Presumptive treatment of malaria is common practice in malaria endemic resource-limited settings. With the changing epidemiology of malaria and the introduction of artemisinin-based combination therapy (ACT), there is increasing need for parasite-based malaria case management to prevent unnecessary use of anti-malarial medicines, improve patient care in parasite-positive patients and identify parasite-negative patients in whom another diagnosis must be sought. Although parasitological confirmation by microscopy or alternatively by malaria rapid diagnostic tests (RDTs) is recommended in all patients suspected of malaria before treatment, gaps remain in the implementation of this policy in resource-limited settings. There is need to evaluate the use of RDTs among highly active anti-retroviral therapy (HAART)-treated people living with HIV (PLHIV).Plasmodium falciparum antigen in peripheral blood. Patients with positive RDT results had microscopy performed to determine the parasite densities and were followed for clinical signs and symptoms during the subsequent six months.Within an urban prospective observational research cohort of 559 PLHIV initiated on HAART and cotrimoxazole prophylaxis between April, 2004 and April, 2005, 128 patients with sustained HIV-RNA viral load < 400 copies/ml for four years were evaluated, in a cross-sectional study, for asymptomatic malaria infection using a histidine-rich protein-2 (HRP-2) RDT to detect P. falciparum antigenaemia. All the patients with positive HRP2 RDT results showed malaria parasites on thick film with parasite densities ranging from 02-15 malaria parasites per high power field. None of the patients with positive RDT results reported signs and symptoms of malaria infection during the subsequent six months.Of the 128 asymptomatic patients screened, only 5 (4%) had asymptomatic falciparum antigenaemia among PLHIV after long-term HAART and cotrimoxazole prophylaxis. Parasite-based malaria diagnosis (PMD) is recommended among PLHIV that are on long-term anti-retroviral therapy. RDTs should be utilized to expand PMD in similar settings where microscopy is unavailable.In an urban area of low to moderate stable malaria transmission, there was low HRP2 P.  Presumptive treatment of malaria is common practice among individuals presenting with fever in malaria endemic settings including people living with HIV/AIDS (PLHIV) -3. With Plasmodium falciparum antigenaemia  years. After four years of HAART with sustained viral suppression, the median CD4 count was 327  cells/μL. Haematological parameters  were within the normal ranges  of asymptomatic r survey . It is ir survey ,15. All r survey . Resultsr survey ,4, there% of asymThe use of the simple rapid tests to detect malaria antigenaemia among PLHIV emphasizes the utility of RDTs in parasite-based malaria diagnosis (PMD) in this population. The findings of this study will contribute to the implementation of RDTs for PMD among PLHIV in areas where microscopy is not accessible . Moreovefalciparum parasitaemia. However, there is need to study the recovery of the host's immunological responses to P. falciparum infection among PLHIV in the setting of long-term HAART and cotrimoxazole prophylaxis.During follow up, none of the patients with asymptomatic parasitaemia reported development of clinical signs and symptoms of malaria infection during the subsequent six months. It is of interest to note that HIV-infected adults did not develop clinical symptoms of malaria despite the increased risk of malaria infections associated with the HIV-associated immune suppression ,4,19. Thfalciparum antigenaemia among PLHIV after long-term successful HAART and cotrimoxazole prophylaxis. Parasite-based malaria diagnosis (PMD) is recommended among PLHIV that are on successful long-term anti-retroviral therapy. RDTs should be utilized to expand the use of PMD in similar settings where microscopy is unavailable.In an urban area of low to moderate stable malaria transmission, there was low HRP2 P. The authors declare that they have no competing interests.DN made substantial contribution to the conception, design, data collection, analysis and drafting of the manuscript. ANK contributed to the data analysis, interpretation and revision of the manuscript. BC contributed to the data collection, data interpretation and revision of the manuscript. DJK made substantial contribution to the conception, study design, and interpretation of data. MRK contributed to the conception, design, data interpretation and revision of the manuscript. All authors read and approved the final manuscript.


Staphylococcus aureus (S. aureus) causes significant morbidity and mortality. The aim of the research was designed a novel DNA vaccine encoding the Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein to provide protection against both influenza and secondary infection with S. aureus. The DNA vaccine vector efficiently expressed the encoded antigen in mammalian cells, as determined by RT-PCR, Western blotting and immunofluorescence analysis. Mice were immunized with the vaccine by intramuscular injection before challenge with IAV and S. aureus. The pulmonary and the splenocyte culture IFN-γ levels were significant higher in immunized mice than their respective controls. Although the antibody titer in the HI test was low, the sera of mice immunized with the novel vaccine vector were effective in neutralisation assay in vitro. The vaccine could reduce the loss of body weight in mice during IAV challenge. Both Western blotting and RT-PCR showed that the vaccine markedly enhanced toll like receptor 2 (TLR2) expression in splenocytes after the secondary infection with S. aureus. The survival rate of mice with high TLR2 expression (pEGFP/Ag85A-HA2 or iPR) was significantly increased compared with mice immunized with pEGFP/HA2 after challenge with S. aureus. However, the pulmonary IL-10 concentration and S. aureus titer were significantly decreased in immunized mice, and expression of TLR2 was increased after challenge with S. aureus. These results demonstrated that Ag85A could strengthen the immune response to IAV and S. aureus, and TLR2 was involved in the host response to S. aureus.Secondary pneumonia due to  Staphylococcus aureus (S. aureus) is a significant cause of mortality associated with influenza A virus (IAV) infection[S. aureus would be highly advantageous. HA is synthesized as a precursor HA0, which is cleaved into the HA1 and HA2 subunits. While the membrane distal domain (HA1) can be highly variable, the stalk region of HA, which contains the core fusion machinery constituted primarily by the HA2 subunit, is relatively conserved. Antibodies targeting the HA2 subunit can provide broad protection against both seasonal and pandemic influenza A infections[S. aureus infections.Secondary pneumonia due to nfection,2. Antibnfection. Howevernfection. Therefofections. Howeverfections is one oMycobacterium tuberculosis has been shown to have a marked immunomodulatory effect in combination with influenza vaccines and can enhance antigen-specific antibody production. Sera from mice vaccinated with BCG exhibit antibacterial activity[M. tuberculosis and an immunodominant antigen of BCG, can increase T helper type 1 (Th1) cytokine responses[S. aureus, we therefore hypothesized that immune reactions to Ag85A may be able to eliminate this bacteria[The Bacille Calmette-Guérin (BCG) vaccine against activity. Ag85A, esponses. As it ibacteria,11. Althbacteria, TherefoM. tuberculosis secreted antigen Ag85A may serve as a good immune adjuvant for HA2. The efficacy of this vaccine in preventing morbidity after challenge with IAV and mortality in mice was evaluated after challenge with IAV and S. aureus, and cytokine profiles were determined in lung homogenates and cultured splenocytes. We also explored the role of TLR2 upregulation in post-influenza pneumonia.We constructed a novel influenza vaccine construct expressing a conserved region of the HA protein linked with Ag85A to evaluate whether pEGFP/Ag85A-HA2, pEGFP-C2, pEGFP/Ag85A and pEGFP/HA2 were successfully transfected in HEK293 cells. Strong green fluorescent signals were clearly seen in the cells transfected with all four plasmids Figure A. The reThe mice were inoculated with pEGFP/Ag85A-HA2, pEGFP/HA2, pEGFP/Ag85A, PBS, pEGFP/C2 or iPR. Development of antibodies against HA was evaluated by hemagglutination inhibition (HI) assays. The HI titers were represented as group means of log 2 dilution. As shown in Table P < 0.05). High NAb titers were detected in mice immunized with iPR, pEGFP/Ag85A-HA2 and pEGFP/HA2. The NAb titer from the pEGFP/Ag85A-HA2 vaccinated group was higher than those of the pEGFP-C2, pEGFP/HA2 and pEGFP/Ag85A vaccinated groups (P < 0.05).Development of neutralizing antibodies (NAbs) against HA was evaluated by the neutralisation assay. The titer of each serum sample was expressed as the dilution (log 2 transformed) that decreased the CPE due to the inoculating virus by 50% in MDCK cells. As shown in Figure P < 0.05). The influenza virus titers of samples from the pEGFP/Ag85A-HA2 vaccinated group was lower than that of the pEGFP-C2, pEGFP/HA2 and pEGFP/Ag85A vaccinated groups (P < 0.05).However, influenza virus loads decreased in mice immunized with iPR, pEGFP/HA2 or pEGFP/Ag85A-HA2 Table  . However, HA induced higher IFN-γ titers in the pEGFP/Ag85A-HA2 group . Splenocytes from mice vaccinated with pEGFP/Ag85A-HA2 produced 1.5-fold higher IFN-γ in response to HA than those from mice vaccinated with pEGFP/HA2 (P < 0.05). HA induced markedly lower IFN-γ levels than did conA (data not shown). Figure  . The pulmonary IFN-γ levels of mice vaccinated with pEGFP/HA2 were significantly higher than those vaccinated with pEGFP-C2 or PBS (P < 0.05). The pulmonary levels of the anti-inflammatory cytokine IL-10 were significantly higher in the PBS group and the pEGFP-C2 vaccinated group than those in the other groups at day 2 after infection with S. aureus (P < 0.05). However, the levels of IL-10 were lower in the pEGFP/Ag85A-HA2 vaccinated group on day 2 after infection with S. aureus (P < 0.05)  . The survival rate was high in the pEGFP/Ag85A-HA2 and iPR groups, while it was low in other groups. There were significant differences in the survival rates between the pEGFP/Ag85A-HA2 group or iPR group and other groups (P < 0.05) . The survival rate of mice vaccinated with pEGFP/HA2 was significantly higher than that of mice vaccinated with pEGFP-C2 or PBS. Additionally, we did not find a significant difference between the pEGFP/Ag85A-HA2 group and iPR group (P > 0.05)  . Splenocytes from mice vaccinated with pEGFP/Ag85A-HA2 produced 1.5-fold higher IFN-γ in response to HA than those from mice vaccinated with pEGFP/HA2 (P < 0.05). The weight loss of mice vaccinated with pEGFP/HA2 was lower than that of mice vaccinated with pEGFP-C2 or PBS after IAV challenge. The survival rate of mice vaccinated with pEGFP/HA2 