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1.
Proc Natl Acad Sci U S A ; 120(4): e2213887120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669098

RESUMO

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons. All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.


Assuntos
Cilióforos , Paramecium tetraurellia , Edição de Genes , Genoma , Cilióforos/genética , Paramecium tetraurellia/metabolismo , Núcleo Celular/metabolismo , DNA de Protozoário/genética
2.
Proc Natl Acad Sci U S A ; 120(4): e2213985120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669106

RESUMO

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.


Assuntos
Cilióforos , Infestações por Ácaros , Humanos , Elementos de DNA Transponíveis/genética , Edição de Genes , Cilióforos/genética , Transposases/genética , Transposases/metabolismo , Células Germinativas/metabolismo , Infestações por Ácaros/genética , DNA de Protozoário/genética
3.
Cell Host Microbe ; 31(1): 112-123.e4, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36521488

RESUMO

The parasite Cryptosporidium hominis is a leading cause of the diarrheal disease cryptosporidiosis, whose incidence in the United States has increased since 2005. Here, we show that the newly emerged and hyper-transmissible subtype IfA12G1R5 is now dominant in the United States. In a comparative analysis of 127 newly sequenced and 95 published C. hominis genomes, IfA12G1R5 isolates from the United States place into three of the 14 clusters (Pop6, Pop13, and Pop14), indicating that this subtype has multiple ancestral origins. Pop6 (IfA12G1R5a) has an East Africa origin and has recombined with autochthonous subtypes after its arrival. Pop13 (IfA12G1R5b) is imported from Europe, where it has recombined with the prevalent local subtype, whereas Pop14 (IfA12G1R5c) is a progeny of secondary recombination between Pop6 and Pop13. Selective sweeps in invasion-associated genes have accompanied the emergence of the dominant Pop14. These observations offer insights into the emergence and evolution of hyper-transmissible pathogens.


Assuntos
Criptosporidiose , Cryptosporidium , Humanos , Estados Unidos , Cryptosporidium/genética , Criptosporidiose/parasitologia , DNA de Protozoário/genética , Genoma , Recombinação Genética , Genótipo , Fezes/parasitologia
5.
BMC Infect Dis ; 22(1): 963, 2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36577945

RESUMO

BACKGROUND: Visceral leishmaniasis is caused by the Leishmania donovani species complex that can spread to internal organs and leading to death if not treated on time. Diagnosis of leishmaniasis is based on clinical signs and symptoms, microscopy, serological and molecular techniques. Because of a broad spectrum of diverse clinical manifestations and similarities of the responses to different species, identification to the species level is often difficult for the proper patient treatment and management. Therefore, the objective of this study was to evaluate the PCR- RFLP assay of the ITS1 region for identification of L. donovani species from clinical smear slide patient samples. METHOD: DNA extraction was performed on a total of 90 smear slide samples using phenol-chloroform method. The PCR detection limit was determined by L. donovani reference strain DNA. The ITS1 region was amplified at 320 bp using LITSR/L5.8S genus specific primers and then the ITS1-PCR products were subjected to RFLP assay for confirmation of L. donovani species using HaeIII restriction enzyme. RESULTS: Of the total samples ITS1-PCR revealed the true positive, false positive, true negative, and false negative results of 42 (46.7%), 6 (6.7%), 37 (41.1%) and 5 (5.6%), respectively. Considering microscopy as the gold standard, the sensitivity, specificity, positive predictive values, and negative predictive values of the ITS1- PCR technique was 89.4%, 86.0%, 87.5%, and 88.1% respectively. All ITS1-PCR positive clinical samples were confirmed as L. donovani species by PCR-RFLP patterns. CONCLUSION: In conclusion, the ITS1- RFLP method is highly sensitive and more specific for identification of L. donovani species in the smear negative clinical samples of visceral leishmaniasis patients. There is also significant association and degree of agreement between the two methods. For direct identification of L. donovani species from clinical samples, irrespective of genus and species level, PCR-RFLP is more recommendable than a microscope.


