P. falciparum blood stage antigens in samples from 160 Ugandans collected at two time points during high malaria transmission and two time points following a dramatic reduction in transmission. Results demonstrated that, for the antigens tested, (i) the rate of decay of total IgG following infection declined with age and was driven consistently by the decrease in IgG3 and occasionally the decrease in IgG1; (ii) the proportion of IgG3 relative to IgG1 in the absence of infection increased with age; (iii) the increase in avidity index (the strength of association between the antibody and antigen) following infection was largely due to a rapid loss of non-avid compared to avid total IgG; and (iv) both avid and non-avid total IgG in the absence of infection increased with age. Further studies are required to understand the functional differences between IgG1 and IgG3 in order to determine their contribution to the longevity of protective immunity to malaria. Measuring changes in antibody avidity may be a better approach of detecting affinity maturation compared to avidity index due to the differential expansion and contraction of high and low avidity total IgG.Understanding how immunity to malaria is affected by declining transmission is important to aid vaccine design and understand disease resurgence. Both IgG subclasses and avidity of antigen-specific responses are important components of an effective immune response. Using a multiplex bead array assay, we measured the total IgG, IgG subclasses, and avidity profiles of responses to 18  IgG is an important component of immunity to malaria, with function defined by the specific recognition of antigens, and tropism of the constant region (Fc) for variant Fc receptors . AvidityP. falciparum antigen targets generally diminish in the absence of re-infection, which is thought to contribute to loss of immunity  and avidity, and responses to only a limited number of P. falciparum blood stage antigens in the same individuals before and after decreases in malaria transmission (>90%) due to indoor residual spraying of insecticide (IRS). We compared the net changes in antibody responses during this period to gain a broader insight into how IgG subclasses and avidity index are acquired with age, and how they influence a reduction in IgG levels in the absence of infection.In this study, we measured antibody levels to total IgG, IgG1–4 and avidity index (AI) to 18 Study participants were part of a cohort in Nagongera, eastern Uganda described in detail elsewhere . In 2011via passive surveillance. P. falciparum infection was identified by active surveillance at 1–3 monthly intervals using both microscopy and LAMP (Plasma samples were selected from study participants (n=160) and classified into three age groups; 1–4 (n=40), 5–11 (n=92) and >18 years (n=28). Time points were selected at 12 and 6 months pre-IRS , and at 6 and 12 months post-IRS . Study participants were provided with ITN at enrollment and monitored for parasite prevalence and malaria incidence . Malariaand LAMP .Ethical approval was obtained from the Makerere University School of Medicine Research and Ethics Committee (REC REF 2011-203), the Uganda National Council for Science and Technology (HS 1074), the LSHTM ethics committee (reference # 6012), and the University of California, San Francisco Committee on Human Research (reference 027911).Written informed consent was obtained from a parents or guardian of each child enrolled in the study and also from participating adults.P. falciparum blood stage antigens were assessed in addition to tetanus toxoid (TT)  as a non-malaria control. The P. falciparum antigens broadly fell into three categories; (i) infected red blood cell, (ii) merozoite apical organelle, and (iii) merozoite surface antigen associated proteins. The recombinant antigens were expressed in Escherichia coli either as glutathione S-transferase (GST) fusion proteins or as histidine tagged constructs  and assayed as previously described  as the secondary antibody with a streptavidin-Phycoerythrin labeled tertiary component , similar to what was previously described , 60. TheTo measure the avidity index, the total IgG assay was modified to include a Guanidine Hydrochloride (GuHCl) step to dissociate antibodies with low avidity. Briefly, 50µl GuHCl was added to one of the duplicate wells for each sample, after the plasma incubation step. PBS was added to the second well. After washing R-Phycoerythrin-conjugated AffiniPure F (ab’) 2 Goat anti-human IgG was added and processed as per the standard MagPix assay described above. The signal detected in the well treated with GuHCl represented high avidity antibodies. Avidity index was calculated as a percentage of the high avidity antibodies of the total antibody as shown below.10 transformed MFI and AI at T2 and T4 respectively.The net changes in total IgG, IgG1–4, avidity index (AI) and the avid IgG levels (the antibody remaining bound after GuHCl treatment) between the peak and lowest malaria transmission were derived from a paired difference between log10 transformed MFI) for total IgG, IgG1–4, avidity index and avid IgG with 90 days since last infection were assessed using the generalized estimation equation (GEE) to allow for repeated measures per individual including all 4 time points. Infections were defined by microscopy or loop mediated isothermal amplification (LAMP) for the smear negative slides.Associations between antibody levels  differences for total IgG, IgG1–4, AI and avid IgG levels between age categories where compared in a one-way ANOVA, not assuming normal distribution  with pairwise comparisons between all age groups.Analysis were performed and figures generated using Stata version 14 , GraphPad Prism version 7  and R-studio .The antigen panel used in the study was comprised of previously validated markers of seroincidence , 61, 62.P. falciparum antigens evaluated, with median responses higher for IgG3 compared to IgG1 to all antigens except HSP40 ag1 and MSP1-19, for which responses were similar, and to AMA-1 and Rh2_2030 which were higher for IgG1 compared to IgG3 (10MFI) were reduced for most of the P. falciparum antigens between the times of peak malaria transmission (T2) and lowest transmission (T4) , between T2 and T4. The median avidity index increased for most of the P. falciparum infection to relate antibodies at all 4 time points (T1–T4) to the time since an individual’s last infection. Waning of antibodies was estimated using log-linear regression, i.e. assuming an exponential rate of decay and accounting for repeated measures using generalized estimating equations (GEE). Total IgG responses decreased following infection for all 18 P. falciparum antigens  to the duration of time since an individual’s last infection. Changes were estimated using log-linear or linear regression for antibody levels and avidity index respectively, i.e. assuming an exponential rate of change. The IgG avidity index increased significantly (p=<0.0001) following infection for all antigens except AMA-1  −0.001–0.025), Rh2_2030 (CI -0.002-0.028) and Rh5.1 (CI 0.004-0.022)  verses older (>18 years) age groups for the majority of the antigens (10MFI IgG3 to IgG1 was determined and compared across the three age categories at T4 (to represent the antibody profile in the relative absence of recent infection). A ratio below 1 implied a higher proportion of IgG1 and a ratio above 1 a higher proportion of IgG3. For the 1–4 year age category the median IgG3/IgG1 ratio was below 1 for all antigens except for GLURP RII , EBA181 RIII-V , MSP2 Dd2 , and AMA-1, for which avidity index was higher for age categories 1–4 compared with 5–11 and with >18 years Figure 5P. falciparum blood stage antigens to gain broad insight into the dynamics of antibody levels and avidity when malaria transmission is reduced.We measured responses to a panel of 18 P.falciparum , and high avidity antibodies by long lived plasma cells (LLPC). Infection drives expansion of the SLPC and non-avid antibodies, and the rapid contraction of SLPCs following infection leads to rapid preferential decay of the low avidity antibodies. Therefore, the increase in avidity index we observed may indicate affinity maturation per se and/or the relative contributions of SLPCs and LLPCs following infection. A study conducted in the setting of ongoing malaria transmission and infection in Nigeria found that antibody avidity was stable among most individuals over time ; this fi+ LLPC with age and exposure.The implication of early acquisition of the predominantly IgG1 response we observed is not well understood, however there is increasing evidence to suggest that IgG3 may be more protective than other subtypes against malaria. A stronger association between IgG3 and a reduced risk of malaria compared to IgG1 was reported against PfRH5 and PfRipr, . In addi+ LLPC cannot be the cells that switched to IgG1. Rather, naïve B cells, IgG3+ MBC, or IgM+ MBC are likely to be the source of IgG3+LLPC that gradually accumulate with age  due to the nature of the CH domains in the Fc region . Therefowith age . Some stwith age  suggesti+ LLPC with age. Previous studies have indicated a requirement of high antibody titers to maintain immunity to malaria , Uganda National Council for Science and Technology (HS 1074), LSHTM ethics committee (reference # 6012), University of California, San Francisco Committee on Human Research (reference 027911). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.GD, MK, PR, BG, and CD designed the surveys. JR, EA, and JN conducted the surveys. IR, BG, and HM-K contributed to data analysis and interpretation. KT and JB supplied antigen constructs. KT generated and supplied protein reagents. IS and KT designed the assays. KT, IS, BG, IR, and CD designed the study. IS wrote the first draft of the manuscript with support from KT, BG, and CD. All authors contributed to the article and approved the submitted version.This study was supported by funds from the National Institutes of Health as part of the East African International Centers of Excellence in Malaria Research (ICMER) program (U19AI089674) and a Fogarty International Centre (FIC) Training in malaria research in Uganda award (D43TW7375). Additional support was provided by a Bloomsbury SET Award  under the UKRI Connecting Capabilities Fund (CCF). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium infections. The development of an effective vaccine against the malaria parasite would mark a breakthrough in the fight to eradicate the disease. Over time, natural infection elicits a robust immune response against the blood stage of the parasite, providing protection against malaria. In recent years, we have gained valuable insight into the mechanisms by which IgG acts to prevent pathology and inhibit parasite replication, as well as the potential role of immunoglobulin M (IgM) in these processes. Here, we discuss recent advances in our understanding of the mechanisms, acquisition, and maintenance of naturally acquired immunity, and the relevance of these discoveries for the development of a potential vaccine against the blood stage of Plasmodium falciparum.Malaria remains a significant contributor to the global burden of disease, with around 40% of the world’s population at risk of  Plasmodium falciparum. In 2018, an estimated 228 million cases occurred globally, resulting in 405,000 deaths, of which most were children  iewed in , 11, andiewed in , 12–15. P. falciparum antigens, including their development and maintenance, key molecular and immunological mechanisms of parasite inhibition, and a potential role for immunoglobulin M (IgM) in the protective response. This review provides an overview of these discoveries and highlights their relevance for vaccine development.The main antigenic targets of blood stage parasites can be divided into two categories: i) parasite variant surface antigens on the cell membrane of infected erythrocytes and ii) proteins that are located on the merozoite surface or secreted by merozoites during erythrocyte invasion  on the cell membrane of the infected erythrocyte. These proteins play crucial roles in both malaria pathogenesis and immune evasion. VSAs belong to multigenic families, most notably P. falciparum erythrocyte membrane protein 1 (PfEMP1), repetitive interspersed repeats (RIFIN), and subtelomeric variant open reading frame (STEVOR). PfEMP1 can bind to specific host receptors lining the vascular endothelium, resulting in sequestration of infected erythrocytes in capillaries, thereby preventing parasite clearance by the spleen  and Duffy binding-like domains  that can be further divided into 147 subtypes –32. ThesP. falciparum strain 3D7 were used, which does not include several known PfEMP1 domains that are associated with severe malaria and does not necessarily match the parasite strains that these children have been exposed to. An extensive study of antibody responses against 456 DBLα domain variants from local parasite isolates among 232 children (0.9–3.2 years old) in Papua New Guinea showed that IgGs against 85 DBLα1 and DBLα2 variants were associated with 70–100% reduction in severe malaria  were detected in children in Tanzania as early as 1 year after birth, but levels did not increase with age . No diffP. falciparum genome  living in Papua New Guinea , or by promoting Fc-mediated effector functions, including complement activation, opsonic phagocytosis, and antibody-dependent cellular cytotoxicity.One of the priorities for malaria vaccine development is the establishment of correlates of protection and the standardization of P. falciparum challenge, demonstrating that neutralization is likely to be the primary mechanism of action  responses  and explains why some degree of exposure is necessary to maintain immunity  were higher compared to previously malaria-naive travelers . These rimmunity . Howeverexposure . Findingexposure . In thisP. falciparum are variant surface antigens on the plasma membrane of the infected erythrocytes and antigens expressed by merozoites. Antibody responses against PfEMP1 variants develop early in human life and seem to be the main driver of protection against severe malaria in young children. Protection against malaria in older children is achieved once antibodies against merozoite antigens reach sufficiently high levels. A vaccine based on a combination of these two classes of antigens may elicit antibodies that have an additive effect, or possibly even act synergistically, toward the prevention of all clinical manifestations of malaria.The main targets of naturally acquired immune responses against The main obstacle for the development of a vaccine targeting PfEMP1 is the identification of conserved epitopes, given the enormous sequence variation among this protein family. The CIDRα1 domain has the most potential as a vaccine candidate against severe malaria, given its role in the pathogenesis of severe malaria through binding to EPCR, and because of structural conservation of the EPCR binding site.Correlations with protection have been observed for antibody responses against many different merozoite antigens. Broader antibody responses provide better protection, with near full protection for responses against combinations of three to four antigens. What remains unclear is which of these (combinations of) antigens should be moved forward for vaccine development. Since many of these antigens are polymorphic, the identification of conserved epitopes has high priority. In addition, several candidates that have been identified have unknown functions and further characterization of these proteins will provide more insight into the merozoite invasion process.Finally, recent studies have revealed mechanisms of antibody-mediated parasite inhibition. Understanding how antibodies function against different target antigens is important to be able to induce the most effective immune response by vaccination and to establish correlates of protection. Nature has provided us with proof-of-concept that protection against blood stage infection is achievable. Our increased understanding of naturally acquired immunity therefore provides hope for the development of a malaria blood stage vaccine.The first draft of the manuscript was prepared by SG and RR, and revised by EB. AB, SB, and GB further edited the manuscript and provided intellectual contributions to the design of the figures. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health [grant numbers AI128466 and AI133274]. SG and AB are supported by Graduate Research in Immunology Program training grant NIH T32 AI138944. RR is supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number TL1TR002647.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum and Plasmodium vivax. We review the main targets of self-antibody responses in malaria as well as the current, but still limited, knowledge of their role in disease pathogenesis or protection.Autoantibodies are frequently reported in patients with malaria, but whether they contribute to protection or to pathology is an issue of debate. A large body of evidence indicates that antibodies against host-self components are associated to malaria clinical outcomes such as cerebral malaria, renal dysfunction and anemia. Nonetheless, self-reactive immunoglobulins induced during an infection can also mediate protection. In light of these controversies, we summarize here the latest findings in our understanding of autoimmune responses in malaria, focusing on  Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, and Plasmodium malariae. Since research emphasis has been placed on P. falciparum and P. vivax, parasites that are responsible for most of malaria cases, here we will focus in these two species.Despite substantial progress in control efforts over the past decades, malaria still accounts for significant morbidity and mortality, mainly in underdeveloped countries. In 2018, an estimated 228 million cases of malaria occurred worldwide with 405,000 deaths, largely in Africa  and human histamine-releasing factor (HRF) , which exhibits homology with human vitronectin  and showed that the immunodominant response stimulated in the primary exposure to this protein has progressed to encompass lesser epitopes with repeated and prolonged exposure.It is widely known that in early immune responses, epitopes of the initial antigens are recognized by the acquired immune system, but during infection, epitopes other than the dominant ones may also become immunogenic and be targets of T and B cells. This reactivity to newer endogenous epitopes is termed “epitope spreading” and may be induced against other epitopes in the same autoantigen (intramolecular epitope spreading) or against epitopes in other self-antigens (intermolecular epitope spreading) that are released after T- or B-cell-mediated bystander . Evidence in this line has been provided by a study with B cells from non-immune donors stimulated with a recombinant version of CIDR1. The recombinant protein was able to promote in vitro proliferation, increase in B-cell size, and expression of immunoglobulins and cytokines in those cells  is a clinical syndrome of severe falciparum malaria characterized by impaired consciousness assessed by Blantyre Coma Score ≤ 2 in children , whose levels were shown to be higher in Kenyan children with CM than in those with uncomplicated disease or uninfected   integrity is necessary to allow antibody influx into the brain  antibodies, which were found to tag nRBCs exposing phosphatidylserine (Fernandez-Arias et al., P. vivax infections (Mourão et al., P. vivax in comparison to P. falciparum. But despite this, P. vivax causes a greater loss of nRBCs. Thus, it is possible that the mechanisms leading to nRBCs removal in P. vivax malaria are distinct from those observed from P. falciparum. More work is needed to elucidate this.Autoantibodies against RBCs have also been described for P. vivax-infected patients such as band 3 (Mourão et al., Different erythrocytic antigens have been shown to be recognized by self-reactive immunoglobullins from anemic P. vivax malaria is spectrin, although it is still unclear how anti-spectrin antibodies bind to an inner component of RBC membrane. Since in silico analysis revealed that human spectrin primary structure shares homology with a P. vivax hypothetical protein, it is possible that molecular mimicry drives autoimmune response against human spectrin (Mourão et al., Other RBC protein that has also been considered a target for autoimmune responses during P. vivax-infected children and adolescents from Pará, a State located in Brazilian Amazon (Ventura et al., P. vivax and P. falciparum coexists. It is important to mention that despite no significant association has been found in both studies, higher frequency of anti-RBCs antibodies has been reported in patients with malaria (Fernandes et al., On the other hand, no association between anti-RBCs antibodies and anemia has been observed in a study conducted with P. falciparum parasitemia and can block in vitro and in vivo RBCs' cytoadherence (Hogh et al., P. vivax and P. falciparum, an issue that should be target of future investigation.In other reports, a beneficial role has been attributed to anti-RBCs antibodies. This is the case of a study carried out in an area of intense transmission of malaria in Liberia, where it has been shown that immune responses to band 3 neoantigens are correlated with lower As can be noted by the findings mentioned above, the literature concerning self-reactive antibodies against RBCs suggest a dual role for these immunoglobulins in malaria-associated anemia. But crucial gaps remain to be addressed .Box 1Which mechanisms are behind the generation of self-reactive antibodies in malaria?P. falciparum and P. vivax malaria?What are the self-antigens that trigger auto-immune responses in How self-reactive antibodies penetrate the blood-brain barrier, a high selective barrier that protects the central nervous system from invaders? Do they cross independently or do plasma cells secrete them?Do self-reactive antibody responses change with anti-malarial therapy?Is there any association between autoantibodies that persist after parasite clearance and long-term complications?What is the prevalence and the magnitude of autoantibody responses in different epidemiological settings?Can anti-self-antibody blockage prevent pathology?These scientific breakthroughs will allow the use of autoantibodies as signatures to predict disease severity or protection, as well as provide insights toward the best vaccination strategies. Furthermore, they will open new therapeutic possibilities to treat malarial anemia.in vitro and in vivo models. Since few reports have associated autoantibodies to protection, this is an issue that should also be better investigated. Additionally, it would be of interest to determine the prevalence and the magnitude of self-reactive responses in cohorts from different epidemiological settings, an analysis that should be extended including prospective studies. The role of self-immunoglobulins isotypes and IgG subclasses is another gap that should also be addressed. A better knowledge of all these points (Studies regarding