was higher than that of mice vaccinated with pEGFP-C2 or PBS. However, the weight loss after IAV challenge and the survival rate after S. aureus challenge in the pEGFP/HA2 group were lower than those in the iPR group. These result confirmed that HA2 could provide limited protection against IAV and S. aureus challenge.IFN-γ is a representative Th1 cytokine associated with inhibition of IAV proliferation. Analysis of cytokine production showed that the pulmonary IFN-γ levels in the pEGFP/HA2 group were significantly higher than those of the pEGFP-C2 and PBS groups after IAV challenge. HA induced higher IFN-γ titers in the pEGFP/Ag85A-HA2 group and pEGFP/ HA2 group (P < 0.05). These results showed that pEGFP/Ag85A-HA2 could induce high Th1 response. The weight loss of mice vaccinated with pEGFP/Ag85A-HA2 was lower than that of mice vaccinated with pEGFP/HA2. The IAV titer in the pEGFP/Ag85A-HA2 group was lower than those of the pEGFP-C2, pEGFP/HA2 and pEGFP/Ag85A group. These results demonstrated that Ag85A could enhance the protective effects against IAV. The survival rate after S. aureus challenge in the pEGFP/Ag85A-HA2 group was higher than that in the pEGFP/HA2 group. The S. aureus titers of the pEGFP/Ag85A-HA2 group and iPR group were lower than that of the pEGFP/HA2 group. The higher survival rate and the lower S. aureus titer after challenge in the pEGFP/Ag85A-HA2 group was probably due to Ag85A-mediated immune activation, which enhanced the protective effects of HA2 against IAV challenge. The increased protection against IAV challenge decreased the influenza virus titer. As IAV can suppress the functions of immune cells[S. aureus challenge.The cytokine analysis also showed that the pulmonary IFN-γ levels in the pEGFP/Ag85A-HA2 vaccinated group were significantly higher than that in the pEGFP/HA2 vaccinated group. Splenocytes from mice vaccinated with pEGFP/Ag85A-HA2 produced 1.5-fold higher IFN-γ in response to HA than those from mice vaccinated with pEGFP/HA2 . These results demonstrated that TLR2 was involved in the host response to S. aureus and enhanced elimination of the bacteria, thereby reducing the incidence of post-influenza pneumonia.IL-10 is an important mediator of the enhanced susceptibility to staphylococcal pneumonia. A high Interactions between TLR2 and their ligands typically results in cellular activation and the production of inflammatory cytokines. IL-10 could inhibit production of inflammatory cytokines by inhibition of MyD88 pathway. It is possible that IL-10 inhibit immune reaction by affecting the expression of TLR2. Infection of IAV could increase the level of IL-10. Our results here showed that TLR2 expression in mice vaccinated with pEGFP/Ag85A-HA2 or iPR was also higher than pEGFP-C2 and IL-10 level in mice vaccinated with pEGFP/Ag85A-HA2 or iPR was lower than that of mice vaccinated with pEGFP-C2. These results demonstrated that IL-10 could affect the expression of TLR2.BCG vaccine has been used widely. Ag85A is an immunodominant antigen of BCG. Therefore, the immune reactions against Ag85A could be easily induced. Our results showed that Ag85A could enhance immune responses to HA2. Ag85A should be investigated as a new adjuvant for influenza vaccines.S. aureus challenge was associated with vaccine-induced TLR2 expression. Altogether, our findings indicate that Ag85A could enhance immune responses to HA2 and should be investigated as a new adjuvant for influenza vaccines.The pEGFP/Ag85A-HA2 vaccine induced Th1 type responses in lungs and splenocytes of inoculated mice. Increase survival after IAV infection and reduced bacterial load after pEGFP/Ag85A was constructed by first cloning the Ag85A sequence (1–250 amino acids of N-terminus),25 from Preparation of inactivated influenza A/PR/8/34 (iPR) virus was carried out by heating at 60°C for 1 h as described previously. The vacHEK293 cells (CRL-1573™) were obtained from the American Type Culture Collection (ATCC) Purified eukaryotic expression plasmids pEGFP/Ag85A-HA2, pEGFP/Ag85A, pEGFP-C2 and pEGFP/HA2 were transfected into HEK293 cells by using Lipofectamine™ 2000 transfection reagent  according to the manufacturer’s instructions. Each plasmid was transfected in triplicate wells, and PBS was applied as a mock transfection control. Plasmid expression in all cell groups was assessed by fluorescence microscopy 48 h after transfection.′-AAAACCTGACCTCCCTTGA-3′ and 5′-GTGCAGCCTGGTGACATT-3′; pEGFP/Ag85A-HA2, 5′-CCCTCGAGCATGCAGCTTGTTGACAG-3′ and 5′-CGCGGATCCTCATATTTCTGAAATTCTAAT-3′; pEGFP/Ag85A, 5′-CCCTCGAGCATGCAGCTTGTTGACAG-3′ and 5′-CGGGATCCTTAGCCGTTGCCGCAGTACAC-3′-pEGFP/HA2, 5′-CCGCTCGAGTATTGAAGGGGGATGGA-3′ and 5′-CGCGGATCCTCATATTTCTGAAATTCTAAT-3′. Control PCRs using GAPDH primers were performed on cDNA from the pEGFP/Ag85A-HA2, pEGFP/Ag85A, pEGFP-C2 and pEGFP/HA2 groups. Test PCRs of the pEGFP-C2 group were performed with pEGFP/HA2 primers, pEGFP/Ag85A primers and pEGFP/Ag85A-HA2 primers. Test PCRs of the pEGFP/Ag85A-HA2, pEGFP/Ag85A and pEGFP/HA2 groups were performed with their respective primers. All PCRs were performed simultaneously in the same conditions: 5 min at 95°C, followed by the appropriate number of cycles consisting of 30 sec at 95°C, 30 sec at 50°C and 2 min at 72°C. After amplification, 10 μl of each PCR product was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. PCR amplification and sequencing were performed using an ABI Prism 377XL DNA sequencer .Total RNA of all groups was isolated as recommended by the manufacturer . Contaminating genomic DNA was then eliminated from the RNA preparations by using RNase-free DNase I . RT reactions were performed with EasyScript First-Strand cDNA Synthesis SuperMix . cDNAs were amplified by PCR using 2× TransTaq High Fidelity (HiFi) PCR SuperMix  in a 50 μl reaction mixture in accordance with the manufacturer’s recommendations. The following PCR primer pairs were used: mouse GAPDH (531), 5Cells of all group were lysed in RIPA sample buffer  per the manufacturer’s instructions, and supernatants were collected to analyze expression of plasmids. The samples were separated by SDS-PAGE (10%), and the proteins were transferred to PVDF membranes, which were blocked for 2 h in 5% nonfat dry milk in TBST (TBS containing 0.05% Tween-20) and hybridized at 4°C overnight in TBST with the primary antibodies . After washing the membranes three times in TBST, for 15 min each time, they were incubated with 1:10,000 dilutions of HRP-conjugated secondary antibodies . The membranes were washed three times in TBST, for 15 min each time, and the blots were visualized with an electrochemiluminescence (ECL) system .A total of 84 female BALB/c mice, aged four to six weeks old, were purchased from the animal care center of Sichuan University and divided into 6 groups, with 14 mice in each group. They were bred under pathogen-free conditions. Each group of mice was injected separately with one of the following: pEGFP/Ag85A-HA2, pEGFP/Ag85A, pEGFP/HA2, pEGFP/c2, PBS, iPR. The mice were immunized by intramuscular injection of the quadriceps of both legs (50 μg in 50 μL PBS for each leg) twice with a 3 week interval between injections. iPR preparations were diluted 1:2 in PBS before mice were injected with 50 μL of the inoculum. Mice in the positive control group were also injected intramuscularly with 50 μL of the iPR inoculum. Body weight was monitored daily, and mice losing more than 25% of their initial weight were sacrificed and scored as dead. All experiments were performed with institutional approval.Seven days after the booster immunization, the mice were challenged with IAV, as described previously,23. In bS. aureus (ATCC 25923) as described previously[After IAV challenge, the numbers of survival mice in all groups were showed in Table eviously,23. The Specimen collection was performed according to the method reported by Zhang et al. and TamuS. aureus titration, Western blotting and cytokine measurements, spleens and lungs were collected from four randomly chosen mice of each group under anesthesia with chloroform at 48 h after S. aureus challenge. The lungs were harvested and homogenized at 4°C in five volumes of sterile isotonic saline with a Dounce homogenizer. The lung homogenates were collected for S. aureus titration and cytokine measurements. The collected spleen samples were lysed in RIPA sample buffer  as described by manufacturer’s instructions, and supernatants were collected to analyze expression of TLR2.For et al.[The HA inhibition (HI) assay was performed according to the method reported by Zhang et al.. In brieThe neutralisation assay was performed as previously reported. In brieSpleens were collected 8 days after the virus challenge. Spleens were removed aseptically and processed as follows. Cells were segregated, washed, adjusted to a concentration of 5 × 106 cells per mL, and grown in a 96-well flat-bottom plate (5 × 105 cells per well) in RPMI 1640 medium, supplemented with HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), glutamine and 10% heat-inactivated fetal calf serum (FCS). Cells were stimulated with 0.5 μg concanavalin A (ConA) or 1.25 μg recombinant HA of A/PR/8/34  separately. Cells were incubated at 37°C in a humidified CO2 incubator, and supernatants were harvested after 72 h. Supernatants were pooled and stored frozen at −20°C until assay. Experiments were repeated three times, and the results of one experiment are shown.S. aureus. For cytokine measurements, lung homogenates from each group were diluted 1:2 in RIPA buffer . Homogenates were centrifuged at 1500 × g for 15 min at 4°C as described as instructed by the manufacturer of the RIPA buffer, and supernatants were pooled and stored at −20°C until assays were performed.Lung homogenates were collected on day 8 after infection with A/PR/8/34 and day 2 after infection with IFN-γ and IL-10 from lung homogenates and IFN-γ from supernatants of splenocyte cultures were measured using specific enzyme-linked immunosorbent assays  in accordance with the manufacturer’s recommendations.The IAV titer assay was performed according to the method reported by Zhang et al.. In brieS. aureus challenge. The whole lungs were harvested and homogenized at 4°C in five volumes of sterile isotonic saline with a Dounce homogenizer. Serial 10-fold dilutions in sterile isotonic saline were made from whole lung homogenates, and 50 μL volumes were plated onto Luria-Bertani (LB) agar plates. LB agar plates were incubated at 37°C, and the colonies were counted after 16 h to determine the cfu.Whole lungs were collected from four randomly selected mice by cardiac bleeding under anesthesia with chloroform at 48 h after Spleens were lysed in RIPA sample buffer  per the manufacturer’s instructions, and supernatants were collected to analyze expression of TLR2 at 48 h after S. aureus challenge. The samples were separated by SDS-PAGE (10%), and the proteins were transferred to PVDF membranes, which were blocked for 2 h in 5% nonfat dry milk in TBST (TBS containing 0.05% Tween-20) and hybridized at 4°C overnight in TBST with the primary antibodies  . After washing the membranes three times in TBST, for 15 min each time, they were incubated with 1:10,000 dilutions of HRP-conjugated secondary antibodies . The membranes were washed three times in TBST, for 15 min each time, and the blots were visualized with an electrochemiluminescence (ECL) system .