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Background At the COVID-19 spring 2020 pandemic peak in Spain, prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCWs) from Hospital Clínic de Barcelona was 11.2%. Methods A follow-up survey 1 month later (April-May 2020) measured infection by rRT-PCR and IgM, IgA, and IgG to the receptor-binding domain of the spike protein by Luminex. Antibody kinetics, including IgG subclasses, was assessed until month 3. Results At month 1, the prevalence of infection measured by rRT-PCR and serology was 14.9% (84/565) and seroprevalence 14.5% (82/565). We found 25 (5%) new infections in 501 participants without previous evidence of infection. IgM, IgG, and IgA levels declined in 3 months (antibody decay rates 0.15 [95% CI, .11–.19], 0.66 [95% CI, .54–.82], and 0.12 [95% CI, .09–.16], respectively), and 68.33% of HCWs had seroreverted for IgM, 3.08% for IgG, and 24.29% for IgA. The most frequent subclass responses were IgG1 (highest levels) and IgG2, followed by IgG3, and only IgA1 but no IgA2 was detected. Conclusions Continuous and improved surveillance of SARS-CoV-2 infections in HCWs remains critical, particularly in high-risk groups. The observed fast decay of IgA and IgM levels has implications for seroprevalence studies using these isotypes., SARS-CoV-2 prevalence in 578 health care workers increased from 11.2% at the COVID-19 pandemic peak to 14.9% 1 month later. IgM, IgG, and IgA levels declined over 3 months of follow-up with 68.33%, 3.08%, and 24.29% of seroreversions, respectively.

Pregnant women constitute a promising sentinel group for continuous monitoring of malaria transmission. To identify antibody signatures of recent Plasmodium falciparum exposure during pregnancy, we dissected IgG responses against VAR2CSA, the parasite antigen that mediates placental sequestration. We used a multiplex peptide-based suspension array in 2,354 samples from pregnant women from Mozambique, Benin, Kenya, Gabon, Tanzania, and Spain. Two VAR2CSA peptides of limited polymorphism were immunogenic and targeted by IgG responses readily boosted during infection and with estimated half-lives of

Coinfection withPlasmodium falciparumand helminths may impact the immune response to these parasites since they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens fromP. falciparumand 11 antigens from helminths (Ascaris lumbricoides, hookworm,Trichuris trichiura,Strongyloides stercoralisandSchistosomaspp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined byP. falciparumand helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water and sanitation variables, individuals exposed/infected withP. falciparumand helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (p≤ 0.05). There was a positive association between exposure/infection toP. falciparumand exposure/infection to helminths or the number of helminth species, andvice versa(p≤ 0.001). In addition, children coexposed/coinfected tended (p= 0.062) to have higherP. falciparumparasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host.GRAPHICAL ABSTRACT

BackgroundGlobal malaria mortality estimates are hindered by the low reliability of the verbal autopsy (VA) and the clinical records, the most common sources of information used to estimate malaria-specific mortality. We aimed to determine the accuracy of these tools, as well as of the minimally invasive autopsy (MIA), a needle-based postmortem sampling method, to identify malaria-specific mortality in a large series of deceased patients from Mozambique, using complete autopsy as the gold standard.MethodsObservational study that included 264 deaths, occurring at a tertiary level hospital in Mozambique, from 1 November 2013 to 31 March 2015 (17 months-long period). Clinical data were abstracted, a computer coded VA was completed using the clinical data as source of information, and an MIA followed by a complete autopsy were performed. Screening for malaria infection was conducted postmortem to all participants using molecular and histological techniques (PCR and immunohistochemistry).FindingsMalaria infection was considered the cause of death in 6/264 (2.3%) cases: 2/54 children (3.7%, both less than 5 years old) and 4/57 (7.0%) maternal deaths. The sensitivity and specificity of the VA, the clinical data and the MIA to identify malaria-specific deaths were 33.3% and 96.1%, 66.7% and 96.1%, and 100% and 100%, respectively. In addition, malaria was identified as a possible contributor in 14 additional patients who died of other diseases. These cases were also accurately identified by the MIA (sensitivity 82.4%, specificity 100%).InterpretationThe high sensitivity and specificity of the MIA in identifying malaria may help to improve current estimates of malaria-specific mortality in endemic areas.

