Msp2 Gene Research Articles

Detection of high frequency of MAD20 allelic variants of Plasmodium falciparum merozoite surface protein 1 gene from Adama and its surroundings, Oromia, Ethiopia

BackgroundOne of the major challenges in developing an effective vaccine against asexual stages of Plasmodium falciparum is genetic polymorphism within parasite population. Understanding the genetic polymorphism like block 2 region of merozoite surface protein-1 (msp-1) gene of P. falciparum enlighten mechanisms underlining disease pathology, identification of the parasite clone profile from the isolates, transmission intensity and potential deficiencies of the ongoing malaria control and elimination efforts in the locality. Detailed understanding of local genetic polymorphism is an input to pave the way for better management, control and elimination of malaria. The aim of this study was to detect the most frequent allelic variant of the msp-1 gene of P. falciparum clinical isolates from selected health facilities in Adama town and its surroundings, Oromia, Ethiopia.MethodsOne hundred thirty-nine clinical isolates were successfully amplified for msp-1 gene using specific primers. Nested PCR amplification was conducted targeting K1, MAD20, and R033 alleles followed by gel electrophoresis for fragment analysis. Based on the detection of a PCR fragment, infections were classified as monoclonal or multiple infections.Results19 different size polymorphism of msp-1 gene were identified in the study, with 67(48%) MAD20, 18 (13%) K-1 and 18 (13%) RO33 allelic family. Whereas, the multiple infections were 21(15%), 8 (5.8%), 4(2.9%), 3(2.2%) for MAD20 + K-1, MAD20 + RO33, K-1 + RO33, and MAD20 + K-1, RO33, respectively. The overall Multiplicity of infection (MOI) was 1.3 and the expected heterozygosity (He) was 0.39 indicating slightly low falciparum malaria transmission.ConclusionThe status of msp-1 allele size polymorphism, MOI and He observed in the study revealed the presence of slightly low genetic diversity of P. falciparum clinical isolates. However, highly frequent MAD20 allelic variant was detected from clinical isolates in the study area. Moreover, the driving force that led to high predominance of MAD20 allelic variant revealed in such malaria declining region demands further research.

Detection of Anaplasma phagocytophilum in European brown hares (Lepus europaeus) using three different methods.

European brown hare (Lepus europaeus Pallas 1778) is a broadly distributed lagomorph species in Europe, recognized as a host for Ixodes ricinus and reservoir of a wide range of pathogens with zoonotic potential. Even though Lepus europaeus represents an important game animal in Central Europe, the data available on Anaplasma phagocytophilum in this lagomorph are scarce. In this study, three populations of brown hare from distinct localities in the Czech Republic were analysed for the presence of Anaplasma phagocytophilum DNA. We used standard qPCR, targeting the msp2 gene and adapted the same assay also for digital droplet PCR. Out of 91 samples, these two methods identified 9 and 12 as positive, respectively. For taxonomic analysis, we amplified the groEL gene from five of six samples that were found positive by both methods. In phylogenetic analyses, this haplotype belongs to ecotype 1, and to the subclade with isolates from cervids and I.ricinus. Our findings underline the importance of correct result interpretation and positivity cut-off set-up for different detection methods of A.phagocytophilum. This bacterium is characterized by a high intraspecific variability and highly sensitive detection itself, is not enough. Detailed molecular typing is necessary to define the zoonotic potential of different strains and their natural reservoirs.

Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.

