RESUMEN
BACKGROUND: Babesiosis is an emerging tick-borne infection in humans. The increasing numbers of reported cases of transfusion-associated babesiosis (TAB), primarily caused by Babesia microti, represents a concern for the safety of the US blood supply. STUDY DESIGN AND METHODS: This study investigated kinetics of parasitemia and innate immune responses and dynamics of antibody responses during B. microti infection in rhesus macaques (RMs) using blood smears, quantitative polymerase chain reaction (qPCR), flow cytometry, and indirect fluorescent antibody testing. A total of six monkeys were transfused with either hamster or monkey-passaged B. microti-infected red blood cells (two and four monkeys, respectively) simulating TAB. RESULTS: The prepatent period in monkeys inoculated with hamster-passaged B. microti was 35 days compared with 4 days in monkeys transfused with monkey-passaged B. microti; the latter monkeys also had markedly higher parasitemia levels. The duration of the window period from the first detected parasitemia by qPCR analysis to the first detected antibody response ranged from 10 to 17 days. Antibody responses fluctuated during the course of the infection. Innate responses assessed by the frequencies of monocytes and activated B cells correlated with the kinetics and magnitude of parasitemia. On Day 14, additional activation peaks were noted for CD14+CD16+ and CD14-CD16+ monocytes and for CD11c+ myeloid dendritic cells, but only in animals transfused with monkey-passaged B. microti. Parasitemia persisted in these immunocompetent animals, similar to human infection. CONCLUSION: The results suggest that transfusion-associated transmission of B. microti leads to rapid onset of parasitemia (Day 4) in RMs, detectable antibody response 14 days later, and persistent parasitemia.
Asunto(s)
Babesiosis/transmisión , Macaca mulatta/inmunología , Reacción a la Transfusión , Animales , Anticuerpos Antiprotozoarios/sangre , Babesiosis/diagnóstico , Babesiosis/inmunología , Cricetinae , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Haplorrinos , Cinética , Macaca mulatta/sangre , Macaca mulatta/parasitología , Parasitemia/sangre , Parasitemia/diagnóstico , Parasitemia/transmisión , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Almost all of the reported US tick-borne and transfusion-associated Babesia cases have been caused by Babesia microti, which is endemic in the Northeast and upper Midwest. We investigated a case caused by B. duncani (formerly, the WA1-type parasite), in a 59-year-old California resident with sickle cell disease (HbSS) whose only risk factor for infection was receipt of red blood cell transfusions. CASE REPORT: The patient's case was diagnosed in September 2008: intraerythrocytic parasites were noted on a blood smear, after a several-month history of increasing transfusion requirements. Molecular and indirect fluorescent antibody (IFA) analyses were negative for B. microti but were positive for B. duncani (IFA titer, 1:1024). The complete 18S ribosomal RNA gene of the parasite was amplified from a blood specimen; the DNA sequence was identical to the sequence for the index WA1 parasite isolated in 1991. The patient's case prompted a transfusion investigation: 34 of 38 pertinent blood donors were evaluated, none of whom tested positive by B. microti IFA. The implicated donor-a 67-year-old California resident-had a B. duncani titer of 1:4096; B. duncani also was isolated by inoculating jirds (Mongolian gerbils) with a blood specimen from March 2009, more than 10 months after his index donation in April 2008. The patient's case was diagnosed more than 4 months after the implicated transfusion in May 2008. CONCLUSIONS: This patient had the third documented transfusion case caused by B. duncani. His case underscores the fact that babesiosis can be caused by agents not detected by molecular or serologic analyses for B. microti.
Asunto(s)
Anemia de Células Falciformes , Babesia , Babesiosis , Donantes de Sangre , Transfusión de Eritrocitos , ARN Protozoario , ARN Ribosómico 18S/sangre , Anciano , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/parasitología , Anemia de Células Falciformes/terapia , Animales , Babesia/genética , Babesia/aislamiento & purificación , Babesiosis/sangre , Babesiosis/genética , Babesiosis/transmisión , California , Eritrocitos/parasitología , Gerbillinae , Humanos , Masculino , Persona de Mediana Edad , ARN Protozoario/sangre , ARN Protozoario/genética , ARN Ribosómico 18S/genéticaRESUMEN
The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Proteínas de la Membrana/genética , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Mutación Puntual , Adolescente , Adulto , Animales , Niño , Preescolar , República Democrática del Congo , Femenino , Humanos , Italia , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias , TanzaníaRESUMEN
Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis.
