unprotected from malaria during the last 4-to-8 weeks of gestation, which is a pivotal period for fetal weight gain. The aim of the study was to compare the efficacy and safety of 3-dose versus 2-dose IPTp-SP for the prevention of placental malaria and associated low birth weight (LBW).
We conducted a parallel-group, open-label, individually randomized controlled superiority trial involving 814 women of all gravidity, enrolled from April 2006 through March 2008. All women were seen at least 3 times and received either 2 (n = 401) or 3 (n = 413) doses of IPTp-SP. The primary endpoint measured was placental malaria, LBW, preterm births, and maternal anemia were secondary endpoints, and severe maternal skin reactions and neonatal jaundice were safety endpoints.
Among the 96% of study subjects who were followed up until delivery, the prevalence of placental malaria was 2-fold lower in the 3-dose group (8.0%) than in the 2-dose group (16.7%); the adjusted prevalence ratio (APR) was 0.48 (95% confidence interval [CI], 0.32-0.71). LBW and preterm births were also reduced; the prevalence of LBW was 6.6% in the 3-dose group versus 13.3% in the 2-dose group (APR, 0.50; 95% CI, 0.32-0.79), and the prevalence of preterm births was 3.2% versus 8.9% (APR, 0.37; 95% CI, 0.19-0.71). No significant reductions in maternal anemia or differences in safety endpoints were observed.
Adding a third dose of ITPp-SP halved the risk of placental malaria, LBW, and preterm births in all gravidae, compared with the standard 2-dose regimen, in this area of highly seasonal transmission with low levels of SP resistance. Clinical Trials Registration: ISRCTN 74189211.
[Show abstract][Hide abstract] ABSTRACT: Antibodies that protect against Plasmodium falciparum (Pf) malaria are only acquired after years of repeated infections. The B cell biology that underlies this observation is poorly understood. We previously reported that "atypical" memory B cells are increased in children and adults exposed to intense Pf transmission in Mali, similar to what has been observed in individuals infected with HIV. In this study we examined B cell subsets of Pf -infected adults in Peru and Mali to determine if Pf transmission intensity correlates with atypical memory B cell expansion.
In this cross-sectional study venous blood was collected from adults in areas of zero (U.S., n = 10), low (Peru, n = 18) and high (Mali, n = 12) Pf transmission. Adults in Peru and Mali were infected with Pf at the time of blood collection. Thawed lymphocytes were analyzed by flow cytometry to quantify B cell subsets, including atypical memory B cells, defined by the cell surface markers CD19(+) CD20(+) CD21(-) CD27(-) CD10(-). In Peru, the mean level of atypical memory B cells, as a percent of total B cells, was higher than U.S. adults (Peru mean: 5.4% [95% CI: 3.61-7.28]; U.S. mean: 1.4% [95% CI: 0.92-1.81]; p<0.0001) but lower than Malian adults (Mali mean 13.1% [95% CI: 10.68-15.57]; p = 0.0001). In Peru, individuals self-reporting ≥1 prior malaria episodes had a higher percentage of atypical memory B cells compared to those reporting no prior episodes (≥1 prior episodes mean: 6.6% [95% CI: 4.09-9.11]; no prior episodes mean: 3.1% [95% CI: 1.52-4.73]; p = 0.028).
Compared to Pf-naive controls, atypical memory B cells were increased in Peruvian adults exposed to low Pf transmission, and further increased in Malian adults exposed to intense Pf transmission. Understanding the origin, function and antigen specificity of atypical memory B cells in the context of Pf infection could contribute to our understanding of naturally-acquired malaria immunity.
