Malaria is a major cause of morbidity and mortality. Treatment of malaria in a timely manner could avert deaths. Treatment ultimately relies on the rapid and accurate diagnosis. Fluorescence in situ hybridization (FISH), a cytogenetic technique based on detection of specific nucleic acid, has the potential to address the limitations of the current diagnostic approaches. This study investigates further the performance of FISH for the diagnosis of malaria in a rural setting in Western Kenya.Blood samples from 302 patients presenting with fever (temperature ≥ 37.5 °C) were examined for malaria using the Giemsa microscopy (GM), rapid diagnostic test (RDT), polymerase chain reaction (PCR) and FISH.The sensitivity and specificity of FISH was 85.6% and 96.2% respectively, while the corresponding values for GM were 82.2% and 100% respectively. RDT and PCR had sensitivities of 91.1% and 98.9%, respectively with their specificities being 89.6 and 100%, respectively. The positive predictive values for RDT, GM, FISH and PCR were 78.8%, 100%, 90.6% and 100%, respectively. The negative predictive values for RDT, GM, FISH and PCR were 96.0%, 93.0%, 94.0% and 99.5%, respectively. Their respective diagnostic accuracies were 90.1%, 94.7% 93.0% and 99.7%.The present study demonstrates that the specificity and reproducibility of FISH assays are high, thus adding to the growing evidence on the potential of the technique as an effective tool for the detection of malaria parasites in remote settings. Malaria is one of the most important public health problems worldwide, and a leading cause of morbidity and mortality in many developing tropical countries, with young children and pregnant women being the most affected groups within populations . An estiIn Kenya, about 52% of the population is at risk of contracting malaria . The goaFollowing WHO’s recommendation that all cases of suspected malaria be confirmed using parasite-based diagnostic testing, either microscopy or rapid diagnostic test (RDT) before commencement of treatment, there has been an impressive increase in the proportion of suspected malaria cases receiving a diagnostic test since 2010, especially in the African Region . HoweverMalaria RDTs provide a valuable tool especially in field studies as they are able to counter the inadequacies of other diagnostic approaches. In countries where malaria is endemic, they provide an alternative approach in settings where microscopy is not feasible . MoreovePlasmodium spp. [Currently, polymerase chain reaction (PCR) assay is used chiefly for research purposes or for speciation in reference laboratories. This method involves detecting circulating malaria parasites by detecting parasite DNA through amplification of ribosomal RNA genes. This can detect parasites at low parasitaemia, even below 5 parasites/μl of blood, for all five human ium spp. . Howeverium spp. . These dP. falciparum antigen have been recently developed. None of these tests however, are used routinely as they are far too complicated or expensive for use in routine diagnosis especially in a developing countries including Kenya. There is therefore a need for sensitive, easily accessible and affordable diagnostic tools to facilitate targeted treatment and management of malaria. In addition, new diagnostics that can detect low levels of parasitaemia in asymptomatic individuals and, in the case of P. vivax, in the blood of asymptomatic individuals who may serve as reservoirs for mosquito infections, are much needed [In Kenya, Giemsa microscopy (GM) is traditionally the gold standard for diagnosis. Under optimum conditions, microscopy can detect 20–50 parasites per μL blood, but such sensitivity is rarely achieved in routine diagnosis. On the other hand, parasite nucleic acids can be detected using PCR. Although this technique may be slightly more sensitive than smear microscopy, it is of limited utility for the diagnosis of acutely ill patients in the standard healthcare setting due to its unavailability and long turnaround time to results. PCR results are often not available quickly enough to be of value in establishing the diagnosis of malaria in such patients. Other alternative laboratory methods for the detection of malaria parasites including the quantitative buffy-coat centrifugal haematology system, immunofluorescence and ELISA tests for the detection of One of the promising molecular techniques in malaria diagnosis is the fluorescent in-situ hybridization (FISH) assay. This is a cytogenetic technique that is able to detect and localize the specific nucleic acid (DNA or RNA) sequences by hybridizing with complementary sequences labeled with fluorescent probes. The assay gives results within 45 mins and can circumvent the drawbacks characterising PCR such as requirement of sophiticated equipment, highly trained personnel and prolonged time to results. Specific FISH assays that can be deployed in peripheral laboratories of limited resource nations where poverty-related diseases including malaria and tuberculosis are endemic, have been developed , 22. The2 and has a population of 968,909 [The study was conducted in Ahero sub-County Hospital, Kisumu County of former Nyanza province, Western Kenya. The County covers an area of 2085.9 kmThis was a descriptive cross-sectional study in which blood samples were collected from patients presenting with fever of ≥37.5 °C and examined for malaria parasites using rapid diagnostic test (RDT) kit, Giemsa microscopy (GM), polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH).The minimum required sample size for the study was calculated using the formula by Lwanga and Lemeshow .\documenWhere;n is the sample sizez∝/2 is the normal standard deviate for a given level of significance p is the prevalence of infection expressed as a proportiond is the desired level of precision (0.05).Malaria prevalence around Lake Victoria is estimated to be 26.7% . Using 5Patients visiting the outpatient clinics of Ahero sub-County Hospital in October 2016 and presenting with fever (≥ 37.5 °C) and with suspected malaria were enrolled sequentially into the study after informed consent was obtained. Exclusion criteria were lack of consent from the caregivers and symptoms associated with signs of severe malaria as defined by WHO . Also exFrom each enrolled subject, 2 ml of venous blood sample was drawn and examined for the presence of malaria parasites using the GM, RDT, PCR and FISH.Giemsa microscopy was performed as outlined in the WHO guidelines . Blood fBlood samples were tested for malaria parasite antigen using SD Bioline Malaria Ag P.f/Pan , RDT kits as outlined in the manufacturers’ instructions inserts.P. falciparum/P. vivax FISH combo and Plasmodium Genus FISH Kit . Briefly, thin smears were prepared from EDTA whole blood mixed with smear preparation reagent (SPR) (3 parts blood: 1 part SPR by volume). Each smear was prepared from 4 μl of the mixture, dried in the air and fixed in methanol. Hybridization buffer with probe mix (12 μl) was added to each fixed smear. The smear was covered with a cover slip and placed in a humid chamber at 37 °C for 30 min to allow for hybridization to occur. The smear was then washed with wash buffer twice, rinsed with rinse buffer and dried in the dark. A drop of plasmodium counterstain was added to the dry smear. The smear was then covered with a cover slip and examined using a fluorescence microscope (magnification: × 1000) fitted with a green filter (excitation and emission of 560 nm and 630 nm respectively).The FISH assays were conducted on all the samples as per the manufacturer’s instructions supplied as part of the Briefly, PCR analyses were performed on purified DNA using primers (Forward 5′- GCTCTTTCTTGATTTCTTGGATG -3′ and Reverse 5’-AGCAGGTTAAGATCTCGTTCG-3′) for plasmodium genus using Rotor gene Q real time PCR cycler.p-value of less than 0.05.Data were entered in Microsoft Excel spread sheet and imported into Stata 13.0 for statistical analysis. Performance of the diagnostic methods was assessed with the combination of PCR and GM results serving as the gold standard for malaria detection. All but one sample which were positive by GM were also positive by PCR. The sample was taken as part of the reference standard. Demographic attributes of the study participants were summarized using appropriate descriptive statistics. The number of true positives (TP), true negatives (TN), false positives (FP) and false negatives (FN) were computed. Sensitivity was calculated as TP/ (TP + FN). Specificity was calculated as TN/ (TN + FP). The positive predictive value (PPV) was calculated as TP/(TP + FP) and negative predictive value (NPV) as TN/ (FN + TN). The accuracy of the test, defined as proportion of all tests that gave a correct result, was calculated as (TP + TN)/number of all tests. Variations in proportions of outcomes based on different diagnostic techniques were tested using chi square (χ2) test. For all statistical tests, the threshold for statistical significance was set at a The density of malaria parasite was determined as follows :\documenCohen’s κ was used to determine if there was agreement between FISH, Giemsa-microscopy, microscopy and/or PCR and RDT in the diagnoses of malaria.A total of 302 children were enrolled in the study with the majority being females (65.6%). Their ages ranged from one to 79 months with the median (interquartile range) age being 21 (9–33) months. Children less than 6 months of age constituted 18.2%; between 6 and 24 months, 40.4%; between 25 and 59 months, 39.7%; and >60 months, 1.7% of the study participants.The prevalence of infections with malaria parasites among the samples tested using the four diagnostic techniques under study is shown in Table Plasmodium infections diagnosed using PCR and/or microscopy were regarded as the “true positives” with the rest being regarded as “true negatives”. The sensitivity and specificity of FISH was, 85.6% and 96.2% respectively, while that of microscopy was 82.2% and 100% respectively. RDT and PCR had sensitivities of, 91.1% and 98.9% respectively. The corresponding specificities were 89.6% and 100%.The findings on the predictive values of the tests are shown on Table The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the tests are shown in Table Diagnostic accuracy was expressed as a sum of the proportion of correctly diagnosed samples (true positives (TP) and true negatives (TN)) out of all the samples tested. The overall diagnostic accuracy of FISH was 93.0% while that of microscopy was 94.7%. RDT and PCR had an accuracy of 90.1% and 99.7% respectively, (Table p < 0.001). Similarly, high levels of agreement in the results were observed when Giemsa-microscopy and PCR were assessed singly against FISH . In addition, a moderate level of agreement was observed between the results obtained from RDT and those obtained by FISH .There was high level of agreement in the