Asunto(s)
Angiostrongylus cantonensis , ADN de Helmintos/líquido cefalorraquídeo , Eosinofilia/parasitología , Meningitis/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones por Strongylida/diagnóstico , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , ADN de Helmintos/aislamiento & purificación , Eosinofilia/patología , Femenino , Humanos , Lactante , Masculino , Meningitis/patología , Persona de Mediana Edad , Infecciones por Strongylida/parasitología , Adulto JovenRESUMEN
The rat lungworm (Angiostrongylus cantonensis) is a parasitic nematode that causes rat lungworm disease. It is the leading cause of eosinophilic meningitis and is a zoonotic health risk. We confirmed the presence of A. cantonensis using species-specific, quantitative PCR in 18 of 50 (36%) giant African land snails (Lissachatina fulica) collected from Miami, Florida, US in May 2013. These snails were collected from seven of 21 core areas that the Florida Department of Agriculture and Consumer Services monitor weekly. Rat lungworms have not previously been identified in these areas. Duplicate DNA extractions of foot muscle tissue from each snail were tested. Of the seven core areas we examined, six were positive for A. cantonensis and prevalence of infection ranged from 27% to 100%. Of the 18 positive snails, only five were positive in both extractions. Our results confirm an increase in the range and prevalence of rat lungworm infection in Miami. We also emphasize the importance of extracting sufficient host tissue to minimize false negatives.
Asunto(s)
Angiostrongylus cantonensis/fisiología , Caracoles/parasitología , Infecciones por Strongylida/veterinaria , Animales , Florida/epidemiología , Reacción en Cadena de la Polimerasa/veterinaria , Prevalencia , Infecciones por Strongylida/epidemiología , Infecciones por Strongylida/transmisiónRESUMEN
Although the geographic origin of malaria cases imported into the United States can often be inferred from travel histories, these histories may be lacking or incomplete. We hypothesized that mitochondrial haplotypes could provide region-specific molecular barcodes for tracing the origin of imported Plasmodium vivax infections. An analysis of 348 mitochondrial genomes from worldwide parasites and new sequences from 69 imported malaria cases diagnosed across the United States allowed for a geographic assignment of most infections originating from the Americas, southeast Asia, east Asia, and Melanesia. However, mitochondrial lineages from Africa, south Asia, central Asia, and the Middle East, which altogether contribute the vast majority of imported malaria cases in the United States, were closely related to each other and could not be reliably assigned to their geographic origins. More mitochondrial genomes are required to characterize molecular barcodes of P. vivax from these regions.
Asunto(s)
Genoma Mitocondrial/genética , Malaria Vivax/epidemiología , Plasmodium vivax/aislamiento & purificación , Vigilancia de la Población , Secuencia de Bases , Teorema de Bayes , Genoma de Protozoos/genética , Haplotipos , Humanos , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Filogenia , Plasmodium vivax/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Viaje , Estados Unidos/epidemiologíaRESUMEN
There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs.
Asunto(s)
Cartilla de ADN , Malaria/diagnóstico , Plasmodium falciparum/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Colorantes Fluorescentes , Humanos , Malaria/genética , Malaria/parasitología , Plasmodium falciparum/genética , ARN Ribosómico 18S/genética , Sensibilidad y EspecificidadRESUMEN
Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.
Asunto(s)
Malaria Vivax/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Humanos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Plasmodium vivax/patogenicidad , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
BACKGROUND: Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection. METHODOLOGY AND SIGNIFICANT FINDINGS: We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites. CONCLUSIONS: The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.
Asunto(s)
Bioensayo/métodos , Malaria/parasitología , Parásitos/genética , Plasmodium knowlesi/genética , Plasmodium knowlesi/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Zoonosis/parasitología , Animales , Secuencia de Bases , Reacciones Cruzadas/inmunología , Cartilla de ADN/metabolismo , Genoma de Protozoos/genética , Haplorrinos/parasitología , Humanos , Límite de Detección , Parásitos/aislamiento & purificación , Plasmodium vivax/genética , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Especificidad de la EspecieAsunto(s)
Angiostrongylus cantonensis/crecimiento & desarrollo , Enfermedades del Simio Antropoideo/parasitología , Meningitis/veterinaria , Pongo pygmaeus , Infecciones por Strongylida/veterinaria , Animales , Enfermedades del Simio Antropoideo/tratamiento farmacológico , Enfermedades del Simio Antropoideo/inmunología , Resultado Fatal , Femenino , Meningitis/tratamiento farmacológico , Meningitis/inmunología , Meningitis/parasitología , Metilprednisolona/uso terapéutico , Infecciones por Strongylida/tratamiento farmacológico , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitologíaRESUMEN
We compared a nested PCR assay and microscopic examination of Giemsa-stained blood films for detection and identification of Plasmodium spp. in blood specimens. PCR was more sensitive than microscopy and capable of identifying malaria parasites at the species level when microscopy was equivocal.
Asunto(s)
Malaria/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Protozoario/sangre , ADN Protozoario/genética , Genes Protozoarios , Humanos , Malaria/parasitología , Microscopía , Parasitología/métodos , Parasitología/estadística & datos numéricos , Plasmodium/genética , Plasmodium/aislamiento & purificación , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published by December 2004. The limits of detection and efficiency of each real-time PCR assay were determined using DNA extracted from stool samples spiked with serially diluted cultured E. histolytica trophozoites. The ability of each assay to correctly distinguish E. histolytica from E. dispar was evaluated with DNA extracted from patients' stools and liver aspirates submitted for confirmatory diagnosis. Real-time PCR allowed quantitative analysis of the spiked stool samples, but major differences in detection limits and assay performance were observed among the evaluated tests. These results illustrate the usefulness of comparative evaluations of diagnostic assays.