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Rapid detection of lactate dehydrogenase and genotyping of Plasmodium falciparum in saliva of children with acute uncomplicated malaria.

ABSTRACT: The diagnosis of malaria in biological fluids other than blood using non-invasive, rapid diagnostic techniques provides a valuable approach in case management and epidemiological studies of malaria. Rapid detection of Plasmodium falciparum lactate dehydrogenase (pLDH) in saliva samples from 130 of 144 children with microscopically confirmed P. falciparum infection was evaluated using Optimal-IT dipsticks. Genotyping of parasites was also performed in saliva and blood samples from a cohort of patients by polymerase chain reaction (PCR). The sensitivity of the dipstick in whole-blood, whole-saliva, or supernatant of spun saliva samples was 97.2%, 77.9%, and 48.4%, respectively. The sensitivity of the dipstick in whole-saliva samples was significantly higher than in supernatant of spun saliva samples (P < 0.0005). Mutant T76 allele was detectable in 60% and 57% of blood and saliva samples, respectively. This finding shows rapid detection of pLDH in patient saliva.

SUBMITTER: Gbotosho GO

PROVIDER: S-EPMC2929040 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Rapid detection of lactate dehydrogenase and genotyping of Plasmodium falciparum in saliva of children with acute uncomplicated malaria.

Gbotosho Grace O GO Happi Christian T CT Folarin Onikepe O Keyamo Ochuko O Sowunmi Akintunde A Oduola Ayoade M J AM

The American journal of tropical medicine and hygiene 20100901 3

The diagnosis of malaria in biological fluids other than blood using non-invasive, rapid diagnostic techniques provides a valuable approach in case management and epidemiological studies of malaria. Rapid detection of Plasmodium falciparum lactate dehydrogenase (pLDH) in saliva samples from 130 of 144 children with microscopically confirmed P. falciparum infection was evaluated using Optimal-IT dipsticks. Genotyping of parasites was also performed in saliva and blood samples from a cohort of pat ...[more]

PMID: 20810809

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Project description:Artemisinin-based combination therapy (ACT) is widely recommended as first-line therapy for uncomplicated Plasmodium falciparum malaria worldwide. Artemisinin resistance has now been reported in Southeast Asia with a clinical phenotype manifested by slow parasite clearance. Although there are no reliable reports of artemisinin resistance in Africa, there is a need to better understand the dynamics of parasite clearance in African children treated with ACT in order to better detect the emergence of artemisinin resistance.Data from a cohort of Ugandan children four to five years old, enrolled in a longitudinal, randomized, clinical trial comparing two leading ACT, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP), were analysed. For all episodes of uncomplicated P. falciparum malaria over a 14-month period, daily blood smears were performed for three days following the initiation of therapy. Associations between pre-treatment variables of interest and persistent parasitaemia were estimated using multivariate, generalized, estimating equations with adjustment for repeated measures in the same patient.A total of 202 children were included, resulting in 416 episodes of malaria treated with AL and 354 episodes treated with DP. The prevalence of parasitaemia on days 1, 2, and 3 following initiation of therapy was 67.6, 5.6 and 0% in those treated with AL, and 52.2, 5.7 and 0.3% in those treated with DP. Independent risk factors for persistent parasitaemia on day 1 included treatment with AL vs DP (RR?=?1.34, 95% CI 1.20-1.50, p?<?0.001), having a temperature ?38.0°C vs?<?37.0°C (RR?=?1.19, 95% CI 1.05-1.35, p?=?0.007) and having a parasite density >20,000/?L vs <4,000/?L (RR?=?3.37, 95% CI 2.44-4.49, p?<?0.001). Independent risk factors for having persistent parasitaemia on day 2 included elevated temperature, higher parasite density, and being HIV infected.Among Ugandan children, parasite clearance following treatment with AL or DP was excellent with only one of 752 patients tested having a positive blood slide three days after initiation of therapy. The type of ACT given, pre-treatment temperature, pre-treatment parasite density and HIV status were associated with differences in persistent parasitaemia, one or two days following therapy.Current Controlled Trials Identifier NCT00527800.

Project description:BACKGROUND:Malaria is a major public health problem in Cameroon. The study of the genetic diversity within parasite population is essential for understanding the mechanism underlying malaria pathology and to determine parasite clones profile in an infection, for proper malaria control strategies. The objective of this study was to perform a molecular characterization of highly polymorphic genetic markers of Plasmodium falciparum, and to determine allelic distribution with their influencing factors valuable to investigate malaria transmission dynamics in Cameroon. METHODS:A total of 350 P. falciparum clinical isolates were characterized by genotyping block 2 of msp-1, block 3 of msp-2, and region II of glurp gene using nested PCR and DNA sequencing between 2012 and 2013. RESULTS:A total of 5 different genotypes with fragment sizes ranging from 597 to 817 bp were recorded for GLURP. Overall, 16 MSP-1 genotypes, including K1, MAD20 and RO33 were identified, ranging from 153 to 335 bp. A peculiarity about this study is the RO33 monomorphic pattern revealed among the Pfmsp-1 allelic type. Again, this study identified 27 different Pfmsp-2 genotypes, ranging from 140 to 568 bp in size, including 15 belonging to the 3D7-type and 12 to the FC27 allelic families. The analysis of the MSP-1 and MSP-2 peptides indicates that the region of the alignment corresponding K1 polymorphism had the highest similarity in the MSP1and MSP2 clade followed by MAD20 with 93% to 100% homology. Therefore, population structure of P. falciparum isolates is identical to that of other areas in Africa, suggesting that vaccine developed with K1 and MAD20 of Pfmsp1 allelic variant could be protective for Africa children but these findings requires further genetic and immunological investigations. The multiplicity of infection (MOI) was significantly higher (P < 0.05) for Pfmsp-2 loci (3.82), as compare with Pfmsp-1 (2.51) and heterozygotes ranged from 0.55 for Pfmsp-1 to 0.96 for Pfmsp-2. CONCLUSION:High genetic diversity and allelic frequencies in P. falciparum isolates indicate a persisting high level of transmission. This study advocate for an intensification of the malaria control strategies in Cameroon. Trial registration This study was approved by Cameroon National Ethics Committee. It is a randomized controlled trial retrospectively registered in NIH U.S. National Library of Medicine, ClinicalTrials.gov on the 28/11/2016 at https://clinicaltrials.gov/ct2/show/NCT02974348 with the registration number NCT02974348.

Project description:BackgroundArtemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana.MethodsSix months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana's Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing.ResultsOf the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates.ConclusionsThe observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Project description:BACKGROUND:Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. METHODS:The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. RESULTS:Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. CONCLUSIONS:This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

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Rapid detection of lactate dehydrogenase and genotyping of Plasmodium falciparum in saliva of children with acute uncomplicated malaria.

Publications

Rapid detection of lactate dehydrogenase and genotyping of Plasmodium falciparum in saliva of children with acute uncomplicated malaria.

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