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In Italy, surveillance through mandatory notification of cases to the National Surveillance System (NSS) has shown evidence of underreporting over the years. To evaluate the overall quality of malaria dataset, Hospital Discharge Records (HDRs) were analyzed as a second data source. Malaria cases by NSS and by HRDs were compared and analyzed from 2011-through 2017. The impact of cases was estimated by annual rates per 100,000 residents. Cases reported to NSS and to HDRs were 5,149 and 6,446, respectively. The annual rate recorded by NSS increased from 1.2 per 100,000 in 2011 to 1.4 per 100,000 in 2017, a similar trend was shown by HDRs, from 1.4 per 100,000 in 2011 to 1.6 per 100,000 in 2017. Every year, the number of NSS cases was lower than HDRs cases suggesting moderate underreporting of the mandatory notification. In both data sources adult males aged 25 to 44, and non-Italian travellers visiting friends and relatives were the most affected groups; Plasmodium falciparum was the prevalent agent identified, being the imported cases originated mainly from sub-Saharan Africa. As places of diagnosis and care, both data sources indicated hospitals located in Northern Italy in over 70% of cases. Although the comparison of malaria cases highlighted some underreporting by NSS, a fair agreement between the two institutional information systems was observed. The use of both data sources improves the performance of malaria surveillance in Italy, essentially for early warning systems in case of locally-acquired events and primary prevention in international travellers. [ABSTRACT FROM AUTHOR]

Interference with transforming growth factor-β-mediated pathways helps several parasites to survive for long periods in immunocompetent hosts. Macrophages and dendritic cells infected by Toxoplasma , Leishmania and Plasmodium spp. produce large amounts of transforming growth factor-β and induce the differentiation of antigen-specific T-regulatory cells. Mechanisms not mediated by antigen-presentation could also account for the expansion of T-regulatory cells in parasitic diseases and they also might be mediated through transforming growth factor-β-receptor activated pathways. We explored the properties of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites, Trichinella spiralis muscle larvae to expand the pool of T-regulatory cells in a population of polyclonally activated T cells in the absence of accessory cells, and compared their effects to those induced by Plasmodium falciparum extracts. Similarly to P. falciparum, L. infantum extracts activate the latent soluble form of transforming growth factor-β and that bound to the membrane of activated T lymphocytes. The interaction of the active cytokine with transforming growth factor-β receptor induces Foxp3 expression by activated lymphocytes, favoring their conversion through the T-regulatory phenotype. Both Toxoplasma gondii and L. infantum extracts are able to induce transforming growth factor-β production by activated T cells in the absence of accessory cells. [ABSTRACT FROM AUTHOR]

Plasmodium vivax was endemic in temperate areas in historic times up to the middle of last century. Temperate climate P. vivax has a long incubation time of up to 8e10 months, which partly explain how it can be endemic in temperate areas with a could winter. P. vivax disappeared from Europe within the last 40e60 years, and this change was not related to climatic changes. The surge of P. vivax in Northern Europe after the second world war was related to displacement of refugees and large movement of military personnel exposed to malaria. Lately P. vivax has been seen along the demilitarized zone in South Korea replication a high endemicity in North Korea. The potential of transmission of P. vivax still exist in temperate zones, but reintroduction in a larger scale of P. vivax to areas without present transmission require large population movements of P. vivax infected people. The highest threat at present is refugees from P. vivax endemic North Korea entering China and South Korea in large numbers. [ABSTRACT FROM AUTHOR]

Summary: Background: The pathogenesis of malaria is the result of complex interactions between parasites, host and environment. Several studies have assessed the role of genetic characteristics of Plasmodium falciparum infection in the clinical severity of malaria infection comparing different genotypic determinants in mild and severe cases. The genes encoding the polymorphic merozoite surface proteins 1 (msp-1) and 2 (msp-2) and the dihydrofolate reductase (dhfr) of malaria parasites have been extensively used as markers to investigate the genetic diversity and the population structure of P. falciparum. The aim of this study was to assess the epidemiological, clinical, host- and parasite-related determinant factor of the genetic diversity of P. falciparum infections in travellers returning to Italy. Methods: Between 1998 and 2001, we have retrospectively studied 64 inpatients all returning from African malaria-endemic countries. Designation of severe malaria was determined by using the World Health Organization (WHO) definition. P. falciparum infections detected by species-specific PCR were genotyped at the msp-1 and msp-2 loci and clones were determined. PCR and enzyme-digestion methods were used to screen the mutation occurring at codon 108. Results: Multiple P. falciparum genotypes were detected in 32 patients (50%). The number of genotypes was correlated to different host characteristics. No association was found between allelic number of msp-1 or msp-2 and season of travel, absence of antimalarial prophylaxis, length of stay or blood parasitemia. At multiple analysis adjusted for few confounding variables, two variables showed a significant association with multiplicity of P. falciparum genotypes: male gender (p=0.018) and severity of disease (p=0.044). Conclusion: In our study all but one patients with severe malaria had a infection with a multiplicity of P. falciparum clones. At multivariate analysis the male gender, and the occurrence of severe malaria were significanly more commonly detected in patients affected by imported malaria with multiple clones. [Copyright &y& Elsevier]

