Malaria and typhoid co-infections can be a serious public health issue in tropical countries leading to incorrect diagnosis due to overlapping clinical presentations of malaria and typhoid and hence, causing a delay in implementing the appropriate treatment regimen for these concurrent infections. This study reports a case of six-year-old female child co-infected with severe malaria (Plasmodium falciparum) and typhoid (Salmonella typhi) diagnosed by rapid malaria antigen test (RMAT) and blood culture respectively. Further, analysis of the chloroquine resistance gene Pfcrt for the falciparum demonstrated the presence of K76T mutant allele in pfcrt gene with high IC50 (150nM) for chloroquine (CQ) drug. The present case highlights the significance of timely identification and treatment of co-infections and also provides information about the circulating P. falciparum clinical strains.

In November 2018, we diagnosed a cluster of falciparum malaria cases in three Chilean travelers returning from Nigeria. Two patients were treated with sequential intravenous artesunate plus oral atovaquone/proguanil (AP) and one with oral AP. The third patient, a 23-year-old man, presented with fever on day 29 after oral AP treatment and was diagnosed with recrudescent falciparum malaria. The patient was then treated with oral mefloquine, followed by clinical recovery and resolution of parasitemia. Analysis of day 0 and follow-up blood samples, collected on days 9, 29, 34, 64, and 83, revealed that parasitemia had initially decreased but then increased on day 29. Sequencing confirmed Tyr268Cys mutation in the cytochrome b gene, associated with atovaquone resistance, in isolates collected on days 29 and 34 and P. falciparum dihydrofolate reductase mutation Asn51Ile, associated with proguanil resistance in all successfully sequenced samples. Molecular characterization of imported malaria contributes to clinical management in non-endemic countries, helps ascertain the appropriateness of antimalarial treatment policies, and contributes to the reporting of drug resistance patterns from endemic regions.

Integrated community case management (iCCM) of malaria complements public health services to improve access to timely diagnosis and treatment of malaria. ICCM relies on standardized test-and-treat algorithms implemented by community health workers using malaria rapid diagnostic tests (RDTs). However, due to a changing epidemiology of fever causes in Africa, positive RDT results might not correctly reflect malaria. In this study, we modeled diagnostic predictive values for all malaria-endemic African regions as an indicator of the programmatic usefulness of RDTs in iCCM campaigns on malaria.
Positive predictive values (PPVs) and negative predictive values (NPVs) of RDTs for clinical malaria were modeled. Assay-specific performance characteristics stem from the Cochrane Library and data on the proportion of malaria-attributable fevers among African febrile children aged <5 years were used as prevalence matrix.
Average country-level PPVs vary considerably. Ethiopia had the lowest PPVs (histidine-rich protein II [HRP2] assay, 17.35%; parasite lactate dehydrogenase [pLDH] assay, 39.73%), and Guinea had the highest PPVs (HRP2 assay, 95.32%; pLDH assay, 98.46%). On the contrary, NPVs were above 90% in all countries (HRP2 assay, ≥94.87%; pLDH assay, ≥93.36%).
PPVs differed considerably within Africa when used to screen febrile children, indicating unfavorable performance of RDT-based test-and-treat algorithms in low-PPV settings. This suggests that the administration of antimalarials alone may not constitute causal treatment in the presence of a positive RDT result for a substantial proportion of patients, particularly in low-PPV settings. Therefore, current iCCM algorithms should be complemented by information on other setting-specific major causes of fever.

More than twenty years ago the reverse vaccinology paradigm came to light trying to design new vaccines based on the analysis of genomic information in order to select those pathogen peptides able to trigger an immune response. In this context, focusing on the proteome of Trypanosoma cruzi, we investigated the link between the probabilities for pathogen peptides to be presented on a cell surface and their distance from human self. We found a reasonable but, as far as we know, undiscovered property: the farther the distance between a peptide and the human-self the higher the probability for that peptide to be presented on a cell surface. We also found that the most distant peptides from human self bind, on average, a broader collection of HLAs than expected, implying a potential immunological role in a large portion of individuals. Finally, introducing a novel quantitative indicator for a peptide to measure its potential immunological role, we proposed a pool of peptides that could be potential epitopes and that can be suitable for experimental testing. The software to compute peptide classes according to the distance from human self is free available at http://www.iasi.cnr.it/~dsantoni/nullomers.

On the surface of the Leishmania promastigote, phosphoglycans (PG) such as lipophosphoglycan (LPG), proteophosphoglycan (PPG), free phosphoglycan polymers (PGs), and acid phosphatases (sAP), are dominant and contribute to the invasion and survival of Leishmania within the host cell by modulating macrophage signaling and intracellular trafficking. Phosphoglycan synthesis depends on the Golgi GDP-mannose transporter encoded by the LPG2 gene. Aiming to investigate the role of PG-containing molecules in Leishmania infantum infection process, herein we describe the generation and characterization of L. infantum LPG2-deficient parasites. This gene was unexpectedly identified as duplicated in the L. infantum genome, which impaired gene targeting using the conventional homologous recombination approach. This limitation was circumvented by the use of CRISPR/Cas9 technology. Knockout parasites were selected by agglutination assays using CA7AE antibodies followed by a lectin (RCA 120). Five clones were isolated and molecularly characterized, all revealing the expected edited genome, as well as the complete absence of LPG and PG-containing molecule expression. Finally, the deletion of LPG2 was found to impair the outcome of infection in human neutrophils, as demonstrated by a pronounced reduction (~83%) in intracellular load compared to wild-type parasite infection. The results obtained herein reinforce the importance of LPG and other PGs as virulence factors in host-parasite interactions.

