Distribution and temporal dynamics of P. falciparum chloroquine resistance transporter mutations associated with piperaquine resistance in Northern Cambodia

Abstract Background Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009-2017. Methods The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative PCR was used to estimate pfpm2 and pfmdr1 copy number. Results Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being out-competed by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single copy pfpm2. Conclusions The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

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P. falciparum and Its Molecular Markers of Resistance to Antimalarial Drugs

10.5772/intechopen.98372 ◽

2021 ◽

Author(s):

Peter Hodoameda

Keyword(s):

Molecular Markers ◽

South America ◽

Copy Number ◽

Antimalarial Drugs ◽

Chloroquine Resistance ◽

Multidrug Resistance Protein 1 ◽

Parasite Genome ◽

Chloroquine Resistance Transporter ◽

The Pacific ◽

Resistance Protein

The use of molecular markers of resistance to monitor the emergence, and the spread of parasite resistance to antimalarial drugs is a very effective way of monitoring antimalarial drug resistance. The identification and validation of molecular markers have boosted our confidence in using these tools to monitor resistance. For example, P. falciparum chloroquine resistance transporter (PfCRT), P. falciparum multidrug resistance protein 1 (PfMDR1), P. falciparum multidrug kelch 13 (pfk13), have been identified as molecular markers of resistance to chloroquine, lumefantrine, and artemisinin respectively. The mechanism of resistance to antimalarial drugs is mostly by; (1) undergoing mutations in the parasite genome, leading to expelling the drug from the digestive vacuole, or (2) loss of binding affinity between the drug and its target. Increased copy number in the pfmdr1 gene also leads to resistance to antimalarial drugs. The major cause of the widespread chloroquine and sulfadoxine-pyrimethamine resistance globally is the spread of parasites resistant to these drugs from Southeast Asia to Africa, the Pacific, and South America. Only a few mutations in the parasite genome lead to resistance to chloroquine and sulfadoxine-pyrimethamine arising from indigenous parasites in Africa, Pacific, and South America.

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No Plasmodium falciparum Chloroquine Resistance Transporter and Artemisinin Resistance Mutations, Haiti

Emerging Infectious Diseases ◽

10.3201/eid2411.180738 ◽

2018 ◽

Vol 24 (11) ◽

pp. 2124-2126 ◽

Cited By ~ 1

Author(s):

Jeanne P. Vincent ◽

Kanako Komaki-Yasuda ◽

Alexandre V. Existe ◽

Jacques Boncy ◽

Shigeyuki Kano

Keyword(s):

Plasmodium Falciparum ◽

Artemisinin Resistance ◽

Chloroquine Resistance ◽

Resistance Mutations ◽

Chloroquine Resistance Transporter

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Polymorphism Analysis of pfmdr1 and pfcrt from Plasmodium falciparum Isolates in Northwestern Nigeria Revealed the Major Markers Associated with Antimalarial Resistance

Diseases ◽

10.3390/diseases9010006 ◽

2021 ◽

Vol 9 (1) ◽

pp. 6

Author(s):

Ruqayya Adam ◽

Muhammad M. Mukhtar ◽

Umar F. Abubakar ◽

Hajara A. Damudi ◽

Abdullahi Muhammad ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Management Strategies ◽

Haplotype Diversity ◽

Resistance Management ◽

Chloroquine Resistance ◽

Polymorphism Analysis ◽

Resistance Mutations ◽

Chloroquine Resistance Transporter ◽

Antimalarial Resistance ◽

Additional Sequence

Suspicion of failure in the effectiveness of artemisinin-based combination therapies (currently the first-line treatment of malaria, worldwide) is leading to the unofficial use of alternative antimalarials, including chloroquine and sulfadoxine/pyrimethamine, across northern Nigeria. To facilitate evidence-based resistance management, antimalarial resistance mutations were investigated in Plasmodium falciparum multidrug resistance-1 (pfmdr1) and chloroquine resistance transporter (pfcrt), in isolates from Kano, northwestern Nigeria. Out of the 88 samples genotyped for pfmdr1N86Y mutation using PCR/restriction fragment length polymorphism, one sample contained the 86Y mutation (86Yfrequency = 1.14%). The analysis of 610 bp fragments of pfmdr1 from 16 isolates revealed two polymorphic sites and low haplotype diversity (Hd = 0.492), with only 86 Y mutations in one isolate, and 184 F replacements in five isolates (184Ffrequency = 31.25%). The analysis of 267 bp fragments of pfcrt isolates revealed high polymorphism (Hd = 0.719), with six haplotypes and seven non-synonymous polymorphic sites. Eleven isolates (61.11%) were chloroquine-resistant, CQR (C72V73I74E75T76 haplotype), two of which had an additional mutation, D57E. An additional sequence was CQR, but of the C72V73M74E75T76 haplotype, while the rest of the sequences (33.33%) were chloroquine susceptible (C72V73M74N75K76 haplotype). The findings of these well characterized resistance markers should be considered when designing resistance management strategies in the northwestern Nigeria.

