Last data update: Sep 16, 2024. (Total: 47680 publications since 2009)
Records 1-13 (of 13 Records) |
Query Trace: Dragan L [original query] |
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Therapeutic efficacy of artemether-lumefantrine and artesunate-amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Mali, 2015-2016.
Diarra Y , Koné O , Sangaré L , Doumbia L , Haidara DBB , Diallo M , Maiga A , Sango HA , Sidibé H , Mihigo J , Nace D , Ljolje D , Talundzic E , Udhayakumar V , Eckert E , Woodfill CJ , Moriarty LF , Lim P , Krogstad DJ , Halsey ES , Lucchi NW , Koita OA . Malar J 2021 20 (1) 235 BACKGROUND: The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali. METHODS: Children between 6 and 59 months of age with uncomplicated Plasmodium falciparum infection and 2000-200,000 asexual parasites/μL of blood were enrolled, randomly assigned to either AL or ASAQ, and followed up for 42 days. Uncorrected and PCR-corrected efficacy results at days 28 and 42. were calculated. Known markers of resistance in the Pfk13, Pfmdr1, and Pfcrt genes were assessed using Sanger sequencing. RESULTS: A total of 449 patients were enrolled: 225 in the AL group and 224 in the ASAQ group. Uncorrected efficacy at day 28 was 83.4% (95% CI 78.5-88.4%) in the AL arm and 93.1% (95% CI 89.7-96.5%) in the ASAQ arm. The per protocol PCR-corrected efficacy at day 28 was 91.0% (86.0-95.9%) in the AL arm and 97.1% (93.6-100%) in the ASAQ arm. ASAQ was significantly (p < 0.05) better than AL for each of the aforementioned efficacy outcomes. No mutations associated with artemisinin resistance were identified in the Pfk13 gene. Overall, for Pfmdr1, the N86 allele and the NFD haplotype were the most common. The NFD haplotype was significantly more prevalent in the post-treatment than in the pre-treatment isolates in the AL arm (p < 0.01) but not in the ASAQ arm. For Pfcrt, the CVIET haplotype was the most common. CONCLUSIONS: The findings indicate that both AL and ASAQ remain effective for the treatment of uncomplicated malaria in Sélingué, Mali. |
Detection of malaria parasites in samples from returning US travelers using the Alethia® Malaria Plus LAMP assay.
Ljolje D , Abdallah R , Lucchi NW . BMC Res Notes 2021 14 (1) 128 OBJECTIVE: In this study, the performance of a commercially available malaria LAMP assay (Alethia® Malaria Plus LAMP) was evaluated using retrospective clinical samples obtained from travelers returning to the United States of America (USA). Recently, several laboratories in non-malaria endemic countries evaluated the use of the loop mediated isothermal amplification (LAMP) assays for the diagnosis of imported malaria cases. These tests are simpler than polymerase-chain reaction (PCR)-based assays and were shown to have high sensitivity. Much of malaria diagnoses in the USA, is undertaken at the state level using mainly microscopy and rapid diagnostic tests (RDTs). However, molecular tools offer greater sensitivity over microscopy and RDTs. A reliable, easy to perform molecular assay can provide a test of choice for the accurate detection of malaria parasites in places where expert microscopy is lacking and/or for the detection of low-parasite density infections. RESULTS: The Alethia® Malaria Plus LAMP assay was easy to use, had similar test performances as the real-time PCR reference test and results were obtained faster (within 1 h) than the reference test. The sensitivity of the assay was 100% with a kappa score of 1 when compared to the reference PET-PCR assay. |
Artemether-Lumefantrine Efficacy for the Treatment of Uncomplicated Plasmodium falciparum Infection in Choco, Colombia after 8 Years as First-Line Treatment.
Olivera MJ , Guerra AP , Cortes LJ , Horth RZ , Padilla J , Novoa J , Ade MP , Ljolje D , Lucchi NW , Marquino W , Renteria M , Yurgaky W , Macedo de Oliveira A . Am J Trop Med Hyg 2020 102 (5) 1056-1063 Artemether-lumefantrine (AL) is the first-line treatment for uncomplicated Plasmodium falciparum infection in Colombia. To assess AL efficacy for uncomplicated falciparum malaria in Quibdo, Choco, Colombia, we conducted a 28-day therapeutic efficacy study (TES) following the WHO guidelines. From July 2018 to February 2019, febrile patients aged 5-65 years with microscopy-confirmed P. falciparum mono-infection and asexual parasite density of 250-100,000 parasites/microL were enrolled and treated with a supervised 3-day course of AL. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28. We attempted to use polymerase chain reaction (PCR) genotyping to differentiate reinfection and recrudescence, and conducted genetic testing for antimalarial resistance-associated genes. Eighty-eight patients consented and were enrolled: four were lost to follow-up or missed treatment doses. Therefore, 84 (95.5%) participants reached a valid endpoint: treatment failure or ACPR. No patient remained microscopy positive for malaria on day 3, evidence of delayed parasite clearance and artemisinin resistance. One patient had recurrent infection (12 parasites/microL) on day 28. Uncorrected ACPR rate was 98.8% (83/84) (95% CI: 93.5-100%). The recurrent infection sample did not amplify during molecular testing, giving a PCR-corrected ACPR of 100% (83/83) (95% CI: 95.7-100%). No P. falciparum kelch 13 polymorphisms associated with artemisinin resistance were identified. Our results support high AL efficacy for falciparum malaria in Choco. Because of the time required to conduct TESs in low-endemic settings, it is important to consider complementary alternatives to monitor antimalarial efficacy and resistance. |
Screening for Pfhrp2/3-Deleted Plasmodium falciparum, Non-falciparum, and Low-Density Malaria Infections by a Multiplex Antigen Assay.