PLoS ONE 01/2011; 6(1):e15983. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast milk contains pro- and anti-inflammatory cytokines and chemokines with potential to influence immunological maturation in the child. We have shown previously that country of birth is associated with the cytokine/chemokine profile of breast milk. In this study we have investigated how these differences in breast milk affect the cellular response of cord blood mononuclear cells (CBMCs) and intestinal epithelial cells (IECs, cell line HT-29) to microbial challenge. Ninety-five women were included: 30 from Mali in West Africa, 32 Swedish immigrants and 33 native Swedish women. CBMCs or IECs were stimulated in vitro with breast milk, alone or in combination with lipopolysaccharide (LPS) or peptidoglycan (PGN). Breast milk in general abrogated the LPS-induced down-regulation of surface CD14 and Toll-like receptor (TLR)-4 expression on CB monocytes, while inhibiting the PGN-induced TLR-2 up-regulation. However, breast milk from immigrant women together with LPS induced a lower CBMC release of interleukin (IL)-6 (P = 0·034) and CXCL-8/IL-8 (P = 0·037) compared with breast milk from Swedish women, while breast milk from Swedish women and Mali women tended to increase the response. The same pattern of CXCL-8/IL-8 release could be seen after stimulation of IECs (HT-29). The lower CBMC and IEC (HT-29) responses to microbial compounds by breast milk from immigrant women could be explained by the fact that breast milk from the immigrant group showed a divergent pro- and anti-inflammatory content for CXCL-8/IL-8, transforming growth factor-β1 and soluble CD14, compared to the other two groups of women. This may have implications for maturation of their children's immune responses.
[Show abstract][Hide abstract] ABSTRACT: Immunity to Plasmodium falciparum (Pf) malaria is only acquired after years of repeated infections and wanes rapidly without ongoing parasite exposure. Antibodies are central to malaria immunity, yet little is known about the B-cell biology that underlies the inefficient acquisition of Pf-specific humoral immunity. This year-long prospective study in Mali of 185 individuals aged 2 to 25 years shows that Pf-specific memory B-cells and antibodies are acquired gradually in a stepwise fashion over years of repeated Pf exposure. Both Pf-specific memory B cells and antibody titers increased after acute malaria and then, after six months of decreased Pf exposure, contracted to a point slightly higher than pre-infection levels. This inefficient, stepwise expansion of both the Pf-specific memory B-cell and long-lived antibody compartments depends on Pf exposure rather than age, based on the comparator response to tetanus vaccination that was efficient and stable. These observations lend new insights into the cellular basis of the delayed acquisition of malaria immunity.
[Show abstract][Hide abstract] ABSTRACT: Abs are central to malaria immunity, which is only acquired after years of exposure to Plasmodium falciparum (Pf). Despite the enormous worldwide burden of malaria, the targets of protective Abs and the basis of their inefficient acquisition are unknown. Addressing these knowledge gaps could accelerate malaria vaccine development. To this end, we developed a protein microarray containing approximately 23% of the Pf 5,400-protein proteome and used this array to probe plasma from 220 individuals between the ages of 2-10 years and 18-25 years in Mali before and after the 6-month malaria season. Episodes of malaria were detected by passive surveillance over the 8-month study period. Ab reactivity to Pf proteins rose dramatically in children during the malaria season; however, most of this response appeared to be short-lived based on cross-sectional analysis before the malaria season, which revealed only modest incremental increases in Ab reactivity with age. Ab reactivities to 49 Pf proteins measured before the malaria season were significantly higher in 8-10-year-old children who were infected with Pf during the malaria season but did not experience malaria (n = 12) vs. those who experienced malaria (n = 29). This analysis also provided insight into patterns of Ab reactivity against Pf proteins based on the life cycle stage at which proteins are expressed, subcellular location, and other proteomic features. This approach, if validated in larger studies and in other epidemiological settings, could prove to be a useful strategy for better understanding fundamental properties of the human immune response to Pf and for identifying previously undescribed vaccine targets.
