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Background Plasmodium falciparum resistance to sulphadoxine-pyrimethamine (SP) threatens the efficacy of intermittent preventive treatment (IPTp) for malaria in pregnancy in Africa. We conducted a meta-analysis to assess the impact of SP resistance on IPTp-SP effectiveness. Methods We searched databases (1990 to March-01-2018) for clinical studies (aggregated data) or surveys (individual-participant data) containing information on low birthweight (LBW, primary outcome) and malaria by IPTp-SP dose, and for studies reporting SP-resistance molecular markers. We performed random-effects meta-analysis (clinical studies) or multivariate log-binomial regression (surveys) to obtain summarized dose-response data (Relative-Risk-Reduction:RRR) and multivariate meta-regression to explore modifying effects of SP-resistance (dhps substitutions A437G, K540E, A581G). Findings Of 1097 records, 57 studies were included in the aggregated-data meta-analysis (59,457 births). The RRR for LBW declined with increasing prevalence of Pfdhps-K540E (P-trend=0.0060) but not with Pfdhps-A437G (P-trend=0.35). The RRR in areas of high (Pfdhps-K540E >90%, n=11), moderate (Central/West Africa:Pfdhps-A437G≥90% or East/southern Africa:Pfdhps-K540E 30-90%, n=16) and low SP-resistances (n=30) were 7% (95% CI 0-13), 21% (14-29) and 27% (21-33) respectively (P-trend=0.0054, I2=69.5%). In the individual-participant analysis of 13 surveys (42,394 births), IPTp-SP was associated with reduced LBW in areas with Pfdhps-K540E>90% & Pfdhps-A581G=10% (pooled Pfdhps-A581G prevalence:37%, range 29-46) (RRR=0.5%, -16-14, n=3). Interpretation The effectiveness of IPTp-SP is reduced in areas with high SP-resistance, but IPTp-SP remains associated with reduced LBW in areas where Pfdhps-K540E prevalence exceeds 90%. IPTp-SP is not effective in areas with ≥37% prevalence of the highly-resistant sextuple Pfdhps-A581G-containing genotype.

Objectives. To estimate the impact of hypothetical antimalarial and nutritional interventions (which reduce the prevalence of low midupper arm circumference [MUAC]) on the incidence of low birth weight (LBW). Methods. We analyzed data from 14 633 pregnancies from 13 studies conducted across Africa and the Western Pacific from 1996 to 2015. We calculated population intervention effects for increasing intermittent preventive therapy in pregnancy (IPTp), full coverage with bed nets, reduction in malaria infection at delivery, and reductions in the prevalence of low MUAC. Results. We estimated that, compared with observed IPTp use, administering 3 or more doses of IPTp to all women would decrease the incidence of LBW from 9.9% to 6.9% (risk difference = 3.0%; 95% confidence interval = 1.7%, 4.0%). The intervention effects for eliminating malaria at delivery, increasing bed net ownership, and decreasing low MUAC prevalence were all modest. Conclusions. Increasing IPTp uptake to at least 3 doses could decrease the incidence of LBW in malaria-endemic countries. The impact of IPTp on LBW was greater than the effect of prevention of malaria, consistent with a nonmalarial effect of IPTp, measurement error, or selection bias.

In pregnancy-associated malaria, infected erythrocytes accumulate in the placenta. It is unclear if in polyclonal infections this results in distinct peripheral and placental parasite populations. We used long amplicon deep sequencing of Plasmodium falciparum var2csa ID1-DBL2X from 15 matched peripheral and placental samples collected at delivery from a high transmission area to determine genetic homology. Despite substantial sequence variation and detecting 23 haplotypes, the matched pairs mostly contained the same genetic variants, with 11 pairs sharing 100% of their variants, whereas others showed some heterogeneity. Thus, at delivery, peripheral and placental parasites appear to intermix and placental genotypes can be inferred through peripheral sampling.

This paper summarises key advances in defining the infectious reservoir for malaria and the measurement of transmission for research and programmatic use since the Malaria Eradication Research Agenda (malERA) publication in 2011. Rapid and effective progress towards elimination requires an improved understanding of the sources of transmission as well as those at risk of infection. Characterising the transmission reservoir in different settings will enable the most appropriate choice, delivery, and evaluation of interventions. Since 2011, progress has been made in a number of areas. The extent of submicroscopic and asymptomatic infections is better understood, as are the biological parameters governing transmission of sexual stage parasites. Limitations of existing transmission measures have been documented, and proof-of-concept has been established for new innovative serological and molecular methods to better characterise transmission. Finally, there now exists a concerted effort towards the use of ensemble datasets across the spectrum of metrics, from passive and active sources, to develop more accurate risk maps of transmission. These can be used to better target interventions and effectively monitor progress toward elimination. The success of interventions depends not only on the level of endemicity but also on how rapidly or recently an area has undergone changes in transmission. Improved understanding of the biology of mosquito-human and human-mosquito transmission is needed particularly in low-endemic settings, where heterogeneity of infection is pronounced and local vector ecology is variable. New and improved measures of transmission need to be operationally feasible for the malaria programmes. Outputs from these research priorities should allow the development of a set of approaches (applicable to both research and control programmes) that address the unique challenges of measuring and monitoring transmission in near-elimination settings and defining the absence of transmission.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA's ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99 g; 95% Confidence Interval [CI]: -466.43 g, -69.55 g) and higher odds of low birthweight (< 2500 g) (Odds Ratio [OR] 5.41; 95% CI: 0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was conducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickettsia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more antigens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