′-AAAACCTGACCTCCCTTGA-3′ and 5′-GTGCAGCCTGGTGACATT-3′. The PCR primers for mouse TLR2 (410) were 5′-TTTGCTCCTGCGAACTCC-3′ and 5′-CAGCTTAAAGGGCGGGTC-3′. PCRs of pEGFP/Ag85A-HA2, pEGFP/Ag85A and pEGFP/HA2 groups were performed with GADPH primers and TLR2 primers. All PCRs were carried out simultaneouly in the same conditions simultaneouly. For all PCRs, the conditions were 5 min at 95°C, followed by 20 cycles consisting of 30 seconds at 95°C, 30 seconds at 60°C, and 2 min at 72°C. After amplification, 10 μl of each PCR product was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. PCR amplification and sequencing using an ABI Prism 377XL DNA sequencer .Reverse transcription and PCR amplification of the TLR2 transcript were performed as described by the manufacturer  of the EasyScript First-Strand cDNA Synthesis SuperMix and 2× TransTaq High Fidelity (HiFi) PCR SuperMix in a 50-μl reaction mixture. The PCR primers for mouse GAPDH (531) were 5Data are presented as means ± SD. Group–group comparisons were analyzed using t-tests. Survival rates were determined using the Kaplan-Meier method. P values < 0.05 were considered significant.All mouse experiments were performed in strict accordance with China legislation on animal experiments and approved by the Ethical Review Committee of Society for Laboratory Animal of Sichuan province (SLAS).The authors declare that they have no competing interests.JD carried out most of the experiments and wrote the manuscript. DP, BW, LR, BZ, KC, and SL did part of the experiment and participated in manuscript preparation. JS, HL and YK participated in antibody detection and lung virus titration. ZJ participated in its design and coordination. ML was the main designer of the experiment and revised the manuscript. All authors read and approved the final manuscript.


Malaria and HIV/AIDS are among the principal causes of morbidity and mortality worldwide, particularly in resource-limited settings such as sub-Saharan Africa. Despite the international community’s efforts to reduce incidence and prevalence of these diseases, they remain a global public health problem. Clinical manifestations of malaria may be more severe in HIV infected patients, which have higher risks of severe malaria and malaria related death. Co-infected pregnant women, children and international travelers from non-malaria endemic countries are at higher risk of clinical complications. However, there is a paucity and conflicting data regarding malaria and HIV co-infection, particularly on how HIV infection can modify the response to antimalarial drugs and about drug-interactions between antiretroviral agents and artemisinin-based combined regimens. Moreover, consulting HIV-infected international travelers and physicians specialized in HIV care and travel medicine should prescribe an adequate chemoprophylaxis in patients travelling towards malaria endemic areas and pay attention on interactions between antiretrovirals and antimalarial prophylaxis drugs in order to prevent clinical complications of this co-infection.This review aims to evaluate the available international literature on malaria and HIV co-infection in adults providing a critical comprehensive review of nowadays knowledge. Plasmodium; five species of Plasmodium  can infect human and among these species, P. falciparum infection may be fatal.2Malaria is one of the most important causes of morbidity and mortality in tropical regions, especially in sub-Saharan Africa and South-East Asia.World Malaria Report 2010, there were 225 million cases of malaria and an estimated 781 000 deaths in 2009. Most deaths occur among children living in Africa where a child dies every 45 seconds for malaria and this disease accounts for approximately 20% of all childhood deaths.2According to the Human Immunodeficiency Virus (HIV) is a retrovirus that can infect immune competent cells causing an impairment of host defense. After the introduction of Highly Active Antiretroviral Therapy (HAART) in 1996, the epidemiology of HIV infection has changed and clinical evolution turned a fatal disease in a treatable chronic infection with an improved quality of life and a reduction of morbidity and mortality.5Plasmodium, may be more severe and, although malaria is not the main cause of death among HIV-infected patients,10Clinical presentation of malaria in HIV-infected people, even if partially immune to vice-versa and data about drug-interactions between antimalarial  and antiretroviral drugs are missing.11Moreover, despite the importance of proper treatments against both infections, there is a lack of data regarding treatment issue: conflicting data are presented in literature about the effects of antimalarial drugs (for treatment and chemoprophylaxis) on antiretroviral agents and Purpose of this article is to review the current knowledge on clinical tools of malaria and HIV co-infection in adults, focusing on pregnant women and international travellers, and to explore the interactions between antiretrovirals and antimalarial drugs, suggesting future research priorities.Sub-Saharan Africa represents the region most heavily affected by both malaria and HIV. In this setting, the overlap of these two infections is common and it would be important to understand their interactions and their correct management in order to limit their clinical burden.12The influences between HIV and malaria are bidirectional and synergistic,15P. falciparum malarial fever were indirectly proportional to CD4+ T-cell count,17Looking at HIV-infection during malaria, it seems that HIV infection worsens the capability to control parasitaemia because of deterioration of immune responses to malaria parasites; several studies reported an association between HIV infection and higher levels of malaria parasitaemia. Whitworth et al. demonstrated that higher levels of parasitaemia were more frequently detected among HIV-positive patients compared to those mono-infected , moreover they found an association between lower CD4+ T-cell count and higher levels of parasitaemia (p=0.0076), and lower CD4+ T-cell count and risk of clinical manifestations of malaria.The intensity of malaria transmission and the population’s level of acquired immunity may influence the clinical impact of malaria. Two major levels of malaria endemicity are described: i) areas of high (stable) malaria transmission, where most of adults have developed enough immunity that lead to poor clinical manifestations of malaria infection and where the reported incidence of malaria was ≥ 1/1000 per year in 2009;16HIV-positive patients with malaria presented higher incidence of anaemia,23Looking now at the consequences of malaria infection on HIV disease progression, several studies showed an increased HIV-RNA replication in patients with malaria. Hoffman et al found 7-fold higher levels of HIV-1 RNA in patients with malaria than in those with only HIV infection (p <0.0001)26Based on data presented, we suggest a strict monitoring of co-infected patients in order to improve the outcomes of these two infections, in particular for people most at risk of complications, such as pregnant women, in particular in stable malaria transmission areas where adults’ parasitaemia values are often below threshold of detection.P. falciparum, especially in sub-Saharan Africa12It is estimated that each year about 24 million pregnant women are infected by Consequences of this co-infection are described in both directions: HIV may promote more severe malaria clinical manifestations and, on the other hand, malaria can favours HIV RNA replication threatening antiretroviral treatment effectiveness.32In co-infected pregnant women, all the adverse pregnancy outcomes are present. HIV impaired malaria outcomes inducing chronic parasitaemia, higher parasite densities and fever, reflecting in more severe clinical malaria manifestations.37As previously said, malaria promotes HIV RNA replication: a study showed that women had about 2-fold increase in HIV-1-RNA both in peripheral and placental blood, with a consequent higher possibility of mother-to-child transmission of HIV,12An appropriate control of both infections is a priority either for women and for their infants. The main recommendations are: a prompt HAART initiation in pregnant women as soon as the eligibility criteria are met, or an effective antiretroviral prophylaxis if criteria for initiating ART are not present, according to World Health Organization (WHO) specific guidelines.34According to the World Tourism Organization (WTO), an increasing number of international travellers has been reported, from 50 million/year after second World War to 980 million/year during 2011.43This is mainly due to the introduction of HAART and the improved quality of life allowed a steady increase of international travels, especially to tropical areas.Several cases of imported malaria, as well severe cases with fatal outcome, have been reported;WHO recommends a viro-immunological parameters check (CD4+ T cell count and HIV viraemia at least) and a clinical examination before travelling.Plasmodium drug sensitivity in the specific area;10Then, physicians involved in HIV care as well as in travel medicine should consider several factors in pre-travel counselling for HIV-infected travellers such as reason for travel , travel risk (pre-arranged or organized travel), travel destination, season and malarial epidemic cycle as well as etc.), adequate chemoprophylaxis and, in selected circumstances, stand-by emergency treatment in case of evocative symptoms should be strongly recommended.Therefore the importance of behavioral preventive measures  has been reported,60For example, Mermin J and colleaguesP. falciparum clearance in HIV-infected patients treated with artemisinin-based regimens has been reportedP. falciparum, at concentration used in clinical practice.P. falciparum and may reduce malarial clinical manifestation, but, in order to better clarify this hypothesis, its underlying biological mechanisms and the role of antiretroviral drugs or classes need further investigations.Therefore some authors suggested that a continuation of prophylaxis with co-trimoxazole in malaria endemic areas is beneficial even when patients have been immune restored.Pharmacological interactions between antiretroviral and antimalarial drugs has been previously extensively reviewed,Table 1.Most of potential clinically significant interactions between antimalarial and antiretrovirals drugs are showed in stand-by emergency treatment (SBET) option for HIV-infected subjects receiving NNRTI-based regimens, while mefloquine could be considered as SBET drug in those prescribing PI-based HAART.82Despite several reports and reviews on malaria and HIV co-infection, few data are present in literature about the use of malaria chemoprophylaxis in HIV infected travellers.Malaria and HIV infections are two of the most important infectious diseases worldwide, particularly in sub-Saharan Africa: their overlapping epidemiology as well their impact in clinical practice needs to be continuously updated.HIV-infected adults, especially if international travelers, even with a restored immunity, are more likely to have severe clinical manifestations of malaria compared to travellers without HIV infection. Moreover co-infection in pregnant women contributes to develop clinical malaria in women, maternal severe anaemia, grater rates of stillbirth and preterm delivery, low birth weight, foetal grow retardation and increased risk of infant mortality.The wider implementation of co-trimoxazole prophylaxis and antiretroviral therapy could change the impact of malaria in HIV-infected patients.The benefits of the continuation of co-trimoxazole prophylaxis to prevent malaria, the antimalarial effects of several antiretroviral agents, as well the interactions between antiretroviral and antimalarial drugs, need to be further investigated. The proper malaria prophylaxis and treatment in HIV-infected pregnant women also needs to be defined. Several clinical trials are ongoing to address several of these opened questions: the results of these trials are needed to create specific guidelines for the prevention and management of malaria and HIV co-infection and to help clinicians involved in travel medicine as well in HIV care.


Plasmodium falciparum parasitemia (APFP) has been reported to be highly prevalent in Sub-Saharan Africa, a region heavily burdened by malaria, yet, the impact of APFP on the immunological reference values have not yet been established. This study was aimed at i) determine the prevalence of APFP in children and adolescents living in a region highly endemic for malaria in southern Mozambique and its impact on the immuno-hematological indices and ii) determine the factors independently associated with APFP.Asymptomatic A cross sectional study was conducted in a rural area highly endemic for Malaria in southern Mozambique during the dry season. Apparently healthy children and adolescents were selected for the study.Plasmodium falciparum was detected in 56.5% (194/343) of study subjects. APFP was more frequent in males and was associated with lower values of hemoglobin and platelets measurements. Parasitized and not parasitized individuals were similar in terms of lymphocyte counts, CD4+ and CD8+ T cells counts. Platelet count was the parameter with strongest association with APFP  in children and its performance in guiding clinical suspicion was moderate . Contrarily, in adolescents, the predictive value of platelets counts was low (AUC: 0.55).Blood samples were collected from 348 participants. Overall, our finding demonstrated that APFP is highly prevalent in regions endemic for malaria in southern Mozambique and was associated with lower hematological parameters but unaltered lymphocyte counts, CD4+ and CD8+ T cells counts. Platelets count was of moderate performance in guiding clinical suspicion of APFP in children but not in adolescents. Plasmodium falciparum parasitemia (APFP), defined as the presence of malaria parasites in peripheral blood in absence of symptoms, has been described to be prevalent in regions highly endemic for malaria. Previous studies conducted in Mozambique and other sub-Saharan African countries showed that a large proportion of individuals living in malaria endemic regions harbour asymptomatic malaria disease [Asymptomatic  disease -5.The spectrum of clinical consequences associated with APFP is not fully understood, but it is well known that APFP causes several haematological alterations, with anaemia and thrombocytopaenia being the most prominent . HoweverImmunological measurements, including CD4+ and CD8 T+ cells counts, play a key role in the diagnosis, prognosis and monitoring of a wide variety of diseases -11. The A previous report from our group demonstrated that children living in southern Mozambique presented almost the double amount of CD4+ T cells counts when compared to children from western countries , raisingIn a time when HAART is being rapidly scaled-up globally, there is a need to determine the impact of APFP on the profile of immunological parameters, particularly on the profile of CD4+ T cells counts in regions where both malaria and HIV are highly prevalent.We conducted this study aiming at determining: i) the prevalence of APFP in a region highly endemic for malaria, ii) the impact APFP on the immuno-hematological indices, and iii) the factors independently associated with APFP.The study was conducted in Calanga, a small village on the eastern coast of southern Mozambique. The houses are precarious with neither piped water, nor electric power supply. Calanga has an estimated population of 9451 inhabitants and is located in an area with high endemicity and transmissibility of malaria. The raining season starts from November through April. The main occupation of the population is subsistence-level farming and cattle grazing. A smaller proportion of the population, mainly adults, works as seasonal sugar cane cutters or fishermen. At the time of our study, only one health facility in Segera, a small community that is part of the village, was available to serve all of the surrounding area.Between August and September 2005, we consecutively enrolled individuals attending 1st (1st - 5th grades) and 2nd (6th - 7th grades) levels of the primary education in Calanga. Consent to participate in the study was obtained through parents or guardians. Before enrollment, information about study objectives and procedures was provided and explained to local authorities, students and their guardians in the local language. Study staff read and explained the consent form in the local language for illiterate parents or guardians. After consent was signed, students were submitted to a clinical evaluation by a physician. Individuals presenting any sign or symptoms of disease such as fever were excluded from the study. The study was approved by Mozambique’s National Health Bioethics Committee. This study was part of a larger project entitled “Human Biological Variability: Implication for Physical Education, Sports, Preventive medicine and Public Health” .Venous blood (10 ml) was collected in all subjects who consented to participate in the study. Blood was collected from the cubital fossa into a 5 ml K3EDTA tube and a 5 ml Serum Separation Tube . Specimens were delivered to the laboratory within four hours of collection.Plasmodium falciparum, P. malarie, P. ovale and P. vivax using light microscopy. Parasite density was estimated by means of a semi-quantitative scale [Upon delivery of samples at the laboratory, a thick blood smear was mounted from anticoagulated whole blood. All blood smears were stained using the Giemsa protocol and screened for ve scale .Hematology parameters, including white blood cell counts and differentials, were determined using an automated five-part differential hematology analyzer . All samples were processed within six hours of collection.TM flow cytometer . MultiTEST reagents, TruCOUNT tubes and MultiSet software  were employed in a lyse-no wash protocol to determine absolute and percent values for T cell subsets  [Immunophenotyping was performed on fresh whole blood using a FACSCalibur8 ratio) .Data was analyzed using the statistics package STATA 9.0 . The Mann Whitney test was used to compare the study groups regarding numerical variables. Associations between categorical variables were determined using the Pearson Chi-square test.P-value less than 0.25 on univariate analysis were included in the initial multivariate model. A P value ≤ 0.05 was considered of statistical significance on the final model.Logistic regression analysis was employed to determine the variables independently associated with APFP, controlling for confounders. The main confounders used for multivariate analyses were: gender, hematocrit, hemoglobin concentration and platelet count. The analysis was built using backwards stepwise method and Log Likelihood Ratio Test. For this purpose, all variables with a Receiver Operational Characteristics (ROC) curve analysis, sensitivity, specificity, and predictive values (positive and negative) were used to determine the performance of platelets count in predicting APFP. The gold standard method for APFP diagnosis relied on blood smear. ROC curve analysis was also used to define the best cut-off point of platelet count to predict APFP. The Area Under the ROC curve (AUC) was computed by the trapezoidal rule.For the purpose of comparison of immuno-hematological values, we stratified the participants by age, using the cut-off of 12 years of age. Those younger than 12 years were denoted as children and those older than 12 years were denoted as adolescents.P. falciparum was the dominant species of Plasmodium in this study and was evidenced in 195 individuals, giving an overall prevalence of APFP of 56.5% (95% IC 51.3% - 62.8%). P. malarie infection was evidenced in 7 samples (2.6%). No sample was positive for either P. ovale or P. vivax. For the purposes of this study, the terminology parasitized denotes infection by P. falciparum.Consent to participate and blood samples were obtained from 348 subjects. Of these, 55% (190/348) were male and the median age of study subjects was 11 years old (IQR: 8–13). versus 47.3%, p=0.007). In both children and adolescents, the prevalence of APFP was higher in boys compared to girls (71.9% versus 56.3% for children and 56.0% versus 43.5% for adolescents), and these differences were statistically significant for children (p=0.044) but not for adolescents (p= 0.091).Table versus non-parasitized 152 cm, p=0.036), weight  and BMI  . In addition, both children and adolescents with APFP presented significantly lower hematocrit levels . Platelet count in parasitized children was lower than in non-parasitized children (p=0.00), but this difference was not observed in adolescents (p= 0.283).As shown in Table Parasitized and non-parasitized children and adolescents were comparable regarding Mean Corpuscular Hemoglobin Concentration (MCHC) levels (p=0.458 for children and p=0.923 for adolescents) Regarding total leukocytes and its subsets counts, there were no differences between parasitized and non-parasitized children and adolescents, except for CD8+ T cells percentage count in adolescents for which parasitized individuals presented higher counts  and adolescents (57.7% versus 44.5% for parasitized and non-parasitized). This difference was statistically significant