autoantibodies and plasmodial infections have indicated that those molecules may play a dual role in malaria . Howevere points  may alloLM, GC-O, and EB conceptualized and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malaria, which is responsible for a substantial amount of deaths in endemic countries, has been shown to have both direct and indirect effects on the hematological parameters. Notwithstanding, some hematological parameters among populations living in malaria endemic regions have not been described consistently, as a standard for measuring malaria burden. Based on the above fact, this study was designed to assess some hematological changes and their diagnostic values in malaria infected patients. A total of 160 malaria positive adult patients, together with 81 healthy control adults were recruited for the study. For the malaria positive group, the female to male ratio was 1.38 : 1. Specifically, 74.38%, 10.00%, and 15.62% of those in the malaria positive group had mild, moderate, and severe parasitaemia, respectively. Leukemia, anemia, and thrombocytopenia were found to be significantly associated with malaria and were all estimated to be specific for the diagnosis of malaria. Anemia was, however, estimated to be both sensitive and specific for malaria diagnosis. Therefore, anemia offers the most diagnostic value in the malaria infected patients of this study. Plasmodium falciparum accounts for up to 99.7% of malaria cases in 2017 [Global malaria cases reduced from the year 2017 (estimated 219 million cases) compared to what was reported in 2010 (estimated 239 million cases) [ in 2017 . For the in 2017 , 6. More in 2017 . Therefo in 2017 , 9–11. S in 2017 , 13. The in 2017 –17. Desp in 2017 . TherefoμL were 13.8 times (odds ratio) more likely to have malaria [9/L and platelet count < 100 × 109/L) in pediatric age groups [Plasmodium, compared to nonparasitized controls [Anemia, as well as alterations in other hematological factors, is shown to be generally affected by malaria –21. This malaria . Similare groups . A studyPlasmodium falciparum malaria infection were evaluated, changes in white blood cells were found to be less severe than thrombocytopenia [Hematological factors are equally affected by malaria in adults as well as in pediatric groups. In a study where blood cells and platelets in ytopenia . Thromboytopenia . In anotytopenia . Also, rytopenia .Plasmodium falciparum malaria [Plasmodium falciparum malaria, as compared to those with acute Plasmodium falciparum malaria [Plasmodium falciparum malaria [Plasmodium falciparum infection was higher [In severe malaria, some of the hematological alterations differed with the developed complications . Other f malaria . Notwith malaria . However malaria . Among c malaria . There ws higher . As repos higher . Many ots higher , 23, 31.s higher . In endes higher . For thes higher . Howevers higher . The sens higher , 35.This study was carried out in the Bamenda Regional Hospital (BRH), the chief government hospital in the North West Region of Cameroon. Known to be one of the ten regional headquarters in Cameroon, Bamenda is located along 10.15 longitude and 5.96 latitude. Bamenda is also situated at a height of 1258 meters above sea level. There are two seasons in Bamenda, the dry and the rainy seasons, with a balanced rainfall per year being 2064 mm (and 172 mm per month). The peak of dry season occurs in January. Meanwhile, the peak of the rainy season is in September. The BRH is part of the Bamenda Health District (BHD), which is made up of many public, private, and mission health facilities located within the 17 health areas in the BHD. The BRH, therefore, functions as the referral hospital in the region, with an estimated 337,036 inhabitants [This study was carried out within a period of three months, from February 2018 to April 2018. The inclusion criteria for the study were all adult (≥18 years) patients who tested malaria positive by the microscopic method. However, healthy malaria negative adults were included in the study as the control group. All those with an established diagnosis of systemic infections, typhoid fever, and meningitis were excluded from the study.The ethical clearance for this study was gotten from the Ethical Review Committee of the University of Bamenda. Signed informed consent was acquired from those who accepted to be enrolled in the study.Approximately 2-3 mL of venous blood samples were collected into EDTA anticoagulated test tubes, from patients who were sent to the BRH laboratory for a malaria test. Blood films (thick and thin) were prepared within a period of 30 minutes, following the techniques recommended by Cheesbrough .μL of blood, moderate parasitaemia was 1,000 to 9,999 parasites/μL of blood, and severe parasitaemia ≥10,000 parasites/μL of blood. This was based on a previous study by Sumbele et al. [The prepared blood films were processed and stained with 3% Giemsa staining technique . Two expe et al. .All malaria positive blood samples and those of the control group were analyzed for blood cell counts. Blood counts were performed using an automated hematology analyzer, the Beckman Coulter counter (URIT-3300), following the manufacturer's instructions. The following hematological parameters were considered for this study: white blood cell counts (WBC), percentage of lymphocytes (LYM%), percentage of monocytes (MON%), percentage of neutrophils (NEUT%), hemoglobin measurements (Hb), red blood cell distribution width (RDW-CV), and platelet counts (PLT).Baseline characteristics of hematological parameters in patients with and without malaria were determined using the statistical package Stata Software version 12.14. These baseline characteristics include the range, mean, and standard deviation, all at 95% confidence interval. A fourfold (2 × 2) contingency table displaying the frequency distribution for each hematological parameter was entered into GraphPad Prism version 8.2.1. In each of the four cells, the contingency table had frequencies for normal and abnormal hematological values of both the positive and negative malaria cases. Chi-square (and Fisher's exact) test was used to calculate sensitivity (%), specificity (%), predictive values (%), likelihood ratios, odds ratios, relative risk, and attributable risk. The level of reliability of the results was determined by the confidence interval, which in this study was at 95%. Frequencies of hematological parameters with statistical significance were further combined and analyzed in contingency tables as described above. This was done to assess the association between combined hematological variables and malaria status.Plasmodium falciparum species. All the malaria positive cases were P values of the hematological parameters in those with and without malaria are shown in P < 0.0001), HGB levels (P=0.0006), and platelet counts (P=0.0164) in those with malaria as compared to those without malaria. There were no significant differences between the mean values of LYM%, MON%, NEUT%, and RDW_CV, in patients with malaria, compared to those without malaria.The mean hematological values, confidence intervals, and 3/μL)-26.67%, monocytopenia (MON% < 1%)-0% and thrombocytopenia (<100 × 103/μL)-23.48% were all significantly low. But anemia (Hb < 12 g/dL) had a statistically significant sensitivity of 68.12%. However, leucopenia (WBC < 4 × 103/μL), monocytopenia (Mon% < 1%), and thrombocytopenia (PLT < 100 × 103/μL) had significantly high specificity of 94.92%, 95.06%, and 90.63%, respectively. Anemia (Hb < 12 g/dL) had a significant specificity (63.64%). The sensitivity and specificity for lymphopenia (lym% < 20%), neutrophilia (Neut% ˃ 70%), and low RDW_CV (RDW_CV < 11.5%) were not statistically significant. The likelihood ratio for the diagnosis of malaria using leukopenia, anemia, and thrombocytopenia was 5.244, 1.873, and 2.505, respectively.With 95% confidence interval, the sensitivity, specificity, likelihood ratio, and positive and negative predictive values were estimated for the diagnosis of malaria using the hematological parameters. Statistically, the sensitivity of leucopenia  for the three variables: WBC < 4 × 103/μL, Hb < 12 g/dL, and PLT < 100 × 103/μL.A combination of positive tests for the following hematological parameters was carried out: WBC < 4 × 10 12 g/dL . This co3/μL, Hb < 12 g/dL, and PLT < 100 × 103/μL, are the hematological parameters with the highest odds ratios of 6.788, 3.739, and 2.967, respectively. These parameters also had the highest relative risk values of 1.445 (WBC < 4 × 103/μL), 1.629 (HGB < 12 g/dL), and 1.319 (PLT < 100 × 103/μL). Their values for the attributable risk were equally among the highest: 0.2844 (WBC < 4 × 103/μL), 0.2974 (HGB < 12 g/dL), and 0.2026 (PLT < 100 × 103/μL).The odds ratio, relative risk, and attributable risk of the hematological parameters for the diagnosis of malaria, at 95% confidence interval, are shown in Among the malaria positive cases, up to 74.38% (119/160) had mild parasitaemia; meanwhile, only 10% (16/160) had moderate parasitaemia, and 15.62% (25/160) had severe parasitaemia. In line with expectation, mild parasitaemia was most prevalent among the study subjects who were all adults. Studies have observed that in malaria endemic areas, immunity against malaria develops with time . Therefoγ, causing gender differences in response to malaria [In this study, up to 63.87% (76/119) of those with mild and 68.75% (11/16) of those with moderate parasitaemia were females. But 76.00% (19/25) of those with severe parasitaemia were males. There was a higher parasite density in males. Since the economy of the study area has its man power highly concentrated in the primary sector, the men mostly work out doors throughout the day. But the women rather rush home to focus on house chores during the dusk period of the day. Therefore, the men become more exposed to mosquito bites, increasing their chances of being repeatedly infected with malaria. Malaria infections were also reported to be more severe in males compared to females, by other authors , 43. Acc malaria . In that malaria . Likewis malaria . However malaria . In cont malaria . The difP=0.0004) reduction in WBC count in those with malaria, as compared to those without malaria. This finding agrees with other findings where WBC counts, among other hematological parameters, were found to be significantly lower in malaria infected patients [3/μL) were significantly low, it had a high specificity (94.92%) and PPV (92.31%) for the diagnosis of malaria. Consequently, findings from this study suggest that although leucopenia do not necessarily signify a positive malaria test (because of low sensitivity), there is a higher probability that those without leukopenia will test negative for malaria. Relative to the other hematological parameters, leukopenia also had the highest likelihood ratio (5.24) for malaria diagnosis. This implies that those with leukopenia may have a higher chance of testing malaria positive. The odds ratio (6.788) for leukopenia is very high, also indicating that those with leukopenia have a higher chance of testing positive for malaria. Additionally, the relative risk of 1.445 indicates that the study participants with leukopenia have a 44.5% higher risk of testing positive for malaria than those without leukopenia.As shown in patients , 16, 31.patients , 26. In There was equally a statistically significant reduction in hemoglobin levels (HGB < 12 g/dL) in those with malaria, in comparison to those without malaria. Anemia in malaria infected children and adults have been frequently observed over the years , 26, 27.P=0.0164) reduced in malaria positive cases, in comparison to those without malaria. In line with studies among malaria infected children in western Kenya, platelets, alongside other hematological parameters, were found to be significantly lower [Plasmodium falciparum malaria in adults [As shown in ly lower . The sigly lower , 27, 28.ly lower , 52. Thrly lower , and hasly lower . Similarly lower . Findingn adults .As shown in The probability of having a positive malaria test was increased when the hematological variables with significant change  were comThe absence of significant differences between the mean values of LYM% in patients with malaria compared to those without malaria contradicts other findings where mild atypical lymphocytosis was found to accompany malaria , 28. HowThis study which sought to assess some hematological changes and their diagnostic values in malaria infected patients found out that hemoglobin levels, white blood cell counts, and platelet counts were significantly reduced among the malaria positive individuals. Based on this outcome, it is recommended that in malaria endemic areas predominated by mild parasitaemia, leukemia, thrombocytopenia, and anemia could be used as prognosticators for infections with malaria. This recommendation can be more appropriate for the adult group.