Evaluation of pfmdr-1 Polymorphisms and Parasites’ Population Diversity in Children with Acute Uncomplicated Malaria 5 Years Post-Adoption of Artemisinin-Based Combination Therapies

Mutatons on pfmdr1 gene have been implicated in drug resistance to chloroquine and the partner drugs in artemisinin-based combination therapies (ACTs), hence the need to evaluate the impact of ACTs five years after its adoption in Nigeria on pfmdr1 polymorphisms and parasite diversity. Parasite genomic DNA was isolated from children below 5 years in Ibadan in 2010. Nested PCR followed by restriction fragment length polymorphism (RFLP) detected pfmdr1 Y86, F184 and Y1246 mutant alleles were present in 27%, 56% and 48% of the isolates respectively, while nested PCR evaluated polymorphic regions of MSP-1, MSP-2 and GLURP genes and monoclonal infections were observed in 81.6%, 51.6% and 5.6% with multiplicity of infection being 1.8, 2.0 and 2.4 respectively. This study showed a relative decline in the prevalence of Y86, F184 and Y1246 mutant alleles, but no significant change in the parasite population diversity of P.falciparum in children in Ibadan, Nigeria.

Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle

BackgroundSerious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle.MethodsMolecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia.ResultsA total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83–81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98–100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle.ConclusionWe present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

Detection of Tick-Borne Pathogens in Red Deer (Cervus elaphus), United Kingdom.

Deer represent a major vertebrate host for all feeding stages of the hard tick Ixodes ricinus in the United Kingdom (UK), and could play a role in the persistence of tick-borne pathogens. However, there have been few studies reporting the presence of Babesia spp. and Anaplasma phagocytophilum in deer in the UK, and those that detected Babesia were unable to confirm the species. To address this, we have investigated blood samples from red deer (Cervus elaphus) for the presence of tick-borne pathogens. Total DNA was extracted from haemolysed blood that was removed from clotted blood sampled from culled, captive red deer. Babesia spp. were detected with a pan-piroplasm PCR that amplifies a fragment of the 18S rRNA gene. Species were identified based on identity with published sequences. Anaplasma phagocytophilum was detected with a probe-based PCR targeting the msp2 gene. In addition, residual serum samples from a subset of animals were tested for the presence of anti-flavivirus antibodies. Of 105 red deer samples tested from three locations in the United Kingdom, 5 were positive for piroplasm and 5 were positive for A. phagocytophilum. Co-infection with both pathogens was detected in two samples from one location. No evidence for antibodies against West Nile virus were detected. However, 12% of sera tested were positive for tick-borne encephalitis virus antibodies.

Therapeutic efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in four malaria endemic states of India

BackgroundMalaria is a major public health problem in India and accounts for about 88% of malaria burden in South-East Asia. India alone accounted for 2% of total malaria cases globally. Anti-malarial drug resistance is one of the major problems for malaria control and elimination programme. Artemether-lumefantrine (AL) is the first-line treatment of uncomplicated Plasmodium falciparum in north eastern states of India since 2013 after confirming the resistance against sulfadoxine-pyrimethamine. In the present study, therapeutic efficacy of artemether-lumefantrine and k13 polymorphism was assessed in uncomplicated P. falciparum malaria.MethodsThis study was conducted at four community health centres located in Koraput district of Odisha, Bastar district of Chhattisgarh, Balaghat district of Madhya Pradesh and Gondia district of Maharashtra state. Patients with uncomplicated P. falciparum malaria were administered with fixed dose combination (6 doses) of artemether-lumefantrine for 3 days and clinical and parasitological response was recorded up to 28 days as per World Health Organization protocol. Nucleotide sequencing of msp1 and msp2 gene was performed to differentiate between recrudescence and reinfection. Amplification and sequencing of k13 propeller gene region covering codon 450–680 was also carried out to identify the polymorphism.ResultsA total 376 malaria patients who fulfilled the enrolment criteria as well as consented for the study were enrolled. Total 356 patients were followed up successfully up to 28 days. Overall, the adequate clinical and parasitological response was 98.9% and 99.4% with and without PCR correction respectively. No case of early treatment failure was observed. However, four cases (1.1%) of late parasitological failure were found from the Bastar district of Chhattisgarh. Genotyping of msp1 and msp2 confirmed 2 cases each of recrudescence and reinfection, respectively. Mutation analysis of k13 propeller gene showed one non-synonymous mutation Q613H in one isolate from Bastar.ConclusionsThe study results showed that artemether-lumefantrine is highly effective in the treatment of uncomplicated P. falciparum malaria among all age groups. No functional mutation in k13 was found in the study area. The data from this study will be helpful in implementation of artemether-lumefantrine in case of treatment failure by artesunate plus sulfadoxine-pyrimethamine.