in children (p=0.002) but not in adolescents (p=0.095) , subsequent investigation of prediction of APFP in children was focused on platelets counts.3. At this cut-off, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 60.2%, 70.4%, 78.7% and 49.4%, respectively.The performance of platelets counts for predicting APFP was higher in children , with only a minor proportion of samples positive for P. malarie  and no circulation of either P. ovale or P. vivax. This was an expected finding since P. falciparum represents the main cause of malaria in our setting. The real prevalence of APFP might be even higher in Calanga as microscopic diagnosis of APFP underestimates the real prevalence of parasites when compared to molecular diagnosis [In sub-Saharan Africa, a region heavily burdened by malaria and where APFP has been reported to be highly prevalent -3, littlP. falciparum in older age [The prevalence of APFP in boys was higher compared to girls. This result corroborates findings from previous studies conducted in Kenya ,19. The Regarding hematological findings, results from this study demonstrated an association between APFP and anemia and thrombocytopenia, in line with data from previous reports . AlthougApart from the hematological parameters, the impact of APFP on immunological indices has not been previously studied in-depth and published literature in this regard is scarce . The undP. falciparum infection in afebrile individuals when compared to febrile individuals, suggests that the modifications induced by P. falciparum on the life cycle of platelets are of lower magnitude in asymptomatic individuals. This fact may be related with the acquisition of immunity. In addition to the impact on the immunohematological profile, the presence of APFP is of additional concern as individuals with APFP: i) represent an important reservoir of transmissible parasites ,23, ii) APFP is highly prevalent in regions highly endemic for malaria in Southern Mozambique and was associated with lower haematological values in school children but less in school adolescents. APFP represents an important cause of anaemia and thrombocytopenia in school children and adolescents in our settings. Contrarily, APFP was not associated with modifications on the immunological measurements commonly used to monitor HIV infection, such us CD4+ and CD8+ T cells counts. Thrombocytopenia was of moderate value in guiding clinical suspicion of APFP in children but not in adolescents.Authors declare they have no competing interestsESG participated in the study design, data collection, data analysis, and writing the manuscript. AP participated in the study design, data collection, data analysis and writing the manuscript. IVJ participated in the study design, data collection, data analysis and writing the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/244/prepub


P. falciparum infections during the first years of life affect the risk of severe malaria. A matched case-control study was nested within a large birth cohort set up to study the immunoepidemiology of pneumococci on the Kenyan coast. Infection patterns in three-monthly blood samples in cohort children admitted to hospital with severe malaria were compared to controls matched on age, residential location and time of sampling. P. falciparum detected at least once from birth conferred an increased risk of severe malaria and particularly if multiclonal infections, as characterized by genotyping of a polymorphic antigen gene, were ever detected. The results show for the first time that children with severe malaria have more infections early in life compared to community controls. These findings provide important insights on the immunity to severe disease, knowledge essential for the development of a vaccine against severe malaria.Children in malaria endemic areas acquire immunity to severe malaria faster than to mild malaria. Only a minority of children suffers from severe malaria and it is not known what determines this. The aim of this study was to establish how  P. falciparum malaria continues to cause an intolerably high burden of morbidity and mortality on children living in malaria endemic areas Severe The age distribution of severe malaria indicates that resistance to serious complications develops much earlier than immunity to uncomplicated and asymptomatic infections Three major syndromes of severe malaria have been identified in children: cerebral malaria severe malarial anemia, and respiratory distress as a sign of acidosis P. falciparum parasite is a highly polymorphic organism with an extensive genetic diversity and natural infections are often composed of several genetically distinct clones expressing different antigen variants. Based on the knowledge from malaria therapy of neuro-syphilis P. falciparum populations is by genotyping the highly polymorphic merozoite surface protein-2 gene (msp2), encoding a thoroughly studied potential vaccine candidate antigen msp2 genotype and different infections can be studied within individuals over time.The P. falciparum msp2 genotypes in three-monthly samples collected from birth in cohort children who subsequently were admitted with severe malaria and from controls matched for age, time of sampling and area of residency. Moreover, serum samples were analysed with regards to antibodies to whole P. falciparum schizont extract as a marker of previous parasite exposure The aim of this study was to investigate whether the pattern of exposure and particularly number of infections and different clones in early childhood affect the risk of severe malaria. A large birth cohort established in Kilifi District on the coast of Kenya, offered the opportunity to study the infection patterns from birth in relation to severe malaria outcome. In a nested case-control design we have investigated the parasite prevalence and the number and types of P. falciparum malaria and transmission typically has two seasonal peaks (June-August and November-December). Spatial differences in transmission intensity and entomological inoculation rate in the area have been described The study was conducted in Kilifi District along the Kenyan coast. Approval for the study was granted by the Kenyan Medical Research Institute National Ethics Committee and the Central Ethical Review Board Stockholm, Sweden. Kilifi District is mesoendemic for Kilifi District Hospital (KDH) serves as a first referral centre for more than 500 000 people. All children admitted to KDH are investigated with a malaria slide and blood cultures, except those admitted after accidents or for elective procedures. Clinical and laboratory data are recorded systematically at admission.2 around KDH and currently tracks a population of about 240000 people. All homesteads in KHDSS are visited 4–6 monthly to collect information on births, migrations and deaths The Kilifi Health and Demographic Surveillance system (KHDSS), established in 2000, covers 891 kmA birth cohort, the Kilifi Birth Cohort (KBC) was set up within the KHDSS in 2001 to study the immunoepidemiology of pneumococcal disease. Children either born at KDH or presenting to the vaccination clinic within their first month of life were recruited and subsequently followed with three-monthly visits for two years. The study was explained to parents or guardians by team members fluent in Swahili or Giriama and parents were also given written versions of the information. At each visit, axillary temperature was recorded together with sampling of 2 ml venous blood (without anticoagulant) and a thick and thin film for malaria microscopy. The blood samples were centrifuged and blood clots and serum samples were stored at −80°C. Children unwell during the study visits were attended to by medical staff and referred to KDH. Parents/guardians who did not bring their children for follow-up visits were visited in their homes the following day and again invited to attend the three-monthly sampling.P. falciparum parasites detected by microscopy together with either one or more of the following signs (syndromes) (1) impaired consciousness defined as Blantyre Coma Score <5 A matched case-control study was nested within the Kilifi Birth Cohort. Cases were children admitted to KDH between April 2002 and January 2010 with Controls were cohort children who had not developed severe malaria up to the time point when the matched case had its episode, and who were individually matched to cases on age (+/−4 months), sampling time (+/−3 months) and residential location. Three controls were sought for each case.P. falciparum densities were defined as the number of asexual parasites/µl of whole blood, based on an estimated leukocyte count of 8000/µl or 5 million erythrocytes/µl, respectively. Each blood film was evaluated separately by two expert microscopists and discrepancies resolved by the results from a third microscopist. The slides from the cohort visits were read during 2010. Slides taken at the time of admissions were read promptly as part of the acute clinical management.Thick and thin blood films were prepared from finger prick blood and stained with Giemsa. Slides were examined under light microscopy and parasites were counted against 200 leukocytes or 500 erythrocytes. msp2 gene was performed by fluorescent PCR followed by capillary electrophoresis msp2 domain, followed by two separate nested reactions with fluorescent primers targeting the two allelic types of msp2: FC27 and IC , and fragment analysis in a DNA sequencer  and GeneMapper software (Applied Biosystems).DNA was extracted by processing frozen blood clots by high-speed shaking in a cell disruptor P. falciparum schizont extract were assessed by Enzyme-linked immunosorbent assay (ELISA) using the A4 parasite line Antibodies against P. falciparum msp-2 gene depending on availability of samples. The number of concurrent clones was categorized as 1 clone or ≥2 clones . Patterns of infections in three-monthly samples were defined in three categories; parasite negative in all samples, parasite positive with single clone and parasite positive with multiple clones in at least one sample. Conditional logistic regression, including risk sets where at least one matched control was available for each case, was used to analyze the risk of being a case given previous parasite exposure. This analysis included the 42 risk sets where the case was no older than 2 years and 3 months at the time of severe malaria episode and thus had samples until three months before the admission. The analyses were adjusted for the logarithm of the number of visits, which was approximately linearly related to the logarithm of the odds of becoming a case. An unconditional case-control study was performed within the 61 cases according to syndrome; severe malaria with impaired consciousness versus non-cerebral severe malaria, using an exact logistic regression due to the small sample size, with a model adjusted for age (in six-month