Abstract: The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I–75E) pfcrt point mutation as molecular marker for CQR. [Copyright &y& Elsevier]

Malaria due to Plasmodium vivax is globally widespread and is associated with substantial morbidity. The parasite was previously prevalent in temperate areas from which it has been eradicated, however there is a risk of re-introduction because of increased international travel and migration. Following the occurrence of an autochthonous case of P. vivax malaria in Italy after decades of malaria eradication, we applied a molecular approach to compare parasites involved in the introduced case and to determine whether a highly polymorphic gene marker could be useful to tag a P. vivax isolate geographically. To this end, the sequence encompassing the interspecies conserved blocks 5 and 6 of the gene encoding for merozoite surface protein 1 (msp-1) was determined in 16 P. vivax isolates from different regions, and analysed along with 24 pvmsp-1 sequences downloaded from published data. Results have shown that: (i) parasites from the introduced case and the putative source of infection identified following epidemiological investigation, although very similar, differed in three nucleotide substitutions, of which one non synonymous; ii) some geographical isolates looked tightly clustered (e.g. Korean and Punjab isolates), but others were less so. [Copyright &y& Elsevier]

• Treatment failure of intravenous artesunate and oral dihydroartemisinin-piperaquine was observed in a malaria case • Molecular markers of resistance to artemisinin or piperaquine were not detected • Drug malabsorption or poor manufacturing practices could be responsible for treatment failure • Surveillance of antimalarial resistance in imported malaria cases is warranted A 68-year-old man returning from Republic of Côte d'Ivoire (Ivory Coast) was diagnosed with severe Plasmodium falciparum malaria and treated with intravenous artesunate followed by oral dihydroartemisinin-piperaquine (DHA-PPQ). A month later the patient experienced a new P. falciparum episode; analysis of pfmsp-1 and pfmsp-2 revealed that the infection was caused by a genetic strain identical to the strain that caused the initial episode, indicating resurgence of the previous infection. No mutations in genes associated with resistance to artemisinin derivatives (pfk13) or piperaquine (pfexonuclease, pfplasmepsin 2/3) were detected, suggesting that treatment failure could have been caused by drug malabsorption or poor drug manufacturing practices. A second treatment with atovaquone-proguanil was successful in eliminating the infection, with no further relapses. To our knowledge, this is the first description of a treatment failure with both artesunate and DHA-PPQ in a traveler returning from a malaria-endemic region. Analysis of molecular markers of resistance to antimalarial drugs revealed mutations associated with resistance to sulfadoxine (pfdhps) and pyrimethamine (pfdhfr) , highlighting the important contribution of surveillance of imported malaria cases to the monitoring of drug resistance globally. [ABSTRACT FROM AUTHOR]

Abstract: Dihydropteroate synthase gene of Plasmodium vivax was recently identified. In the present study, the sequences of the dyhydropteroate synthase gene of 68 P. vivax isolates from various geographic areas were compared. Sequencing revealed limited polymorphism at codons 383 and 553 in all analyzed samples. Interstrain analysis showed several genotypic variations in the tandem repeats domain which produce length polymorphism between different parasite isolates. [Copyright &y& Elsevier]

[Display omitted] • Artemisinin-based combination therapy for the treatment of malaria cases in Cameroon. • Drug resistance remains one of the greatest challenges of malaria control. • Next-generation sequencing is an effective method for monitoring drug-resistant Plasmodium falciparum parasites. • Artemisinin-based combination therapy is still effective in Cameroon. • The high prevalence of dhps / dhfr mutations suggests active surveillance of sulfadoxine-pyrimethamine efficacy is important. Recent studies showed the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. In this study, we used targeted amplicon deep sequencing of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes, k13 , crt , mdr1 , dhfr , and dhps , and the cytochrome b gene (cytb) in Plasmodium falciparum. No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt , Pfmdr1 , Pfdhfr , and Pfdhps. Notably, a high frequency of other nonsynonymous mutations was observed across all the genes, except for Pfcytb , suggesting continued selection pressure. The results from this study supported the continued use of artemisinin-based combination therapy and administration of sulfadoxine-pyrimethamine for intermittent preventive therapy in pregnant women, and for seasonal chemoprevention in these study sites in Cameroon. [ABSTRACT FROM AUTHOR]