Background: Malaria diagnosis relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia.Methods: We used PfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n=951) and pregnant women at first antenatal care (ANC) visit (n=87) enrolled in the Saint Joseph Catholic Hospital (Monrovia) from March to July 2019 to assess the frequency of false-negative RDT results. True false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy resultsResults: One hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and increased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n=5) and 8% (n=7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the sixteen RDT-negative but microscopy-positive samples which were available for molecular testing. Conclusion: There is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates the appropriate performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

Large-scale programs targeting Plasmodium falciparum (Pf) elimination can exert strong selection pressures on the parasite population. To better understand the impact that elimination initiatives can have on Pf genetic structure and gametocyte carriage, we applied amplicon-based sequencing of two polymorphic Pf genes and quantitative reverse-transcription PCR targeting gametocyte-specific genes to Pf isolates collected in Magude District (Southern Mozambique) before and after an elimination initiative. The 71% reduction of Pf prevalence achieved in 2 years was followed by reductions in Pf genetic diversity and increases in between-infection similarity. These genetic shifts were accompanied by increases in the relative transcript number of the female mature gametocyte marker pfs25, the pfap2g transcription factor that drives gametocytogenesis and the sexual ring marker pfgexp02, suggesting the parasite ability of adapting its sexual investment during elimination initiatives. Reactive interventions that target Pf sexual stages may be required to achieve complete interruption of transmission.

BackgroundAt the peak of the COVID-19 pandemic in Spain, cumulative prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCW) from Hospital Clínic de Barcelona was 11.2%.MethodsA follow-up survey one month after the baseline (April-May 2020) measured SARS-CoV-2 infection by real time reverse-transcriptase polymerase chain reaction (rRT-PCR) and IgM, IgA, IgG and subclasses to the receptor-binding domain of the SARS-CoV-2 spike protein by Luminex. Prevalence of infection was defined by a positive SARS-CoV-2 rRT-PCR and/or antibody seropositivity.ResultsThe cumulative prevalence of infection at month 1 was 14.9% (84/565) and the seroprevalence 14.5% (82/565) for IgM and/or IgG and/or IgA. We found 25 (5%) new infections in participants without previous evidence of infection at baseline (501) and two participants seroreverted for IgM and/or IgG and/or IgA. Among seropositive participants at baseline, IgM and IgA levels generally declined at month 1 (antibody decay rates of 0.49 (95% CI, 0.40-0.60) and 0.34 (95% CI, 0.26-0.44)), respectively. Eight percent of the participants seroreverted for IgM and 11% for IgA. Subjects reporting COVID-19-like symptoms and laboratory and other technicians had higher risk of infection. The most frequent subclass responses were IgG1 and IgG2, followed by IgG3, with higher levels of IgG1, and only IgA1 but no IgA2 was detected.ConclusionsOur findings highlight the importance of a continuous and improved surveillance of SARS-CoV-2 infections in HCW, particularly in high risk groups. The decay of IgA and IgM levels have implications for seroprevalence studies using these isotypes.

Antibody transplacental transfer is essential for conferring protection in newborns against infectious diseases. This transfer may be affected by gestational age and maternal infections, although the effects are not consistent across studies. We measured total IgG and IgG subclasses by quantitative suspension array technology against fourteen pathogens and vaccine antigens, including target of maternal immunization, in 341 delivering HIV− and HIV+ mother-infant pairs from southern Mozambique. Maternal antibody levels were the main determinant of cord antibody levels. HIV broadly reduced the placental transfer and cord levels of IgG and IgG1, but also IgG2 to half of the antigens. Plasmodium falciparum exposure and prematurity were negatively associated with cord antibody levels and placental transfer but this was antigen-subclass dependent. These findings suggest maternal infections may impact the efficacy of maternal immunization and confirm the lower transfer of antibodies as one of the causes underlying increased susceptibility to infections in HIV-exposed infants.