Plasmodium parasites. Progress in the use ofnon-invasive methods has rendered investigations into wild ape populationspossible. Present knowledge is mainly focused on parasite diversity andphylogeny, with still large gaps to fill on malaria parasite ecology.Understanding what malaria infection means in terms of great ape health isalso an important, but challenging avenue of research and has been subject torelatively few research efforts so far. This paper reviews current knowledgeon African great ape malaria and identifies gaps and future researchperspectives.Humans and African great apes (AGAs) are naturally infected with severalspecies of closely related malaria parasites. The need to understand theorigins of human malaria as well as the risk of zoonotic transmissions andemergence of new malaria strains in human populations has markedly encouragedresearch on great ape  Plasmodium species infecting chimpanzees (Pan troglodytes) and gorillas (Gorilla spp.), which are closely relatedto those infecting humans . To this day, itremains difficult to date the actual origins of the malaria parasites whichinfect humans  as well as theiremergence in human populations. However, the expansion of P. falciparum for instance, the most virulent Plasmodium parasite, is believed to have started in Africa at leastthousands or even tens of thousands of years ago  and to have played a significant role in recent human evolution. Malaria is currentlygeographically widespread and constitutes a heavy burden for human publichealth . Malaria in great apes, our closestevolutionary relatives, has thus increasingly been subject to research asthis leads to a better understanding of human malaria and thesignificance and implications of malaria parasite infection for great apepopulation health.African great apes (AGAs) have been known to naturally host malaria parasites sincethe early 1920s, when researchers first described variousPlasmodium species in chimpanzees and gorillas, and the study oftheir phylogenetic relationships . Most of the current knowledge concernsmalaria parasite diversity, host specificity and evolutionary history. Thereis still relatively little understanding of the epidemiology, ecology andbiology of great ape malaria, but with increasing efforts being made inrecent years to investigate these aspects. Here, we review up-to-dateknowledge about the natural history of malaria parasite infection in wildAGAs, highlight the gaps in the knowledge and conclude with some perspectives forfurther investigation.Research on AGA malaria parasites was initially limited to the study ofcaptive and laboratory individuals . However, the development ofmolecular diagnostic tools and, later on, the ability to use non-invasivesamples, i.e. the discovery that malaria parasite genetic material can befound in faecal samples of primates ,opened up opportunities for further research in wild ape populations. Thisallowed for more accurate identifications, the discovery of additionalPlasmodium species, considered similar to the human parasites, wereidentified in West and Central Africa: P. falciparum-like andP. ovale- or vivax-like parasites in chimpanzees andgorillas and P. malariae-like parasites in chimpanzees . These three specieswere subsequently named P. reichenowi  , P. schwetzi   and P. rodhaini  . P. falciparum and P. reichenowi, considered relatively distinct fromother known Plasmodium species, were grouped separately under theLaverania subgenus .Malaria parasites naturally infecting AGAs were initially described in the1920s by Reichenow  and then studied further andexperimented on by various researchers such as Blacklock and Adler , Schwetz , Rodhain , Brumpt , Garnham, Bray  and Contacos . Based on morphological features, three distinctPlasmodium species have been shown to be endemic inwild-living chimpanzees: three Laverania species –P. reichenowi  and two newly discovered Laverania species, P. gaboni  and P. billcollinsi; andthree non-Laverania species – P. ovale- , vivax-  and malariae-like parasites . All four chimpanzee subspecies are naturally infectedwith all known chimpanzee Laverania parasites and with at least oneof the non-Laverania parasites . Western lowland gorillas(Gorilla gorilla gorilla) also naturally harbour three parasitespecies belonging to the Laverania subgenus, i.e. P. praefalciparum, P. adleri and P. blacklocki , and two non-Laverania species,i.e. P. vivax-  and ovale-like parasites . A P. praefalciparum, orfalciparum-like, sequence (an undistinguishable sequence) wasalso detected in Cross River gorillas (Gorilla gorilla diehli), and P. vivax-like parasites were detected in easternlowland gorillas (G. b. graueri) . Despitesignificant search efforts ,natural infections of wild-living bonobos (Pan paniscus) have notbeen reported so far .From 2009 onwards, with the increased use of molecular diagnostic tools and, lateron, the development of non-invasive methods to detect malaria infectionthrough the amplification of parasite DNA from faecal samples, large-scalestudies on malaria parasites infecting wild great apes flourished. These studies ledto the description of a higher diversity of parasites than previouslythought and gave clearer insight into prevalence rates and the possibleorigins and evolutionary history of malaria infection in humans. To date, atleast six Plasmodium spp. infection in wild apes were estimated usingdetection rates from larger sets of faecal samples. Parasites were clearlyfound to be endemic in the four chimpanzee subspecies and in lowlandgorillas (G. g. gorilla and G. b. graueri), with, according todifferent studies, estimated prevalence levels ranging from 32 % to48 % for P. t. ellioti, P. t. troglodytes, P. t. schweinfurthii and G. g. gorilla, in which faecal detection rates were correctedfor the sensitivity of the diagnostic test, sample degradation and redundantsampling ;15.42 % for P. t. troglodytes and 21.28 % for G. g. gorilla faecal detection rates ; and 33 % forP. t. verus, in which the point prevalence rate was based on the proportion ofpositive individuals tested by faecal detection . The vast majority of parasites harboured by AGAs belong to theLaverania clade with non-Laverania strains rarelyfound . The specific prevalence rates of P. vivax-like parasites inchimpanzees (P. t. troglodytes and P. t. schweinfurthii) andlowland gorillas (G. g. gorilla and G. b. graueri) seem torange from 4 to 8 %, close to the infection rates in endemic humanpopulations . Co-infection withtwo or more Plasmodium parasite species is prevalent , e.g. present in 55 % of positive chimpanzee samplestested by Liu et al., (2010). Bear in mind that these prevalencerates are likely to represent underestimates due to the lower sensitivity offaecal testing compared to blood testing . In some chimpanzee and gorilla communities, nomalaria parasites were detected so far , which could be the result of therelatively low sensitivity of faecal detection combined with the small samplesize . Additionally, lowprevalence or absence of malaria parasites in certain communities could alsobe explained by environmental factors – for example, factors that lead to a scarcityof efficient vectors or demographic factors such as variations in hostdensity.Before large-scale screenings on wild great ape populations were carriedout, the natural prevalence of malaria parasites in some chimpanzee andgorilla populations was suspected to be high given the frequent detectionin sporadic blood or tissue samples  as well as in faecal samples  from wild-living chimpanzees  and western gorillas. This was confirmed when prevalence levelsof Laverania parasites, P. reichenowi was known as the closest relative of P. falciparum . Consequently, it was the firstgreat ape Plasmodium species to be molecularly characterised. Anumber of phylogenetic analyses have led to various hypotheses on the originof the human P. falciparum: (1) one is the co-speciation hypothesis, in which the two Plasmodium species diverged from acommon ancestor at the same time as the chimpanzee and human lineagesdiverged, about 6 to 10 million years ago ; and (2) another is the hypothesis of a more recent chimpanzee originthrough a single host transfer of P. reichenowi from chimpanzee tohuman. The latter theory was supported by the low genetic polymorphism ofP. falciparum which was fully included into the broader genetic diversity ofmalaria parasite lineages, which the authors collectively considered asforming the species P. reichenowi . However, recent broad-scaleinvestigations based on the detection of malaria parasite DNA in faecal samples of wildAGA populations resulted in the genetic characterisation of a wide array ofother Plasmodium species. This showed that the entire diversity ofhuman-derived P. falciparum sequences is encompassed by the gorillaP. praefalciparum clade  and led to the substantial indicationthat P. falciparum originated from a relatively recent zoonotictransmission of P. praefalciparum from gorillas to humans .Until the discovery of other Laverania parasites are concerned, many questionsremain on their evolutionary origins and the phylogenetic relationships between the parasites hosted bynon-human primates (NHPs) and those hosted by humans. Phylogenetic analyses initially suggested thatP. vivax originated in Asia from an ancestral macaquePlasmodium species . P. simium, a New World monkeyparasite, has also been designated as a potential ancestor of P. vivax given their high genetic similarity, which is suggestive of a recenthost transfer between New World monkeys and humans, although the directionof this cross-species transmission remains uncertain . Finally, the near fixation of theDuffy-negative phenotype in Sub-Saharan human populations, which confersresistance against P. vivax, supports the hypothesis of an Africanorigin of this parasite with positive selection of the Duffy negativitythrough long-term exposure . The P. vivaxstrains found in captive AGAs are genetically distinct from those circulatingin humans . They includethe monophyletic lineage formed by the human P. vivax parasitesin their diversity, but they lack host specificity amongst the AGAs theyinfect, i.e. P. t. troglodytes, P. t. ellioti, P. t. schweinfurthii, G. g. gorilla and G. b. graueri . This supports the idea that the human