RESUMEN
INTRODUCTION: Chagas disease caused by Trypanosoma cruzi (T cruzi) found in the Americas is often missed during the early stage of infection due to lack of sensitive diagnostic tests. The classic immunological and parasitological tests often fail in the acute phase due to the nonspecific and low antibody level nature of the infection and in the chronic phase due to low levels of trypanosomes in the blood. For successful control strategies, there must be a sensitive and specific diagnostic test. OBJECTIVE/METHODS: We have demonstrated the possibility (proof of concept) of detecting T cruzi-specific repeat DNA via polymerase chain reaction (PCR) by (1) spiking 15 urine samples collected from volunteers free of prior infection with 3 different concentrations of T cruzi (3 strains), Trypanosoma brucei, and Trypanosoma rhodesiense (African strain) genomic DNA and (2) from filtered collected clinical samples from Argentina. Three sets of primers were used. RESULTS: Our approach detected repeat DNA specific for T cruzi strains from 1 clinical sample by 2 sets of primer and from spiked urine by all 3 sets of primer but not the African species. A serial dilution (spiking) also was performed on T cruzi strains to detect sensitivities of the assay. One set of primers constantly detected satellite DNA for all T cruzi strains from 70 pg/µl to 175 fg/µl. CONCLUSIONS: We were able to demonstrate the feasibility of detecting T cruzi-specific DNA from filtered urine samples by sensitive and specific PCR assay. Besides the evident increased sensitivity and specificity of primers, our approach can be used to explore Chagas prevalence in endemic areas - especially in congenital Chagas newborn screening - and in the acute phase.
Asunto(s)
Enfermedad de Chagas , ADN Protozoario , Reacción en Cadena de la Polimerasa , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/orina , ADN Protozoario/orina , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Sensibilidad y Especificidad , Argentina/epidemiología , Especificidad de la EspecieRESUMEN
Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.
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ADN Protozoario/orina , Leishmania/genética , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/orina , ADN Protozoario/aislamiento & purificación , Humanos , Leishmania/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
ABSTRACT Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.
Asunto(s)
Humanos , ADN Protozoario/orina , Leishmania/genética , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/orina , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , ADN Protozoario/aislamiento & purificación , Sensibilidad y Especificidad , Leishmania/aislamiento & purificaciónRESUMEN
The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.
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Enfermedades Endémicas , Leishmania infantum/aislamiento & purificación , Leishmania major/aislamiento & purificación , Leishmania tropica/aislamiento & purificación , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Visceral/diagnóstico , Adolescente , Adulto , Anciano , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/orina , Estudios de Casos y Controles , Niño , Preescolar , ADN Protozoario/sangre , ADN Protozoario/orina , Femenino , Humanos , Irán , Leishmania infantum/clasificación , Leishmania infantum/genética , Leishmania infantum/inmunología , Leishmania major/clasificación , Leishmania major/genética , Leishmania major/inmunología , Leishmania tropica/clasificación , Leishmania tropica/genética , Leishmania tropica/inmunología , Leishmaniasis Cutánea/sangre , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/orina , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/orina , Masculino , Persona de Mediana Edad , Filogenia , Reacción en Cadena de la Polimerasa , Estudios ProspectivosRESUMEN
The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per µL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients.
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ADN de Cinetoplasto/aislamiento & purificación , ADN de Cinetoplasto/orina , Leishmania infantum/genética , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/orina , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Orina/parasitología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Adolescente , Adulto , Anciano , Brasil , Niño , Creatinina/sangre , Creatinina/orina , ADN de Cinetoplasto/sangre , ADN de Cinetoplasto/genética , ADN Protozoario/sangre , ADN Protozoario/aislamiento & purificación , ADN Protozoario/orina , Femenino , VIH/patogenicidad , Humanos , Leishmania infantum/patogenicidad , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Urea/sangre , Urea/orina , Adulto JovenRESUMEN
Plasmodium vivax is the most prevalent cause of human malaria in the world and can lead to severe disease with high potential for relapse. Its genetic and geographic diversities make it challenging to control. P. vivax is understudied and to achieve control of malaria in endemic areas, a rapid, accurate, and simple diagnostic tool is necessary. In this pilot study, we found that a colorimetric system using AuNPs and MSP10 DNA detection in urine can provide fast, easy, and inexpensive identification of P. vivax. The test exhibited promising sensitivity (84%), high specificity (97%), and only mild cross-reactivity with P. falciparum (21%). It is simple to use, with a visible color change that negates the need for a spectrometer, making it suitable for use in austere conditions. Using urine eliminates the need for finger-prick, increasing both the safety profile and patient acceptance of this model.