Severe malaria (SM) is a major public health problem in malaria-endemic countries. Sequestration of Plasmodium falciparum (Pf) infected erythrocytes in vital organs and the associated inflammation leads to organ dysfunction. MicroRNAs (miRNAs), which are rapidly released from damaged tissues into the host fluids, constitute a promising biomarker for the prognosis of SM. This study applied next-generation sequencing to evaluate the differential expression of miRNAs in SM compared to uncomplicated malaria (UM). Six miRNAs were associated with in vitro Pf cytoadhesion, severity in Mozambican children and Pf biomass. Relative expression of hsa-miR-4497 quantified by TaqMan-RT-qPCR, was higher in SM children plasmas compared to that of UM (pPf biomass (p=0.033). These findings suggest that different physiopathological processes in SM and UM lead to differential expression of miRNAs and pave the way to future studies aiming to assess the prognostic value of these miRNAs in malaria.

Background Innovative approaches are needed to limit antimalarial resistance evolution. Understanding the role of intermittent preventive treatment in pregnancy (IPTp) on the selection for resistance and the impact such selection has on pregnancy outcomes can guide future interventions. Methods Plasmodium falciparum isolates (n = 914) from 2 randomized clinical trials were screened for pfmdr1 copy number variation and pfcrt, pfmdr1, pfdhfr, and pfdhps resistance markers. The trials were conducted between 2010 and 2013 in Benin, Gabon, Kenya, and Mozambique to establish the efficacy of IPTp-mefloquine (MQ) compared with IPTp-sulphadoxine-pyrimethamine (SP) in human immunodeficiency virus (HIV)-uninfected and to IPTp-placebo in HIV-infected women. Results In HIV-uninfected women, the prevalence of pfcrt mutants, pfdhfr/pfdhps quintuple mutants, and pfmdr1 copy number was similar between women receiving IPT-SP and IPTp-MQ. However, prevalence of pfmdr1 polymorphism 86Y was lower in the IPTp-MQ group than in the IPTp-SP group, and within the IPTp-MQ group it was lower at delivery compared with recruitment. No effect of IPTp-MQ on resistance markers was observed among HIV-infected women. The carriage of resistance markers was not associated with pregnancy outcomes. Conclusions Selection of wild-type pfmdr1 polymorphism N86 by IPTp-MQ highlights the strong selective pressure IPTp can exert and the opportunity for using negative cross-resistance in drug choice for clinical treatment and IPTp.

Maternal factors and exposure to pathogens have an impact on infant health. For instance, HIV exposed but uninfected infants have higher morbidity and mortality than HIV unexposed infants. Innate responses are the first line of defense and orchestrate the subsequent adaptive immune response and are especially relevant in newborns. To determine the association of maternal HIV infection with maternal and newborn innate immunity we analyzed the cytokine responses upon pattern recognition receptor (PRR) stimulations in the triad of maternal peripheral and placental blood as well as in cord blood in a cohort of mother-infant pairs from southern Mozambique. A total of 48 women (35 HIV-uninfected and 13 HIV-infected) were included. Women and infant innate responses positively correlated with each other. Age, gravidity and sex of the fetus had some associations with spontaneous production of cytokines in the maternal peripheral blood. HIV-infected women not receiving antiretroviral therapy (ART) before pregnancy showed decreased IL-8 and IL-6 PRR responses in peripheral blood compared to those HIV-uninfected, and PRR hyporesponsiveness for IL-8 was also found in the corresponding infant's cord blood. HIV infection had a greater impact on placental blood responses, with significantly increased pro-inflammatory, T H 1 and T H 17 PRR responses in HIV-infected women not receiving ART before pregnancy compared to HIV-uninfected women. In conclusion, innate response of the mother and her newborn was altered by HIV infection in the women who did not receive ART before pregnancy. As these responses could be related to birth outcomes, targeted innate immune modulation could improve maternal and newborn health.