The histidine-rich protein 2 of Plasmodium falciparum is the most common malaria antigen targeted by rapid diagnostic tests for the specific diagnosis of P. falciparum . Recently, pfhrp2 gene deletions have been documented in P. falciparum isolates from South America and some multiple endemic countries in Africa and Asia. Parasites with such gene deletions can produce false negative diagnostic results using HRP2-based rapid diagnostic kits. In the present work, the prevalence of P. falciparum parasites lacking pfhrp2 , pfhrp3 , which produces a second P. falciparum antigen that is recognized by PfHRP2 -based rapid diagnostic tests, and their flanking genes was evaluated in 135 P. falciparum isolates from Gash Barka region and in 9 isolates from Debub region, in Eritrea. In the analyzed samples, 56% (81/144) of isolates were pfhrp2 / pfhrp3 positive, while 9.7% (14/144) showed deletion of exon 2 of pfhrp2 gene and 43% (62/144) of isolates lacked the pfhrp3 gene. These results suggest that the pfhrp2 and pfhrp3 deletion phenomenon is present in a considerable proportion in the study areas, thus making the HRP2/3 based rapid diagnostic tests not completely reliable for malaria diagnosis in Eritrea. [ABSTRACT FROM AUTHOR]

The introduction of artemisinin-based combination therapy has led to extraordinary results in malaria control, however the recent emergence of partial resistance to artemisinin therapy in Southeast Asia jeopardizes these successes. This study aimed at investigating resistance to the antimalarial drugs by evaluating the polymorphisms in the PfK13 , Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolates obtained from patients in Eritrea. [ABSTRACT FROM AUTHOR]

Polymorphic genetic markers and especially microsatellite analysis can be used to investigate multiple aspects of the biology of Plasmodium species. In the current study, we characterized 7 polymorphic microsatellites in a total of 281 Plasmodium vivax isolates to determine the genetic diversity and population structure of P. vivax populations from Sudan, Madagascar, French Guiana, and Armenia. All four parasite populations were highly polymorphic with 3–32 alleles per locus. Mean genetic diversity values was 0.83, 0.79, 0.78 and 0.67 for Madagascar, French Guiana, Sudan, and Armenia, respectively. Significant genetic differentiation between all four populations was observed. [ABSTRACT FROM AUTHOR]

Abstract: A new seminested polymerase chain reaction (sn-PCR)–based protocol was developed and used to detect and identify Plasmodium species in 1226 whole-blood samples from patients (872 Italians and 354 foreigners) with at least 1 symptom compatible with clinical malaria. The results were compared with those obtained by microscopy: 187 samples were positive by microscopy for malaria parasites and 196 were positive by sn-PCR. When compared to microscopy, the sn-PCR detected different malaria parasite species in 11 cases. In 4 of 11 cases, the sn-PCR identified 1 additional malaria parasite species not observed microscopically, suggesting increased sensitivity. In 4 samples with levels of parasitemia too low for accurate identification of species by microscopy, the sn-PCR detected 2 P. falciparum, 1 P. ovale, and 1 P. falciparum plus P. ovale. Moreover, 9 negative samples by microscopy were positive by sn-PCR. Follow-up analysis demonstrated a parasite clearance of P. falciparum DNA up to 3 days after the disappearance of parasitemia at microscopy. In conclusion, sn-PCR–based diagnosis of malaria appears to be a useful tool when the results of conventional techniques are negative in the presence of a syndrome consistent with malaria, yielding accurate species identification and consequential correct treatment. [Copyright &y& Elsevier]

Abstract: A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with Ki values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication. [Copyright &y& Elsevier]

Abstract: A variegate group of metallodrugs was evaluated in vitro for antimalarial activity through the pLDH test. The panel comprised one mononuclear gold(III) complex, (Aubipy), three dinuclear gold(III) compounds (Auoxo4, Auoxo5 and Auoxo6), three ruthenium(III) complexes (NAMI A, PMRU20, PMRU27), one ruthenium(II) complex (PMRU52), one bismuth(III) compound (Bismuth citrate), antimony trichloride (SbCl3 ) and arsenic trioxide (As2O3 ). This panel, although relatively small, was built up in such a way to include a variety of metal centers, structural motifs and metal coordination environments. In general, the tested compounds turned out to contrast effectively Plasmodium falciparum growth in vitro. In two cases, i.e. NAMI A and antimony trichloride, IC50 values in the high nanomolar range were measured. Notably, the antiplasmodial effects appear not to be correlated to in vitro anticancer properties. The mechanistic and pharmacological implications of the obtained results are discussed. [Copyright &y& Elsevier]