results of a combination of Giemsa-microscopy and PCR when assessed against FISH  that tested positive for malaria showed that the parasite levels ranged between 173 and 229,533 parasites/μL of blood. The geometric mean malaria parasite density was 15,631 parasites/μL of blood. The majority of samples  had high levels of parasitaemia (≥5000 parasites/μl of blood). The number of cases with low (1–999 parasites/μL of blood) and medium malaria parasite densities (1000–4999 parasites/μL of blood) were 14 (18.9%) and 7 (9.5%) respectively of the samples that were positive for malaria parasites.Plasmodium sp. infection in blood smears when compared to Giemsa microscopy, PCR and RDTs. FISH is a potential test for use in malaria endemic resource-limited settings. In particular, the use of light emitting diode (LED) as a source of light makes the technique invaluable in such settings, which characterize many sub-Saharan African countries [Plasmodium species. The FISH assays detected all stages of the malaria parasites present in blood including gametocytes [The current study was undertaken to evaluate the fluorescence in situ hybridization (FISH) based technique for detecting ountries . It has ountries . Prior tountries –31. Thisountries . The preetocytes .The potential of FISH as a vital tool for monitoring the efficacy of treatment for malaria is being considered. This is based on the fact that it detects only live parasites. FISH detects specific 18S rRNA fragments in live parasites whereas PCR, RDT and Giemsa microscopy do not distinguish between live and dead parasites. In case of microscopy, parasite detection and speciation is based on morphology only , whereasP. falciparum and P. vivax by GM respectively [In the study, it was noted that the FISH assay had the higher sensitivity (86%) when compared to the Giemsa-microscopy (82%) diagnostic approach which is routinely used in diagnosis centres across the country. On the other hand, it had lower sensitivity when evaluated against the RDT (91%). As expected, PCR registered the highest sensitivity as compared to all other diagnostic approaches (99%). The findings in this study corroborates those of a similar study by Shah and others who reported that FISH assays were more sensitive than GM. Shah and her team found that the sensitivities of P-Genus, PF and PV FISH assays to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of ectively . In the ectively . Therefoectively .Trypanosoma cruzi, Babesia duncani, B. microti and Leishmania major), 7 bacteria and 11 viruses [Mycobacterium tuberculosis infections in resource-constrained settings. They established that sensitivities for the Mycobacterium spp. and Mycobacterium tuberculosis FISH assay of sputum were 82% and 89%; and specificities were 91% and 95%, respectively [In the current study, specificities were highest in both PCR and GM (100%), whereas RDT showed the lowest specificity (90%). The specificity of FISH assay (96%) was hence comparable to that of PCR and GM. Another study reported a much lower specificity for FISH assays and RDT (88.2% and 97.6% respectively) . The dis viruses . Similarectively .In this study, the diagnostic accuracy of FISH assay was 93.0%, which was higher than that of RDTs (90%) though not statistically significant. Diagnostic accuracy of FISH assay was comparable to that of microscopy which was 95%. Although PCR had diagnostic accuracy of about 100% its utility in malaria endemic areas is limited by the complexity of the requisite methodologies in addition to the high costs, in terms of the facilities required and the advanced level of staff training needed. Moreover, maintenance of PCR equipment is also critical, further lowering its suitability for diagnosis of malaria in remote settings and in routine clinical work . On the On evaluation of the agreement between the various diagnostic approaches studied, FISH assays showed a high level of reliability in the diagnoses of malaria with the agreement with Giemsa-microscopy, and also microscopy and/or PCR being rated as high. Moderate level of agreement was found between FISH and RDT. In their research, Shah et al. also demonstrated the reproducibility of the FISH assays .The present study has a number of limitations. The study did not assess the variations in the performance of the FISH assays based on the levels of parasitaemia in blood (parasite densities). Considering that FISH assays detect only live parasites while other diagnostic approaches including Giemsa microscopy detect both live and dead parasites there may be a difference in performance of the various malaria diagnostic tools . The stuPlasmodium species as well as in the mixed infections cases.This study highlights the potential use of FISH assay as an effective diagnostic tool for the detection of malaria parasites in resource-constrained settings. The specificity and reproducibility of FISH assay were found to be high. The adoption of this assay may well promote better clinical practices such as monitoring the efficacy of malaria treatment in addition to avoiding unnecessary, or minimizing empirical treatments, as it detects live parasites only. This, in turn, will lower the risk of development of drug resistance, ultimately resulting in substantial cost savings from avoidance of unwarranted treatments of patients. There remains a need to conduct further similar studies in a field setup and in different malaria epidemiological zones so as to further assess the effectiveness of this diagnostic tool. The performance of the tool also needs to be evaluated against various


Kohat district is one of the medium intensity malaria transmission areas in Pakistan where asymptomatic carriers are likely to form a reservoir of infection. This study was done to explore the possibility of using microscopy, rapid diagnostic testing (RDT), real time polymerase chain reaction (RT-PCR) and RT-PCR followed by endpoint fluorometry (EPF) for detection of malaria in asymptomatic immediate family members of patients of malaria (homestead) and in a sample from the general population of Kohat.This cross-sectional study was done at Combined Military Hospital Kohat and Molecular Lab of Riphah International University, Islamabad from Jan to Dec 2015. A total of 1000 individuals including 200 microscopy positive patients of malaria, 400 asymptomatic immediate family members (homestead) of the active patients of malaria and 400 apparently healthy controls were tested by microscopy, RDT and RT-PCR. At the end of RT-PCR the result were read by EPF.p = 0.0002) and the relative risk of malaria was 4.0 times higher (95% CI 1.87–8.57).In the 200 malaria microscopy positive patients, 190 (95%) were RDT positive and all were RT-PCR positive. In the 400 individuals from the homestead of malaria patients, 6 (1.5%) individuals were malaria microscopy positive while RDT failed to pick any positive and 32 (8%) were RT-PCR positive for malaria. EPF of all the RT-PCR positive results were positive and the negative results were negative. The difference in the frequency of malaria in the homestead versus general population was very significant  contains supplementary material, which is available to authorized users. Malaria is one of the common causes of death from infectious diseases, but remains the main global cause of death from parasitic infectious diseases . AsymptoThis cross-sectional study was done at Combined Military Hospital (CMH) Kohat and Molecular Lab, Riphah International University, Islamabad from Jan to Dec 2015 to identify a suitable approach for detection of asymptomatic carriers of malaria in Kohat district of Pakistan. The study was approved by the ethical review committee of Riphah International University, Islamabad. A total of 1000 individuals were included in this study. This included 200 consecutive patients of malaria who presented to CMH Kohat and were confirmed on microscopy, 400 apparently asymptomatic individuals from the immediate family members (homestead) of active patients of malaria and 400 apparently healthy individuals randomly picked from the same resident community with similar socio-economic status. A written consent for participation was obtained from all of the study participants. Apparently healthy individuals not resident in the area for at least 2 years and those with history of fever in the last 2 months were excluded from the study.Each individual was tested by microscopy for malaria, RDT, RT-PCR and EPF. Microscopy was done on Leishman stained thin smears. Each smear was examined by an experienced microscopist for 3–5 min. The slides of individuals who were negative on microscopy but were positive by RT-PCR were re-examined for at least 100 high power fields. The specie of malaria parasite was identified when visible on microscopy and the number of parasites per µL was calculated . All micRapid diagnostic testing for malaria was done as per manufacturer’s instructions . It is a pan malaria RDT that can detect all species. DNA extraction was done from peripheral blood collected in EDTA by Chelex™ method . Red cells in 300 µL blood sample were lysed by distilled water. The process of red cell lysis was repeated more than once if any visible trace of haemoglobin was left behind. White cells including the malarial parasite were pelleted after centrifugation at 10,000 rpm for 2 min. The pellet was incubated at 95 °C for 20 min in 300 µL of 7% Chelex™. The clear supernatant was used as source of DNA.Plasmodium specific 18S rRNA gene (SSUrRNA gene) [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale). The following primers and TaqMan probe were used:PCR amplification was done by real time method targeting the genus NA gene) , 9. The Forward: 5′-ACATGGCTATGACGGGTAACG-3′Reverse: 5′-TGCCTTCCTTAGATGTGGTAGCTA-3′TaqMan Probe: 6FAM 5′-TCAGGCTCCCTCTCCGGAATCGA-3′-BHQ12, 100 mg/mL gelatine and 0.1–0.3 µg of genomic DNA (2 µL of extracted DNA). Real time PCR was done on Rotor-Gene 6000 . Thermal cycling comprised initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 95 °C for 20 s and annealing/extension at 60 °C for 1 min. Green fluorescence was read at the 60 °C step.DNA amplification was done in 20 µL reaction mixture containing 5 pM of each primer and the probe , 0.5 units of Taq polymerase , 30 mM of each dNTP, 10 mM Tris HCl (pH 8.3), 50 mM KCL, 1.5 mM MgClThe results of RT-PCR were recorded as cycle threshold (Ct). Samples with Ct ≤ 35 were taken as positive while all others were taken as negative. In each batch of PCR known positive and negative DNA for malaria were also included.http://grcpk.com/gti-pcr-reader-g/). The instrument measures green fluorescence in 0.2 mL PCR reaction vials. The amount of fluorescence is recorded on a computer software and the results are expressed in relative fluorescence units (RFU) after subtracting background fluorescence of a known negative sample.At the end of RT-PCR the