Background Malaria epidemiological and immunological data suggest that parasite tolerance wanes in the absence of continuous exposure to the parasite, potentially enhancing pathogenesis. The expansion of control interventions and elimination campaigns raises the necessity to better understand the host factors leading to susceptibility or tolerance that are affected by rapid changes in malaria transmission intensity (MTI). Mediators of cellular immune responses are responsible for the symptoms and pathological alterations during disease and are expected to change rapidly upon malaria exposure or cessation. Methods The plasma concentrations of 30 cytokine, chemokine and growth factors in individuals of all ages from a malaria endemic area of southern Mozambique were compared between 2 years of different MTI: 2010 (lower, n = 234) and 2013 (higher, n = 143). The effect of the year on the correlations between cytokines, chemokines and growth factors and IgGs to Plasmodium falciparum (markers of exposure) was explored. The effects of age, sex, neighbourhood and parasitaemia on analyte levels and their interactions with year were also assessed. Results An inverse correlation of several cellular immune mediators with malarial antibodies in 2013, and a lack of correlation or even a positive correlation in 2010 were observed. Most cytokines, chemokines and growth factors, regardless of their immune function, had higher concentrations in 2010 compared with 2013 in P. falciparum-infected and uninfected subjects. Age and neighbourhood showed an effect on analyte concentrations. Conclusions The results show a different regulation of the cellular immune response in 2010 vs 2013 which could be related to a loss of immune-tolerance after a decline in MTI in 2010 and previous years, and a rapid re-establishment of tolerance as a consequence of more continuous exposure as MTI began increasing in 2012. Cellular immune mediators warrant further investigation as possible surrogates of MTI-associated host susceptibility or tolerance.

An expatriate to Ivory Coast (supposedly allergic to artemether-lumefantrine) was diagnosed with severe malaria in Spain. Parasitemia increased from 2% up to 21% within 24 h under quinine (10 mg/kg) and clindamycin (450 mg/8 h) combination treatment. Molecular profiling of the patient revealed the presence of molecular markers of quinine and other antimalarials resistance. Additionally, multiple copies of pfpm2 gene were also noticed in the patient sample, despite the absence of piperaquine drug pressure in Ivory Coast. Parasitemia was cleared with artesunate (2.4 mg/kg) under a desensitization protocol. Nevertheless, detection of early treatment failure is needed mainly in cases of suspected antimalarial resistance.

Afebrile Plasmodium falciparum infections usually remain undetected and untreated in the community and could potentially contribute to sustaining local malaria transmission in areas aiming for malaria elimination.Thirty-two men with afebrile P. falciparum infections detected with rapid diagnostic test (RDTs) were followed for 28 days. Kaplan-Meier estimates were computed to estimate probability of parasite positivity and of reducing parasitemia by half of its initial level by day 28. Trends of parasite densities quantified by microscopy and real-time quantitative polymerase chain reaction (qPCR) were assessed using Poisson regression models, and the microscopy-to-qPCR positivity ratio was calculated at each time point. Three survival distributions (Gompertz, Weibull, and gamma) were used to evaluate their strength of fit to the data and to predict the median lifetime of infection.The cumulative probability of parasite qPCR positivity by day 28 was 81% (95% confidence interval [CI], 60.2-91.6). Geometric mean parasitemia at recruitment was 516.1 parasites/μL and fell to100 parasites/μL by day 3, reaching 56.7 parasites/μL on day 28 (P.001). The ratio of P. falciparum-positive samples by microscopy to qPCR decreased from 0.9 to 0.52 from recruitment to day 28. The best model fit to the data was obtained assuming a Gompertz distribution.Afebrile P. falciparum infections detectable by RDT in semi-immune adults fall and stabilize at low-density levels during the first 4 days after detection, suggesting a rapid decline of potential transmissibility in this hidden parasite reservoir.NCT02698748.

Background Malaria programmes use Plasmodium falciparum histidine-rich protein-2 (PfHRP2) based rapid diagnostic tests (RDTs) for malaria diagnosis. The deletion of this target antigen could potentially lead to misdiagnosis, delayed treatment and continuation of active transmission. Methods Plasmodium falciparum isolates (n = 1162) collected in Southern Mozambique were assessed by RDTs, microscopy and/or 18SrRNA qPCR. pfhrp2 and pfhrp3 deletions were investigated in isolates from individuals who were negative by RDT but positive by microscopy and/or qPCR (n = 69) using gene-specific PCRs, with kelch13 PCR as the parasite DNA control. Results Lack of pfhrp2 PCR amplification was observed in one of the 69 isolates subjected to molecular analysis [1.45% (95% CI 0.3–7.8%)]. Conclusions The low prevalence of pfhrp2 deletions suggests that RDTs will detect the vast majority of the P. falciparum infections. Nevertheless, active surveillance for changing deletion frequencies is required.