As fragment-based drug discovery has become mainstream, there has been an increase in various screening methodologies. Protein-observed 19F (PrOF) NMR and 1H CPMG NMR are two fragment screening assays that have complementary advantages. Here, we sought to combine these two NMR-based assays into a new screening workflow. This combination of protein- and ligand-observed experiments allows for a time- and resource-efficient multiplexed screen of mixtures of fragments and proteins. PrOF NMR is first used to screen mixtures against two proteins. Hit mixtures for each protein are identified then deconvoluted using 1H CPMG NMR. We demonstrate the benefit of this fragment screening method by conducting the first reported fragment screens against the bromodomains of BPTF and Plasmodium falciparum (Pf) GCN5 using 467 3D-enriched fragments. The hit rates were 6%, 5% and 4% for fragments binding BPTF, PfGCN5, and fragments binding both proteins, respectively. Select hits were characterized, revealing a broad range of affinities from low µM to mM dissociation constants. Follow-up experiments supported a low-affinity second binding site on PfGCN5. This approach can be used to bias fragment screens towards more selective hits at the onset of inhibitor development in a resource- and time-efficient manner.

Following the diagnosis of a falciparum malaria case imported from Djibouti and not detected by a pfHRP2-based rapid diagnostic test (RDT), we investigated the prevalence of the pfhrp2/pfhrp3-deleted parasites in Djibouti using 378 blood samples collected between January and May 2019, from Djiboutian patients with suspected malaria. Malaria diagnosis by quantitative PCR confirmed the presence of Plasmodium falciparum for 20.9% (79/378) samples while RDTs did not detect HRP2 antigen in 83.5% (66/79) of these samples. Quantitative PCRs targeting the pfhrp2/pfhrp3 genes confirmed the absence of both genes for 86.5% of P. falciparum strains. The very large number (86.5%) of falciparum parasites lacking the pfhrp2/pfhrp3 genes observed in this study, now justifies the use of non-HRP2 alternative RDTs in Djibouti. In this area and in most countries where HRP2-based RDTs constitute the main arsenal for falciparum malaria diagnosis, it is important to implement a systematic surveillance and to inform biologists and clinicians about the risk of malaria misdiagnosis. Further investigations are needed to better understand the mechanism of selection and diffusion of the pfhrp2/pfhrp3-deleted parasites.

We report a case of severe congenital toxoplasmosis that involved an atypical T. gondii genotype in a newborn baby from Alagoas state in Northeastern Brazil. A pregnant woman presented IgM and IgG anti-T. gondii antibodies, as detected by the chemiluminescence immunoassay on the second trimester of pregnancy. A mouse bioassay was performed using umbilical cord blood and one isolate was obtained. The isolate was designated TgCTBrAL1 and genetic characterization revealed genotype ToxoDB #162. Genotype results of the rhoptry genes, ROP5 and ROP18, could predict the high virulence of the isolate in mice, which was confirmed by an in vivo virulence assay. This is the first report of generating a T. gondii isolate from a newborn baby with congenital toxoplasmosis in Northeastern Brazil.

Combating malaria is almost a never-ending battle, as Plasmodium parasites develop resistance to the drugs used against them, as observed recently in artemisinin-based combination therapies. The main concern now is if the resistant parasite strains spread from Southeast Asia to Africa, the continent hosting most malaria cases. To prevent catastrophic results, we need to find non-conventional approaches. Allosteric drug targeting sites and modulators might be a new hope for malarial treatments. Heat shock proteins (HSPs) are potential malarial drug targets and have complex allosteric control mechanisms. Yet, studies on designing allosteric modulators against them are limited. Here, we identified allosteric modulators (SANC190 and SANC651) against P. falciparum Hsp70-1 and Hsp70-x, affecting the conformational dynamics of the proteins, delicately balanced by the endogenous ligands. Previously, we established a pipeline to identify allosteric sites and modulators. This study also further investigated alternative approaches to speed up the process by comparing all atom molecular dynamics simulations and dynamic residue network analysis with the coarse-grained (CG) versions of the calculations. Betweenness centrality (BC) profiles for PfHsp70-1 and PfHsp70-x derived from CG simulations not only revealed similar trends but also pointed to the same functional regions and specific residues corresponding to BC profile peaks.

Community-based active case detection of malaria parasites with conventional rapid diagnostic tests (cRDTs) is a strategy used most commonly in low-transmission settings. We estimated the sensitivity of this approach in a high-transmission setting in Western Kenya. We tested 3,547 members of 912 households identified in 2013-2014 by index children with (case) and without (control) cRDT-positive malaria. All were tested for Plasmodium falciparum with both a cRDT targeting histidine-rich protein 2 and with an ultrasensitive real-time polymerase chain reaction (PCR). We computed cRDT sensitivity against PCR as the referent, compared prevalence between participant types, and estimated cRDT detectability as a function of PCR-estimated parasite density. Parasite prevalence was 22.9% by cRDTs and 61.5% by PCR. Compared with children aged < 5 years or adults aged > 15 years, geometric mean parasite densities (95% CI) were highest in school-age children aged 5-15 years (8.4 p/uL; 6.6-10.6). The overall sensitivity of cRDT was 36%; among asymptomatic household members, cRDT sensitivity was 25.5% and lowest in adults aged > 15 years (15.8%). When modeled as a function of parasite density, relative to school-age children, the probability of cRDT positivity was reduced in both children aged < 5 years (odds ratio [OR] 0.48; 95% CI: 0.34-0.69) and in adults aged > 15 years (OR: 0.35; 95% CI: 0.27-0.47). An HRP2-detecting cRDT had poor sensitivity for active P. falciparum case detection in asymptomatic community members, and sensitivity was lowest in highly prevalent low-density infections and in adults. Future studies can model the incremental effects of high-sensitivity rapid diagnostic tests and the impacts on transmission.