Drug-resistant Plasmodium falciparum malaria is a major public health problem. An elevated pfmdr1 gene copy number (CN) is known to decrease parasite sensitivity to the commonly used antimalarial mefloquine (MFQ). To understand the relationship between pfmdr1 CN and mefloquine resistance, we evaluated pfmdr1 transcript levels in three P. falciparum strains with different CNs in the presence and absence of MFQ. Parasite strains with multiple pfmdr1 gene copies exhibited higher relative transcript levels than single-copy parasites, and MFQ induced pfmdr1 expression above the levels without treatment in all three strains evaluated. Concomitant morphology analyses of the sampled cultures revealed that MFQ treatment of synchronized ring-stage parasites induced a delay in parasite maturation through the intraerythrocytic cycle. pfmdr1 expression peaks in the ring stage, and MFQ could be causing increased transcription by delaying parasite maturation. However, pretreatment with mefloquine did not affect the artemisinin in vitro half-maximal inhibitory concentration (IC50). These results suggest that MFQ-induced increases in pfmdr1 expression are the direct result of the maturation delay at the ring stage but that this change in expression does not affect the antimalarial activity of artemisinin.

Evaluation of antimalarial efficacy is difficult because recurrent parasitemia can be due to recrudescence or reinfection. PCR is used to differentiate between recrudescences and reinfections by comparing parasite allelic variants before and after treatment. However, PCR-corrected results are susceptible to misclassification: false recrudescences, due to reinfection by the same variant present in the patient before treatment; and false reinfections, due to variants that are present but too infrequent to be detected in the pre-treatment PCR, but are then detectable post-treatment. This article aims to explore factors affecting the probability of false recrudescences and proposes a Monte Carlo uncertainty analysis to adjust for both types of misclassification. Higher levels of transmission intensity, increased multiplicity of infection, and limited allelic variation resulted in more false recrudescences. The uncertainty analysis exploits characteristics of study data to minimize bias in the estimate of efficacy and can be applied to areas of different transmission intensity.

Subtype C human immunodeficiency virus type 1 (HIV-1C) continues to cause the majority of new cases of mother-to-child transmission (MTCT), and yet there are limited data on HIV-1C transmission. We amplified env from plasma RNA for 19 HIV-1C MTCT pairs, 10 transmitting in utero (IU) and 9 transmitting intrapartum (IP). There was a strong genetic bottleneck between all mother-infant pairs, with a majority of transmission events involving the transmission of a single virus. env genes of viruses transmitted to infants IP, but not IU, encoded Env proteins that were shorter and had fewer putative N-linked glycosylation sites in the V1-V5 region than matched maternal sequences. Viruses pseudotyped with env clones representative of each maternal and infant population were tested for neutralization sensitivity. The 50% inhibitory concentration of autologous serum was similar against both transmitted (infant) and nontransmitted (maternal) viruses in a paired analysis. Mother and infant Env proteins were also similar in sensitivity to soluble CD4, to a panel of monoclonal antibodies, and to heterologous HIV-1C sera. In addition, there was no difference in the breadth or potency of neutralizing antibodies between sera from 50 nontransmitting and 23 IU and 23 IP transmitting HIV-1C-infected women against four Env proteins from heterologous viruses. Thus, while a strong genetic bottleneck was detected during MCTC, with viruses of shorter and fewer glycosylation sites in env present in IP transmission, our data do not support this bottleneck being driven by selective resistance to antibodies.

HIV infections are initiated by a limited number of variants that diverge into a diverse quasispecies swarm. During in utero mother-to-child transmission (IU MTCT), transmitted viral variants must pass through multiple unique environments, and our previously published data suggest a nonstochastic model of transmission. As an alternative to a stochastic model of viral transmission, we hypothesize that viral selection in the placental environment influences the character of the viral quasispecies when HIV-1 is transmitted in utero. To test this hypothesis, we used single-template amplification to isolate HIV-1 envelope gene (env) sequences from both peripheral plasma and the placentas of eight nontransmitting (NT) and nine IU-transmitting participants. Statistically significant compartmentalization between peripheral and placental HIV-1 env was detected in one of the eight NT cases and six of the nine IU MTCT cases. In addition, viral sequences isolated from IU MTCT placental tissue showed variation in env V1 loop lengths compared to matched maternal sequences, while NT placental env sequences did not. Finally, comparison of env sequences from NT and IU MTCT participants indicated statistically significant differences in Kyte-Doolittle hydropathy in the signal peptide, C2, V3, and C3 regions. Our working hypothesis is that the hydropathy differences in Env associated with IU MTCT alter viral cellular tropism or affinity, allowing HIV-1 to efficiently infect placentally localized cells.