Assuntos
Leishmania donovani , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Cutânea/diagnóstico , Polimorfismo de Fragmento de Restrição , DNA de Protozoário/genética , Hospitais
6.
BMC Vet Res ; 18(1): 449, 2022 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-36564739

RESUMO

BACKGROUND: Cryptosporidium is the most common protozoan that can infect a wide variety of animals, including mammals and birds. Fecal samples of six saffron finches, Sicalis flaveola, from a commercial establishment were screened for the presence of Cryptosporidium by the modified Ziehl-Neelsen technique and nested PCR of the 18S rRNA gene followed by sequencing of the amplified fragments. RESULTS: The species Cryptosporidium galli was identified in all six saffron fiches, in addition to Cryptosporidium andersoni in one of the birds, indicating a mixed infection. Only two birds had feathers that were ruffled and dirty with feces. Concomitant infection with Isospora spp. was observed in all birds. CONCLUSIONS: Saffron finches are a possible host of C. andersoni and this is the first report of this species in a captive bird and the third report of parasitism by C. galli in Sicalis flaveola.


Assuntos
Crocus , Criptosporidiose , Cryptosporidium , Tentilhões , Passeriformes , Animais , Cryptosporidium/genética , Crocus/genética , Filogenia , Fezes , DNA de Protozoário/genética , Mamíferos/genética
7.
BMC Infect Dis ; 22(1): 944, 2022 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-36527077

RESUMO

BACKGROUND: Transmission-blocking vaccines (TBVs) target the sexual stages of malaria parasites to reduce or interrupt the transmission cycle in human and mosquito populations. The genetic diversity of TBVs candidate antigens, Pvs25 and Pvs28, in Plasmodium vivax could provide evidence for the development of TBVs. METHODS: Dry blood spots from P. vivax patients were collected from Dandong, Suining, Hainan, Nyingchi, Tengchong, and Yingjiang in China. The pvs25 and pvs28 genes were amplified and sequenced. The genetic diversity of pvs25 and pvs28 were analyzed using DNASTAR, MEGA6, and DnaSP 5.0 programs. RESULTS: A total of 377 samples were collected, among which 324 and 272 samples were successfully amplified in the pvs25 and pvs28 genes, respectively. Eight haplotypes were identified in Pvs25, for which the predominant mutation was I130T with 100% prevalence. A variety of 22 haplotypes in Pvs28 were identified. The number of GSGGE/D repeats of Pvs28 was a range of 4-8, among which, high (7-8) and low (4-5) copy numbers of tandem repeats were found in haplotypes H2 and H17, respectively. The nucleotide diversity of pvs28 (π = 0.00305 ± 0.00061) was slightly higher than that of pvs25 (π = 0.00146 ± 0.00007), thus they were not significantly different (P > 0.05). The Tajima's D value of pvs25 was positive whereas pvs28 was negative, which indicated that both genes were affected by natural selection. CONCLUSION: The genetic diversity of pvs25 and pvs28 genes in China was relatively limited, which provided valuable information for TBVs design and optimization.


Assuntos
Vacinas Antimaláricas , Malária Vivax , Animais , Humanos , Plasmodium vivax , Vacinas Antimaláricas/genética , Antígenos de Protozoários/genética , DNA de Protozoário/genética , Antígenos de Superfície/genética , Polimorfismo Genético , Malária Vivax/prevenção & controle , Malária Vivax/parasitologia , Proteínas de Protozoários/genética , Variação Genética
8.
PLoS One ; 17(12): e0279730, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36584086

RESUMO

Although a diversity of trypanosome species have been detected in various animal taxa from human African trypanosomosis (HAT) foci, cattle trypanosomosis has not been addressed in HAT foci of west and central African countries including Chad. This study aimed to determine the prevalence of pathogenic trypanosome species in cattle from three HAT foci of the south of Chad. Blood samples were collected from 1466 randomly selected cattle from HAT foci of Mandoul, Maro, and Moïssala in the south of Chad. For each animal, the sex, age and body condition were recorded. Rapid diagnostic test (RDT) was used to search Trypanosoma brucei gambiense antibodies while the capillary tube centrifugation (CTC) test and PCR-based methods enabled to detect and identify trypanosome species. From the 1466 cattle, 45 (3.1%) were positive to RDT. The prevalence of trypanosome infections revealed by CTC and PCR-based method were respectively 2.7% and 11.1%. Trypanosomes of the subgenus Trypanozoon were dominant (6.5%) followed by T. congolense savannah (2.9%), T. congolense forest (2.5%) and T. vivax (0.8%). No animal was found with DNA of human infective trypanosome (T. b. gambiense). The overall prevalence of trypanosome infections was significantly higher in animal from the Maro HAT focus (13.8%) than those from Mandoul (11.1%) and Moïssala HAT foci (8.0%). This prevalence was also significantly higher in animal having poor body condition (77.5%) than those with medium (11.2%) and good (0.5%) body condition. The overall prevalence of single and mixed infections were respectively 9.4% and 1.6%. This study revealed natural infections of several pathogenic trypanosome species in cattle from different HAT foci of Chad. It showed similar transmission patterns of these trypanosome species and highlighted the need of developing control strategies for animal African trypanosomosis (AAT) with the overarching goal of improving animal health and the economy of smallholder farmers.