P. vivaxdeveloped from within a Plasmodium species which also infectedAfrican apes and that all present human P. vivax parasitesoriginated from an ancestor which spread out of Africa . For P. malariae- andovale-like parasites, larger-scale investigations of infectionsin wild NHPs together with phylogenetic analyses are still needed to clarifythe evolutionary relationship of these parasites with human strains.As far as non-Anopheles genus (An.), of which approximately 40 species(out of 430) are thought to be competent for malaria parasite transmission. They have been studied extensively and mapped indetail . The ecology of mosquito species, including habitat and hostpreferences, varies and determines their importance as a vector in givenhabitats . For instance, An. gambiae is considered as one ofthe most efficient vectors in open landscapes in Sub-Saharan Africa . Additionally, An. moucheti, alsohighly anthropophilic, is entirely restricted to forested habitats andconsidered a significant vector of human malaria in this type of environment .It is well established that mosquitoes are responsible for the transmission ofmalaria between humans. These malaria vectors belong to theP. reichenowi , with a notableexception being An. gambiae. Bray (1958a) had managed to infect An. gambiae with the formerlycalled P. schwetzi; however, the low levels of sporozoites in thesalivary glands had suggested that these were not the natural vectors.Another experiment succeeded in infecting humans with P. schwetzivia Anopheles balabacensis bites .Until very recently, not a single vector responsible for AGA malaria parasitetransmission had been identified. Early experiments had shown thesusceptibility of some known human malaria vector species to P. falciparum that the question of AGA malaria vectors has been increasingly raiseddue to the concern for future zoonotic transfers of malariaparasites from NHPs to humans and thus the importance of identifying speciesthat can act as bridges . At thepresent time, several studies have thus specifically searched for mosquitospecies responsible for the transmission of malaria parasites betweenchimpanzees or gorillas in their natural habitat. The first study testedfemale Anopheles (P. t. schweinfurthii) natural nesting and feeding sites in Uganda, but it did notdetect any Plasmodium parasites . The failure of detectionmight, however, have been linked to the small number of specimens tested. Asecond study, which screened a larger number of female Anopheles . Finally, the analysis of 2415 female Anopheles captured in the sameareas in Gabon led to the discovery of both chimpanzee and gorillaLaverania and non-Laverania parasites in An. vinckei, An. moucheti and An. marshallii , with the highestprevalence rate in An. vinckei (Laverania parasites.It is mainly since the discovery of the gorilla origin of Anopheles in the primary forest (at a camp site), whereas they wereabundant in the neighbouring villages . Mosquitoes of the Uranotaenia genus, however,were dominant in the primary forest but rarely found in other areas.This suggests that the malaria vectors responsible for the transmissionof malaria parasites in the resident chimpanzee population differ from thosetransmitting parasites in humans and in other AGA populations. Obviously,other mosquito genera and other dipterans should not be excluded aspossible vectors. Biting midges (genus Culicoides), for example,transmit Hepatocystis spp., which are bat and monkey malariaparasites , and are known to be abundant in the TNP .It is important to note that the prevalence rates of different mosquito speciesmight differ between geographical sites, suggesting a variability of vectorsdepending on theinvestigated sites.For example, mosquito trappingsperformed in and around the forest of the Taï National Park (TNP) inCôte d'Ivoire to study the distribution of mosquito genera in habitatsvarying from human settlements to a primary forest only identified one singleSince the discovery of AGA malaria parasites, the marked similarities theyshare with those infecting humans have raised the question of whetherthe exchange of these parasites between AGAs and humans could occur,experimentally and/or naturally.P. rodhaini  , the human-borneP. malariae ,the chimpanzee-borne P. schwetzi   and the human-borne P. vivax. In contrast, attempts totransfer the Laverania species P. reichenowi fromchimpanzees to humans failed . Themarked host specificity of P. reichenowi might be due to the humanloss of the common primate Sia  N-glycolylneuraminic acid, which is thepreferred target of P. reichenowi for erythrocyte binding, asopposed to P. falciparum, which prefers the precursor of theN-glycolylneuraminic acid, the Sia N-acetylneuraminic acid . In addition, the transfer of P. falciparum to chimpanzees only seemedpossible in splenectomised individuals, or limited to the liver orto a very low parasitaemia in intact individuals .Early experiments involving captive chimpanzees and humans  demonstratedthat, for some strains, the infection of humans with chimpanzee parasites, orvice versa, is possible. This was the case for the chimpanzee-borneP. falciparum gave poor results, but captivebonobos  and chimpanzees  living in areas of high humanP. falciparum endemicity have been found to be infected with theseparasites. These infections were probably acquired from humans . There is, however, noinformation on the viability of such infections and thus the potential forvector-based transmission between apes. Similarly, P. malariae,which has never been detected in wild bonobos so far, was found in captiveindividuals , and a P. ovale strain, identical to the humanP. ovale variant type, was found in a captive chimpanzee .For certain human malaria parasites, natural  transmissionto AGAs seems to occur. De facto, early experimental attempts at infectingchimpanzees with Plasmodium  havemanaged to naturally cross the species barrier at least once . This, together with some of the old experimental results reportedabove, instinctively leads to the assumption that natural zoonotic transfersof AGA malaria parasites might still occur, leading to new host switches orjust to regular transmission events in the same manner that a P. knowlesi infection in humans originates from regular zoonotic transferevents from Southeast Asian macaques .Interestingly, the AGA malaria parasite vectors found byMakanga et al. (2016) are mosquito speciesthat also seem to feed on other mammalian species, including humans. Theycould thus, in theory, act as vectors for interspecies transmissions. Studiesled in Cameroon  and Gabon  searched for evidence of zoonotic transfers of apeLaverania species by testing large numbers of human blood samples were detected inthese samples, implying that zoonotic transfers might be rare events.As for natural zoonotic transmission of AGA parasites to humans, it appearsfrom the phylogenetic relationships between known malaria parasites that,historically, ape Laverania species P. vivaxis ongoing. Since wild chimpanzees in West and Central Africa were confirmedhosts of P. vivax-like strains, it was hypothesised that wild apescould act as a natural reservoir , thereby explaining the occasional infection of travellersdespite the low prevalence of P. vivax in the human populations ofthese regions . Moreover, a P. vivax-likeparasite isolated from a traveller coming back from a Central African forestwas recently shown to cluster with the AGA isolates outside of the humanlineage, confirming the occurrence of cross-species transmissions . This is supported by the fact that P. vivax-likeparasite DNA was found in an anthropophilic mosquito species,Anopheles moucheti . Given the close resemblance to their human counterparts andpast experimental findings, it is likely that, similarly to P. vivax, zoonotic transfers of P. ovale- and malariae-likeparasites from chimpanzees also take place. It would be useful tocharacterise more of these parasites in order to search for evidence of suchtransmissions .There is, however, current evidence that cross-species transmission fromwild apes to humans of the non-Laverania parasites from chimpanzeesto gorillas and vice versa . Cross-species transmission between AGAs and other NHPs sharingtheir habitat is probably exceptional. So far, the only published findingshowing such potential is that of P. praefalciparum detected in a singlecaptive greater spot-nosed monkey, Cercopithecus nictitans . Yet, during asubsequent study which tested blood from a significant number of wildspot-nosed monkeys . P. vivax hasalso specifically been searched for in blood samples . Similar results were obtained after screening specimens from sixmonkey species from Uganda . Hepatocystis parasites comply with a broad pattern of host-specificity, with distinct lineagesthat specialise on monkeys and bats . So far, there is no published evidence of transmission of NHPmalaria parasites such as Hepatocystis spp. to AGAs. Moreover,amongst 30 chimpanzee carcasses from the TNP (Côte d'Ivoire) that weretested for malaria parasites, no Hepatocystis spp. were found . Interestingly, however, a liversample from one of these chimpanzees tested positive for a saurianPlasmodium parasite.Transmission of host-specific parasites between different AGA genera hasonly, so far, been observed in captivity, where great ape species cohabitclosely, e.g. transmission of Extrinsic factors, such as environmental factors (habitat type and climate),group size and host density as well as possibly behavioural factors, suchas sleeping site choice, most likely influence the exposure to malariaparasites and thus the frequency of infections in specific AGA communities.However, only very few studies so far have investigated their effects, whichremain largely unknown.Plasmodiumdetection in chimpanzee and gorilla faecal samples was tested in a singlestudy so far, but no significant effect was found . Yet, it wasshown that rainfall can affect Plasmodium parasite prevalence inthe vectors of AGA malaria parasites, with increased prevalence and thusexposure during the rainy season . Prevalence in mosquitoes was also shown to vary with canopyheight, with higher rates at mid-height compared to ground and canopy level.The influence of monthly rainfall on the probability of Anopheles mosquitoes. Mosquitoes were also less abundant in siteswith more nests .Moreover, an experimental study showed that tree species preferred bychimpanzees for nest building might be selected due to their repulsiveproperties against flying arthropods .There is very limited information on the effects of host group size andsleeping site, mainly drawn from studies on New World primates. In contrastto the encounter-dilution effect, which predicts a decreased risk ofexposure to an infected vector with increasing group size , malaria prevalence in New World primates wasfound to increase with average sleeping group size, suggesting that malariamight be a significant cost associated with larger group sizes . However, data on primate malaria and group sizeare still too scarce to draw any firm conclusions. Sleeping site selection inthese primates also seems to play a role in malaria infection rates byreducing the risk of attack by the vector . In chimpanzees, two studies looked at malaria and sleepingsites indirectly by capturing mosquitoes at natural nest sites. While onestudy failed to detect any patterns of association between the choice of nest siteand mosquito densities ,the second study found patterns compatible with mosquito avoidance. Chimpanzees seemed tochoose nest sites in drier and higher places compared to their feedingsites, and