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The impact of HIV-associated immunosuppression on the Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) of HIV patients in Akure, Nigeria

Bulletin of the National Research Centre ◽

10.1186/s42269-020-00401-0 ◽

2020 ◽

Vol 44 (1) ◽

Author(s):

Iyabo Adepeju Simon-Oke ◽

Adeola Olanireti Ade-Alao ◽

Foluso Ologundudu

Keyword(s):

Plasmodium Falciparum ◽

Malaria Prevalence ◽

Cd4 Count ◽

Chloroquine Resistance ◽

Hiv Care ◽

Transporter Gene ◽

Hiv Patients ◽

Chloroquine Resistance Transporter ◽

Hiv Test ◽

Low Prevalence

Abstract Background The study evaluated the prevalence of malaria and Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) in HIV patients attending Specialist Hospital, Akure. This study was carried out between April and June 2019. Three hundred and seventeen (317) patients attending the antiretroviral clinic (ART) were involved, out of which 89 (28.08%) were males and 228 (71.92%) were females. HIV test was done using the Unigold® HIV test kit, malaria test was done using thick and thin blood smear, CD4 test was done using the Partec® CD4 counter and PCR was used to detect the presence of plasmodium falciparum mutant gene. The data obtained from this analysis was subjected to Pearson’s Chi-square test. Results The overall result showed low prevalence of malaria (23.03%) in the sampled patients. Highest malaria prevalence (31.0%) was recorded in HIV patients with CD4 count between 200–500 cells/μl of blood, with the males recording 24.7% malaria prevalence. The age group 20–29 years recorded the highest prevalence of 27.3%. A higher prevalence 91.1% of PfCRT gene in HIV-positive and (40.0%) in HIV-negative patients was recorded with 100% prevalence in patients with CD4 count ≤ 200. This shows that the low prevalence of malaria recorded in this study could be credited to good health-seeking attitude of HIV patients and the upscale of HIV care and treatment centres. Conclusion The high prevalence of PfCRT gene shows that treatment of malaria with chloroquine is still being practised despite the availability of artemisinin-based combination therapy (ACTs) as the recommended regimen for malaria treatment.

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Dynamic Subspecies Population Structure of Vibrio Cholerae in Dhaka, Bangladesh

10.21203/rs.3.rs-364004/v1 ◽

2021 ◽

Author(s):

Mohammad Islam ◽

Tania Nasreen ◽

Kevin Y. H. Liang ◽

Fatema-Tuz Johura ◽

Paul C. Kirchberger ◽

...

Keyword(s):

Vibrio Cholerae ◽

Temporal Dynamics ◽

Sampling Period ◽

Amplicon Sequencing ◽

Rapid Expansion ◽

Capital City ◽

Dhaka City ◽

Physiochemical Parameters ◽

Subspecies Level ◽

Abundance And Distribution

Abstract Cholera has been endemic to the Ganges delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae, the most important being the pandemic generating lineage (PG) consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG and other lineages belonging to the V. cholerae species in surface water in and around Dhaka city, we used qPCR and high throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months and physiochemical parameters were measured in situ. Total abundance of V. cholerae was found to be relatively stable throughout the six months sampling period, with 2×105 to 4×105 genome copies/L at six sites and around 5 ×105 genome copies/L at the site located in the most densely populated part of Dhaka city. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24-92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated to salinity, conductivity and total dissolved solids (TDS), while it was negatively correlated to dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0-0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.

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Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

10.21203/rs.3.rs-22444/v3 ◽

2020 ◽

Author(s):

Nonlawat Boonyalai ◽

Brian A Vesely ◽

Chatchadaporn Thamnurak ◽

Chantida Praditpol ◽

Watcharintorn Fagnark ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Drug Susceptibility ◽

Antagonistic Activity ◽

Drug Combinations ◽

Chloroquine Resistance ◽

Phenotypic Characterization ◽

Field Isolates

Abstract Background High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 ( pfpm2 ), exonuclease ( pfexo ) and chloroquine resistance transporter ( pfcrt ) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.Results The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovoquone-proguanil combinations revealed synergistic antimalarial activity.Conclusions Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

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Mitochondrial DNA length variation and heteroplasmy in populations of white sturgeon (Acipenser transmontanus).

Genetics ◽

10.1093/genetics/132.1.221 ◽

1992 ◽

Vol 132 (1) ◽

pp. 221-228 ◽

Cited By ~ 6

Author(s):

J R Brown ◽

A T Beckenbach ◽

M J Smith

Keyword(s):

Copy Number ◽

Single Copy ◽

Repeat Sequence ◽

White Sturgeon ◽

Acipenser Transmontanus ◽

Length Variation ◽

Chi Square ◽

Mutation Pressure ◽

Frequency Distributions ◽

D Loop

Abstract Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.