Plasmodium falciparum immature gametocytes are not observed in peripheral blood. However, gametocyte stages in organs such as bone marrow have never been assessed by molecular techniques, which are more sensitive than optical microscopy. We quantified P falciparum sexual stages in bone marrow (n = 174) and peripheral blood (n = 70) of Mozambican anemic children by quantitative polymerase chain reaction targeting transcripts specific for early (PF14_0748; PHISTa), intermediate (PF13_0247; Pfs48/45), and mature (PF10_0303; Pfs25) gametocytes. Among children positive for the P falciparum housekeeping gene (PF08_0085; ubiquitin-conjugating enzyme gene) in bone marrow (n = 136) and peripheral blood (n = 25), prevalence of immature gametocytes was higher in bone marrow than peripheral blood (early: 95% vs 20%, P < .001; intermediate: 80% vs 16%; P < .001), as were transcript levels (P < .001 for both stages). In contrast, mature gametocytes were more prevalent (100% vs 51%, P < .001) and abundant (P < .001) in peripheral blood than in the bone marrow. Severe anemia (3.57, 95% confidence interval 1.49-8.53) and dyserythropoiesis (6.21, 95% confidence interval 2.24-17.25) were independently associated with a higher prevalence of mature gametocytes in bone marrow. Our results highlight the high prevalence and abundance of early sexual stages in bone marrow, as well as the relationship between hematological disturbances and gametocyte development in this tissue.

There are no large-scale ex vivo studies addressing the contribution of Plasmodium falciparum in the bone marrow to anaemia. The presence of malaria parasites and haemozoin were studied in bone marrows from 290 anaemic children attending a rural hospital in Mozambique. Peripheral blood infections were determined by microscopy and polymerase chain reactions. Bone marrow parasitaemia, haemozoin and dyserythropoiesis were microscopically assessed. Forty-two percent (123/290) of children had parasites in the bone marrow and 49% (111/226) had haemozoin, overlapping with parasitaemia in 83% (92/111) of cases. Sexual and mature asexual parasites were highly prevalent (62% gametocytes, 71% trophozoites, 23% schizonts) suggesting their sequestration in this tissue. Sixteen percent (19/120) of children without peripheral infection had haemozoin in the bone marrow. Haemozoin in the bone marrow was independently associated with decreased Hb concentration (P = 0·005) and was more common in dyserythropoietic bone marrows (P = 0·010). The results of this ex vivo study suggest that haemozoin in the bone marrow has a role in the pathogenesis of malarial-anaemia through ineffective erythropoiesis. This finding may have clinical implications for the development of drugs targeted to prevent and treat malarial-anaemia.

The combination of fosmidomycin and clindamycin (F/C) is effective in adults and older children for the treatment of malaria and could be an important alternative to existing artemisinin-based combinations (ACTs) if proven to work in younger children. We conducted an open-label clinical trial to assess the efficacy, safety, and tolerability of F/C for the treatment of uncomplicated P. falciparum malaria in Mozambican children

