Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.
According to current estimates, Plasmodium malariae is not very common in Senegal, as more than 98% of malaria cases are suspected to be due to Plasmodium falciparum. However, it is possible that other malarial species are being under-reported or misdiagnosed. This is a report of a case of P. malariae in a 30-year-old man previously hospitalized with acute kidney injury after treatment with quinine and re-hospitalized three months later. He was diagnosed with renal cortical necrosis post malaria treatment. Plasmodium malariae was identified with light microscope and confirmed using species-specific small-subunit rRNA (ssrRNA) amplification.The patient was treated for malaria with intravenous quinine for seven days, followed by three days of oral treatment; the bacterial infection was treated using ceftriaxone during the first hospitalization and ciprofloxacin associated with ceftriaxone the second time. He also had four rounds of dialysis after which he partially recovered the renal function. Given the complications that can be caused by P. malariae infection, it should be systematically looked for, even if the predominant species is P. falciparum in Senegal.
BackgroundThe World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. In addition to providing quick and accurate detection of Plasmodium parasite proteins in the blood, these tests can be used as sources of DNA for further genetic studies. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs.MethodsThe proximal portion of the nitrocellulose membrane of discarded RDTs was used for DNA extraction. This genomic DNA was amplified using HRM to genotype the molecular markers involved in resistance to sulfadoxine-pyrimethamine: dhfr (51, 59, 108, and 164) and dhps (436, 437, 540, 581, and 613). Additionally, the msp1 and msp2 genes were amplified to determine the average clonality between Grande-Comore (Comoros) and Thiès (Senegal).ResultsA total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR. A high prevalence of resistance mutations were observed in the dhfr gene at codons 51, 59, and 108 as well as in the dhps gene at codons 437 and 436. A novel mutant in dhps at codon positions 436Y/437A was observed. The dhfr I164L codon and dhps K540 and dhps A581G codons had 100 % wild type alleles in all samples.ConclusionThe utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting). RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0861-6) contains supplementary material, which is available to authorized users.
In the Union of Comoros, interventions for combating malaria have contributed to a spectacular decrease in the prevalence of the disease. We studied the current distribution of Plasmodium species on the island of Grande Comore using nested PCR. The rapid diagnostic tests (RDTs) currently used in the Comoros are able to identify Plasmodium falciparum but no other Plasmodium species. In this study, we tested 211 RDTs (158 positive and 53 negative). Among the 158 positive RDTs, 22 were positive for HRP2, 3 were positive only for pLDH, and 133 were positive for HRP2 and pLDH. DNA was extracted from a proximal part of the nitrocellulose membrane of RDTs. A total of 159 samples were positive by nested PCR. Of those, 156 (98.11%) were positive for P. falciparum, 2 (1.25%) were positive for P. vivaxI, and 1 (0.62%) was positive for P. malariae. None of the samples were positive for P. ovale. Our results show that P. falciparum is still the most dominant species on the island of Grande Comore, but P. vivax and P. malariae are present at a low prevalence.
BackgroundEmergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction–restriction fragment length polymorphism (PCR/RFLP) methodology.MethodsFifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR–RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries.ResultsA high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania.ConclusionHere the results demonstrate that HRM is a rapid, sensitive, and field-deployable alternative technique to PCR–RFLP genotyping that is useful in populations harbouring more than one parasite genome (polygenomic infections). In this study, a high levels of resistance polymorphisms was observed in both dhfr and dhps, among samples from Tanzania and Sénégal. A routine monitoring by molecular markers can be a way to detect emergence of resistance involving a change in the treatment policy.
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