Proceedings of the National Academy of Sciences 03/2010; 107(15):6958-63. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunity to the asexual blood stage of Plasmodium falciparum is complex and likely involves several effector mechanisms. Antibodies are thought to play a critical role in malaria immunity, and a corresponding in vitro correlate of antibody-mediated immunity has long been sought to facilitate malaria vaccine development. The growth inhibition assay (GIA) measures the capacity of antibodies to limit red blood cell (RBC) invasion and/or growth of P. falciparum in vitro. In humans, naturally acquired and vaccine-induced P. falciparum-specific antibodies have growth-inhibitory activity, but it is unclear if growth-inhibitory activity correlates with protection from clinical disease. In a longitudinal study in Mali, purified IgGs, obtained from plasmas collected before the malaria season from 220 individuals aged 2 to 10 and 18 to 25 years, were assayed for growth-inhibitory activity. Malaria episodes were recorded by passive surveillance over the subsequent 6-month malaria season. Logistic regression showed that greater age (odds ratio [OR], 0.78; 95% confidence interval [95% CI], 0.63 to 0.95; P = 0.02) and growth-inhibitory activity (OR, 0.50; 95% CI, 0.30 to 0.85; P = 0.01) were significantly associated with decreased malaria risk in children. A growth-inhibitory activity level of 40% was determined to be the optimal cutoff for discriminating malaria-immune and susceptible individuals in this cohort, with a sensitivity of 97.0%, but a low specificity of 24.3%, which limited the assay's ability to accurately predict protective immunity and to serve as an in vitro correlate of antibody-mediated immunity. These data suggest that antibodies which block merozoite invasion of RBC and/or inhibit the intra-RBC growth of the parasite contribute to but are not sufficient for the acquisition of malaria immunity.
Infection and immunity 11/2009; 78(2):737-45. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiological observations in malaria endemic areas have long suggested a deficiency in the generation and maintenance of B cell memory to Plasmodium falciparum (Pf) in individuals chronically reinfected with the parasite. Recently, a functionally and phenotypically distinct population of FCRL4(+) hyporesponsive memory B cells (MBCs) was reported to be expanded in HIV-infected individuals with high viral loads. In this study, we provide evidence that a phenotypically similar atypical MBC population is significantly expanded in Pf-exposed Malian adults and children as young as 2 years of age as compared with healthy U.S. adult controls. The number of these atypical MBCs was higher in children with chronic asymptomatic Pf infections compared with uninfected children, suggesting that the chronic presence of the parasite may drive expansion of these distinct MBCs. This is the first description of an atypical MBC phenotype associated with malaria. Understanding the origin and function of these MBCs could be important in informing the design of malaria vaccines.
The Journal of Immunology 09/2009; 183(3):2176-82. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The World Health Organization (WHO) recently recommended that the time to first malaria episode serve as the primary end point in phase III malaria vaccine trials--the first of which will be held in Africa. Although common red blood cell (RBC) polymorphisms such as sickle hemoglobin (HbS) are known to protect against malaria in Africa, their impact on this end point has not been investigated.
A longitudinal study of 225 individuals aged 2-25 years was conducted in Mali. The association between common RBC polymorphisms and the time to first malaria episode was evaluated.
Among children aged 2-10 years, sickle cell trait (HbAS) was associated with a 34-day delay in the median time to first malaria episode (P= .017) Cox regression analysis showed that greater age (hazard ratio [HR], 0.87 [95% CI, 0.80-0.94]; (P= .001), HbAS (HR, 0.48 [95% CI, 0.26-0.91]; (P= .024), and asymptomatic parasitemia at enrollment (HR, 0.35 [95% CI, 0.14-0.85]; (P= .021) were associated with decreased malaria risk.
Given the delay in the time to first malaria episode associated with HbAS, it would be advisable for clinical trials and observational studies that use this end point to include Hb typing in the design of studies conducted in areas where HbAS is prevalent.
The Journal of Infectious Diseases 09/2008; 198(9):1265-75. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: From June 2003 to May 2004 we carried out a comparative study of two malaria prophylaxis regimens for pregnant women. The purpose was to compare the efficacy of two regimens using chloroquine (CQ) or sulfadoxine-pyrimethamine (SP) during pregnancy and delivery in a village located in an endemic area of Mali. The study was carried out in Faladié (District of Kati) located 80 km from Bamako. Prophylaxis was administered during the second and third trimesters of pregnancy (except the 9th month for SP). A total of 301 pregnant women were enrolled including 150 in the CQ group and 151 in the SP group. At the onset of the study, the two groups were comparable with regard to socio-demographic and malaria factors. At the time of delivery, malaria infection was reduced by 43.3% in the CQ group (P < 10-6), and by 79.1% in the SP group (p < 10-6). The anemia rate was reduced by 57.5% in the CQ group (Ch2 of McNemar = 0.017), and by 74.8% in the SP group (Ch2 of McNeamar = 0.025). The incidence of placental infection was 20.6 % in the CQ group versus 8.3 % in the SP group (p = 4.10-3). Overall 16.7% of newborns presented low birth weight at delivery including 70.4% in the CQ group. The findings of this study suggest that intermittent presumptive treatment using SP is more effective than intermittent presumptive treatment using CQ in protecting both the mother and newborn against intra-uterine malaria transmission and its consequences.