Background. Accurate diagnosis of malaria infection during pregnancy remains challenging because of low parasite densities and placental sequestration of Plasmodium falciparum. The performance of different methods to detect P. falciparum in pregnancy and the clinical relevance of undetected infections were evaluated. Methods. P. falciparum infections were assessed in 272 Mozambican women at delivery by microscopy, placental histology, quantitative polymerase chain reaction (qPCR) and detection of histidine-rich protein 2 (HRP2) in plasma by enzyme-linked immunosorbent assay (ELISA) and a rapid diagnostic test (RDT). Association between infection and delivery outcomes was determined. Results. Among the 122 women qPCR-positive for P. falciparum in peripheral and/or placental blood samples, 87 (71.3%) did not receive a positive diagnosis by peripheral microscopy, 75 (61.5%) by HRP2 ELISA, and 74 (60.7%) by HRP2 RDT in plasma. Fifty-seven of the 98 qPCR-positive placental infections (58.2%) were not detected by histology. Women who were qPCR-positive but negative in their peripheral blood by microscopy or HRP2 RDT in plasma (n 5 62) were at increased risk of anemia, compared with negative women (n 5 141; odds ratio, 2.03; 95% confidence interval, 1.07‐3.83; P 5 .029). Conclusions. Microscopy, placental histology and HRP2-based plasma diagnostic methods fail to identify the majority of the P. falciparum infections detected by qPCR in peripheral and placental blood. Undetected infections were associated with maternal anemia, highlighting the urgent need for more accurate malaria diagnostic tools for pregnant women to avoid the negative clinical impact that hidden infections can have during pregnancy. Clinical Trials Registration. NCT00209781.

This study aimed to evaluate whether intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) had an effect on the acquisition of IgG against Plasmodium falciparum variant surface antigens (VSA) and growth-inhibitory antibodies in Manhica, Mozambique. In addition, we assessed factors affecting the magnitude of these responses and the association between antibody levels and protection against malaria. IgG to VSA expressed by MOZ2, R29 and E8B parasite isolates were measured in plasma samples collected at 5, 9, 12 and 24 months of age by flow cytometry. Growth-inhibitory antibodies in dialyzed plasmas using GFP-D10 parasites were measured by flow cytometry at 12 and 24 months. IPTi-SP did not significantly modify the levels of IgG against VSA nor the growth-inhibitory capacity of antibodies up to 2 years of age. Age but not previous episodes of malaria influenced the magnitude of these responses. In addition, anti-VSA IgG levels were 7% higher in children with current P. falciparum infection and were associated with neighborhood of residence. Children aged 24 months had 10% less parasite growth than those aged 12 months (95% CI 0.88-0.93, P

Pregnant women are susceptible to Plasmodium falciparum parasites that sequester in the placenta. The massive accumulation of infected erythrocytes in the placenta has been suggested to trigger the deleterious effects of malaria in pregnant women and their offspring. The risk of malaria is also high during the postpartum period, although mechanisms underlying this susceptibility are not known. Here, we aimed to identify host factors contributing to the risk of postpartum infections and to determine the origin of postpartum parasites by comparing their genotypes with those present at the time of delivery. To address this, blood samples were collected at delivery ( n = 402) and postpartum ( n = 354) from Mozambican women enrolled in a trial of intermittent preventive treatment in pregnancy (IPTp). P. falciparum was detected by real-time quantitative PCR (qPCR), and the parasite merozoite surface protein 1 ( msp-1 ) and msp-2 genes were genotyped. Fifty-seven out of 354 (16%) women were infected postpartum as assessed by qPCR, whereas prevalence by optical microscopy was only 4%. Risk of postpartum infection was lower in older women (odds ratio [OR] = 0.34, 95% confidence interval [CI] = 0.15 to 0.81) and higher in women with a placental infection at delivery (OR = 4.20, 95% CI = 2.19 to 8.08). Among 24 women with matched infections, 12 (50%) were infected postpartum with at least one parasite strain that was also present in their placentas. These results suggest that parasites infecting pregnant women persist after delivery and increase the risk of malaria during the postpartum period. Interventions that reduce malaria during pregnancy may translate into a lower risk of postpartum infection.

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n = 48), European adults (naïve travelers, n = 22; expatriates with few prior malaria exposures, n = 15) and Mozambican adults with long-life malaria exposure (n = 99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-142 (P = 0.030) and a P. falciparum isolate (P = 0.002), as well as higher IL-12 (P = 0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P≤0.0090), IL-2 (P≤0.0379) and IL-8 (P≤0.0233). Children also had higher IL-12 (P = 0.0001), IL-4 (P = 0.003), IL-1β (P = 0.024) and TNF (P = 0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A TH1/pro-inflammatory response was the hallmark of non-immune subjects.