reaction vials of all positive and negative samples were read in a fluorometer  and one RT-PCR negative sample were run by the RT-PCR protocol. The dilutions and the negative sample were run under identical RT-PCR conditions in five separate reaction tubes each. At the end of RT-PCR EPF was measured as described earlier. The results of Ct values and the corresponding EPF of each dilution were cross tabulated. The cost of DNA extraction, RT-PCR and the technician time were calculated in US$ as per the local market price.In order to validate the EPF results tenfold dilutions  of a RT-PCR positive DNA sample  individuals. All of the six had P. vivax malaria. The parasite density ranged from 70 to 500/µL. Microscopy did not show malarial parasite in any of the 400 apparently healthy controls.In the 200 microscopy positive patients 196 (98%) had In the 200 lab detected patients of malaria 190 (95%) were positive on RDT. RDT failed to pick any positive case out of the 800 subjects from the homestead and the population controls.All of the microscopy positive patients of malaria were positive on RT-PCR. In the 400 subjects from the homestead group 32 (8%) were positive for malaria Table  whereas The results of EPF validation assay showed good correlation between the RT-PCR and the EPF results . The relative risk of malaria in the household of an index patient was 4.0 times higher than in the general population (95% CI 1.87 to 8.57).There was a very significant difference between the frequency of individuals carrying malarial parasite in the homestead of active patients of malaria (8%) and the general population (2%) (P. vivax while the remaining 20% are caused by P. falciparum. The cumulative overall transmission rate of malaria in the Khyber Pakhtunkhwa province is 4.3/1000 population [Malaria is the leading cause of morbidity and mortality due to vector born disease in Pakistan . An estipulation .In geographical areas with intense transmission of malaria the population develops high level of immunity and malaria remains “stable” with no epidemics. On the other hand when the malaria transmission is intermittent due to seasonal variations the infection is “unstable”. In such populations the level of immunity is low, therefore, epidemics are common and the disease may also run a severe course . MalariaDistrict Kohat is located in Khyber Pakhtunkhwa and WHO has defined it as a region of low to moderate transmission setting where the annual malaria transmission rate is < 10%/1000 population . It is eIn this study RDTs failed to detect low level parasitaemia in both symptomatic and asymptomatic patients of malaria. Similarly, thin film microscopy also lacked the sensitivity to pick low level parasitaemia. Similar observations have also been reported in an earlier study from Pakistan . MicroscDetection of low-density parasite count by PCR in asymptomatic patients has far-reaching implications for areas with moderate transmission in Pakistan. It is important that the asymptomatic patients are identified and treated otherwise malaria control cannot be successful in these areas of Pakistan. Mathematical models frequently predict erroneous prevalence rates based on detection of malaria in asymptomatic individuals by routine slide examination. Strategies solely relying on microscopy and RDT results can end up in failure. This study augments the reason to use PCR for accurate estimates of the infectivity levels of the sub microscopic reservoir in the asymptomatic individuals . Major hThe frequency of asymptomatic carriers of malaria is significantly higher in the homestead of patients of malaria than in the general population. Microscopy and RDT are not suitable for their detection. They are best detected by RT-PCR or PCR with EPF.


Anopheles bites were never explored together in a study focusing on the acquisition of malaria antibody responses among infants living in endemic areas.Five hundred and sixty-seven Beninese infants were weekly followed-up from birth to 18 months of age. Immunoglobulin G (IgG), IgG1 and IgG3 specific for 5 malaria antigens were measured every 3 months. A linear mixed model was used to analyze the effect of each variable on the acquisition of antimalarial antibodies in 6-to18-month old infants in univariate and multivariate analyses. Placental malaria, nutrition intakes and sickle-cell trait did not influence the infant antibody levels to P. falciparum antigens. In contrary, age, malaria antibody levels at birth, previous and present malaria infections as well as exposure to Anopheles bites were significantly associated with the natural acquisition of malaria antibodies in 6-to18-month old Beninese infants. This study highlighted inescapable factors to consider simultaneously in an immuno-epidemiological study or a vaccine trial in early life.To our knowledge, effects of age, placental malaria infection, infections during follow-up, nutritional habits, sickle-cell trait and individual exposure to  Plasmodium falciparum. Parasite, host, environmental and behavioral factors are essential to be examined for a better understanding of the acquisition of clinical immunity and thus clinical outcomes of malaria infections. To our knowledge, this was not exhaustively investigated in previous studies. Indeed, Dobaño et al. evaluated the age dependence of naturally acquired antibodies to Merozoite Surface Protein (MSP) 1-19, Apical Membrane Antigen 1 (AMA1), and Erythrocyte Binding Antigen 175 only on the basis of past and present parasite exposure, occurrence of previous episodes and neighborhood of residenceet al. showed that malaria specific antibody responses increased with age in children under 5 years oldet al. clearly showed that host age, independent of cumulative exposure, represents a determinant key of the quantitative and qualitative immunoglobulin G (IgG) response to P. falciparum, certainly through an immune system maturation processNaturally-acquired immunity to malaria develops slowly following repeated exposures to et al., concluded that malaria antibody responses differed according to the areas where distinct Anopheles species evolveet al. have observed significant associations between parasite prevalence, altitude, as well as recent rainfalls and the prevalence of antibodies specific for MSP1-19 in individuals between 3 to 45 years oldet al. showed a lower risk to be seropositive to P. falciparum for people living at more than 1.5 km of lakes and riversEnvironmental factors also influence malaria specific antibody levels. Sarr Other factors may be incriminated such as malnutrition. Indeed, low levels of IgG directed to SPf66 and the Ring-infected Erythrocyte Surface Antigen have been shown in wasted children7et al. showed that the HbAS protection occurred during the first 10 years of life. The authors proposed that this innate protection would allow the host to be exposed safely to malaria antigens capable of inducing malaria specific immunityThe genotype AS of hemoglobin (Hb) is the main host genetic factor that confers a resistance to severe malariaet al. showed that the development of malaria specific antibody responses was impaired during the first year of an infant born to a mother with placental malaria at deliveryet al., showed that infants born to mothers with pregnancy-associated malaria without MSP1-driven cord blood lymphocyte responses had a lower functional activity of anti-MSP1 antibodies from 18 to 30 months in comparison to the other infantset al, did not found any difference in anti-MSP1 and anti-AMA1 IgG levels between infants born to mothers with or without placental malariaFinally, placental malaria could influence the development of immunity in newborns since studies suggested that offspring of women with placental malaria are more susceptible to malaria during infancy11P. falciparum blood-stage antigens from birth to 18 months and ii) to understand what are the key factors that could influence the emergence of antibody responses in infants.The present work is based on an epidemiological study already described16The results presented in the From birth to 6 months of age, maternal IgG, IgG1 and IgG3 are decreasing in infant blood and after 6 months of age the infant antibodies are produced. This production is increasing with age as we can visualize that at 6 months the level of specific IgG, IgG1 or IgG3 and total IgG are lower than at 18 months of age. For AMA1, the concentration of specific IgG/IgG1/IgG3 is stronger than for all the other antigens. The same effect is still observed but instead to see a decrease/increase at 6 months of age, the phenomenon is observed at 12 months of age . Although the sickle-cell trait was negatively associated with the acquisition of IgG3 to MSP2-FC27, this effect was not significant (P = 0.26). IgG levels to P. falciparum in infants aged more than 6 months were positively associated with maternal  and to MSP3 (P = 0.004)), cord ) and 3-month-infant  antibody levels with the strongest effect observed in cord blood. Placental malaria infection was not associated with specific IgG levels.The nutritional status was not associated with antibody levels (−3 for each antibody response) was detected between age and infections. This result is consistent with the fact that an infection occurring later in the follow-up had a stronger effect on the antibody response than an infection occurring early after birth when the immune system can be considered as less mature  to highlight the factors that could influence the acquisition of these antibody responses in infant.The purposes of this work were i) to explore the acquisition of IgG, IgG1 and IgG3 responses to P. falciparum antibody responses allowing 2 complementary interpretations: i) even with a close survey, an infant can develop unperceived infections and ii) malaria specific immune response would subtly develop in infant following submicroscopic infections. The strong effect of the environmental variable corroborates both explanations: P. falciparum exposure was related to the specific antibody response in 6–18 months old infants. This variable was inseparable from the presence of anopheleset al. showed that protection could be induced by sub-clinical infections with very low circulating parasitesP. falciparum during sero-epidemiological studies.Infants with no detected malaria infection presented low but nonzero et al., did not show any correlation between malaria antibody levels and memory B cell frequenciesAn infection occurring in the 3-to-6-month period before the antibody measurement could be followed or not by an infection in the 3-month period. In case of no infection occurring during the 3-month period before the antibody measurement, there was a positive “residual effect” on the level of total and cytophilic IgG, of the infection having occurred in the last 3-to-6 month period. In case of an additional infection during the 3-month