Assuntos
Trypanosoma , Tripanossomíase Africana , Moscas Tsé-Tsé , Animais , Bovinos , Humanos , Chade/epidemiologia , Prevalência , Tripanossomíase Africana/epidemiologia , Tripanossomíase Africana/veterinária , Trypanosoma/genética , DNA de Protozoário/genética
9.
Sci Rep ; 12(1): 19084, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36351984

RESUMO

In Egypt, Blastocystis sp. is not yet on the diagnostic list of parasitology reports, and information about its subtypes (STs) is scarce. This study investigated its prevalence and its STs/alleles, performed phylogenetic analysis, and considered the distribution of risk factors associated with Blastocystis sp. infections in West Ismailia, Ismailia governorate. Sociodemographic data, exposure factors, and previous parasitic infection status were recorded for symptomatic and asymptomatic individuals. Microscopy, polymerase chain reaction, sequencing, and phylogenetic analysis for Blastocystis sp. isolated from fecal samples were performed. Eighty Blastocystis sp.-infected individuals (15.3%) were examined. The age of the individuals ranged between 0.60 and 85.0 (mean 17.10 ± 15.70), the male/female ratio was 33/47, and the asymptomatic/symptomatic ratio was 55/25. The findings demonstrate clear evidence of direct contact with animals, poor water quality, and previous parasitic infections. Eleven samples yielded three Blastocystis STs (ST1: allele 4, ST2: alleles 9 and 12, and ST3: allele 34), with ST3 (45.5%) representing the most common subtype. Phylogenetic analysis with a robust bootstrap revealed three distinct clades for isolates of each subtype. This study updates the epidemiological knowledge of the distribution of Blastocystis sp. STs in Egypt and expands the current understanding of the prevalence, risk factor frequencies, and genetic diversity of this protist in the studied area.


Assuntos
Infecções por Blastocystis , Blastocystis , Animais , Feminino , Masculino , Blastocystis/genética , Filogenia , Egito/epidemiologia , DNA de Protozoário/genética , Variação Genética , Infecções por Blastocystis/epidemiologia , Infecções por Blastocystis/parasitologia , Fezes/parasitologia , Prevalência
10.
BMC Infect Dis ; 22(1): 807, 2022 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-36310166

RESUMO

BACKGROUND: Plasmodium vivax apical membrane antigen-1 (pvama-1) is an important vaccine candidate against Malaria. The genetic composition assessment of pvama-1 from wide-range geography is vital to plan the antigen based vaccine designing against Malaria. METHODS: The blood samples were collected from 84 P. vivax positive malaria patients from different districts of Khyber Pakhtunkhwa (KP) province of Pakistan. The highly polymorphic and immunogenic domain-I (DI) region of pvama-1 was PCR amplified and DNA sequenced. The QC based sequences raw data filtration was done using DNASTAR package. The downstream population genetic analyses were performed using MEGA4, DnaSP, Arlequin v3.5 and Network.5 resources. RESULTS: The analyses unveiled total 57 haplotypes of pvama-1 (DI) in KP samples with majorly prevalent H-14 and H-5 haplotypes. Pairwise comparative population genetics analyses identified limited to moderate genetic distinctions among the samples collected from different districts of KP, Pakistan. In context of worldwide available data, the KP samples depicted major genetic differentiation against the Korean samples with Fst = 0.40915 (P-value = 0.0001), while least distinction was observed against Indian and Iranian samples. The statistically significant negative values of Fu and Li's D* and F* tests indicate the evidence of population expansion and directional positive selection signature. The slow LD decay across the nucleotide distance in KP isolates indicates low nucleotide diversity. In context of reference pvama-1 sequence, the KP samples were identified to have 09 novel non-synonymous single nucleotide polymorphisms (nsSNPs), including several trimorphic and tetramorphic substitutions. Few of these nsSNPs are mapped within the B-cell predicted epitopic motifs of the pvama-1, and possibly modulate the immune response mechanism. CONCLUSION: Low genetic differentiation was observed across the pvama-1 DI among the P. vivax isolates acquired from widespread regions of KP province of Pakistan. The information may implicate in future vaccine designing strategies based on antigenic features of pvama-1.