categories) and number of visits.Statistical analyses were performed using PRISM GraphPad and R version 2.13.1. Parasite prevalence at the cohort visits was based on results from microscopy as well as PCR genotyping of the P. falciparum, and 93 children  fulfilled the criteria of severe malaria by a strict syndrome definition  had been admitted to Kilifi District Hospital until January 2010. In 265 of the total 2366 (11%) admissions, a blood film was positive for finition . Among tfinition . No chilfinition  and did P. falciparum parasites were detected by PCR or microscopy in at least one of the three-monthly visits in 33% of the cases and 15% of the controls . The number of clones in multiclonal infections ranged from 2–3 clones in the cases and 2–6 clones in the controls. The cumulative number of clones, including all samples, ranged from 0–6 in cases and 0–15 clones in controls. The number of clones was not correlated to parasite densities . Children admitted with impaired consciousness had only one single clone in samples prior to admission; whereas children with respiratory distress and/or anemia (without impaired consciousness) had up to 6 clones cumulatively in three-monthly visits.controls . Among tmsp2 allele (same type and fragment size) before admission and 5 of 10 cases had a new msp2 allelic type (FC27 or IC) at admission. The same alleles (at bp precision) were detected in consecutive three-monthly visits in 3 control children (illustrated in File S1). Additional information of msp2 genotyping profiles and allele frequencies in visits and admissions are presented in the Supporting information  and particularly if a multiclonal infection was ever detected  (P = 0.085). Assessment of the risk of severe malaria with regards to parasite status at different ages, number of parasite positive visits, or to cumulative number of clones was restricted by limited data for subgroup analysis.Detection of = 0.022) . ParasitP. falciparum schizont extract (assessed as a marker of ever being exposed) were detected in at least one of the three most recent samples before admission, in 75% of cases and 70% of controls, respectively  . Within P. falciparum infections, especially infections with multiple clones, in three monthly samples during the two first years of life was associated with an increased risk of admission with severe malaria. The infection patterns in the cohort children who had been admitted to hospital with severe malaria were compared to the profiles in children of the same age, residential location and time of sampling. Although the background cumulative parasite rates were relatively low, detection of antibodies to schizont extract suggested an overall high exposure to P. falciparum parasites in both cases and controls. This high seroprevalence confirms that a majority of cases had been exposed to P. falciparum prior to the severe disease episode, thus arguing against the notion that severe malaria occurs predominantly after first encounters with the parasite.In this matched case-control study nested within a birth cohort on the coast of Kenya, detection of The three-monthly assessments provide only snapshots of the infection dynamics P. falciparum at least once. In the youngest children, antibodies might have been partly maternally derived and a more in depth analysis of the dynamics of anti-malarial antibodies in these children is ongoing.However, the finding of similar and high rates of preexisting antimalarial antibodies in the closely matched cases and controls indicates that similar proportions of children have been exposed to An alternative explanation for the more frequent prior detection of parasites in children developing severe malaria is that it reflects less good control of parasite densities in those individuals following exposure to malaria. It should be noted that most prior infections were asymptomatic at the cohort visits suggesting a state of partial immunity, at least to the infecting parasite. Children thus succumb to severe malaria despite having previously been able to harbor parasites without fever. We therefore tested whether subsequent episodes of severe disease represented exposure to new infections.msp2 gene provides more sensitive and detailed data on different P. falciparum populations than just assessing parasite prevalence by microscopy or PCR. The genotyping confirmed that the infections at the time of the severe malaria admissions were all different from the parasites detected in previous three-monthly visits. The finding of multiclonal P. falciparum infections in the cohort visits might be a result of several genotypes transmitted in single mosquito inoculations but can also reflect accumulation of repeated infections. However, most PCR positive samples in these children were composed of single clones. Also in high transmission areas where infections are mainly multiclonal, children younger than 1 year are more often infected with fewer clones Genotyping of the P. falciparum clones has, on the contrary, been associated with a reduced risk of uncomplicated malaria The risk of severe malaria was highest in children who had a multiclonal infection detected in at least one sample since birth. In previous studies of uncomplicated malaria from this and other areas of low or moderate transmission intensity, multiclonal infections in asymptomatic individuals at base line have predicted an increased risk of subsequent malaria The design of this study was unique in the sense that the three-monthly sampling within this large birth cohort allowed for genotyping of the infections encountered before the episodes of severe malaria. Although the study was running over several years, only a limited number of children had developed severe malaria within the cohort. Since Kilifi District Hospital is the only referral hospital in the district and the systematic recording of all pediatric hospital admissions is well established, we believe that the linkage was successful in capturing the majority of admissions of severe malaria within the cohort. Only including children with the major syndromes of severe malaria restricted the number of cases, however a high specificity was achieved Immunity to severe non-cerebral malaria has been suggested to be acquired after only one or two infections Knowledge of how and when children develop protective immunity to severe malaria is important for the development of a vaccine and important also for design of other control measures. Although findings need to be confirmed, the results suggest that infection patterns differ between children who develop severe malaria when compared with children who do not, and also may differ in children who develop different syndromes of severe malaria. Although epidemiological evidence shows that immunity develops faster to severe than to mild disease, our results argue against the notion that children who succumb to severe malaria are previously naïve to the infection.Figure S1Distribution and mean age at admissions with severe malaria within the cohort during the study period 2002–2010. Severe malaria admissions were included up to 2010, i.e. 2 years after inclusions ended.(TIF)Click here for additional data file.Figure S2Distribution of alleles of A) FC27 types and B) IC types in admissions and three-monthly visits. The y-axes determine the number of alleles of the same fragment length at base pair precision.(TIF)Click here for additional data file.File S1Additional information of msp2 genotyping profiles and allele frequencies in visits and admissions.(DOCX)Click here for additional data file.


Patient immune status is thought to affect the efficacy of anti-malarial chemotherapy. This is a subject of some importance, since evidence of immunity-related interactions may influence our use of chemotherapy in populations with drug resistance, as well as assessment of the value of suboptimal vaccines. The study aim was to investigate relationship between antibodies and anti-malarial drug treatment outcomes.Some 248 children aged 0.5 and 15 years were recruited prior to the high malaria transmission season. Venous blood (5 ml) was obtained from each child to measure antibody levels to selected malaria antigens, using ELISA. Blood smears were also performed to assess drug efficacy and malaria infection prevalence. Children were actively followed up to record clinical malaria cases.IgG levels to MSP3 were always higher in the successfully treated group than in the group with treatment failure. The same observation was made for GLURP but the reverse observation was noticed for MSP1-19. Cytophilic and non-cytophilic antibodies were significantly associated with protection against all three antigens, except for IgG4 to MSP1-19 and GLURP.Acquired anti-malarial antibodies may play an important role in the efficacy of anti-malarial drugs in younger children more susceptible to the disease. Plasmodium falciparum malaria is acquired over several years, in response to numerous disease episodes.. In vitro studies have shown that antibodies against some malaria vaccine candidates  play a protective role against malaria; P = 0.01), IgG1 , and IgG2  against MSP3 were all strongly associated with drug efficacy. The same trend was observed for IgG  and IgM  against GLURP. For the SP group, with the exception of IgG3  against MSP3, none of the other antigens showed any relationship between efficacy and antibody levels  were compared according to each drug's efficacy. Children were assumed to be "protected" if they were able to clear the parasites during the follow-up period, and "unprotected" if they failed to clear the parasites during this period. It appears that in the CQ group, total IgG  of the antibody levels was found to increase rapidly with age  with LTF levels of 69.5% for CQ and 14.6% for SP. These deceptors . The proeceptors .In malaria endemic zones, it is of paramount importance to give treatment as soon as the diagnosis is made. But it is not yet clear, however, how early treatment could modify the development of protection to further infections, and knowledge of how a primary infection with the malaria parasite may influence the immune response to later infections is scarce. Nevertheless, these results suggest that a background humoral immune response could be beneficial to children during anti-malarial chemotherapy. This information needs to be taken into account not only for drug treatment policy, but also for malaria vaccine development.The authors declare that they have no competing interests.AD participated in the design of the study, collected data, coordinated the study, performed statistical analysis and wrote the first draft of the manuscript. ABT, AO, ATK, MT, DD, AST and BSS participated in the design of the study, coordinated and participated in the drafting of the manuscript. IN and IS participated in the lab work and data interpretation. All the authors read and approved the final version.