147 Serological and molecular detection of Anaplasma in East Texas horses

Anaplasma phagocytophilum (AP) is a tick-borne bacteria that cycles between the tick, Ixodes scapularis and the hematopoietic cells of vertebrate hosts. AP is responsible for the disease equine granulocytic anaplasmosis, a generally transient infection characterized by fever, depression, inappetence, ventral edema petechiae, icterus, ataxia, recumbency, muscle stiffness, and, in severe cases, death. Current infection with AP is detected through PCR analysis of an animal's immune cell DNA, while serum antibody titers determine if a horse has previously been infected with AP. Other regions and countries have reported wide ranging prevalence of serum reactive horses, with 17 to 67% of apparently healthy horses being serum reactive and 8% of these being PCR positive. There were 130 clinically healthy horses aged from 1 to 29 years old included in this survey, 70 from Texas, and 11 from New Jersey, and 49 from New York. Horses from Texas were chosen based on location in the state and high potential for tick exposure, the latter 2 states were included as populations with known tick exposure. From each horse, blood was collected for serum and DNA. DNA was screened by nested PCR for the 23s rRNA gene of the Anaplasmataceae family. Samples positive for 23s were analyzed by conventional PCR for the msp2 gene of AP. Lastly, serum was analyzed by immunofluorescence antibody test to detect antibodies against AP. Seventeen horses were positive for 23s, with 4, 3, and 10 from Texas, New Jersey, and New York, respectively. Of these, 2 Texas horses were positive for msp2. Further, 107 horses tested positive for antibodies against AP, with 47, 11, and 49 from Texas, New Jersey, and New York, respectively. The observed 80% seroprevalence is similar to that reported from other countries and indicates high exposure to tick populations.

Molecular and recombinant characterization of major surface protein 5 from Anaplasma marginale

Anaplasmosis is a tick-borne disease caused by the intracellular rickettsia Anaplasma marginale, which affects cattle and other ruminants in both tropical and subtropical regions of the world, and also causing tremendous economic losses due to decreasing livestock production. The major surface protein 5 (MSP5) of A. marginale is an immunodominant and highly conserved protein encoding by a single gene. In the present study, the complete full-length of the msp5 coding sequence of A. marginale Thailand strain was cloned and determined at a size of 633 bp. Phylogenetic analysis based on neigh-joining (NJ) method showed that the msp5 sequence Thailand strains were clearly distributed in 3rd clade and conserved when compared with other strains. The results showed 9 haplotypes of the msp5 genes, and the entropy analysis of MSP5 amino acid sequences displayed 92 high entropy peaks with value ranging from 0.198 to 0.845 Additionally, a recombinant MSP5 of A. marginale (rAmMSP5) was over-expressed in the E. coli BL21 Star™ (DE3) host cell, affinity purified, and found in SDS-PAGE at a molecular weight of 26 kDa. The antigenicity of rAmMSP5 (26 kDa) and AmMSP5 (19 kDa) was recognized by rabbit anti-rAmMSP5 antisera and A. marginale-infected cattle sera. Both rAmMSP5 and AmMSP5 were perceived by these sera manifesting that recombinant and native AmMSP5 have conserved epitopes. Immunofluorescence technique using rabbit anti-rAmMSP5 antisera exhibited that the AmMSP5 is distributed on both the membrane and the outside of infected erythrocytes. Therefore, the recombinant MSP5 could be used for the development of immunodiagnostic assays and vaccine purposes for controlling anaplasmosis.