Médecine tropicale: revue du Corps de santé colonial 11/2007; 67(5):477-80.
[Show abstract][Hide abstract] ABSTRACT: After the first efficacy trial of Intermittent Preventive therapy (IPT) using Sulfadoxine-pyrimethamine (SP), the Malian National Malaria Control Program (NMCP) recommended this new strategy on a large scale to reduce malaria burden during pregnancy during the transition period of the implementation.
From July 2003 to February 2005, we conducted a community-based study in the rural village of Bancoumana
with 8,000 inhabitants located 60 km of Bamako in the Savannah transmission area of Mali. Pregnant women with all parities were enrolled. At enrollment, sociodemographic characteristics such as age, marital status, parity, height, and gestational age were determined and hemoglobin level, as well as peripheral parasitemia was measured. At delivery, peripheral and placental parasitemia were determined, as well as hemoglobin level. Newborn were weighed and examined for congenital abnormalities and potential adverse events. Out of 410 pregnant women enrolled, 378 delivered: 189 in each treatment group.
The mean age of women was 24.26 ± 6.2 years. At enrollment, groups were comparable regarding anemia and peripheral parasitemia. During the follow-up period, women in SP group were less likely to get malaria infection (p=0,005), but no statistical difference was found in anemia prevalence (p>0.05).
At delivery, the prevalence of anemia was higher in Chloroquine (CQ) group (39%) compared to SP group (28.1%); (p =0.003). The two groups were comparable regarding peripheral and placental parasitemia and the rate of low birth weight. The rate of singleton stillbirths (4.1%) and preterm delivery (4.2%) were not statistically significant between groups. Anemia was associated with age (P =0.003) and parity (P= 0.001); primigravidae
and young women (<20 years) were more affected. Preliminary results of this community based trial indicate a reduction of malaria adverse effect with SP compared to CQ, therefore IPT/SP implementation efforts should be maintained by WHO and NMCP in Mali.
American Society of Tropical Medicine and Hygiene 54th Annual meeting, Washington DC; 12/2005
[Show abstract][Hide abstract] ABSTRACT: Malaria during pregnancy contributes to maternal anemia and low birth weight. In East Africa, several studies have demonstrated that intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine (SP) is more efficacious than weekly chloroquine (CQ) chemoprophylaxis in preventing these adverse consequences. To our knowledge, there are no published trials evaluating IPT in West Africa.
We undertook a randomized controlled trial of weekly CQ chemoprophylaxis, 2-dose IPT with CQ, and 2-dose IPT with SP; 1163 women were enrolled.
In multivariate analyses, when compared with weekly CQ, IPT/SP was associated with a reduction in third-trimester anemia (adjusted odds ratio [AOR], 0.49; P<.001), placental parasitemia (AOR, 0.69; P=.04), and low birth weight (<2500 g) (AOR, 0.69; P=.04). The prevalence of placental infection remained unexpectedly high, even in the IPT/SP group (24.5%), possibly because of the intensity of seasonal transmission. There were no significant differences in stillbirths, spontaneous abortions, or neonatal deaths among the 3 groups.
In Mali, IPT with SP appears more efficacious than weekly chloroquine chemoprophylaxis in preventing malaria during pregnancy. These data support World Health Organization recommendations to administer at least 2 doses of IPT during pregnancy. In intensely seasonal transmission settings in Mali, >2 doses may be required to prevent placental reinfection prior to delivery.
The Journal of Infectious Diseases 01/2005; 191(1):109-16. · 5.85 Impact Factor