period before the antibody measurement, the malaria specific antibody levels were boosted (2 consecutive infections). This is consistent with the development of an immune memory for infants living in an endemic area. Additionally, the quantification of memory B cells CD19+ CD21+ CD27+) would represent a substantial information especially as Ngundu + CD27+ wet al. who followed-up a birth cohort in Kenya and found no age-linked differences in anti-malaria IgG levels between not exposed, exposed-and-not-sensitized and sensitized groups of childrenet al. showed that antimalarial antibody responses decreased during the first year of an infant born to a mother with placental malaria at deliveryet al. also observed that tolerant (exposed-and-not-sensitized) children had impaired functional antibody responses to MSP1-19In the present study, the 6–18 months infants’ levels of antimalarial antibodies were not related to placental malaria. These results are consistent with those of Malhotra P. falciparum IgG, IgG1 and IgG3 levels during the follow-up were not associated with placental malaria. This result means that the infants born with high levels of transplacental transferred maternal antibodies to malaria produced high levels of IgG specific for malaria. If we consider the hypothesis according to which antibodies are markers of exposure, the higher malaria infections would occur in infants the higher the malaria antibody levels would be. It was previously demonstrated in this cohort that infants born to mothers with placental malaria had a higher risk of malaria infection in their 18 first months of ageCord blood malaria antibody levels were positively associated with et al. found no association between antibody levels to MSP1 and MSP2 and nutritional statusNutritional intakes had no effect on malaria specific antibody levels. Genton Sickle-cell trait was not significantly associated with antibody evolution in the univariate or multivariate analyses.In the present work, antibody responses were analyzed independently. The profiles of malaria antibody concentrations in infant are similar over the time as those expressed in arbitrary units or in OD values in other studies2627Anopheles bites). Such factors have to be considered simultaneously in follow-ups of cohorts aimed at elucidating the specific humoral response mainly in the context of vaccine trials.At last, this study demonstrates the huge importance to give to key factors conditioning the antibody response in early life The study took place in Southern Benin where ® EDTA (Ethylene diaminetetraacetic acid) tubes.Briefly, at delivery, (i) a questionnaire collecting information on maternal age, parity, use of Intermittent Preventive Treatment during pregnancy (IPTp) and bed net possession was administered and (ii) placental blood smears were performed and maternal peripheral as well as cord bloods were collected into VacutainerEvery child was visited weekly. In case of fever (axillary temperature ≥37.5 °C) and/or a reported 48-hours-fever episode, a questionnaire and both a rapid diagnostic test (RDT) and a thick blood smear (TBS) were performed. Symptomatic malaria infection (fever and positive TBS and/or RDT) was treated with artemether and lumefantrine combination therapy as recommended by the National Malaria Control Program. Every month, a systematic TBS to detect asymptomatic malaria infection and a qualitative dietary questionnairewww.amanet-trust.org]) - were performed to assess malaria antibody concentrations by ELISA39ELISA was performed as described previouslySupplementary material3334353637Standard curves were established using human IgG, IgG1 and IgG3 purified proteins  to determine the concentration of specific antibodies. Each point was tested in duplicate.http://www.malariaresearch.eu/content/software) was used to analyze automatically the ELISA optical density (OD) leading to antibody concentrations. Discordant duplicates (with a variation coefficient >15%) were technically repeated.The ADAMSEL FLP b039 software (OD that were below detection threshold or over saturation were referred as “Low” and “High” concentrations (μg/mL) respectively. These thresholds depend on standard concentrations, normalization procedures and are therefore specific to each ELISA plate. These censored values were imputed using a (log) linear regression model taking into account confounding variables. The model was fitted using a stochastic expectation maximization algorithmThe outcome variable was time-dependent . Antibody levels were firstly adjusted on the nuisance variables using a linear regression for: health center, prematurity, parity, use of IPTp, bed net possession and maternal age in order to focus on the effects of variables of interest (detailed in “Independent variables” section). The residuals of these adjustments - referred as “adjusted antibody level” - were then used as outcome variable.Birth and 3-month infant samples are strongly represented by maternal antibodies that could interfere with the evaluation of acquired IgG of neonatesP. falciparum in thick placental smears.Placental malaria infection was defined by the presence of The number of malaria attacks between two successive antibody measurements was used either as a quantitative or a qualitative variable. Malaria infections could be feverish or not.The effect of an infection on the antibody levels was tested by two ways: a prospective effect and a boosting effect of a second infection. To evaluate the prospective effect, infections occurring in the 3-month period before the antibody measurements were considered to be associated with antibody levels. To evaluate the boosting of the antibody response in children being infected in the 3-month period before the antibody measurement, we considered whether they were also infected in the 3-to-6 month period (2 consecutive infections: one occurring in the 3-to-6 month period and one in the 3-month period before the antibody measurements).Individual 24-Hour Dietary Recall questionnaires - based on child feeding practicesDdeI enzyme into 3 fragments when normal and into 2 fragments when mutatedGenomic DNA was extracted from buffy coat using QIAamp DNA blood midi kit . The amplified beta globin DNA was then cleaved by the The environmental risk of exposure to malaria was modeled for each child based on climatic, entomological, and characteristics of the children immediate surroundingsDuring the study period, antibodies levels were determined quarterly, and the child’s measurements cannot be considered independent. Therefore, child’s antibody levels presented a two-level hierarchy: antibody measurements (level 1) were clustered within children (level 2)The effect of age on the acquisition of natural antibodies was tested using polynomial models. The best fitted model (Bayesian Information Criterion) was used for further analyses.P ≤ 0.20 in the univariate step. Placental malaria was systematically included in multivariate analyses. An interaction between age and infection was tested in the model. All statistical analyses were performed using Stata, version 11.0 . Statistical significance was set at P < 0.05.Multivariate analysis was performed including the variables with The study protocol was approved by the University of Abomey-Calavi’s institutional review board and the IRD’s Consultative Ethics Committee. All women in this study signed an informed consent before enrollment  with the possibility to withdraw at any time. All the methods were carried out in accordance with the approved guidelines.How to cite this article: Dechavanne, C. et al. Acquisition of natural humoral immunity to P. falciparum in early life in Benin: impact of clinical, environmental and host factors. Sci. Rep. 6, 33961; doi: 10.1038/srep33961 (2016).


Malaria continues to be a great public health concern due to the significant mortality and morbidity associated with the disease especially in developing countries. Microparticles (MPs), also called plasma membrane derived extracellular vesicles (PMEVs) are subcellular structures that are generated when they bud off the plasma membrane. They can be found in healthy individuals but the numbers tend to increase in pathological conditions including malaria. Although, various studies have been carried out on the protein content of specific cellular derived MPs, there seems to be paucity of information on the protein content of circulating MPs in malaria and their association with the various signs and symptoms of the disease. The aim of this study was therefore to carry out proteomic analyses of MPs isolated from malaria positive samples and compare them with proteins of MPs from malaria parasite culture supernatant and healthy controls in order to ascertain the role of MPs in malaria infection.r.Plasma samples were obtained from forty-three (43) malaria diagnosed patients (cases) and ten (10) healthy individuals (controls). Malaria parasite culture supernatant was obtained from our laboratory and MPs were isolated from them and confirmed using flow cytometry. 2D LC-MS was done to obtain their protein content. Resultant data were analyzed using SPSS Ver. 21.0 statistical software, Kruskal Wallis test and Spearman’s correlation coefficient In all, 1806 proteins were isolated from the samples. The MPs from malaria positive samples recorded 1729 proteins, those from culture supernatant were 333 while the control samples recorded 234 proteins. The mean number of proteins in MPs of malaria positive samples was significantly higher than that in the control samples. Significantly, higher quantities of haemoglobin subunits were seen in MPs from malaria samples and culture supernatant compared to control samples.P.falciparum infection. Also while some proteins were upregulated in circulating MPs in malaria samples, others were down regulated.A great number of proteins were observed to be carried in the microparticles (MPs) from malaria samples and culture supernatant compared to controls. The greater loss of haemoglobin from erythrocytes via MPs from malaria patients could serve as the initiation and progression of anaemia in  Microparticles (MPs) also called plasma membrane derived extracellular vesicles (PMEVs) are a heterogeneous group of small sub-membrane fragments or membrane coated vesicles shed from the plasma membrane of various cells during normal cellular activities like growth, senescence, proliferation and apoptosis . MPs carPlasmodium which is a unicellular protozoan parasite. The disease is a major cause of mortality and morbidity in many developing countries especially in Sub-Saharan Africa. It is estimated 3.4 billion people worldwide risk being infected with malaria in 104 countries [Malaria is caused by five species of the genus Plasmodium falciparum is considered to be associated with the dysregulation of the coagulation system which include endothelial damage, lower