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Copy Number Variations of Four Y-Linked Genes in Swamp Buffaloes

Animals ◽

10.3390/ani10010031 ◽

2019 ◽

Vol 10 (1) ◽

pp. 31

Author(s):

Ting Sun ◽

Quratulain Hanif ◽

Hong Chen ◽

Chuzhao Lei ◽

Ruihua Dang

Keyword(s):

Y Chromosome ◽

Copy Number ◽

Water Buffalo ◽

Single Copy ◽

Copy Number Variations ◽

Tetratricopeptide Repeat ◽

Dead Box ◽

Complex Phenotypes ◽

Swamp Buffaloes ◽

Number Variation

Copy number variation (CNV), a significant source of genetic diversity in the mammalian Y chromosome, is associated with the development of many complex phenotypes, such as spermatogenesis and male fertility. The contribution of Y-linked CNVs has been studied in various species, however, water buffalo has not been explored in this area and the genetic information still remains unknown. The aim of the current study was to investigate the CNVs of four Y-linked genes, including, sex determining Region of Y-Chromosome (SRY), ubiquitously transcribed tetratricopeptide repeat gene protein on the chromosome Y (UTY), DEAD-box helicase 3 Y-linked (DDX3Y, also known as DBY), and oral-facial-digital syndrome 1 Y-linked (OFD1Y) in 254 swamp buffaloes from 15 populations distributed across China, Vietnam, and Laos using quantitative real-time PCR (qPCR). Our results revealed the prevalence of a single-copy UTY gene in buffaloes. The DBY and OFD1Y represented CNVs among and within different buffalo breeds. The SRY showed CNVs only in Vietnamese and Laotian buffaloes. In conclusion, this study indicated that DBY, OFD1Y, and SRY showed CNVs, while the UTY was a single-copy gene in swamp buffaloes.

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Survey of Plasmodium falciparum multidrug resistance-1 and chloroquine resistance transporter alleles in Haiti

Malaria Journal ◽

10.1186/1475-2875-12-426 ◽

2013 ◽

Vol 12 (1) ◽

pp. 426 ◽

Cited By ~ 20

Author(s):

Maha A ElBadry ◽

Alexandre Existe ◽

Yves S Victor ◽

Gladys Memnon ◽

Mark Fukuda ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Chloroquine Resistance ◽

Chloroquine Resistance Transporter

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Implications of rRNA Operon Copy Number and Ribosome Content in the Marine Oligotrophic Ultramicrobacterium Sphingomonassp. Strain RB2256

Applied and Environmental Microbiology ◽

10.1128/aem.64.11.4433-4438.1998 ◽

1998 ◽

Vol 64 (11) ◽

pp. 4433-4438 ◽

Cited By ~ 122

Author(s):

Fitri Fegatella ◽

Julianne Lim ◽

Staffan Kjelleberg ◽

Ricardo Cavicchioli

Keyword(s):

Growth Rate ◽

Copy Number ◽

Heterotrophic Bacteria ◽

Single Copy ◽

Rrna Operon ◽

Limiting Factor ◽

E Coli ◽

Rate Of Growth ◽

Rates Of Growth ◽

Low Rate

ABSTRACT Sphingomonas sp. strain RB2256 is a representative of the dominant class of ultramicrobacteria that are present in marine oligotrophic waters. In this study we examined the rRNA copy number and ribosome content of RB2256 to identify factors that may be associated with the relatively low rate of growth exhibited by the organism. It was found that RB2256 contains a single copy of the rRNA operon, in contrast to Vibrio spp., which contain more than eight copies. The maximum number of ribosomes per cell was observed during mid-log phase; however, this maximum content was low compared to those of faster-growing, heterotrophic bacteria (approximately 8% of the maximum ribosome content of Escherichia coli with a growth rate of 1.5 h−1). The low number of ribosomes per cell appears to correlate with the low rate of growth (0.16 to 0.18 h−1) and the presence of a single copy of the rRNA operon. However, on the basis of cell volume, RB2256 appears to have a higher concentration of ribosomes than E. coli (approximately double that of E. coli with a growth rate of 1.5 h−1). Ribosome numbers reached maximum levels during mid-log-phase growth but decreased rapidly to 10% of maximum during late log phase through 7 days of starvation. The cells in late log phase and at the onset of starvation displayed an immediate response to a sudden addition of excess glucose (3 mM). This result demonstrates that a ribosome content 10% of maximum is sufficient to allow cells to immediately respond to nutrient upshift and achieve maximum rates of growth. These data indicate that the bulk of the ribosome pool is not required for protein synthesis and that ribosomes are not the limiting factor contributing to a low rate of growth. Our findings show that the regulation of ribosome content, the number of ribosomes per cell, and growth rate responses in RB2256 are fundamentally different from those characteristics in fast-growing heterotrophs like E. coliand that they may be characteristics typical of oligotrophic ultramicrobacteria.

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