In Ethiopia, information regarding highland malaria transmission is scarce, and no report has been presented from Butajira highland so far whether the appearance of malaria in the area was due to endemicity or due to highland malaria transmission. Thus this study aimed to determine the presence and magnitude of malaria transmission in Butajira.For parasitological survey, longitudinal study was conducted from October to December 2006. The entomological surveys were done from October to December 2006 and continued from April to May 2007. Both parasitological and entomological surveys were done using standard procedures.Plasmodium vivax and the rest 16 (1.5%) were due to Plasmodium falciparum. The highest prevalence 39(3.6%) of the parasite was observed in age groups of above 15 years old. Among the total tested, 25(2.3%) of males and 23(2.1%) of females had malaria infection. Among tested individuals, 38(5.3%) and 10 (2.7%) of infection was occurred in Misrak-Meskan (2100 m a.s.l) and Mirab-Meskan (2280 m a.s.l), respectively which was statistically significant . Although the prevalence pattern of Plasmodium species declined from October to December, the trend was non-significant . The entomological survey showed a collection of 602 larvae and 80 adult Anopheles. Anopheles christyi was the dominant species both in the first (45.3%) and in the second (35.4%) surveys; where as, Anopheles gambiae sensu lato comprised 4.7% and 14.6%, in the first and second surveys, respectively. Anopheles gambiae s.l comprises 55% of the adult collection, and both species were collected more from outdoors (57.5%). The number of An. christyi was higher in Mirab-Meskan (58. 3%) than Misrak-Meskan (41.7%) (P < 0.05).The parasitological result in all the survey months (October-December) showed an overall detection rate of 4.4% (48/1082)  malaria parasite. Among infected individuals, 32 (3.0%) of the infection was due to An. christyi as a secondary vector in the highlands must be further investigated by including blood meal sources detection.Malaria parasite and its vectors were found to be common during transmission periods in the highland fringes of Butajira. Thus, health education about the risk of malaria and its control programme in the area must be given adequate attention to minimize potential epidemics. In addition, the current study should be complemented from sero-epidemiological, prospective longitudinal and retrospective studies along with metrological and ecological factors, and socio-demographic data before concluding in favour of highland malaria transmission in the area. In light of its abundance, which coincided with the malaria transmission seasons, the possible role of  Malaria is one of the world's most serious and complex public health problem. Its transmission is usually associated with topography, climate and socio-economic conditions. The problem of the disease in Africa is aggravated by climate change , povertyDue to Ethiopia's extraordinary diverse topography and climatic conditions, the epidemiology of malaria in the country is more variable and unstable than any other country in Africa. Highland fringe areas at an altitudinal range of 1,500-2,500 metres above sea level (a.s.l.) i.e. northern and central parts and lowland arid areas i.e. eastern and southern parts at altitudinal ranges of below 1,500 m a.s.l. are prone to malaria epidemics. Some areas in western lowland, however, have relatively stable transmissions . A cycliPlasmodium falciparum and Plasmodium vivax are the dominant species responsible for frequent morbidity and mortality cases. A total of 42 species of Anopheline mosquitoes have been documented in the country. The works of Giaquinto-Mira [Anopheles arabiensis and Anopheles quadriannulatus spp B are the two major species of Anopheles gambiae complex that are found in Ethiopia [All of the four human malaria parasites species are occur in Ethiopia, but nto-Mira , Covel [nto-Mira , Verronento-Mira ,10 and Onto-Mira  have conEthiopia  and causThe conditions and nature of highland malaria transmission of the country were the same to the Butajira highland fringe areas in which epidemics were reported several times . HoweverButajira district is located in Gurage zone, Southern Nations, Nationalities, and Peoples Regional state (SNNPR) of Ethiopia. The District borders includes Siliti zone to the south, the Mareko District to the east, the Muhur and Akillial District to the west, and the Sodo District to the north. The estimated size of the district is 56,200 hectares. The district lies at an average altitude of 1,900 m a.s.l. ranging from 1,750 m a.s.l. in the lowlands to 3,400 m a.s.l. in the mountains. The population of the district is estimated to be 216, 968 in 2005 . The District has one Hospital, three Health Centers, 32 functional Health Posts, 11 registered non-governmental Health facilities.Teff (Eragrostos teff), maize, millet, barley and legumes are the main crops that the local people produce. Pepper and khat  are also grown as cash crops. Enset  cultivation is very common and gives the main staple food in the area. Extensive crop cultivation and relatively high population density leave little room for animal husbandry.Annual rainfall ranges between 700 to 1,870 mm. The main rainy season is from June to September, with light rain common around March and April. The warmest months are between January and June with maximum average temperature of 27.6°C and 27°C in February and March, respectively in the last six years . The area has rich fertile soil and farming is the main mode of living. Kebeles  in Butajira District, which have altitudes higher than 2,000 m a.s.l. The selection of the two farming associations was not random but based on the prevalence of malaria which was obtained from Butajira Health Center. From all highland localities, Misrak-Meskan was chosen to determine the altitudinal limit at which malaria was prevalent. In addition to a topographical map, a handheld Global Positioning System (GPS) unit was used to record the elevation of the two Farming Associations. Based on this, the average altitude of Misrak-Meskan Farming Association is 2,100 m a.s.l., while the average altitude of two neighboring localities is 2,500 m and 2,600 m a.s.l., respectively which was not included in the study because they were considered to be beyond Anopheline flight range. The other study site was Mirab-Meskan, which has the average altitude of 2,280 m a.s.l. The proportion of sampled individuals in the two localities was determined using the size of the proportion of the population living in the area.The survey was conducted in two For parasitological surveys, longitudinal study was done from October to December 2006 each month. The entomological surveys were also done from October to December 2006 and continued from April to May 2007. These two separate seasons (from October to December 2006 and April to May 2007) were chosen since sporadic rain which is favourable for vector reproduction is common in Ethiopia before and after the rainy season which is from June to September (8).Considering 95% confidence interval, 5% margin of error and 40% proportion  and 5% contingency using the formula for estimating single proportion, a total of 387 patients were included in this study. However, 366 individuals were given eligible data and blood collection was repeated for the three months. Thus a total of 1082 (98.5%) blood films were done and 16 (1.5%) of them was rejected due to various reasons.The lists of households of the two- selected localities were used as a sampling frame, with an assumption of similar exposure of houses and random malaria infections among inhabitants, a total of 100 households were selected systematically based on the calculated proportion for both localities to the sample size of 366 individuals. The calculated proportion was based on the total population of the two localities obtained from Meskan District Administrative office.If the occupants refused to participate in the survey, the household nearest to it was selected. After obtaining consent, blood samples were taken from all selected family members of the household by fingerpick, and thick and thin smears were prepared. This was done monthly on the same individuals from October through December.Plasmodium and species identification, respectively.Blood film collection was performed in peak malaria transmission season of the country ,13,14 frAnopheles mosquitoes were used to determine the sporozoites infection rate. Samples were collected from resting sites and also from breeding sites. The collected specimens were identified to species level on morphological basis using Verone [Both immature and adult mosquitoes were collected to determine the species composition. Female adult g Verone ,10 for b2) and sampling was made at a rate of 6 dips/m2 (four dips at the margin and two from the middle). One 'sample' was defined as 30 dips  taken over a surface area of 5 m2 for sites in the range of 5-10 m2 and forth [Anophelines sampled from each type of breeding habitat were transferred to separate vials by direct pipetting. The collected samples were immediately preserved in 75% ethanol. The 3rd and 4th instars larvae then, mounted on slides and identified [Larval collection was performed from suspected breeding sites such as artificial ponds, stream margins, swamps, excavation ditches and rain pools using standard dippers (350 ml) twice a month in a major malaria transmission seasons (October-December) and minor rainy seasons (April-May). The surface area of each potential mosquito breeding site was estimated in square meter . The houses were randomly selected from both localities and at each month, samples were taken twice among five houses in each locality. The collection was done for about 20 minutes each by two persons for two days for every visit. For the second collection, houses selected during first collection were not considered. Therefore, for the five months, a total of 50 houses were selected for mosquitoes collection.Adult Collection was done during major malaria transmission period through October to December and minor peak in April to May. The collections were done by space spray (knock down collection), CDC light catches and mouth operated aspirators. Space spray collections were conducted early in the morning from 06:00 to 08:00 among selected houses. A white sheet of cloth and aerosol were used to perform space-spray (knock down) collection.After consent from inhabitants, all occupants, removable house utensils, foods and drinks were removed from the sprayed room. All opening that allowed mosquito escaping, doors and windows were closed: the entire floor was covered with the white cloth sheet. The houses were then sprayed with aerosol for about 10 minutes. After waiting about 10 minutes, after spraying, the sheet was brought outside the room to inspect and collect the fallen mosquitoes. The mosquitoes were picked using forceps and put in box for later identification and for sporozoites rate determination.Anopheles mosquitoes, and then killed with chloroform. Mosquitoes were also collected indoors using aspirator from their resting sites. A flash light was used in locating the mosquitoes in relatively dark parts of the interior of a dwelling. The collected mosquitoes were transferred to a box and killed with chloroform vapor.Dry cell battery- operated CDC light trap were placed indoors near the bed from 18:00 in the evening to 06:00 in the mooring to collect