levels of anticoagulation and the release of pro-coagulant MPs [The degree of anaemia experienced in malaria does not always correspond to the parasitaemia level . This islant MPs .To this end, malaria has been associated with an increase in the level of circulating plasma MPs and plasma concentrations of endothelial MPs (EMPs) which may be proportional to disease severity , 8. The Existing literature indicate that elevated MPs levels have been seen in cancer, sepsis, pulmonary hypertension, idiopathic thrombocytopenic purpura and atherosclerosis . ResearcRegev-Rudzki et al. (2013) postulated that MPs released in malaria are also capable of activating the blood–brain barrier which exacerbates inflammation . The perProteomic analysis on circulating plasma MPs obtained from plasma of malaria positive blood samples once explored will give a general idea of the protein and protein groups borne by these MPs thereby influencing the pathophysiology of malaria infection. This study seeks to examine the protein composition of plasma MPs of malaria samples in order to explore their effect on the pathogenesis of malaria and the possible linkage of circulating plasma MPs to the anaemia.The aim of this study was to carry out proteomic analyses of MPs isolated from malaria positive samples and compare them with proteins of MPs from healthy controls in order to ascertain the role of MPs in malaria infection.The study was a cross-sectional study conducted over a 2-year period, from May 2014 to June, 2016. The samples were collected at the Sunyani Regional Hospital, Ghana and all the laboratory work-up were carried out at the Department of Molecular Medicine, Kwame Nkrumah University of Science and Technology (KNUST), Noguchi Memorial Institute for Medical Research (NMIMR) and the Proteomic and Flow cytometry Core Facilities of the Indiana University School of Medicine.In all, fifty three (53) participants who were out-patients were recruited into the study. There were 43  cases and 10  controls. The age range for controls was 28–47 years while the age range for patients was 1 week to 62 years.Convenience sampling was employed for this study. Three milliliters (3 ml) of blood was collected from 43 laboratory diagnosed malaria patients. The parasite density was confirmed using independently prepared thick film. Control samples were obtained from 10 apparently healthy individuals. The samples were categorized into three (3): mild, moderate and high based on the level of parasitaemia.Briefly, 6 microlitres (6 μl) of blood was placed in the middle of a glass slide for a thick film preparation using the WHO standardized template. The slides were then air-dried and subsequently stained as below.The films were stained with 1:10 diluted Paskem® Giemsa for 10 min, air dried and examined using the Olympus ®CX 31 microscope . Parasite count was calculated using the formulaWhere 6000 is the assumed WBC count per 1 μl of blood.Briefly, EDTA-anticoagulated blood (3 mL) was centrifuged at 160 × g for 5 min, and plasma was stored at −20 °C until further processing. During processing, samples were thawed and centrifuged at 4000 x g for 60 min to obtain platelet-free plasma. The resultant supernatant was then spun for 120 min at 19,000 × g to obtain the microparticle pellet after the supernatant had been discarded . The MP TM flow cytometer. MPs isolated from plasma were gated (Annexin V+) based on their forward (FSC) and side (SSC) scatter distribution as compared to the distribution of synthetic 0.7–0.9 μm SPHERO™ Amino Fluorescent Particles   was added to the thawed MP pellet. Equal volume of Annexin V binding buffer was added to label the MPs. Labeled plasma samples were analyzed on a BD FACSAriawww.uniprot.org [http://string-db.org/cgi/.One hundred microlitres (100 μl) of frozen MPs fractions were thawed and resuspended in 60% methanol and 0.1 M ammonium bicarbonate pH 8.0. Incubation with diothiothreitol followed by cysteine alkylation with iodoacetamide reduced the proteins. The solution was incubated overnight with mass spectrometry grade trypsin  at 37 °C to digest the proteins. The obtained tryptic peptides underwent desalting using reversed phase cartridge by washing in 0.5% acetic acid. The peptides were then eluted with 95% acetonitrile and 0.5% acetic acid. The eluted peptides were lyophilized and re-suspended in 0.5% acetic acid and directly analyzed using 2-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS). The samples were loaded onto a microcapillary strong cation exchange column and fractions were collected using an increasing salt step elution gradient. Afterwards, each fraction was analyzed using the LTQ ion trap mass spectrometer . The acquired MS/MS spectra were searched against protein database available at prot.org . The proExperimental Procedures: MPs were thawed and lyzed in sample buffer and were loaded on SDS-PAGE gels. The presence of the identified proteins in the samples was analyzed by Western blot. Bands at the predicted molecular weight for the proteins were observed .All experiments  were repeated 2–4 times.r. A p-value < 0.05 was considered to be statistically significant. For illustration purposes the samples were categorized into 3 sub-groups according to the parasitaemia based on the old system of classification  to analyze their association with proteins released in MPs.Proteins identified were analyzed under molecular function, biological processes, subcellular location and cellular component using the uniprot database. Resultant data were further analyzed using SPSS 21.0 statistical software. Differences between the medians of the various groups were analyzed by Kruska Willis test. Pairwise correlations were evaluated with Spearman’s correlation coefficient The characteristics of subjects of this study are presented in Table falciparum. This phenomenon was seen in our study where all the patients tested had P. falciparum malaria. Subsequently the MPs types seen also carried P. falciparum markers as well as those of other activated cells .The predominant plasmodium specie found in Ghana is The red blood cell indices showed significant variation between the patients and the control. For example there was significance in haemoglobin concentration, MCV, MCH and RDW as seen in Table A total of 1729 proteins were identified in the malaria positive samples. The culture supernatant (CSN) showed 333 proteins while a total of 234 proteins were identified in the control samples. Seventy-two (72) proteins were common to the malaria positive samples, CSN and the control. The details are shown in Fig. t-test showed significantly higher number of proteins released in MPs from malaria samples. Twenty-nine P. falciparum proteins were identified in at least one malaria positive sample  RAB proteins were identified in MPs from malaria samples exclusively without any being found in the control samples as presented in Table Some of the proteins identified showed strong link to inflammation. Inflammatory proteins seen in malaria MPs but not control sample MPs include Heat shock protein (HSP) 90-alpha, HSP 90-beta, 60 kDa HSP mitochondrial, 70 kDa HSP, HSP beta-1, Transforming growth factor beta-1 induced transcript 1 protein, Macrophage migration inhibitory factor and Transforming growth factor beta-1. Again, inflammatory proteins released in malaria MPs but in reduced quantities included: Heat shock cognate 71 kDa protein, Heat shock 70 kDa protein 1A/1B, Heat shock 70 kDa protein 6, Heat shock 70 kDa protein 1-like.A good number of complement associated proteins were seen in the MPs from the malaria samples. It was observed that the levels of Complement C3 and Complement C4-B, Complement C5, Complement C9, Complement factor B and Complement C1q subcomponent subunit C were significantly elevated in malaria plasma MPs compared to controls  and Mannose-binding lectin serine protease 2  while it positively correlated with Complement C3 . Parasite count correlated negatively with Complement C3  and correlated positively with Complement C1s subcomponent  and C4b-binding protein beta chain .Tables p = 0.037). This finding agrees with a study in the Brazilian Amazon in which out of a total of 7831 people studied, individuals with malaria were seen to have the lowest Hb compared to community controls and malaria-negative febrile patients [P. falciparum for instance is capable of digesting more than 80% of the erythrocyte haemoglobin [r = −0.415, p < 0.001) indicating that the greater the parasite density in a patient the lower the Hb, as was also reported in a study in Thailand [The study identified an array of proteins from the MPs isolated from the malaria positive samples, culture supernatant and healthy controls. The mean haemoglobin (Hb) of the studied subjects was significantly lower than that of the control group  of the patients  to consume haemoglobin during its growth in the erythrocyte [The mean cell volume (MCV) of the patients was significantly lower than that of the control group (Table P. vivax . Therefothrocyte  and the throcyte .p = 0.032) in the patients compared to the control group. It was however consistent with results from a P. vivax malaria study [P.vivax malaria however, the elevated RDW results from the initial size increase of parasitized erythrocytes which is followed by erythrocyte rapture. The same mechanism however cannot be said to be associated with RDW increase in P. falciparum infection since parasitized cells maintain their sizes [The red cell distribution width (RDW) was significantly higher  which is also called precursor to major merozoite surface antigens (PMMSA) or merozoite surface antigen 1 (MSA-1) was identified in MPs from 2 of the malaria samples [Plasmodium falciparum. MSP-1 is well conserved among P. falciparum isolates and hence it was identified in the MP isolated from the culture supernatant [Plasmodium falciparum [ samples . This prernatant . This prlciparum .P. falciparum was identified [Plasmodium falciparum Apical membrane antigen 1 (PfAMA1) which is synthesized during schizogony and transported to micronemes. PfAMA1 translocates onto the merozoite surface prior to erythrocyte invasion when it serves as an adhesion molecule playing a central role in the invasion process [Also, merozoite surface antigen 2 (MSP2) which is a surface coat protein essential for the survival of the blood-stages of entified . This pr process . Ring-in process .P. falciparum actin 1(PfACT1) was expressed in the MPs of 35 samples. PfACT1 is a ubiquitously expressed protein and it is expressed throughout the lifecycle of the Plasmodium. However, its isoform PfACT 2 which is expressed only in the sexual stages of the parasite was not isolated in any of the samples [Glutamic acid-rich protein (GLURP) found in one sample is a molecule located on the surface of merozoites and also in the hepatic stage . This pr samples .P. falciparum has six