Assuntos
Malária Vivax , Plasmodium vivax , Humanos , Plasmodium vivax/genética , Irã (Geográfico) , Paquistão/epidemiologia , DNA de Protozoário/genética , Antígenos de Protozoários/genética , Proteínas de Protozoários/genética , Malária Vivax/epidemiologia , Genética Populacional , Variação Genética , Nucleotídeos , Seleção Genética , Análise de Sequência de DNA
11.
Front Cell Infect Microbiol ; 12: 1024690, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36225232

RESUMO

Humans are exposed to Toxoplasma gondii infection as pet cats gradually become family members and represent an increasing public health risk worldwide. Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this study, real-time fluorescence quantitative loop-mediated isothermal amplification (qLAMP) and visual LAMP detection technologies were established to conduct tests of T. gondii based on the membrane DNA extraction method, and the optimal detection mix was determined by adding the protective reagent trehalose and screening the concentrations of Mg2+ and dNTPs. Paraffin and lyophilization were used to reduce and even remove aerosol pollution, constructing a detailed anti-contamination protocol. Based on the positive standard plasmid DNA, the LODs of qLAMP and visual LAMP were 92 copies/µL and 92 copies/µL, and the standard curve of qLAMP was Y=2.9503X+20.8992 with R2 = 0.99. The applicability of the qLAMP and visual LAMP assays in disease diagnosis was assessed by evaluating 200 clinical cat faeces samples. The assays showed good diagnostic consistency, with kappa values of 1.0 and 0.99 compared with TaqMan qPCR, respectively. Compared with TaqMan qPCR, the diagnostic specificity/sensitivity of qLAMP and visual LAMP were 100%/100% and 100%/80%, respectively. The qLAMP and visual LAMP assays reported here are rapid and simple tests without extensive sample preparation and have a short turnaround time within 60 min, making them suitable for point-of-care testing.


Assuntos
Toxoplasma , Toxoplasmose , Animais , Gatos , DNA de Protozoário/análise , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Parafina , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose/diagnóstico , Trealose
12.
Vector Borne Zoonotic Dis ; 22(10): 512-519, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36201229

RESUMO

Background: Toxoplasma gondii is an obligate intracellular parasite that invades nearly all nucleated cells of a broad spectrum of vertebrate hosts, and which may cause serious disease in immunocompromised patients, as well as in the immunologically incompetent fetus. This study aimed to establish a loop-mediated isothermal amplification (LAMP) technique to rapidly detect T. gondii in the blood infection by targeting the 529 bp repeat element of T. gondii. Materials and Methods: A turbidity monitoring system, together with visual reagent, was used to test the amplification result of the LAMP assay. In addition, the specificity and sensitivity of the LAMP assay were measured. Results: The results suggest that the successfully established LAMP assay profile can detect the DNA of T. gondii at 67°C within 40 min. The limit of detection of the LAMP assay was 101 copies/µL. No cross reaction occurred with Plasmodium vivax, Toxocara cati, Clonorchis sinensi, Spirometra mansoni or Cryptosporidium parvum. We validated the developed LAMP assay by detecting T. gondii in DNA extracted from 353 blood samples collected from domestic cats and dogs. The percentages of positive results in detecting these blood samples by LAMP and conventional PCR were 5.38% and 2.83%, respectively. Conclusions: Our findings show that the developed LAMP assay offers higher analytical sensitivity than conventional PCR and good analytical specificity, minimizes aerosol contamination, and can be applied to on-site rapid detection of T. gondii.