these sites were indeed characterised by lower densities ofAs for intrinsic determinants of malaria infection in AGAs, based on datafrom human malaria, one would expect factors such as age, pregnancy status,co-infections, immune status and genetics to influence susceptibilityto infection as well as levels of parasitaemia and the expression of clinicalmalaria.Plasmodium infection were found to behigher in younger individuals of P. t. verus  and G. g. gorilla as well as in pregnantfemales of P. t. verus .This probably reflects changes in the frequency of newinfections and/or in parasite densities induced by variation insusceptibility to these parasites due to age and pregnancy status.These fluctuations in susceptibility might be the result of immuneprotection acquired in adults through constant exposure to the parasites and, during pregnancy, acombination of immunological and hormonal changes, together with the abilityof P. falciparum-infected erythrocytes to sequester in the placenta. However, variations in the exposureto the vectors and, thus, to malaria parasites might also occur and play arole in these observed patterns. It was, for instance, demonstrated inhumans that pregnant women are more attractive to Anophelesmosquitoes compared to non-pregnant women, probably for physiologicalreasons, e.g. increased exhaled breath and abdominal temperature . Additionally, possiblebehavioural changes, such as nesting habits, that could lead to increasedexposure to the vectors should be considered.As a matter of fact, patterns strikingly similar to those found in humanpopulations from malaria endemic areas have been observed in wild AGAs.Faecal detection rates of Microtus agrestis), that co-infection withseveral microparasites explains more of the variation in the risk of infectioncompared to other factors such as host condition, e.g. age, and exposurerisk, e.g. season .Co-infection interactions can take place via competition for sharedresources, e.g. nutrients, physical site of infection, or the immune systemof the host, e.g. cross-reactivity or immunosuppressant pathogens such as HIV . Resource-mediatedparasite interactions would be likely to occur between malaria parasites andother haemoparasite species which target the same cells, like erythrocytes.For instance, Babesia microti infection inMicrotus agrestis negatively affects host susceptibility toBartonella spp. infection and vice versa . Likewise, anegative association between Plasmodium spp. and Babesiaspp. has been recently observed in wild Malagasy primates(Propithecus verreauxi), where high Babesia spp. infectionlevels in younger individuals also seem to confer protection againstPlasmodium spp. infections, possibly via shared resourcecompetition . Inhibition ofPlasmodium cynomolgi infection by Babesia microti was alsopreviously described in experimental rhesus macaques . In humans, thereis evidence of a pathological interaction between the immunosuppressant HIVvirus and Plasmodium, with HIV-seropositive people being atincreased risk of Plasmodium infection and the development of clinicalmalaria . Malaria parasites and varioushelminth species have also been shown to interact in humans, with either anantagonistic or beneficial relationship, possibly mediated by cross-reactiveimmune responses . In orderto fully understand the patterns of malaria infection in AGAs, it would thusbe necessary to also explore and take into account co-infections withparasites that are likely to interact.In addition, it would be judicious to keep in mind that in a given host, malariaparasites are part of a parasite community, whereas we use the term parasitein its broad ecological sense, designating micro- and macroparasites. This implies that, justlike in an ecosystem, multiple and complex interactions between theseparasites and with their host (i.e. their environment) take place;this can also partly dictate the presence of parasite species or theirdensities in a specific host, as opposed to a single host–parasiterelationship , and perhaps explain  the patterns of malaria parasite detection observed in AGAs. As anillustration, Telfer et al. (2010) actually demonstrated, for thefirst time in a natural population of field vole . The onlyindications on the duration of infections also come from earlyexperimental studies. The infections seemed to be able to last for at least severalmonths, with periods as long as 5 months recorded for P. reichenowiand P. schwetzi and over 1 year for P. malariae .None of the published studies have focused on the consequences of malariainfection on wild great apes' health so far, and the information collected fromexperimental or captive conditions is rather limited. Observations madeduring early experiments on the clinical course of malaria in captivechimpanzees, i.e. mainly the lack of noticeable illness behaviour duringvarious infections with human and possibly chimpanzee strains, led Rodhain  to believe that, in natural conditions, chimpanzees do notsuffer significantly from malaria and that they can be subject toasymptomatic chronic infections lasting several months. On one occasion, hereported a double inoculation with P. malariae, without showing any evidenceof illness for 30 years . P. falciparum infection of captive bonobos in the DemocraticRepublic of the Congo were also not associated with apparent clinical signs orincreased body temperature .Moreover, a young chimpanzee newly transferred to a sanctuary after6 years of captivity in an urban setting developed severe anaemia andhyperthermia concomitant with a high P. reichenowi parasitaemia. The latter case would suggestthat infection with certain malaria parasites can, under certaincircumstances, be detrimental for chimpanzees' health. However,extrapolating these observations to the wild is difficult as stress relatedto captivity could also influence the effect of such infections. Moreover,whether individuals were initially naïve to the Plasmodiumstrains they were diagnosed with (whether human or chimpanzee derived) isgenerally undetermined (strains novel to a chimpanzee's immune system wouldbe more likely to cause illness). One publication also reported the death ofa 1-year-old captive chimpanzee infected with P. reichenowi; however,given the extremely poor and stressful conditions this individual was keptin, whether malaria effectively played a significant role in the fataloutcome is questionable .Reports from sanctuaries or other facilities hosting captive individuals arerare and variable. In Japan, two captive chimpanzees were found to bechronically infected with In contrast, pathogenicity has never been demonstrated in AGAs living innatural conditions so far. The general impression given by the above-mentionedelements, as well as by the absence or rarity of reportedmalaria-like clinical signs or death associated with infection inindividuals observed on a sometimes daily basis  , is that wild-living chimpanzees aremostly undisturbed by malaria infections, or that at least the clinicalimpact and related discomfort are generally mild enough to be invisible toobservers' eyes when not specifically searched for. Also, as pointed out byRayner et al. (2011), the very high prevalence rates of malaria parasiteinfections observed in numerous wild chimpanzee and gorilla populationsindicate that severe outcomes are probably rather rare.As young and pregnant chimpanzees seem to be more susceptible to malariainfection, it is possible that they are also more prone to suffer from thebiological consequences of infection and to develop clinical malaria, asseen in humans . Healthy andnon-pregnant adults probably benefit from a certain degree of acquiredclinical immunity, but it would be reasonable to think that malariainfection would start having some level of detrimental effect when thehost's ability to control parasite density is diminished. Higher levels ofblood parasitaemia are likely to at least induce higher degrees of anaemia, which in turn could cause morbidity or evenmortality. Anaemia is actually recognised as one of the major causes ofmorbidity and mortality related to malaria in young children and pregnantwomen . Moreover, in humans,malaria during pregnancy can have deleterious consequences for thepregnancy'soutcome and the infant's health . Deleterious effects on pregnancy outcomeand/or infant health would mean a general impact on population reproductivesuccess and fitness, thereby shaping population dynamics.var genes, found only inLaverania parasites, are unique to P. falciparum in humansand play an important role in its pathogenesis by coding for proteins whichmediate the cytoadherence of infected erythrocytes . Analysis of varand var-like gene sequences from human and ape Laveraniaspecies showed that many characteristics of these genes are conserved acrossthe different Laverania species and that these evolved well beforethe emergence of all the Laverania species. In addition, thevar2csa genes, known to be associated with the adherence ofP. falciparum-infected erythrocytes to the placenta and placentalmalaria in humans, have also been identified in P. reichenowi andP. gaboni . However, the question of whether theexpression of var genes is also associated with pathogenicityin AGAs remains open. Some significant differences have alsobeen found between var genes from P. gaboni and var genes fromP. falciparum and P. reichenowi, suggesting potentialdisparities in their biology.Further investigation of the relationship between malaria parasites andchimpanzees in natural conditions is important to understand thesignificance of malaria infection for wild great ape populations' health,and it can serve as a model to understand the host–parasite relationship in humanpopulations, where malaria is believed to have acted as an important driverof recent evolution . Wild habituated AGA communitiesare ideal targets to pursue investigations on illness related to malariainfection. In a more indirect way, studying the AGA malaria parasites'genes, which might play a role in pathogenesis, can also provide valuableinformation. For example, the very diverse P. malariae and P. ovalestrains with human strains, and of zoonotic infections in humans living closeto AGA communities.It is apparent that, to date, most of the knowledge on wild AGA malaria isconcentrated around parasite diversity, distribution and phylogeneticrelationships with malaria parasites infecting other species, humans inparticular. Indeed, the majority of research studies ultimately aim atidentifying potential animal reservoirs with ongoing zoonotic transmissionas well as assessing the risk of emergence of new malaria strains in humanpopulations . Many gaps, however, still need to be filled in order to achievethese objectives – e.g. further exploration of vectors, of phylogeneticrelationships of ape-derived The epidemiological determinants and the effects of malaria infection inwild AGAs have been left largely unexplored. This also leaves many questionsthat need to be addressed in order to further understand the basic epidemiology and biologyof malaria parasites in AGAs. What are the influences of other knownintrinsic and extrinsic factors affecting human malaria on infection,parasite density or, perhaps, disease in AGAs? Are there visible patterns ofinfection in specific populations? How frequently and for how long areindividuals infected? What are the pathological consequences of malariainfection? Does infection have a significant impact on great ape health?Could the impact of malaria be sufficiently subtle to be misleading? Is this justlike SIV (simian immunodeficiency virus)infection in chimpanzees, which was believed for many years to berather trivial for their health (non-pathogenic), but whose actualdeleterious impact on their health only became apparent recently when examined atthe level of an entire community ? Does malaria play a role in sociality,demographics, population dynamics and general fitness? Are there anyindications of selective pressures exerted by malaria on AGA evolution ordevelopment of resistance? These are just a fraction of the multiplequestions that still need to be addressed. Using faecal samples has provensuitable and efficient, not only to address broad-scale questions such asthose on the geographic distribution and genetic diversity of great apemalaria parasites, but also to address small-scale epidemiological questionswhich might, in comparison, require a higher  resolution andthus more sensitive screening . As non-invasivemethods to study pathogen infections and monitor health parameters are nowavailable, attempts to answer some of these questions are actuallyconceivable. In the meantime, further exploration, improvement and testingof non-invasive methods need to be pursued in order to enhance sensitivity,facilitate the interpretation of results and broaden research possibilities.