Molecular assays can provide critical information for malaria diagnosis, speciation, and drug resistance, but their cost and resource requirements limit their application to clinical malaria studies. This study describes the application of a resource-conserving testing algorithm employing sample pooling for real-time PCR assays for malaria in a cohort of 182 pregnant women in Kinshasa. A total of 1,268 peripheral blood samples were collected during the study. Using a real-time PCR assay that detects all Plasmodium species, microscopy-positive samples were amplified individually; the microscopy-negative samples were amplified after pooling the genomic DNA (gDNA) of four samples prior to testing. Of 176 microscopy-positive samples, 74 were positive by the real-time PCR assay; the 1,092 microscopy-negative samples were initially amplified in 293 pools, and subsequently, 35 samples were real-time PCR positive (3%). With the real-time PCR result as the referent standard, microscopy was 67.9% sensitive (95% confidence interval [CI], 58.3% to 76.5%) and 91.2% specific (95% CI, 89.4% to 92.8%) for malaria. In total, we detected 109 parasitemias by real-time PCR and, by pooling samples, obviated over 50% of reactions and halved the cost of testing. Our study highlights both substantial discordance between malaria diagnostics and the utility and parsimony of employing a sample pooling strategy for molecular diagnostics in clinical and epidemiologic malaria studies.

Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine.

Malaria in pregnancy is a significant cause of maternal and infant morbidity and mortality. Malawi adopted intermittent preventive treatment with sulfadoxinepyrimethamine (SP) for the control of malaria in pregnancy in 1993. However there is little information on the in-vivo SP efficacy in pregnant women. This study was conducted to determine: prevalence of malaria and anaemia at the first antenatal visit and rate of parasitological failure to SP in pregnancy.A cross-sectional followed by a prospective cohort study was conducted in women attending antenatal care clinic at Montfort Hospital in Lower Shire Valley from June 2004 to February 2005. Women were screened for malaria and anaemia at the first antenatal visit. After taking SP under direct observation, women with malaria parasitaemia were followed up to day 14 to determine parasitological response.Of 961 women screened, 9% had malaria, 77% had anaemia (HB11.0g/dl), 24% had moderate anaemia (HB 7.0-8.9g/dl) and 6% had severe anaemia (HB7.0g/dl). Malaria was significantly more frequent in primigravidae, the second trimester and in the post- rainy season (all p0.05). Moderate anaemia (Hb9.0g/dl) was significantly more common in adolescents and primigravidae (both p0.05). In the14-day follow up study, loss to follow up was 13%. Of the 74 women who completed the follow up, 89% cleared malaria parasites successfully and 11% had parasitological failure. Parasitological failures were all of the R1 type except for one with R2 failure.Anaemia prevalence was high at first antenatal visit in this population. Rate of parasitological failure to SP in pregnancy increased from 5% in 1996 to 11% in 2004.

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Heteroduplex tracking assays (HTAs) of Plasmodium falciparum merozoite surface protein 1 block-2 were used to assess complexity of infection and treatment efficacy in a trial of three antimalarial treatments in 141 Malawian pregnant women. An elevated complexity of infection (COI) was associated with anemia, parasite burden, and human immunodeficiency virus infection but was not associated with age or gravidity. Comparisons of HTA patterns before and after treatment allowed the classification of 20 of 30 (66%) recurrent episodes as either definite treatment failures or reinfections. An elevated COI was strongly associated with treatment failure (P = 0.003). An algorithm was developed to assign a probability of failure for the 10 indeterminate participants, some of whose infections shared a single variant of high prevalence (>10%). By summing these probabilities, treatment efficacy was estimated.