Hsp70s [Plasmodium [Plasmodium [Heat shock 70 kDa protein (Hsp70) was expressed in 35 samples. x Hsp70s . Hsp70 px Hsp70s  and thisasmodium . It is kasmodium .Theileria found in cattle, species of Plasmodium are the only organisms that the gene family translating into Serine-repeat antigen (SERA) protein has been found [P. falciparum development [Precursors of serine-repeat antigen (SERA) proteins are synthesized in the late trophozoites . Among oen found . This prelopment .Plasmodium falciparum ADP-ribosylation factor 1 (PfARF1) which is activated after binding to GTP. In the secretary pathway, PfARF1 regulates vesicular biogenesis and trafficking processes [P. falciparum S-antigen proteins were found in the MPs of 2 samples. They are heat-stable antigens that are serologically diverse among varied isolates of the parasite [Also identified was rocesses . It is arocesses . Hypotheparasite .r = 0.457). It is known that MPs are able to bind to complement component C1q and cause the C3 to be fixed on the MPs after exposure to normal human serum through the classical pathway. These complement-fixed MPs are capable of binding to erythrocytes and remove them from circulation [There was significant difference in the levels of the following complement system proteins released in plasma circulating MPs between the malaria samples and that of control: complement component C6, complement C4-B, complement component C9, complement factor B and Complement C1q subcomponent subunit C. The difference between the patients and the control group in complement component C3 was however not significant but correlation analysis indicated a significant negative correlation between C3 released in MPs and parasite count in the malaria samples. There was significant positive correlation between C3 and haemoglobin  of the interaction of the


Malaria antibody responses measured at delivery have been associated with protection from maternal anaemia and low birth weight deliveries. Whether malarial antibodies present in the first half of pregnancy may protect from these or other poor birth outcomes is unclear. To determine whether malaria antibodies in the first half of pregnancy predict pregnancy outcomes, antibodies were measured to a range of merozoite antigens and to antigens expressed on the surface of parasitized red blood cells (pRBCs) in plasma samples collected at 14–20 weeks of gestation from Malawian women. The latter antibodies were measured as total IgG to pRBCs, and antibodies promoting opsonic phagocytosis of pRBCs. Associations between antibodies and maternal haemoglobin in late pregnancy or newborn size were investigated, after adjusting for potential covariates.Antibodies to pRBC surface antigens were associated with higher haemoglobin concentration at 36 weeks. Total IgG to pRBCs was associated with 0.4 g/l  increase in haemoglobin, and opsonizing antibody with 0.5  increase in haemoglobin for each 10% increase in antibody. These antibodies were not associated with birthweight, placental malaria, or newborn anthropometrics. Antibodies to merozoite antigens and non-placental-binding IEs were not associated with decreased risk of any of these outcomes. In some instances, they were negatively associated with outcomes of interest.Antibodies to placental-binding infected erythrocytes may be associated with higher haemoglobin levels in pregnancy, whereas antibodies to other malaria antigens may instead be markers of malaria exposure.Trial registration clinicaltrials.gov NCT01239693. Registered Nov 10, 2010. Plasmodium falciparum. Many of the associated deaths are due to severe maternal anaemia and low birth weight (LBW) [P. falciparum infections generally peak between 13 and 18 gestation weeks, which is when parasitized red blood cells (pRBCs) begin to sequester in the placenta [Malaria in pregnancy accounts for an estimated 10,000 maternal and 200,000 infant deaths per year worldwide with over 25 million pregnancies at risk of infection in sub-Saharan Africa alone [placenta . Placentplacenta . With replacenta , 8, thatplacenta , 10. By placenta –13.Women who are parasitaemic at first antenatal care visit may have an increased risk of adverse outcomes such as LBW, placental infection, preterm birth and anaemia –17. It iAcquisition of antibodies is dependent on a number of factors such as maternal age, gravidity and presence of other infections such as HIV , 21, 22.Pregnant women residing in malaria-endemic regions often suffer from undernutrition as a result of poverty, seasonal variation in food availability and poor intake of micronutrients, which significantly impact their health and pregnancy outcomes. It was hypothesized that among Malawian women enrolled in a nutrient supplementation study, one or more measures of naturally acquired malaria antibody, taken at first antenatal visit, would be associated with protection from adverse pregnancy outcomes such as maternal anaemia, evidence of malaria on placental histology, or measures of fetal growth in utero.®). The trial design, details of the nutrient supplements, and main findings have been published elsewhere [From February 2011 to August 2012 one thousand three hundred and ninety-one pregnant women were enrolled in the iLiNS-DYAD-M randomized controlled trial, which evaluated whether home fortification of pregnant women’s diets with nutrient supplements could increase birth size. Participants received either iron and folic acid supplements, a multiple micronutrient capsule, or 20 g of lipid-based nutrient supplementation each day from enrolment, from 14 to 20 completed weeks of gestation, until delivery. Dating ultrasounds were performed at enrolment to determine gestation at delivery. All participants received 2 doses of sulfadoxine-pyrimethamine , polymerase chain reaction (PCR), and placental histology at delivery. Placental malaria (PM) was categorized as active infection (presence of pRBCs with or without haemozoin in monocytes and/or fibrin); past infection (haemozoin without pRBCs); or no infection, based on placental histology. Due to the low numbers of active placental infections (n = 31), only past placental infection and no infection were compared.Ethical approval for the trial and the laboratory work was granted by the College of Medicine Research and Ethics Committee of Malawi, and by Tampere University Hospital Ethics Committee, Finland. The Melbourne Health Human Research Ethics Committee approved the laboratory work performed for the study.Plasmodium falciparum isolates CS2 and E8B were selected as representatives of placental-binding and non-placental-binding parasite isolates. CS2 binds to CSA expressed on the placenta while E8B binds to endothelial receptors CD36 and ICAM-1 [d ICAM-1 , 28. Pard ICAM-1 . Pre-mond ICAM-1 .Plasma samples from women who had samples available from study enrolment and at 36 gw were used to measure antibody levels. Flow cytometry based immunoassays were performed as previously described  to deterRecombinant merozoite antigens MSP1 19kD, MSP2, MSP3, PfRh2 (construct PfRh2-2030) (6088–7584 bp) and EBA175 (region III-V) were prepared as detailed . A fluorThe outcome variables of interest included maternal haemoglobin levels  at 36 gw; past placental malaria (PM) compared to no placental malaria; birthweight ; small for gestational age , stunti2), normal (18.5–25 kg/m2) and increased (>25 kg/m2). The SES was calculated based on a household assets adjusted z-score (HHAZ) system [The selected covariates included gravidity, maternal age, HIV, bed net use, body mass index (BMI) at enrolment, malaria microscopy at enrolment, socioeconomic status (SES), study site, season at enrolment and supplementation group, and were recorded as described . These c) system , where at test for normally distributed variables or Mann–Whitney U test for non-normally distributed variables, or test of proportion for categorical variables.Statistical analyses were performed using STATA 13.0 version . Differences in the characteristics between the participants included and excluded in the study were determined by performing a Student’s Univariate and multivariate linear or logistic regression analysis were performed as appropriate to assess the relationship between antibody levels at enrolment and pregnancy outcomes adjusting for the covariates described above. Antibody levels were treated as continuous variables or as categorical variables following categorization into low, middle and high antibody tertiles.Following exclusion of twins, antibody determination was performed for plasma samples collected from 1002 participants at enrolment who also had a sample at 36 gw. Among participants with samples at 36 gw, only 2.8% had preterm births.Socio-demographic characteristics and birth outcome differences between iLiNS study participants who were included and excluded from the present analyses were determined Table . Women iMedian haemoglobin levels at 36 gw were 110 g/l  had active placental malaria, the analysis compared only past PM (present in 36%) to no PM. In adjusted analyses, pregnant women in the high antibody tertile for MSP2 were more likely to have past PM than those in the low antibody tertile, OR 1.83  , preventing an investigation of whether antibody protects against preterm delivery; measurement of antibody responses to single parasite isolates of each phenotype, and single variants of each of a modest number of merozoite proteins; and measurement of a single functional role of antibodies (opsonization of pRBC for phagocytosis). Apart from preterm delivery and associated lower birth weights, other characteristics more common in excluded women included being primigravid, and higher rates of malaria infection by PCR at enrolment. This may have limited our power to find protective associations. Quantitating antibody to other placental-binding parasite isolates,  and undeThe study findings suggest that antibody to pregnancy-specific parasite isolates, measured using two different approaches, may be associated with protection from malarial anaemia, whereas antibodies to merozoite-specific antigens are not. Further studies that prospectively evaluate pregnancy-specific and more general measures of malaria antibody, under a range of transmission settings, will be required to determine whether one or more measures may identify women at risk with sufficient sensitivity and specificity to be considered as a screening tool for high-risk pregnancies in the future. The lack of evidence that naturally-acquired antibodies to one placental binding parasite line were associated with improved fetal growth may reflect the relatively modest impact of malaria on birth weight in this cohort. Demonstration that induction of antibodies by a VAR2CSA based vaccine improves pregnancy outcomes would require careful selection of target populations in which malaria is a major contributor to adverse pregnancy outcomes.