Allelic diversity of merozoite surface protein genes (msp1 and msp2) and clinical manifestations of Plasmodium falciparum malaria cases in Aceh, Indonesia

BackgroundThe malaria control programme in Indonesia has successfully brought down malaria incidence in many parts in Indonesia, including Aceh Province. Clinical manifestation of reported malaria cases in Aceh varied widely from asymptomatic, mild uncomplicated to severe and fatal complications. The present study aims to explore the allelic diversity of merozoite surface protein 1 gene (msp1) and msp2 among the Plasmodium falciparum isolates in Aceh Province and to determine their potential correlation with the severity of malaria clinical manifestation.MethodsScreening of over 500 malaria cases admitted to the hospitals in 11 districts hospital within Aceh Province during 2013–2015, identified 90 cases of P. falciparum mono-infection without any co-morbidity. The subjects were clinically phenotyped and parasite DNA was extracted and polymerase chain reaction (PCR) amplified for the msp1 and msp2 allelic subfamilies.ResultsAnalysis of clinical manifestation revealed that fever-chill is the most frequent symptom. Based on WHO criteria showed 19 cases were classified as severe and 71 as mild malaria. Analysis of msp1 gene revealed the presence of K1 allele subfamily in 34 subjects, MAD20 in 42 subjects, RO33 in 1 subject, and mixed allelic of K1 + MAD20 in 5 subjects, K1 + RO33 in 4 subjects, and MAD20 + RO33 in 4 subjects. Analysis of msp2 gene revealed 34 subjects carried the FC27 allelic subfamily, 37 subjects carried the 3D7 and 19 subjects carried the mixed FC27 + 3D7. Analysis of multiplicity of infection revealed that msp1 alleles is slightly higher than msp2 with the mean of MOI were 2.69 and 2.27, respectively. Statistical analysis to determine the association between each clinical manifestation and msp1 and msp2 alleles revealed that liver function abnormal value was associated with the msp2 mixed alleles (odds ratio (OR):0.13; 95%CI: 0.03–0.53). Mixed msp1 of K1 + RO33 was associated with severe malaria (OR: 28.50; 95%CI: 1.59–1532.30).ConclusionThis study found a strong association between severe malaria in Aceh with subjects carrying the msp1 mixed alleles of K1 and RO33. The liver function abnormal value associated with the msp2 mixed allelic subfamilies. Further study in different geographic areas is recommended.

Incidence of human granulocytic anaplasmosis in returning travellers with fever.

Although tick-borne pathogens have been reported as an important cause of imported fever, the incidence of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), in travellers is unknown. We conducted a prospective cohort study to investigate the aetiologies of fever in returning travellers (November 2017-July 2019). Polymerase chain reaction for msp2 gene amplification and indirect immunofluorescence assay for A. phagocitophilum were performed in all returning travellers with undifferentiated non-malarial fever. Among 141 travellers included, 8 patients were diagnosed with probable or confirmed HGA. The overall incidence rate of HGA was 19.9 cases/1000 person-week of travel. The main destination of travel was Asia, accounting for 62.5% patients with HGA. Co-infections were found in 37.5% of patients with HGA. Diagnosis of HGA and empirical treatment with doxycycline should be considered in travellers with fever.

Evaluation of sporozoite and macroschizont antigen (Spm2) of Theileria annulata for its diagnostic potential

Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle.

Genetic polymorphisms of Plasmodium falciparum isolates from Melka-Werer, North East Ethiopia based on the merozoite surface protein-2 (msp-2) gene as a molecular marker