Background: Although maternal anaemia often stems from malaria infection during pregnancy, its effects on foetal haemoglobin levels are not straightforward. Lower-than-expected cord haemoglobin values in malarious versus non-malarious regions were noted by one review, which hypothesized they resulted from foetal immune activation to maternal malaria. This study addressed this idea by examining cord haemoglobin levels in relation to maternal malaria, anaemia, and markers of foetal immune activation.\ud Methods: Cord haemoglobin levels were examined in 32 malaria-infected and 58 uninfected women in Blantyre, Malawi, in relation to maternal haemoglobin levels, malaria status, and markers of foetal haematological status, hypoxia, and inflammation, including TNF-alpha, TGF-beta, and ferritin. All women were HIV-negative.\ud Results: Although malaria was associated with a reduction in maternal haemoglobin (10.8 g/dL vs. 12.1 g/ dL, p < 0.001), no reduction in cord haemoglobin and no significant relationship between maternal and cord haemoglobin levels were found. Cord blood markers of haematological and hypoxic statuses did not differ between malaria-infected and uninfected women. Maternal malaria was associated with decreased TGF- and increased cord ferritin, the latter of which was positively correlated with parasitaemia (r = 0.474, p = 0.009). Increased cord ferritin was associated with significantly decreased birth weight and gestational length, although maternal and cord haemoglobin levels and malaria status had no effect on birth outcome.\ud Conclusion: In this population, cord haemoglobin levels were protected from the effect of maternal malaria. However, decreased TGF- and elevated ferritin levels in cord blood suggest foetal immune activation to maternal malaria, which may help explain poor birth outcomes.

Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T517A or P519S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds.

We conducted a prevalence study of mutations in Plasmodium falciparum that are associated with antifolate resistance in Blantyre, Malawi. The dihydrofolate reductase 164-Leu mutation, which confers resistance to both pyrimethamine and chlorproguanil, was found in 4.7% of the samples. Previously unreported mutations in dihydropteroate synthase were also found.

The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.

The aromatic diamidines represent a class of compounds with broad-spectrum antimicrobial activity; however, their development is hindered by a lack of understanding of their mechanism of antimicrobial action. DB75 [2,5-bis(4-amidinophenyl)furan] is a trypanocidal aromatic diamidine that was originally developed as a structural analogue of the antitrypanosomal agent pentamidine. DB289, a novel orally active prodrug of DB75, is undergoing phase IIb clinical trials for early-stage human African trypanosomiasis, Pneumocystis jiroveci carinii pneumonia, and malaria. The purpose of this study was to investigate mechanisms of action of DB75 using Saccharomyces cerevisiae as a model organism. The results of this investigation suggest that DB75 inhibits mitochondrial function. Yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75. DB75 localizes (by fluorescence) within the mitochondria of living yeast cells and collapses the mitochondrial membrane potential in isolated yeast mitochondria. Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration. We conclude that the mitochondrion is a cellular target of DB75 in yeast cells and anticipate that the results of this study will aid in the target-based design of new antimicrobial aromatic diamidines.

Pneumocystis jiroveci (human-derived P. carinii) is an opportunistic pathogenic fungus which causes pneumonia and is life-threatening in immunocompromised individuals. Spontaneously acquired resistance to atovaquone, a hydroxynaphthoquinone that is used to treat P. jiroveci infections, was linked to mutations in the mitochondrially encoded cytochrome b gene. Because P. jiroveci cannot be easily cultivated, we have developed Saccharomyces cerevisiae as an alternative system to study atovaquone resistance mutations. In this work, we introduced seven mutations linked with atovaquone resistance in P. jiroveci into the S. cerevisiae cytochrome b gene. The effects of the mutations on the respiratory function and on the sensitivity to the inhibitor were then characterized. Six of the reported mutations lowered the sensitivity of the S. cerevisiae bc1 complex to atovaquone, while one mutation had no effect on the drug resistance. These results were confirmed by monitoring the in vivo resistance of S. cerevisiae mutants which carried both the cytochrome b mutations and a deletion of the ABC transporter genes, allowing the drug to bypass the weakened efflux pump system. S. cerevisiae thus provides an easy-to-use system to characterize in vivo and in vitro cytochrome b mutations reported in pathogens and to assess their role in drug resistance.

The relative importance of acute high-density versus persistent low-density Plasmodium falciparum parasitemia in contributing to the public health problem of malarial anemia remains unclear. The Asembo Bay Cohort Project in western Kenya collected monthly hemoglobin (Hb) and parasitologic measurements and biweekly assessments of antimalarial drug use among 942 singleton live births between 1992 and 1996. A mixed-model analysis appropriate for repeated measures data was used to study how time-varying parasitemia and antimalarial drug exposures influenced mean Hb profiles. Incidence of World Health Organization-defined severe malarial anemia was 28.1 per 1,000 person-years. Among children aged less than 24 months, concurrent parasitemia was significantly associated with lower mean Hb, especially when compared to children with no concurrent parasitemia. Increased densities of the 90-day history of parasitemia preceding Hb measurement was more strongly associated with mean Hb levels than concurrent parasitemia density. While the highest quartile of 90-day parasitemia history was associated with lowest mean Hb levels, children in the lowest 90-day exposure quartile still experienced significantly lower Hb levels when compared to children who remained parasitemia-free for the same 90-day period. The results highlight the importance of collecting and analyzing longitudinal Hb and parasitologic data when studying the natural history of malarial anemia