Small dense low density lipoprotein-cholesterol (sdLDL-C), cholesterol ratios and carotid-femoral pulse wave velocity (cf-PWV) impart risk for all-cause morbidity and mortality independently of conventional cardiovascular disease (CVD) risk factors. This study was designed to identify feasible indicators for predicting arterial stiffness progression.We followed up 816 normotensive participants without diabetes or CVD for nearly 5.0 years. Cholesterol parameters, ratios and other clinical and laboratory data were collected at baseline. cf-PWV were measured at baseline and the end of follow-up.p < 0.001) and △PWV . TG/HDL-C was only one cholesterol ratios that associated with all PWV parameters. sdLDL-C  and TG/HDL-C  could significantly determine the progression of PWV after correction for covariates. High sd-LDL-C quantiles subjects were more likely to develop arterial stiffness progression than low quantiles .PWV progression subjects had higher levels of PWV parameters, sdLDL-C and TG/HDL-C ratio. sdLDL-C and TG/HDL-C were significantly correlated with all PWV parameters. Multiple regression models showed that sdLDL-C was closely associated with follow-up PWV (β = 0.222, We founded that sdLDL-C and TG/HDL-C ratio can independently predict arterial stiffness progression in normotensive subjects, and high level sdLDL-C and TG/HDL-C ratio were associated with a higher risk of arterial stiffness. Arterial stiffness is aggravated with age, which is extensively associated with atherosclerotic vascular diseases. There are several ways of determining arterial stiffness, among which carotid-femoral pulse wave velocity (cf-PWV) is currently considered the gold standard . cf-PWV Several variables affect arterial stiffness, including age gender, blood pressure, high heart rate and life style, a variety of cardiovascular risk factors as well as genetic and systemic inflammation –8.Blood This study aimed to explore the relationship between sdLDL-C, cholesterol ratios and arterial stiffness progression which measured by the gold standard of cf-PWV. We followed up a normotensive population to identify an optimal indicator for predicting early arterial stiffness progression.From September 2010 through March 2011, 1148 consecutive normotensive subjects without known cardiovascular disease according to WHO guidelines were selected for study. Subjects were invited to participate in a study for evaluation of cf-PWV. The inclusion criteria included normal blood pressure (systolic blood pressure [SBP] < 140 and diastolic blood pressure [DBP] < 90 mm Hg) and age of 18 years or higher. Patients with the following conditions were excluded: history of heart failure or cardiomyopathy, coronary heart disease, resting electrocardiogram (ECG) abnormalities (Q waves or left bundle branch block), cerebrovascular or peripheral artery disease and diabetes. Five years later, the subjects were invited to undergo the same arterial examinations. Overall, 816 subjects (71% of those invited) had their second visit from October 2015 through April 2016. Among the 332 subjects who did not have follow-up measurements, 198 lost contacts, 54 refused, and 20 did not return for unknown reasons. All participants provided written informed consent at the first visit.In the present study, family income was classified as < 30,000, 30,000-100,000, and > 100,000 CNY/y. Educational level was categorized as low , middle (3 or 4 years of secondary education), and high (college and university education). Physical activity was classified into three groups using methods presented elsewhere . We defiBlood samples were collected in the morning after overnight fasting for at least 12 h. Blood was drawn into plastic tubes containing EDTA-2Na, and chilled in ice. Plasma was obtained by centrifugation at 4 °C, and kept at 0–4 °C until assays. All measurements were performed within 6 h. Fasting blood glucose(FBG), liver enzyme levels, serum lipid profiles, including TG, TC, LDL-C)and HDL-C were determined on a Olympus AU600 analyzer . sdLDL-C was measured by the method of Hirano et al.  with minSupine blood pressure was assessed using a manual sphygmomanometer. After a 10 min rest period, blood pressure was measured 3 times and the average measurements used for statistical analyses.cf-PWV measurements were obtained under the same conditions, including constant room temperature of 19 °C to 21 °C. We determined cf-PWV using a SphygmoCor automatic device . SphygmoCor offers the possibility of cf-PWV measurements in two steps [p values < 0.05 were considered statistically significant.The statistical software package SPSS18.0  was employed. Continuous variables in normal distribution were compared using Student’s t-test and analysis of variance (ANOVA). Categorical variables were analyzed using the chi-squared test. Patients were divided into 2 groups according to their gender and hypertension or cf-PWV progression. In this study, we defined cf-PWV > 12 m/s as a clinically significant outlier . Subjectp < 0.001). At the end of the follow-up, there were a total of 144 patients with cf-PWV progression, with an average of 12.70 ± 0.64 m/s.A total of 816 consecutive subjects that met the inclusion criteria were enrolled and completed follow-up. The participant population included 476 (58.3%) men, and the average age of participants was 39.1 ± 7.8 years. Baseline blood pressure parameters were as follows: SBP (125.9 ± 9.4 mmHg), DBP (75.2 ± 7.1 mmHg), pulse pressure (PP) (50.7 ± 9.5 mmHg) and MAP (92.1 ± 6.5 mm Hg). Baseline mean cf-PWV was 9.90 ± 0.84 m/s and follow-up was 10.51 ± 1.12 m/s. The annualized average progression of cf-PWV was 0.12 ± 0.08 m/s/year. After 5 years of follow-up, 129 normal blood pressure subjects developed hypertension. Among them, grade Ihypertensive patients were 82, grade II were 38 and grade III were 9. In addition, 60 subjects used drugs to control blood pressure. At the end of follow-up, the cf-PWV of hypertensions were significantly higher than normal subjects , creatinine , sdLDL-C , sdLDL-C/HDL-C ratio , baseline PWV , follow-up PWV , △PWV  and hs-CRP than females ,BMI , blood urea nitrogen . Homoplastically, sdLDL-C/HDL-C had a similar statistical result to sd LDL-C, accordingly for baseline PWV , follow-up PWV  and △PWV . TG/HDL-C was only one cholesterol ratios that associated with all PWV parameters in the multiple regression models.Then, the associations of sdLDL-C and cholesterol ratios with the PWV parameters were adjusted for covariates using multiple regression models. As shown in Table Further analyses were performed to examine sdLDL-C and cholesterol ratios that were associated with PWV progression. At the end of an average of 5 years of follow-up, 144 subjects had PWV progression. We used the Cox regression model to analyze the predictive value of these cholesterol ratios and sdLDL-C for cf-PWV progression. After adjusted the covariates including gender, age, exercise, smoking, drinking, BMI, SBP, MAP, FBG, UA, baseline PWV, TNF-α, hs-CPR and IL-6, in our study population, sdLDL-C and TG/HDL-C were able to independently predict PWV progression after 5 years (Table p = 0.03). Meanwhile, the risk of high TG/HDL-C ratio quantiles was significantly higher than low quantiles . However, there was no significant difference between the sdLDL-C and TG/HDL-C ratio median quantiles and low quantiles subjects with arterial stiffness risk . Meanwhile, TG/HDL-C ratio and sdLDL-C also showed a significant positive correlation. Interestingly, TG/HDL-C was associated with baseline PWV, whereas sdLDL-C and sdLDL-C/HDL-C were not correlated with it.In our normal blood pressure population, high TG/HDL-C ratio subjects have a higher risk of arterial stiffness. This may be associated with the fact that TG/HDL-C can better predict sdLDL-C, which is a kind of atherosclerotic lipid particle that can strongly predict CVD . Under tPreviously, a limited number of studies have addressed the relation between ApoB and arterial PWV. In relatively small study populations, using multiple linear logistic regression models found that ApoB and PWV had independent positive associations after adjusting for cardiovascular risk factors . In addiMany studies have suggested that arteriosclerosis was an inflammatory reaction secondary to vascular injury, and inflammatory factors have a sustained effect on whole process of arteriosclerosis. In healthy individuals, acute systemic inflammation increases arterial stiffness and decreases wave reflections . Azra MaThis study has several limitations. Dyslipidemia in the Chinese population is mainly type IV hyperlipidemia, characterized by hypertriglyceridemia and low HDL-C, which is closely related to the carbohydrate-based diet in the general Chinese population . AdditioIn conclusion, our study showed that sdLDL-C and TG/HDL-C ratio could independently predict arterial stiffness progression after adjusting for other CVD risk factors. Subjects with a high sdLDL-C level and TG/HDL-C ratio had a higher risk of arterial stiffness. However, it remains to be confirmed whether these conclusions can be applied to other populations.