Assuntos
Doenças do Gato , Criptosporidiose , Cryptosporidium , Doenças do Cão , Toxoplasma , Gatos , Cães , Animais , Toxoplasma/genética , DNA de Protozoário/genética , Sensibilidade e Especificidade , Cryptosporidium/genética , Técnicas de Amplificação de Ácido Nucleico/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças do Gato/diagnóstico
13.
Protist ; 173(6): 125912, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36242851

RESUMO

Diverse and dynamic communities of ciliates and other microbes thrive in the natural environment, driving the functioning of aquatic ecosystems. Many microbes are present in very low numbers or are dormant in the 'seedbank', escaping detection in environmental surveys and, consequently, remaining underexplored. Here, we report an extraordinarily rare ciliate that was discovered after persistent exploration of freshwater anoxic sediments - Legendrea loyezae Fauré-Fremiet, 1908, a member of the Family Spathidiidae, Order Haptorida. In this study, we present the sixth account of the ciliate since 1908 and reveal its phylogenetic position with the first 18S rRNA data for the genus. We explain the key morphological features of the species, describing a remarkable behaviour in which the ciliate "shapeshifts'' due to its ability of controlled full extension and retraction of its tube-like tentacles. Our results shed light on the similarity of L. loyezae to another ciliate that was first described as Legendrea bellerophon, later moved under a new genus and named Thysanomorpha bellerophon. We question the validity of this taxonomic decision and, based on morphological characters and tentacle movement, we propose moving T. bellerophon back under Legendrea. This study demonstrates how continued and persistent exploration of natural habitats lead to the discovery of microbial communities and species.


Assuntos
Cilióforos , Ecossistema , Filogenia , DNA de Protozoário/genética , Cilióforos/genética , RNA Ribossômico 18S/genética
14.
Vet Parasitol Reg Stud Reports ; 35: 100776, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36184107

RESUMO

Toxoplasmosis has been reported in Nigeria using several diagnostic tools with high prevalence in humans and some food animals. Rodents have been recognised as vital intermediate hosts of Toxoplasma gondii. However, there is paucity of information on the occurrence of T. gondii in wild rats found in Nigeria. This study aimed at molecular detection of T. gondii in Zyzomys pedunculatus and to evaluate its involvement in the epidemiology of toxoplasmosis in Nigeria. A total of 84 rats were sampled across three states of the North Central Nigeria, and DNA was extracted from the brain, lungs, kidney and intestine of the rats for the detection of T. gondii DNA by nested PCR to amplify the multicopy B1 gene. Sixty-four of the 84 samples (76.2%) were positive for T. gondii out of which 5 samples were sequenced and had an identity score of between 97.73% and 99.35% with the reference B1 gene of T. gondii in GenBank. This study suggests Nigerian wild rats may be an important intermediate hosts of T. gondii and may play a role in the epidemiology and maintenance of T. gondii circulation in Nigeria.


Assuntos
Doenças dos Roedores , Toxoplasma , Toxoplasmose Animal , Animais , DNA de Protozoário/análise , Humanos , Nigéria/epidemiologia , Prevalência , Ratos , Doenças dos Roedores/epidemiologia , Roedores , Toxoplasma/genética , Toxoplasmose Animal/diagnóstico , Toxoplasmose Animal/epidemiologia
15.
Microbiol Spectr ; 10(5): e0262822, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36190410

RESUMO

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Feminino , Humanos , DNA de Cinetoplasto/genética , Parasitemia/diagnóstico , Parasitemia/epidemiologia , Parasitemia/genética , DNA Satélite , Espanha/epidemiologia , DNA de Protozoário/genética , DNA de Protozoário/análise , Transmissão Vertical de Doenças Infecciosas , Doença de Chagas/diagnóstico , Doença de Chagas/epidemiologia , Doença de Chagas/genética , Trypanosoma cruzi/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
16.
EMBO J ; 41(22): e111839, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36221862

RESUMO

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.


Assuntos
Paramecium , Paramecium/genética , Paramecium/metabolismo , Elementos de DNA Transponíveis/genética , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , DNA de Protozoário/genética , DNA de Protozoário/metabolismo
17.
Eur J Protistol ; 85: 125910, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35939868

RESUMO

In the present study, a new freshwater peniculid species, Frontonia apoelegans sp. nov., and two other peniculid species, Frontonia atra (Ehrenberg, 1833) Bütschli, 1889 and Stokesia vernalis Wenrich, 1929, were isolated from Lake Weishan wetland, northern China. Their morphology and infraciliature are described based on live observations and silver staining methods. The SSU rRNA gene sequences are also provided. Frontonia apoelegans sp. nov. is recognized by the following combination of characteristics: two contractile vacuoles located right-dorsally, without collecting canals; peniculi 1 and 2 four-rowed, peniculus 3 three-rowed with leftmost row containing only one kinetosome; 62-76 somatic kineties; three ophryokineties; and four or five postoral kineties. We also provide improved diagnoses for Frontonia atra and Stokesia vernalis based on current and previous reports. Comparisons with sequences of morphologically similar species clearly support the validity of the new species. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Frontonia species with two contractile vacuoles cluster in a single clade, indicating these species may have a common origin. The family Frontoniidae is non-monophyletic whereas the family Stokesiidae remains monophyletic according to our analyses.