endophagic Mosquitoes were surveyed and collected from outdoor resting places among vegetation , in animal shed, in pits, on ground or under logs of wood by the help of aspirator. Battery operated CDC light traps were hung in vicinity of sampled houses from 18:00 in the evening to 06:00 in the mooring twice for each month.Anopheles mosquitoes were dry-stored on silica gel before running the sporozoite ELISA [P. falciparum 0.2 μg per 50 μl PBS was used and for P. vivax 0.025 μg per 50 μl PBS was used. The plates were covered and incubated for 30 minutes after which well contents were dump emptied and filled with 200 μl BB buffer for one hour for further incubation. At the time mosquitoes were ground with 50 μl BB; Igepal 630 with pestle. To avoid contamination between mosquitoes the pestle were cleaned with tissue paper. After removal of buffer, 50 μ l mosquitoes triturate were added to wells of both P. falciparum and P. vivax duplicates test wells. The test was controlled using positive and negative controls. The control procedures were the same as that of the test samples. Then the plates were covered and incubated for 2 hours and washed 2 times with PBS-tween 20. Fifty micro litter aliquots of homologous peroxidase- conjugated MAB (0.05 μ g/50 μ l BB) were dispensed to each well of respective plates. After one hour incubation the plates were emptied and washed with PBS-tween 20 three times. Hundred micro liter substrates were added to each well after washing. Finally the plates were read visually or by 405-415 nm ELISA reader after adding the substrate. Samples were considered positive if the absorbance values exceeded the mean absorbance of seven negative controls [The collected female te ELISA . Two sepcontrols .After discussing the objective and application of the study to Meskan District Health Office and elders of the study area, the inhabitants were contacted to secure their consent in the same way. Blood smear was obtained with finger prick using disposable blood lancet and cotton immersed in 75% alcohol. Treatment of malaria to confirmed cases was given in collaboration with Misrak-Meskan Health Post freely. The study obtained ethical approval from the ethical committee of Biology Department, Addis Ababa University.Data were managed and analysed using a statistical computer programme SPPS version 13.0. Simple descriptive statistics and chi-squire tests were used to assess different variables.A total of 366 study participants were enrolled in the study, but in the last survey conducted in December, 2006, 16(4.4%) were missed due to different reasons. The study population was composed of mainly individuals above 15 years old (67.8%). The age groups vary from nine months to 88 years old. Among the total study participants, 188(51.4%) were males and 178(48.6%) were females.P. vivax and P. falciparum, respectively. Plasmodium vivax was the dominant species detected in each of the survey, even if, the difference was not statistically significant . The decline through the study months was also evident among Misrak-Meskan and Mirab-Meskan and the sexes  of the study area, malaria prevalence was determined. Out of the total 1082 blood film examined in three month surveys 48(4.4%)  were infected and among these 32(3.0%) and 16(1.5%) were due to 2 = 3.72, P < 0.05)  of males and 232.1%) of females had malaria infection  in Misrak-Meskan, which served as a breeding site during the dry season, while the others were collected in different breeding sites (Table Anopheles christyi and An. gambiae s.l. Anopheles christyi was the most abundant during the first three months survey (45.3%) (October to December) as well as in the second survey (April to May) (35.4%), whereas An. gambiae s.l comprised 4.7% in the first survey while it was 14.6% during the second survey season  from outdoors in both from Misrak-Meskan and Mirab-Meskan communities. The number of An.christyi (58. 3%) out number in Mirab-Meskan than Misrak-Meskan (41.7%) (P < 0.05).A total of 80 An. gambiae s.l (44) and An. christyi (36) adult mosquitoes were assayed by ELISA to determine the infection rate, but none were found to be infected with malaria parasites.All the collected (80) Anopheles species composition in the area were also surveyed.This study determined the presence and magnitude of malaria prevalence and the periodic malaria parasite transmission by parasite specific species, sex and age structures of the population, and the study communities. The mosquito breeding sites, sprozoites rate, and the Plasmodium falciparum is the dominant species, accounting for 60-70% of the cases followed by Plasmodium vivax, which is responsible for 30-40% of the cases (17). Furthermore, P.falciparum is known to be the dominant species during the peak malaria transmission (September and October) while P. vivax tends to dominate during the dry season [P. vivax was the dominant cause of infection than P. falciparum. The early appearance of the dry season in September 2006   and the commencement of data collection in October (more dry than September) may have contributed to the dominance of P. vivax during the present survey. Furthermore, the relapsing cases or nature of P. vivax may have increased the prevalence of the species in the area during the survey.In Ethiopia, all of the four human malaria parasites species have occurred. However, he cases . FurtherThe survey also showed that malaria cases declined from October to December 2006. There was also a clear decline in prevalence over the three month period among the locality and sex. This is actually in agreement with the overall malaria epidemiological picture of the country . HoweverAnopheles mosquito vector breeding. The two farming associations have numerous water bodies, such as swamps, rivers, and streams. The Rivers Auffaro and Erinzaaf being the major ones. The overflow of Auffaro River is common during the rainy season; due to which numerous breeding sites created during the summer season near villages of Misrak-Meskan locality.Malaria was observed in both survey localities in the study area, the high number of cases was seen in Misrak-Meskan which might be associated with the presence of swamps near the village and the relatively lower altitude  or the highest test done in the locality may be the sources of bias for higher prevalence record. The swamps that contained water throughout the study period was determined to support It is also known that in hyper or holoendemic conditions, the occurrence of malaria infection in infants or children in stable communities confirms autochthonous transmission and the standard characterization of the epidemiology of malaria is based on an age-prevalence curve. With the prevalence of infection peaking at an early stage with an increases up on the age of 9 years and showing a sudden fall in the age group 10 to 15 years and then slowly declining with age , whereasP. vivax.In the present study, relatively high malaria prevalence with statistically significance was observed in individuals above 15 year of age. This suggests the poor development of immunity to the malaria parasites in the population. Thus, the individuals aged above 15 years would make up the bulk of the population that probably constitutes the main reservoir of infection in the study area since the dominante species identified was It was also reported that in several places that there has been increased spread of malaria up into highland fringes and the climate change is responsible for this. In particular, global warming was indicated in many reports that it is becoming one of the major causes for encroachment and occurrence of malaria epidemics in high altitude areas of Africa, including Ethiopia ,22,23. AAn. gambiae s.l. at an altitude of 2,280 m a.s.l was a confirmation of the report Wayessa et al [An. christyi in the oily swamps of the study area was in agreement with what was reported from Akaki town where it was found in water bodies polluted by organic substances [An. christyi unusually tolerate relatively high level of pollution [An. gambiae s.l., which is the main malaria vector of the country. The abundance of An. christyi especially in the highland swamps where malaria is prevalent, makes it suspect as a possible vector of malaria in that locality. However, since An. christyi was found to breed in almost all the water collections investigated, its low density in adult collections may be related to its poor anthropophilic behaviour [An. arabiensis whose feeding behaviour depends on the availability of host, whether outdoors or indoors [The detection of sa et al  from thesa et al  from thebstances . Earlierehaviour , unlike et al [An. arabiensis in Ziway area, Ethiopia, which gave negative results in both salivary gland dissection and ELISA detection methods. However, the small sample size which was assayed in this study may explain the absence of sporozoite detection.The absence of sporozoite infection in the mosquito samples assayed may have been because of the very low sporozoite rate in the mosquito populations in the study area, which had a relatively low level of malaria prevalence. A similar finding has been reported by Abose et al  regardinAn. gambiae s.l. and An. christyi species were identified. Anopheles christyi larvae were the dominant collection whereas the opposite was true in adult collection. Thus, health education about the risk of malaria and its control programme in the upper highland fringes of Butajira must be given adequate attention and recommended to minimize potential epidemics. The sero-epidemiological study as well as assessing the knowledge, attitudes and practices of these high land communities must be done to assess the extent of malaria endemicity or emergence of highland malaria transmission in addition to current study. In light of its abundance, which coincided with the malaria transmission seasons, the possible relevance of An. christyi as a secondary vector in the highlands should be further investigated by including blood meal sources detection. In addition, the current findings must be also complemented by prospective longitudinal study and retrospective analysis of documented clinical data in the area. This might help to fill the gaps to the current findings, before concluding in favour of the emergence of highland malaria transmission or due to the endemicity of malaria in the highland of Butajira. Furthermore, the actual cause for the rise in malaria incidence in the area cannot be established without detailed study of changes in climate and agro-ecological factors.In conclusion, the parasitological survey showed that malaria parasite was common throughout the survey periods where peak prevalence was seen in the month October, in Mesrak-Meskan community, and in the age groups older than 15 years old. In the entomological survey, All authors have declared that no competing interests exist and the manuscript has not been published before or submitted elsewhere for publication.ST: Conception of the research idea, designing and data collection, data analysis and interpretation, and manuscript reviewing. YB: Data collection, interpretation of the results, and drafting the manuscript. TT: Data collection, interpretation of the results and drafting the manuscript with YB. TM: Designing and data analysis, interpretation, and manuscript reviewing.BP: Conception of the research idea, designing, data analysis and interpretation, and manuscript reviewing. All authors have read and approved the final manuscript.