FMR1 gene, which encodes a protein with a critical role in synaptic plasticity. The molecular abnormality underlying FMR1 silencing, CGG repeat expansion, is well characterized; however, delineation of the pathway from DNA to RNA to protein using biosamples from well characterized patients with FXS is limited. Since FXS is a common and prototypical genetic disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD), a comprehensive assessment of the FMR1 DNA-RNA-protein pathway and its correlations with the neurobehavioral phenotype is a priority. We applied nine sensitive and quantitative assays evaluating FMR1 DNA, RNA, and FMRP parameters to a reference set of cell lines representing the range of FMR1 expansions. We then used the most informative of these assays on blood and buccal specimens from cohorts of patients with different FMR1 expansions, with emphasis on those with FXS . The group with FMRP data was also evaluated comprehensively in terms of its neurobehavioral profile, which allowed molecular–neurobehavioral correlations. FMR1 CGG repeat expansions, methylation levels, and FMRP levels, in both cell lines and blood samples, were consistent with findings of previous FMR1 genomic and protein studies. They also demonstrated a high level of agreement between blood and buccal specimens. These assays further corroborated previous reports of the relatively high prevalence of methylation mosaicism (slightly over 50% of the samples). Molecular-neurobehavioral correlations confirmed the inverse relationship between overall severity of the FXS phenotype and decrease in FMRP levels . Other intriguing findings included a significant relationship between the diagnosis of FXS with ASD and two-fold lower levels of FMRP , in particular observed in younger age- and IQ-adjusted males , compared to FXS without ASD. Those with severe ID had even lower FMRP levels independent of ASD status in the male-only subset. The results underscore the link between FMR1 expansion, gene methylation, and FMRP deficit. The association between FMRP deficiency and overall severity of the neurobehavioral phenotype invites follow up studies in larger patient cohorts. They would be valuable to confirm and potentially extend our initial findings of the relationship between ASD and other neurobehavioral features and the magnitude of FMRP deficit. Molecular profiling of individuals with FXS may have important implications in research and clinical practice.Fragile X syndrome (FXS) is caused by silencing of the  FMR1  gene. The expansion of the mutation leads to atypical methylation, transcriptional silencing, and ultimately deficiency of the FMR1-encoded protein  [FMR1 transcripts [FMR1 is typically fully or mostly methylated, while transcript and protein expression are markedly reduced. FMRP interacts with about 4% of total mammalian brain mRNAs [Fragile X syndrome (FXS) is caused by a full mutation FM, >200 CGGs) expansion in the promoter region of the 00 CGGs enscripts . In contin mRNAs  and reguin mRNAs . As an Xin mRNAs . Levels in mRNAs ,9,10.FMR1 gene expansions are complex, exhibiting size and methylation variability ; skewed X-inactivation may add on another layer of complexity in females. These factors may affect the level of FMRP deficiency and phenotype severity [FMR1 genotypes between blood cells  and fibroblast or buccal epithelial cells  from the same individual [FMR1 and FMRP assays are generally based on blood samples.severity ,11,12,13severity ,15,16, wdividual ,17. As FFMR1 genomic profiles are complex and merit characterization in FXS. In particular, FMRP expression in the brain is the ultimate factor determining the severity of the neurobehavioral phenotype [henotype . Intriguhenotype . Studieshenotype ,21,22.FMR1 and FMRP profiles in FXS is quite compelling, further underscoring the need to improve molecular assays and molecular-phenotypical analyses.In addition to genotypical and molecular phenotypical  complexity, FXS is heterogeneous in its physical and neurobehavioral manifestations . This isFMR1 assays and more accurate FMRP profiles is necessary [FMR1-based molecular methods quantifying genetic and epigenetic features of the mutation with greater sensitivity, specificity, and breadth have been reported [FMR1 assays can be applied across different specimen types, such as blood and buccal epithelium, and combined with FMRP measurements [FMR1 repeat expansions, detect low-level repeat size mosaicism, determine the number and the sequence context of sequences that interrupt the repeat tract, quantify X-inactivation, and measure skewed FMR1 gene silencing that was masked using less sensitive techniques such as Southern blotting [FMR1 protein levels and, consequently, more accurate FMR1–FMRP correlations [Namely, integration of diagnostic genomic data with complementary ecessary . The intecessary . Such liecessary ,36, as wecessary . Recentlreported ,39,40,41urements ,43. Specblotting ,45,46. Ielations ,43,47. Telations .FMR1 and FMRP profiles and their associations with the FXS neurobehavioral phenotype. We compared nine complementary assays for assessing FMR1 DNA, RNA, and protein, using a reference set of well-characterized FMR1 cell lines. We then applied data from the most informative assays to examine present molecular–neurobehavioral relationships in an independent cohort of patients with FXS with and without ASD. In addition to these primary analyses, we also contrasted molecular profiles in matched blood and buccal specimens in order to include their value in informing FXS phenotypical variability.In the present investigation, we report results from a comprehensive evaluation of FMR1 genotypes, from normal to FM; their samples were used to generate genomic DNA (gDNA) for different genomic studies (repeat-primed PCR (RP-PCR), methylation PCR (mPCR), AGG interruption analysis, and Southern blot analysis) and to create lymphoblastoid cell lines and other durable control material for evaluating, comparing, and contrasting FMR1 DNA and protein analyses.Two cohorts were included in the molecular and molecular-phenotypical analyses. The first was a Reference Cohort of 11 individuals, with and without FXS, that provided whole blood specimens at Rush University Medical Center (RUMC) under approved Institutional Review Board (IRB) protocols. These five males and six females represented a range of FMR1 expansions : 78/76 for the entire Clinical Cohort, 84/74 for the FM–ALL Subcohort, and 84/77 for the FM–FMRP Subcohort. As shown in FMR1 CGG range, five with PM, and three with FM, each of whom contributed a lymphoblastoid cell line. These lines were established at RUMC using standard techniques . Transformed cell lines were expanded, cryopreserved, and provided as frozen stocks to the Asuragen team  for further expansion. Cell lines were grown in suspension at 37 °C in RPMI 1640  supplemented with 15% fetal bovine serum, 2 mM L-glutamine, and penicillin and streptomycin. Viability and total cell counts were determined using a standard trypan blue stain procedure and a phase hemacytometer ; bacterial contamination was assessed using a Universal Mycoplasma Detection Kit . After being grown to confluence (~8 × 105 cells/mL), cells were harvested for DNA, RNA, and total protein isolation.The Reference Cohort consisted of three individuals in the normal FMR1 CGG repeat quantification and DNA methylation analysis were not affected by the method of the blood collection or storage; thus, the FTA cards were selected as a preferred sample type for the remaining 22/42 subjects in the study. FMRP analysis, however, was not supported by specimens preserved on FTA cards, which prompted the use of the 903 protein saver cards as an additional specimen collection. Cards were spotted with ~125 µL of whole blood (EDTA tubes) and stored at room temperature. Specimens from each subject were shipped to Asuragen for subsequent molecular and protein testing .Whole blood was collected using: (i) EDTA vacutainer tubes, as the whole liquid blood for initial 20 subjects of the Clinical Cohort; (ii) FTA cards, preserved blood saturated spot cards for all (N = 42) subjects ; and/or (iii) 903 Specimen Collection Paper/Whatman protein saver cards for a total of 36/42 subjects . Both the whole liquid blood and FTA cards were analyzed for the first 20/42 subjects. This approach demonstrated that Matched whole blood and buccal cell specimens were collected from 42 patients from the Clinical Cohort. Buccal cell samples were collected using ORAcollect·DNA  following the manufacturer’s instructions. Blood samples were obtained using standard clinical procedures. All samples were obtained following informed consent and according to protocols approved by the Johns Hopkins Medicine’s IRB.Genomic DNA was isolated from the whole blood specimens , FTA cards, buccal swabs, and cell lines described above using the DNeasy Blood and Tissue Kit  in accordance with the manufacturer’s instructions. The concentration of gDNA was determined using spectrophotometry . The level of intact or non-fragmented DNA was assessed by agarose gel electrophoresis .To isolate multiple analytes  simultaneously from the cell lines, we used the AllPrep DNA/RNA Mini Kit  following the manufacturer’s instructions. Nucleic acid concentration was determined by spectrophotometry . Additionally, RNA integrity was determined on the 2100 Bioanalyzer  using standard procedures. The minimum RNA Integrity Number (RIN) for all samples was 9.6.FMR1 genotypes from cell-line and blood sample gDNA were determined using triplet repeat-primed PCR, followed by fragment analysis of amplicons by capillary electrophoresis (CE) on an ABI 3500xl Genetic Analyzer , and Sanger sequencing. PCR-based CGG repeat genotyping was performed with both two-primer and three-primer FMR1 PCR/CE ((FMR1 gene-specific PCR/CE (GS-PCR/CE) and repeat-primed PCR/CE (RP-PCR/CE), AmplideX® PCR/CE FMR1 Kit ) [FMR1 PCR with products resolved by agarose gel electrophoresis (GS-PCR/AGE) [®, a PCR-based method  [X, USA)) ,21. CellTX, USA) ,41,45,48FMR1 Kit  was used to quantify relative allele-specific DNA methylation of the FMR1 gene [The AmplideX mPCR MR1 gene ,49 on gDMR1 gene . A samplEcoRI and NruI and separated on an agarose gel. After the DNA transfer, the membranes were hybridized with the FMR1-specific StB12.3 genomic probe and imaged according to published procedures [Southern blot analysis was