Background Estimation of Plasmodium falciparum parasitaemia can vary with the method used and time of sampling. Quantitative real time PCR (qPCR) on whole blood or plasma samples has previously been shown to be more sensitive than thick film microscopy. However the efficiencies of each method have not been compared using samples obtained from infants less than one year old. Methods A multiple of statistical approaches were used to compare the performance of qPCR on whole blood or plasma to detect the 18 S ribosomal gene of P. falciparum in 548 samples from children aged 2.5 or 24 months. Parasite prevalence in matched samples was compared using Mcnemar’s test and agreement of positive results quantified as Kappa scores. Parasite prevalences between different age groups were compared by Fisher’s test. Results from analyses by thick film microscopy were also available from children at 24 months and their correlation to each qPCR method examined by the Spearman’s test. Finally the association of P. falciparum infection with the incidence of multiple malaria episodes from contact to 24 months of age was evaluated using negative binomial regression. Results These analyses showed that qPCR from whole blood detected approximately 3-fold more cases of infection than plasma qPCR. Both qPCR methods agreed well with each other although qPCR from plasma had a greater agreement with microscopy (96.85%) than did qPCR from blood (69.7%). At 24 months the prevalence of infection detected by all methods was associated with anaemia (p Conclusions The data presented here demonstrates that low levels of parasitaemia are better detected by qPCR using parasite DNA from whole blood than from plasma. However plasma samples provide a viable substitute when parasite smears are unavailable.

Background. Plasmodium falciparum infection in pregnancy can lead to congenital malaria, which has detrimental health consequences for infants. Human immunodeficiency virus (HIV) might increase cord blood P. falciparum infection by decreasing maternal antimalarial‐specific antibodies. Methods. HIV-negative (n5133) and HIV-positive (n555) Mozambican pregnant women were assessed at delivery for maternal and cord P. falciparum infection by quantitative polymerase chain reaction (qPCR) and P. falciparum‐specific antibodies by enzyme-linked immunosorbent assay and flow cytometry. Results. Prevalence of qPCR-detected cord blood infection was 8.0%. Risk of cord infection was increased in presence of HIV (adjusted odds ratio [AOR], 3.80; P5.04)and placental malaria (AOR, 22.08; P5.002) after adjusting for clinical variables. The odds of having a high immunoglobulin G response to chondrotin sulphate A‐binding infected erythrocytes, parasite lysate, and erythrocyte-binding antigen-175 were reduced among HIV-positive women (P , .001, .048, and .056, respectively) and among women with cord P. falciparum infection (P 5 .009, .04, and .046, respectively). In multivariate analysis including maternal HIV status, placental malaria, and antibody responses, HIV was no longer associated with cord blood infection (P 5 .11). Conclusions. HIV-associated impairment of antibody responses in pregnant women may contribute to a higher transmission of P. falciparum to their infants.

Background. Increased susceptibility to Plasmodium falciparum infection during pregnancy has been attributed to the accumulation of infected erythrocytes in the placenta. This phenomenon is mediated by a var gene coding for VAR2CSA, which adheres to chondroitin sulphate A. However, the contribution of parasites transcribing other var genes to maternal infections has not been well characterized. Methods. Transcription of var2csa and var groups A, B, and C was measured by real-time polymerase chain reaction in 30 placental and 21 peripheral P. falciparum isolates from pregnant women and in 42 isolates from nonpregnant adults and children. Associations of infections with non-var2csa isolates with maternal parasitemia and immune responses were assessed. Results. Placental parasites showed the highest levels of var2csa. ABC var genes were transcribed by 20 (67%) of 30 placental isolates and were associated with higher parasitemia compared with infections by parasites only transcribing var2csa (P = .004). Peripheral isolates from pregnant women transcribed ABC var genes at levels similar to those of parasites infecting nonpregnant adults with clinical malaria (P[varA] = .420, P[varB] = .808, and P[varC] = .619). Conclusions. Transcripts of var2csa are abundant in pregnancy-associated P. falciparum infections; however, ABC var types are also common, especially in peripheral blood, with transcription levels similar to those of infections out of pregnancy. These findings are of interest for the design of malaria vaccines for pregnant women.