BackgroundThe characterization of parasite populations circulating in malaria endemic areas is necessary to evaluate the success of ongoing interventions and malaria control strategies. This study was designed to investigate the genetic diversity of Plasmodium falciparum isolates from the semi-arid area in North East Ethiopia, using the highly polymorphic merozoite surface protein-2 (msp2) gene as a molecular marker.MethodsDried blood spot isolates were collected from patients with P. falciparum infection between September 2014 and January 2015 from Melka-Werer, North East Ethiopia. Parasite DNA was extracted and genotyped using allele-specific nested polymerase chain reactions for msp2.Results52 isolates were collected with msp2 identified in 41 (78.8%) isolates. Allele typing of the msp2 gene detected the 3D7/IC allelic family in 54% and FC27 allelic family in 46%. A total of 14 different msp2 genotypes were detected including 6 belonging to the 3D7/IC family and 8 to the FC27 family. Forty percent of isolates had multiple genotypes and the overall mean multiplicity of infections (MOI) was 1.2 (95%CI 0.96–1.42). The heterozygosity index was 0.50 for the msp2 locus. There was no difference in MOI between age groups. A negative correlation between parasite density and multiplicity of infection was found (p = 0.02).ConclusionPlasmodium falciparum isolates from the semi-arid area of North East Ethiopia are mainly monoclonal with low MOI and limited genetic diversity in the study population.

&lt;strong&gt;Identity and microbial agents related to &lt;em&gt;Dermacentor marginatus&lt;/em&gt; Sulzer (Acari: Ixodidae) with a new record of &lt;em&gt;Rickettsia slovaca&lt;/em&gt; (Rickettsiales: Rickettsiaceae) in Iran&lt;/strong&gt;

Dermacentor marginatus is a known vector of Rickettsia slovaca and Anaplasma ovis distributed across Eurasia. A total of 300 D. marginatus adult male ticks were collected, removed from sheep in East Azerbaijan province, northwestern Iran. The DNA of Rickettsia and Anaplasma were found in 10 (two pools) ticks by PCR amplifying parts of ompA and msp4 genes, respectively. Our study describes the first PCR detection of tick-borne pathogens, R. slovaca and A. ovis, show that they are spread in D. marginatus of Iran highlight their risk for human and animal hosts. Different patterns (ornamentation) were observed. The identity of different patterns in this study was evaluated with cytb, 16S rRNA and ITS2 genes. BLAST analysis of the cytb, 16S rRNA, ITS2 nucleotide sequence was showed 99%, 98%–100% and 98% sequence identity to sequences of D. marginatus, respectively. It was found that all different patterns of this study are in fact a polymorphic species, D. marginatus. These data increase our knowledge of tick and tick-borne microbial agents in Iran.

Allelic diversity of MSP1 and MSP2 repeat loci correlate with levels of malaria endemicity in Senegal and Nigerian populations

BackgroundCharacterizing the genetic diversity of malaria parasite populations in different endemic settings (from low to high) could be helpful in determining the effectiveness of malaria interventions. This study compared Plasmodium falciparum parasite population diversity from two sites with low (pre-elimination) and high transmission in Senegal and Nigeria, respectively.MethodsParasite genomic DNA was extracted from 187 dried blood spot collected from confirmed uncomplicated P. falciparum malaria infected patients in Senegal (94) and Nigeria (93). Allelic polymorphism at merozoite surface protein 1 (msp1) and merozoite surface protein- 2 (msp2) genes were assessed by nested PCR.ResultsThe most frequent msp1 and msp2 allelic families are the K1 and IC3D7 allelotypes in both Senegal and Nigeria. Multiplicity of infection (MOI) of greater that 1 and thus complex infections was common in both study sites in Senegal (Thies:1.51/2.53; Kedougou:2.2/2.0 for msp1/2) than in Nigeria (Gbagada: 1.39/1.96; Oredo: 1.35/1.75]). The heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal (Thies: 0.62; Kedougou: 0.53) than isolates from Nigeria (Gbagada: 0.55; Oredo: 0.50). In Senegal, K1 alleles was associated with heavy than with moderate parasite density. Meanwhile, equal proportions of K1 were observed in both heavy and moderate infection types in Nigeria. The IC3D7 subtype allele of the msp2 family was the most frequent in heavily parasitaemic individuals from both countries than in the moderately infected participants.ConclusionThe unexpectedly low genetic diversity of infections high endemic Nigerian setting compared to the low endemic settings in Senegal is suggestive of possible epidemic outbreak in Nigeria.