performed utilizing gDNA isolated either from the whole blood samples or from the cell lines. DNA was digested with ocedures .FMR1 transcript expression was evaluated via quantitative real-time PCR (qPCR) for all cell line samples. A total of 2.5 µg of purified total mRNA was converted to cDNA using a reverse transcription (RT) protocol. The RT reaction was incubated at 42 °C for 45 min followed by 10 min at 93 °C, and then rapidly cooled to 4 °C. FMR1 mRNA specific primers   for use with the KAPA SYBR Fast qPCR kit  were applied according to the manufacturer’s instructions. Each sample was run at two input concentrations of 31 ng/µL and 6.25 ng/µL, respectively. FMR1 mRNA expression was quantified relative to expression of the control housekeeping gene USP33 [g to remove cell debris. Two 50 µL aliquots of the eluate were independently analyzed in the qFMRP assay using liquid bead array  following the method described by Gustin et al. [FMRP levels were determined using a quantitative FMRP (qFMRP) assay developed by LaFauci et al. . This asn et al. . The FMR(i)Diagnostic and Statistical Manual-5th Edition criteria (DSM-5) , supplem(ii)A FSIQ was determined by the Stanford–Binet Intelligence Scales-5th Edition (SB-5) for 15/31 individuals, the Wechsler Preschool and Primary Scale of Intelligence (WPPSI) for 1/31, the Wechsler Intelligence Scale for Children  for 7/31, The Differential Ability Scales (DAS) for 4/31 and the Mullen Scales of Early Learning for 4/31 . To addr(iii)Adaptive functioning was assessed by using adaptive skill scales, which included the Vineland Adaptive Behavior Scales-Second Edition (VABS–II) for most participants, the Adaptive Behavior Assessment System Second and Third Editions (ABAS-2 and -3) for 10 individuals, and Scales of Independent Behavior-Revised (SIB-R) for 2 participants.(iv)Problem behaviors were assessed by the Aberrant Behavior Checklist-Community Edition (ABC-C) adapted for FXS (ABC-CFX), which applies a subscale scoring algorithm developed specifically for the disorder and yields six subscales : (i) Irr(v)The CGI-S score evaluates the overall impairment of a patient using the clinician’s past experience with patients who have the same diagnosis as a reference. Possible ratings of the CGI-S are as follows: 1—normal, not at all ill, 2—borderline ill, 3—mildly ill, 4—moderately ill, 5—markedly ill, 6—severely ill, and 7—extremely ill. The CGI approach was also applied separately to profile the severity/level of SA (CGI-SANX); based on CGI-SANX scores, two categories were defined: ≥5 (severe) and ≤4 (mild-moderate).(vi)A patient’s use of antipsychotics was determined through health records and marked as “there is” or “there is not” (yes/no) use of this class of drugs.The following assessment tools were employed:t-test for unequal variances and equivalent non-parametric tests (Mann–Whitney) were performed to compare means of two samples. Welch’s t-test maintains type I error rates close to nominal for unequal variances and for unequal sample sizes under normality. Significance was indicated by p ≤ 0.05 and high significance by p ≤ 0.01.Statistical analysis was performed using IBM SPSS Statistics version 25  or JMP version 14 . Descriptive statistics included frequency (percent) of nominal variables and median, mean, standard deviation (SD), and range for continuous variables. Tests of normality and homogeneity of variances were also performed. Depending on data distribution, either parametric or non-parametric tests were applied. The Chi square test was used to test differences between nominal variables (frequencies). Pearson’s correlation coefficient was used as a measurement of the strength of the linear relationship between normally distributed variables. Welch’s FMR1 DNA, RNA, or protein-based assays  or partially methylated . Two of the three FM cell lines were nearly completely methylated consistent with inactivation of FMR1 in FXS. However, one FM cell line (RU08) was unmethylated   or no measurable MM (N = 13) . Of note = 0.02) .FMR1 repeat size and methylation occurs in many patients with pathogenic repeat expansions. Nonetheless, it can also manifest in different tissues from the same patient, though the potential phenotypic consequences of these differences are not well understood. Consequently, we compared FM DNA profiles across matched blood and buccal specimens from the same individual. Mosaicism, as we defined it, was (i) detected with high analytical sensitivity using assays previously known to report mosaicism down to 1–5% of cell equivalents [Heterogeneity in ivalents ,57,58 anp = 0.016 and p = 0.001, respectively, Mann–Whitney) and all individuals with moderate or severe ID were males. Frequency of SA diagnosis was high for both genders (68%), especially for females (100%). Unspecified anxiety was only found in males (38%), particularly among the lowest functioning (data not shown). Not surprisingly, ASD was present more frequently in males with FXS , who also had statistically significantly higher mean ABC-CFX total and CGI-S scores than females . FMRP levels were also significantly lower in males than females .In order to determine the influence of ASD status on neurobehavioral profiles in the FM–FMRP Subcohort, we compared males with and without ASD (FXS + ASD and FXS-only). t-tests showed significant lower levels in the FXS + ASD group .Analyses of the FM–FMRP Subcohort presented above have demonstrated groups with lower cognitive functioning, as well as those with ASD, have lower FMRP levels. Thus, we evaluated in greater detail the relationship between intellectual functioning and ASD, and related behaviors, and FMRP in the male subgroups described in p = 0.013 and p = 0.019, respectively; Welch’s t-test and Mann–Whitney). These differences were also present when males with FXS + ASD and mild or moderate ID were compared with those with ASD and severe ID  affected by the small subsample , and when similar groups without ASD were compared . There was also a statistical trend towards lower FMRP levels in the FXS + ASD subgroup with mild + moderate ID when compared with FXS-only with mild + moderate ID , but no differences when groups with severe ID with and without ASD were compared. Another factor that could contribute to the ASD-lower FMRP association is the presence of social behavior impairment that can be interpreted as or associated with ASD. As described above, the group of males with severe SA, by CGI-SANX scores, had a higher proportion of individuals with ASD. This group also had lower FMRP levels than those with mild-moderate SA .Since previous studies have already reported an inverse relationship between intellectual functioning and FMRP levels and a close association between more severe ID and ASD, we also evaluated the role of level of intellectual impairment in the ASD-lower FMRP relationship. An assessment of ID categories and FMRP levels (in pg FMRP/ng gDNA) showed that males with FXS and either mild or moderate ID had significantly higher levels of FMRP than those with severe ID , adjusted for age-  and FSIQ ; while males with an ABC-CFX total score ≥50 and/or severe CGI-S scores (≥5) also closely linked to FXS + ASD, had significantly lower mean levels of FMRP .To illustrate the potential clinical impact of the FMR1 and FMRP measurements reported here, one high functioning 7-year old male in the FXS-only group was an outlier with a normal FSIQ of 95 and an adaptive VABS-II Composite score of 79. His FMRP level was 7.4; (mean ± SD) all males (4.2 ± 3.3), FXS-only (5.4 ± 4.0). The subject had unmethylated size and methylation mosaicism on his blood sample and a partially methylated PM smear with an additional PM peak on the buccal sample.Finally, we examined the influence of age, overall clinical severity, as measured by CGI-S, and overall problem behavior severity, as determined by ABC-CFX total score, on FMRP levels within the FXS + ASD group. Younger males  had two-fold lower FMRP levels (pg FMRP/ng gDNA)  [FMR1 mRNA and FMRP levels were the main contributors to cognitive impairment and the presence of a normal allele appeared to compensate in some but not all individuals. Pretto et al. [FMR1 RNA and FMRP levels correlated in general with FMR1 methylation in peripheral blood cells and fibroblasts although there were differences in CGG expansion and blood samples showed lower methylation. They also reported FSIQ scores were inversely correlated with level of methylation and directly with FMRP levels. However, other aspects of the neurobehavioral phenotype, such as number of seizures and severity of hyperactivity or autistic behavior were not related to methylation or FMRP levels.Using a combination of sensitive and quantitative multi-omic assays, we found that  alleles ,4,5. FM terature ,16,39. We in FXS ,59,60. Me in FXS ,62. Only the FXS . In our  the FXS , served  earlier ,19. A siS + ASD) . They coo et al.  studied FMR1 methylation, FMR1 mRNA, and FMRP and the more direct phenotypical implications of protein expression, FMRP levels seem to be the most suitable molecular parameter for molecular-neurobehavioral analyses [FMR1 and other genes linked to the behavioral disorder substantially overlap [Considering the relatively consistent relationship between analyses . Our finanalyses ,10,29,36analyses . We founanalyses ,29,36,46analyses , allowed overlap ,24,28,63 overlap . Anxiety overlap ,65, and  overlap .As with ASD in males with severe ID, the relationship between FMRP levels and overall severe problem behavior  was also cofounded with greater cognitive impairment. These findings are in line with the recognition, in recent years, of a more severe neurobehavioral phenotype mainly in males with FXS. This is characterized by severe ID, ASD diagnosis, and severe irritability, aggression, agitation, and self-injury types of often anxiety-driven behavior ,27,66. IFMR1 and FMRP assays, the sample size was relatively modest considering the variability and complexity of the molecular and phenotypical measures. Marked neurobehavioral differences between males and females with FXS, coupled with the well-known genetic differences inherent to X-linked disorders  complicated the molecular and correlational analyses. Given the size of the cohort, the necessity of combining size- and methylation-mosaicism limited the full appreciation of differences between these types of mosaicism. Despite the use of raw score-based z-score calculations for FSIQ, floor effects on cognitive or adaptive skills testing for some subjects were also a limitation for studying range of cognitive impairment. The lack of a formal anxiety measure also limited our ability to subtype anxiety.Although our study, to our knowledge, is the largest molecular–neurobehavioral correlation using multiple and sensitive FMR1 CGG expansion, gene methylation, FMRP levels, and overall neurobehavioral severity, including an ASD diagnosis. The synergy of FMR1 genomic and protein parameters and the concordance of blood and buccal profiles using a mixture of sensitive and quantitative multi-omic assays support the benefit of characterizing molecular profiles particularly in males with FXS in observational and interventional studies. The benefit of stratifying their genotype–phenotype profiles is further strengthened by two-fold higher FMRP levels and lesser clinical severity in a subset of these males with substantial methylation mosaicism. Thus, the benefit also highlights the potential of FMRP levels, in particular, as a prognostic marker in clinical trials and other relevant studies in males with FXS.Our findings underscore the association between