Background. Factors involved in the development of resistance to sulphadoxine-pyrimethamine (SP) by Plasmodium falciparum, particularly in the context of intermittent preventive treatment during pregnancy (IPTp), are not well known. We aimed to determine the impact of IPTp and human immunodeficiency virus (HIV) infection on molecular markers of SP resistance and the clinical relevance of resistant infections. Methods. SP resistance alleles were determined in peripheral (n 5 125) and placental (n 5 145) P. falciparum isolates obtained from pregnant women enrolled in a randomized, placebo-controlled trial of IPTp in Manhic xa, Mozambique. Results. Prevalence of quintuple mutant infections was 12% (23 of 185 isolates) in pregnant women who received placebo and 24% (20 of 85 isolates) in those who received SP (P 5 .031). When the last IPTp dose was administered at late pregnancy, mutant infections at delivery were more prevalent in placental samples (7 [23%] of 30, samples) than in peripheral blood samples (2 [7%] of 30 samples; P 5 .025), more prevalent in women who received IPTp-SP than in those who received placebo (odds ratio [OR], 8.13; 95% confidence interval [CI], 1.69‐ 39.08), and more prevalent in HIV-positive women than in HIV-negative women (OR, 5.17; 95% CI, 1.23‐21.66). No association was found between mutant infections and increased parasite density or malaria-related morbidity in mothers and children. Conclusions. IPTp with SP increases the prevalence of resistance markers in the placenta and in HIV-infected women at delivery, which suggests that host immunity is key for the clearance of drug-resistant infections. However, this effect of IPTp is limited to the period when blood levels of SP are likely to be significant and does not translate into more-severe infections or adverse clinical outcomes.

Background Control of malaria in pregnancy remains a public health challenge. Improvements in its correct diagnosis and the adequacy of protocols to evaluate anti-malarial drug efficacy in pregnancy, are essential to achieve this goal. Methods The presence of Plasmodium falciparum was assessed by real-time (RT) PCR in 284 blood samples from pregnant women with clinical complaints suggestive of malaria, attending the maternity clinic of a Mozambican rural hospital. Parasite recrudescences in 33 consecutive paired episodes during the same pregnancy were identified by msp1 and msp2 genotyping. Results Prevalence of parasitaemia by microscopy was 5.3% (15/284) and 23.2% (66/284) by RT-PCR. Sensitivity of microscopy, compared to RT-PCR detection, was 22.7%. Risk of maternal anaemia was higher in PCR-positive women than in PCR-negative women (odds ratio [OR] = 1.92, 95% confidence interval [CI] 1.09–3.36). Genotyping confirmed that recrudescence after malaria treatment occurred in 7 (21%) out of 33 pregnant women with consecutive episodes during the same pregnancy (time range between recrudescent episodes: 14 to 187 days). Conclusion More accurate and sensitive diagnostic indicators of malaria infection in pregnancy are needed to improve malaria control. Longer follow-up periods than the standard in vivo drug efficacy protocol should be used to assess anti-malarial drug efficacy in pregnancy.

BACKGROUND: Malaria programmes use Plasmodium falciparum histidine-rich protein-2 (PfHRP2) based rapid diagnostic tests (RDTs) for malaria diagnosis. The deletion of this target antigen could potentially lead to misdiagnosis, delayed treatment and continuation of active transmission. METHODS: Plasmodium falciparum isolates (n = 1162) collected in Southern Mozambique were assessed by RDTs, microscopy and/or 18SrRNA qPCR. pfhrp2 and pfhrp3 deletions were investigated in isolates from individuals who were negative by RDT but positive by microscopy and/or qPCR (n = 69) using gene-specific PCRs, with kelch13 PCR as the parasite DNA control. RESULTS: Lack of pfhrp2 PCR amplification was observed in one of the 69 isolates subjected to molecular analysis [1.45% (95% CI 0.3-7.8%)]. CONCLUSIONS: The low prevalence of pfhrp2 deletions suggests that RDTs will detect the vast majority of the P. falciparum infections. Nevertheless, active surveillance for changing deletion frequencies is required.