Assuntos
Cilióforos , Oligoimenóforos , China , Cilióforos/genética , DNA de Protozoário/genética , Genes de RNAr/genética , Oligoimenóforos/genética , Filogenia
18.
Parasitol Res ; 121(10): 2817-2829, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35939148

RESUMO

The development of new molecular methods has significantly improved the detection and identification of avian haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) compared to microscopic examination. Very large numbers of previously hidden Haemosporida species of a wide range of avian hosts have thus been discovered in the last two decades. However, test parameters of the various detection methods remain largely unevaluated. In this study, the merits of microscopy, multiplex PCR, and nested PCR were compared to identify the infection status of three Malagasy bird species. A total of 414 blood samples of Hypsipetes madagascariensis, Foudia omissa and F. madagascariensis, as well as 147 blood smears, were examined for haemosporidian infection. Thirty-four lineages of haemosporidian parasites could be identified, of which six have been detected for the first time. Microscopy, multiplex and nested PCR showed differences in detection rate, most likely due to low parasitemia of chronically infected birds. The combination of both PCR methods yielded the best results. In particular, detection of multiple infections could be greatly improved and will enable more precise prevalence estimates of individual haemosporidian species in wild birds in the future.


Assuntos
Doenças das Aves , Haemosporida , Parasitos , Plasmodium , Infecções Protozoárias em Animais , Animais , Doenças das Aves/diagnóstico , Doenças das Aves/parasitologia , Aves , DNA de Protozoário/genética , Haemosporida/genética , Microscopia , Reação em Cadeia da Polimerase Multiplex , Filogenia , Plasmodium/genética , Infecções Protozoárias em Animais/diagnóstico , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/parasitologia
19.
Res Vet Sci ; 152: 58-60, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-35930934

RESUMO

Toxoplasmosis, a zoonotic infection that is significant for public health (immunocompromised patients, pregnant women) and veterinary medicine (economic losses in the herd), is caused by an intracellular protozoan parasite belonging to the phylum Apicomplexa called Toxoplasma gondii. Consumption of unpasteurized milk and contaminated undercooked meat is a significant source for humans. The present study aimed to determine Toxoplasma gondii DNA in sheep, goats and donkeys Milk kept in East Azerbaijan province using the PCR method based on the B1 gene. For this purpose, 100 milk samples, including 45 sheep, 45 goats and 10 donkeys, were collected from different regions of northwestern Iran using direct milking and then transferred to the Food and Aquatic Health Laboratory under refrigerated conditions. The results showed that out of 100 milk samples examined, 16 samples (16%) were contaminated, and Toxoplasma gondii DNA was detected in 5 (11.11%) sheep, 9 (20%) goats and 2 (20%) donkeys milk specimen, respectively. These findings indicated that Toxoplasma gondii contaminated the raw milk, a human infection source.


Assuntos
Doenças das Cabras , Doenças dos Ovinos , Toxoplasma , Toxoplasmose Animal , Gravidez , Ovinos/genética , Humanos , Feminino , Animais , Toxoplasma/genética , Toxoplasmose Animal/diagnóstico , Toxoplasmose Animal/epidemiologia , Toxoplasmose Animal/parasitologia , Cabras/genética , Equidae/genética , Leite/química , Carneiro Doméstico/genética , Irã (Geográfico)/epidemiologia , Azerbaijão , DNA de Protozoário/genética , DNA de Protozoário/análise , Reação em Cadeia da Polimerase/veterinária , Estudos Soroepidemiológicos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/parasitologia , Doenças das Cabras/epidemiologia , Doenças das Cabras/parasitologia
20.
Korean J Parasitol ; 60(4): 295-299, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36041492

RESUMO

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.


Assuntos
Malária Falciparum , Malária Vivax , Malária , DNA de Protozoário/análise , DNA de Protozoário/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Malária/diagnóstico , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Plasmodium falciparum/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade
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