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

BackgroundThe emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum.MethodsThree SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.ResultsThe KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.ConclusionsThe KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Molecular detection and phylogenetic analysis of Anaplasma ovis in ixodid ticks of small ruminants in Tamil Nadu

Anaplasma ovis is an obligate epicellular rickettsial pathogen transmitted by ticks and causes anaplasmosis in small ruminants. The present study aimed to detect and characterize A. ovis in ixodid ticks of small ruminants in two districts of Tamil Nadu, by using msp4 gene fragment as the target. A total of 2096 ixodid ticks were collected from sheep and goats and identified morphologically. Salivary glands were separated from the ticks and DNA was extracted. Subsequently PCR amplification of msp4 gene was carried out and the 831bp PCR product was sequenced. A total of 29.75% (122/410) of pooled samples of ticks were positive for A. ovis. The nucleotide sequences of msp4 gene were analyzed and phylogenetic tree was constructed. The nucleotide sequences of sheep and goat isolates of A. ovis in this study were 100% identical. They also showed perfect similarity with the sequences of A. ovis in public database. Based on the study, it was concluded that Rhipicephalus haemaphysaloides and Haemaphysalis bispinosa could act as possible vectors for A. ovis.

The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes

ABSTRACT An emerging infectious disease caused by “Anaplasma capra” was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma. Anaplasma capra-positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL, 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright–Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra. Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra-positive, while leukocyte DNA samples were A. capra-negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra-positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., “Anaplasma capra,” in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name “A. capra” as Anaplasma capra sp. nov.

Molecular and serological detection of arthropod-borne pathogens in carnivorous birds from Brazil.

Rickettsiales, Haemosporida and Rhizobiales agents can cause diseases that affect various animal species, including humans. Due to predation behaviour, carnivorous birds may play an important role in spreading these etiological agentes across geographically distant areas, specially if they are migratory. The aim of this study was to investigate the occurrence and to access the phylogenetic relations among Anaplasmataceae (Ehrlichia, Anaplasma, Neorickettsia), Bartonellaceae (Bartonella spp.), and Haemosporida (Plasmodium, Haemoproteus and Leucocytozoon) agents in blood samples from 121 carnivorous birds sampled in the states of São Paulo and Rio de Janeiro, Brazil. Inclusions resembling hemoparasites were not observed in Giemsa-stained preparations. While three animals were seropositive for E. chaffeensis (3.41% [3/88]; 95% CI:1.17-9.55%), five showed antibodies to A. phagocytophilum (5.68% [5/88]; 95% CI: 2.45-12.62%). Despite the detection of rrs gene fragments closely related to E. chaffeensis (4.13% [5/121]; 95% CI: 1.78-9.31%), no positivity was observed in the qPCR based on the genes vlpt for the organism. Similarly, 12 (9.91% [12/121]; 95% CI: 5.76-16.74%) samples were positive in the qPCR for Anaplasma spp. based on groEL gene, but negative in the qPCR for A. phagocytophilum based on msp-2 gene. Three samples were positive in the nPCR for E. canis based on rrs gene. Three samples were positive for Haemoproteus spp. and one for Plasmodium spp. in the nPCR based on cytB gene. Four birds (3.3% [4/121]; 95% CI: 1.29-8.19%) presented co-positivity by Ehrlichia sp. and Anaplasma sp. in molecular assays. One (0.82% [1/121]; 95% CI:0.15-4.53%) bird showed to be seropositive for E. chaffeensis and and positive in PCR for Haemoproteus sp. All birds were negative in the qPCR assay for Bartonella spp. (nuoG). The present work showed the occurrence of Anaplasmataceae agents and hemosporidians in carnivorous birds from southeastern Brazil. The role of these animals in the dispersion of Anaplasmataceae agents should be further investigated.

Plasmodium falciparum Allelic Diversity: A Comparison of DNA Extraction from Isolates Collected on Rapid Diagnostic Tests (Rdts) and Filter Paper

Background:To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity.Methods:Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification.Results:Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs −20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples.Conclusion:These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.