Plucinski MM , Herman C , Jones S , Dimbu R , Fortes F , Ljolje D , Lucchi N , Murphy SC , Smith NT , Cruz KR , Seilie AM , Halsey ES , Udhayakumar V , Aidoo M , Rogier E . J Infect Dis 2018 219 (3) 437-447 Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis worldwide. Methods: We developed a sensitive bead-based multiplex assay for laboratory use which simultaneously detects the pan-Plasmodium pAldo, pan-Plasmodium pLDH, and P. falciparum PfHRP2 antigens. The assay was validated against purified recombinant antigens, mono-species malaria infections, and non-infected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n=1267) were assayed. Results: Of 466 Angolan samples positive for at least one antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of qRT-PCR positivity and parasite density. Eight Angolan samples (1.7%) had either no or very low levels of PfHRP2 but were positive for one or both of the other antigens. PCR analysis confirmed three (0.6%) were P. ovale infections, and two (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3. Conclusions: These are the first reports of P. falciparum Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low density P. falciparum, non-falciparum, and Pfhrp2/3-deleted malaria parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization. |
Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015.
Ljolje D , Dimbu PR , Kelley J , Goldman I , Nace D , Macaia A , Halsey ES , Ringwald P , Fortes F , Udhayakumar V , Talundzic E , Lucchi NW , Plucinski MM . Malar J 2018 17 (1) 84 BACKGROUND: Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola. METHODS: DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated. RESULTS: The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether-lumefantrine (p value 0.03). CONCLUSIONS: The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola's three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether-lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether-lumefantrine treatment arms. |
A next-generation sequencing and bioinformatics protocol for Malaria drug Resistance marker Surveillance (MaRS).
Talundzic E , Ravishankar S , Kelly J , Patel D , Plucinski M , Schmedes S , Ljolje D , Clemons B , Madison-Antenucci S , Arguin PM , Lucchi N , Vannberg F , Udhayakumar V . Antimicrob Agents Chemother 2018 62 (4) The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug resistant parasites. To take advantage of this technology an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called Malaria Resistance Surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and cytochrome b) as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistant crt CV IET genotype was observed in 42% of samples, the highly pyrimethamine resistant triple mutant dhpsIRN in 92% of samples, and the sulfadoxine resistant dhps S GE AA in 26% of samples. The mdr1 N F SND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistant K13 alleles were identified and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the U.S. and aid in the adoption of this system in participating state public health laboratories as well as global partners. |
Molecular Characterization of a Cluster of Imported Malaria Cases in Puerto Rico.
Chenet SM , Silva-Flannery L , Lucchi NW , Dragan L , Dirlikov E , Mace K , Rivera-Garcia B , Arguin PM , Udhayakumar V . Am J Trop Med Hyg 2017 97 (3) 758-760 The Caribbean island of Hispaniola is targeted for malaria elimination. Currently, this is the only island with ongoing transmission of malaria in the Caribbean. In 2015, six patients from Puerto Rico and one from Massachusetts, who traveled to Punta Cana, Dominican Republic, were confirmed to be infected with Plasmodium falciparum. Additional molecular analysis was performed at the Centers for Disease Control and Prevention to characterize the drug-resistant alleles and Plasmodium population genetic markers. All specimens carried wildtype genotypes for chloroquine, sulfadoxine-pyrimethamine, and artemisinin resistance genetic markers. A mutation in codon 184 (Y/F) of Pfmdr-1 gene was observed in all samples and they shared an identical genetic lineage as determined by microsatellite analysis. This genetic profile was similar to the one reported from Hispaniola suggesting that a clonal P. falciparum residual parasite population present in Punta Cana is the source population for these imported malaria cases. |
Molecular diagnosis of Plasmodium ovale by photo-induced electron transfer fluorogenic primers: PET-PCR.
Akerele D , Ljolje D , Talundzic E , Udhayakumar V , Lucchi NW . PLoS One 2017 12 (6) e0179178 Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/mul. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs. |
Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites.