In the Lao PDR, malaria morbidity and mortality have remarkably decreased over the past decade. However, asymptomatic infections in rural villages contribute to the on-going local transmission. The primary objective of this study was to explore the characteristics of infections in a malaria-endemic district of the Lao PDR. The specific objectives were to investigate the prevalence and species of malaria parasites using molecular methods and to assess individual and household parasite levels and the characteristics associated with malaria infection.The study population included 870 participants from 236 households in 10 villages of the Xepon district. Interviews, blood examinations and body temperature measurements were conducted between August and September 2013. A multilevel logistic regression model, with adjustment for clustering effects, was used to assess the association between predictor variables and an outcome variable . The predictive factors included individual-level factors  and household-level factors .Plasmodium falciparum mono-infection was the most common infection , followed by P. falciparum and Plasmodium vivax mixed infections . The majority of infected participants  had no fever episodes in the two previous weeks or a measurable fever (>37 °C) at the time of survey. Living in a household with one other malaria-infected member significantly increased the odds of infection . Among the 40 households that had at least one infected member, nine households were responsible for 40.4 % of the total infections.Fifty-two participants (including 26 children) tested positive (positive rate: 6.0 %): Plasmodium vivax was detected more frequently than it was reported from the district hospital. Most infections were asymptomatic and sub-microscopic and were highly clustered within households. To further eliminate malaria in Xepon and other similar settings in the country, the National Malaria Control Programme should consider household-based strategies, including reactive case detection targeting the household members of index cases.The online version of this article (doi:10.1186/s12936-016-1552-7) contains supplementary material, which is available to authorized users. Plasmodium falciparum has long been the predominant reported infection, but the incidence Plasmodium vivax has been increasing in recent years [The Lao People’s Democratic Republic (Lao PDR) is a lower-middle-income country in Southeast Asia, bordering Thailand, Vietnam, Cambodia, China, and Myanmar. The population of the Lao PDR was 6.5 million in 2015, and 63 % of the population lives in rural areas [nt years . The gront years , 8.Since 1992, the Lao PDR has implemented a nationwide malaria control programme . The curAs a result of the above-mentioned control efforts, there have been remarkable decreases in the rates of mortality and morbidity due to malaria over the past decade. The Lao PDR achieved a >75 % decrease in the incidence of malaria between 2000 and 2015. However, asymptomatic infections continue to be detected in rural, remote villages , 10, whiArtemisinin resistance has been increasingly reported from many areas in the Greater Mekong Sub-region  , posing P. falciparum mono-infection, 10 cases of P. vivax mono-infection, and two cases of co-infections. In 2012, 218 cases were reported, including 214 cases of P. falciparum mono-infection and four cases of P. vivax mono-infection. Xepon district was deliberately selected because reliable demographic data were available on households in the 20 villages in which the Health and Demographic Surveillance System (HDSS) was in place. The HDSS has been described elsewhere [Approximately 90 % of all malaria cases in the Lao PDR occur in the southernmost provinces including Savannakhet province . Of the The primary objective of this study was to explore the characteristics of individuals with malaria infections in a rural district of the Lao PDR. The specific objectives were (1) to investigate the prevalence and species of malaria parasites in the study community using molecular methods, and (2) to assess the individual- and household-level characteristics that are associated with malaria infection.This study was conducted in Xepon district, Savannakhet province, Lao PDR between August and September of 2013. From the 20 villages where the HDSS was in place, 10 villages were deliberately selected as the study site based on the geographical characteristics of these villages: two hillside villages, five roadside villages, and three riverside villages were selected . Finally, the surveyors collected a finger-prick blood sample from the participants that was subjected to RDT . Blood samples were also collected for microscopic examination and PCR. For PCR, blood samples were collected on Whatman FTA Classic Cards .Experienced medical technologists prepared Giemsa-stained thick and thin blood smears and performed the microscopic examinations.cytochrome b gene in the mitochondrial genome of Plasmodium parasites [Plasmodium species identification was performed with species-specific primer sets targeting the same gene by a nested PCR with DNA samples that were identified as being Plasmodium parasite-positive in the screening. SaoAdvanced Universal SYBR Green Supermix  was used for the screening PCR with 2 µl of the extracted DNA and the primer set  was extracted from dried blood spots on the FTA Classic Cards with a QIAamp DNA Mini Kit  in accordance with the manufacturer’s instructions. The extracted DNA was eluted with 50 µl of elution buffer in the kit and preserved at 4 °C until the PCR. The PCR was performed in two steps. First, screening by a real-time PCR was performed with genus-specific primers targeting the arasites . Second,P. falciparum, P. vivax, Plasmodium malariae, and Plasmodium ovale were used. The primary PCR for the identification was conducted by the real-time PCR. The secondary PCR for the identification was conducted using a conventional PCR with the first PCR product diluted 1000 times with Go-Taq DNA Polymerase . The PCR products were visualized by 2 % agarose gel electrophoresis with Gel Red  staining by UV transillumination. All of the DNA extraction procedures and PCRs were conducted at the National Center for Global Health and Medicine, Japan.For the PCR for species identification, primer sets for The primary outcome measure in this study was the malaria infection status principally determined by PCR. The secondary outcome measure was the malaria infection status determined by RDT and then confirmed principally by PCR, i.e., true positives detected by RDT. Malaria infection was defined by a positive test result with or without clinical symptoms. The individual-level predictive factors included age, gender, bed net use on the night before the survey, the experience of working in the forest at night during the previous month, and the experience of a fever during the two weeks prior to the survey. The age and gender variables were determined from the information in the HDSS database because the self-reported ages of the subjects at the study site might not have been reliable. Other variables were measured using a questionnaire-based interview. At the household level, the predictive factors included the number of household members, the person-per-net ratio (defined as the number of household members divided by the number of available bed nets in a household), and living in a household with one other malaria-infected member. These household-level factors were determined according to the information in the HDSS database and the results of a bed net observation survey that was conducted prior to the present study .p < 0.05 was considered statistically significant. All statistical analyses were performed with the STATA MP12 software program .For the analysis of associations between the outcomes and predictive factors, multi-level modelling was used to account for the hierarchical structure of the data; i.e., 870 individuals in 236 households. Thus, a two-level, mixed-effects logistic regression model was used for the multivariate analysis. For the bi-variate analysis, a Chi square test or Fisher’s exact test was used. For other statistical analyses, a Chi square test or Fisher’s exact test was used for a categorical variable and a t test was used for a discrete variable. A P. falciparum was the most common form of infection , followed by mixed infection with P. falciparum and P. vivax . RDT and microscopic examinations could only detect 36.7 % of the PCR-diagnosed infections.Fifty-two participants were confirmed to have malaria parasitaemia: 49 individuals were confirmed by PCR and the other three individuals, whose filter paper samples were not available, were determined by microscopic examination Table . The totApproximately one half of the participants (54.0 %) were female Table , and neaThe majority n = 42, 80.8 %) of the participants with positive test results did not have a fever episode in the two weeks before the survey or a measurable fever (>37 °C) at the time of the survey . The proportion of the population aged over 14 years of age was significantly lower in the participants than in the non-participants . There was also a difference in gender proportion between the participants and non-participants in the age subgroups: among the participants, males accounted for 53.7 % (36/67) in the youngest group, 49.7 % (195/392) in the second youngest group, and 40.6 % (167/411) in the oldest group. In contrast, among the non-participants, males accounted for 51.5 % (102/198) in the youngest group, 45.3 % (82/181) in the second youngest group, and 54.0 % (309/572) in the oldest group.There were significant differences in gender and age between the study participants and non-participants in the study villages Table . The proMost households 89.4 %) possessed at least one bed net (Table  % possesp = 0.235 by Fisher’s exact test). Among the nine households that had at least two infected members, the mean number of members (standard deviation) was 7.2 (2.6), whereas it was 6.7 (2.5) among the 31 households that had only one infected member. The difference (7.2 vs 6.7) was not statistically significant (p = 0.574 by t test).Among the 40 households with at least one infected member, nine households with at least two infected members were responsible for 40.4 % of the total infections   Table . No othe1) Table .Table 6Bp < 0.001 by Fisher’s exact test) (Table p = 0.084 by Fisher’s exact test). Consistent with the results of the bivariate analysis, the multivariate analysis showed that living in a household with at least one additional infected member was significantly associated with an increased risk of infection   Table . No otheP. falciparum mono-infection accounted for the majority of infections (78.8 %). However, the results of the present study also showed a difference in the proportion of P. falciparum and P. vivax infections in the patient records and the present study: P. vivax infections including co-infections with P. falciparum, which were rarely reported from the hospital in 2012 (1.8 %) and 2013 (7.9 %), accounted for 21.2 % of the infections that were detected in the present study. This suggests that the burden of vivax malaria is underestimated in the district.As expected from the patient records of the Xepon District Hospital, the present community-based study showed that Consistent with the findings of a previous study conducted in Savannakhet province , the preThe results of the present study indicated that infections were highly clustered at the household level and that infections were unevenly distributed across households. A considerable number of studies have reported household clustering in countries other than the Lao PDR –19. One For such reactive case detection to be implemented in Xepon district or other similar settings in the Lao PDR, RDT would be used as a screening tool. Although the present study showed that RDT detected only 36.7 % of the true positives confirmed by PCR in the study site and malaria elimination cannot be achieved by screening with the current generation of RDTs , the useAnopheles dirus mosquitoes, which are a main vector in the Lao PDR, were captured in indoor light traps. Although treatment seeking from the private sector is also high in Xepon district, in which approximately 44 % of the malaria cases were reported from the private sector in 2013/14, the private sector has been an integral part of the National Malaria Control Programme in the Lao PDR including Xepon district. Most importantly, there was a marked difference in the transmission intensity between the present study and the Cambodian study (positive rate by RDT: 2.4 vs 0.5 %).The Cambodian study  showed, A number of factors that were previously reported as risk factors from the Lao PDR and/or neighboring countries were not identified as risk factors in the present study site. Although not using a bed net was reported as a risk factor for malaria infection from Savannakhet province , almost A major limitation of this study is the low participation rate: only 47.8 % of the population in the study villages participated. As Table This study has two other limitations. First, because the data collection was confined to half of the population in the 10 villages from the HDSS area, the present sample was not representative of the population of Xepon district. Second, the survey took place between August and September (the rainy season) and, therefore, did not take seasonal malaria epidemiology into account or seasonal variation in the frequency of risk factors such as sleeping away from home, working in the forest at night, and not sleeping under a bed net. However, sleeping away from home is more frequently reported in the rainy season than in the dry season in the Lao PDR .P. falciparum infections were common but that infections involving P. vivax, which was rarely reported from the district healthcare facility, accounted for 21 % of the malaria infections. This suggested that the burden of P. vivax infection is underestimated. Most (63 %) of the infections were asymptomatic and sub-microscopic and were highly clustered within households. To eliminate malaria in Xepon district and other similar settings in the Lao PDR, the National Malaria Control Programme should consider household-based control strategies. The findings of the present study suggest the use of a reactive case detection strategy targeting the household members of index cases.The present community-based study showed that