Lucchi NW , Gaye M , Diallo MA , Goldman IF , Ljolje D , Deme AB , Badiane A , Ndiaye YD , Barnwell JW , Udhayakumar V , Ndiaye D . Sci Rep 2016 6 36808 Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/mul depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis. |
Plasmodium falciparum Drug-Resistant Haplotypes and Population Structure in Postearthquake Haiti, 2010.
Morton LC , Huber C , Okoth SA , Griffing S , Lucchi N , Ljolje D , Boncy J , Oscar R , Townes D , McMorrow M , Chang MA , Udhayakumar V , Barnwell JW . Am J Trop Med Hyg 2016 95 (4) 811-816 Chloroquine (CQ) remains the first-line treatment of malaria in Haiti. Given the challenges of conducting in vivo drug efficacy trials in low-endemic settings like Haiti, molecular surveillance for drug resistance markers is a reasonable approach for detecting resistant parasites. In this study, 349 blood spots were collected from suspected malaria cases in areas in and around Port-au-Prince from March to July 2010. Among them, 121 samples that were Plasmodium falciparum positive by polymerase chain reaction were genotyped for drug-resistant pfcrt, pfdhfr, pfdhps, and pfmdr1 alleles. Among the 108 samples that were successfully sequenced for CQ resistant markers in pfcrt, 107 were wild type (CVMNK), whereas one sample carried a CQ-resistant allele (CVIET). Neutral microsatellite genotyping revealed that the CQ-resistant isolate was distinct from all other samples in this study. Furthermore, the remaining parasite specimens appeared to be genetically distinct from other reported Central and South American populations. |
Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.
Lucchi NW , Ljolje D , Silva-Flannery L , Udhayakumar V . PLoS One 2016 11 (3) e0151437 Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63 degrees C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/muL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. |
Increasing Prevalence of a Novel Triple-Mutant Dihydropteroate Synthase Genotype in Plasmodium falciparum in Western Kenya.
Lucchi NW , Okoth SA , Komino F , Onyona P , Goldman IF , Ljolje D , Shi YP , Barnwell JW , Udhayakumar V , Kariuki S . Antimicrob Agents Chemother 2015 59 (7) 3995-4002 The molecular basis of sulfadoxine-pyrimethamine (SP) resistance lies in a combination of single-nucleotide polymorphisms (SNPs) in two genes coding for Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and P. falciparum dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. The continued use of SP for intermittent preventive treatment in pregnant women in many African countries, despite SP's discontinuation as a first-line antimalarial treatment option, due to high levels of drug resistance, may further increase the prevalence of SP-resistant parasites and/or lead to selection of new mutations. An antimalarial drug resistance surveillance study was conducted in western Kenya between 2010 and 2013. A total of 203 clinical samples from children with uncomplicated malaria were genotyped for SNPs associated with SP resistance. The prevalence of the triple mutant Pfdhfr C50 I51R59N: 108I164 and the double mutant Pfdhps S436 G437E540: A581A613 genotypes was high. Two triple mutant Pfdhps genotypes were found: S436 G437E540G: 581A613 and H: 436 G437E540: A581A613, with the latter, thus far, uniquely found in western Kenya. The prevalence of the S436 G: 437 E: 540 G: 581A613 genotype was low. However, a steady increase in the triple Pfdhps H: 436 G: 437 E: 540A581A613 genotype was observed since its appearance in early 2000. These isolates shared substantial microsatellite haplotypes with the most common double mutant allele suggesting that this triple mutant allele may have evolved locally. Overall, these findings show that the triple H: 436 G437E540: A581A613 mutant may be increasing in this population and could compromise the efficacy of SP for IPTp if it increases the resistant threshold further. |
PET-PCR method for the molecular detection of malaria parasites in a national malaria surveillance study in Haiti, 2011.
Lucchi NW , Karell MA , Journel I , Rogier E , Goldman I , Ljolje D , Huber C , Mace KE , Jean SE , Akom EE , Oscar R , Buteau J , Boncy J , Barnwell JW , Udhayakumar V . Malar J 2014 13 (462) 462 BACKGROUND: Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated. METHODS: DNA from the dried blood spots was extracted using QIAGEN methodology. All 2,989 samples were screened using the PET-PCR assay in duplicate. Samples with a cycle threshold (CT) of 40 or less were scored as positive. A subset of the total samples (534) was retested using a nested PCR assay for confirmation. In addition, these same samples were also tested using a TaqMan-based real-time PCR assay. RESULTS: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7). These same samples were also found to be positive by the nested and TaqMan-based methods. The nested PCR detected an additional positive sample in a subset of 534 samples that was not detected by either PET-PCR or TaqMan-based PCR method. CONCLUSION: While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples. Given the ease of use and lower cost than the nested PCR, the PET-PCR provides an alternative assay for the rapid screening of a large number of samples in laboratory settings. |
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