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Colorimetría/métodos , Malaria Vivax/diagnóstico , Nanopartículas del Metal , Oligonucleótidos , Plasmodium vivax/aislamiento & purificación , Orina/parasitología , Antígenos de Protozoos/genética , Colorimetría/economía , Colorimetría/normas , Reacciones Cruzadas , ADN Protozoario/orina , Oro , Humanos , Malaria Vivax/parasitología , Malaria Vivax/orina , Tamizaje Masivo , Microscopía , Parasitemia/diagnóstico , Parasitemia/parasitología , Proyectos Piloto , Plasmodium vivax/genética , Plasmodium vivax/ultraestructura , Proteínas Protozoarias/genética , Sensibilidad y EspecificidadRESUMEN
Pigs were infected with a Bolivian strain of Trypanosoma cruzi (genotype I) and evaluated up to 150 days postinoculation (dpi) to determine the use of pigs as an animal model of Chagas disease. Parasitemia was observed in the infected pigs during the acute phase (15-40 dpi). Anti-T. cruzi immunoglobulin M was detected during 15-75 dpi; high levels of anti-T. cruzi immunoglobulin G were detected in all infected pigs from 75 to 150 dpi. Parasitic DNA was observed by western blot (58%, 28/48) and polymerase chain reaction (27%, 13/48) in urine samples, and in the brain (75%, 3/4), spleen (50%, 2/4), and duodenum (25%, 1/4), but no parasitic DNA was found in the heart, colon, and kidney. Parasites were not observed microscopically in tissues samples, but mild inflammation, vasculitis, and congestion was observed in heart, brain, kidney, and spleen. This pig model was useful for the standardization of the urine test because of the higher volume that can be obtained as compared with other small animal models. However, further experiments are required to observe pathological changes characteristic of Chagas disease in humans.
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Enfermedad de Chagas/parasitología , Modelos Animales de Enfermedad , Trypanosoma cruzi/fisiología , Animales , Anticuerpos Antiprotozoarios/sangre , Western Blotting , ADN Protozoario/aislamiento & purificación , ADN Protozoario/orina , Femenino , Inmunoglobulina G/sangre , Parasitemia , Porcinos , Factores de Tiempo , Distribución TisularRESUMEN
Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.
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ADN Protozoario/análisis , ADN Protozoario/orina , Técnicas de Amplificación de Ácido Nucleico/métodos , Saliva/parasitología , Trypanosoma brucei gambiense/genética , Tripanosomiasis Africana/diagnóstico , Animales , Chlorocebus aethiops , ADN Protozoario/aislamiento & purificación , Modelos Animales de Enfermedad , Parasitología , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Trypanosoma brucei gambiense/aislamiento & purificación , Tripanosomiasis Africana/parasitologíaRESUMEN
This study compares the utility of a molecular diagnosis of experimental CanL on non-invasive samples (urine, conjunctival (CS), oral (OS) and vulvar (VS) swabs) with that of traditional invasive techniques during the course of infection. Eight dogs were experimen-tally infected with Leishmania infantum and followed monthly for 12 months to assess clinical, clinicopathological, immunological and parasitological variables. Active infection was produced in 100% of the dogs. The animals showed positive bone marrow (BM) cytologies and cultures, clinical signs, clinicopathological abnormalities and a high specific humoral immune response. The infection was detected at 90 days post-infection (p.i.) by real-time quantitative PCR (rtQ-PCR) on BM in all dogs and in blood in 2 dogs, while anti-L. infantum antibody seroconversion occurred between Days 120 and 180 days p.i. The tissue with the highest L. infantum kDNA load, as detected by rtQ-PCR, was BM (range 381.570,000 parasites/ml at the study end), this sample type showing greater sensitivity than peripheral blood (PB). The vulvar swabs used here for the first time to quantify para-site loads in dogs revealed a greater load than oral and conjunctival swabs at one year p.i. Urine samples showed the lowest concentrations of L. infantum DNA (maximum: 8.57 par-asites/ml). Our results suggest that for the early detection of infection, adding to serology a test such as rtQ-PCR on OS or VS improves sensitivity and specificity.
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Enfermedades de los Perros/diagnóstico , Leishmania infantum/genética , Leishmaniasis Visceral/veterinaria , Animales , Médula Ósea/parasitología , ADN Protozoario/genética , ADN Protozoario/orina , Enfermedades de los Perros/parasitología , Perros , Femenino , Estudios de Seguimiento , Leishmania infantum/inmunología , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y EspecificidadRESUMEN
The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the host's body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection.
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ADN Protozoario/sangre , Enfermedades Endémicas , Schistosoma japonicum/genética , Esquistosomiasis Japónica/diagnóstico , Esquistosomiasis Japónica/epidemiología , Adolescente , Adulto , Animales , Sistema Libre de Células , ADN Protozoario/orina , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Filipinas/epidemiología , Reacción en Cadena de la Polimerasa , Schistosoma japonicum/aislamiento & purificación , Esquistosomiasis Japónica/genética , Sensibilidad y Especificidad , Adulto JovenRESUMEN
BACKGROUND: Diagnostic techniques based on PCR for the detection of Plasmodium DNA can be highly sensitive and specific. The vast majority of these techniques rely, however, on the invasive sampling of blood from infected hosts. There is, currently, considerable interest in the possibility of using body fluids other than blood as sources of parasite DNA for PCR diagnosis. METHODS: Urine and faeces were obtained from a Plasmodium knowlesi infected-Japanese macaque (Macaca fuscata) over the course of an experimentally induced infection. P. knowlesi DNA (PkDNA) extracted from urine and faeces were monitored by nested PCR targeting the P. knowlesi specific cytochrome b (cytb) gene. RESULTS: Urinary PkDNA was detected on day 2, but was not amplified using DNA templates extracted from the samples on day 4, day 5 and day 6. Subsequently, urinary PkDNA was detected from day 7 until day 11, and from day 20 until day 30. PkDNA in faeces was detected from day 7 until day 11, and from day 20 until day 37. Moreover, real-time quantitative PCR showed a remarkable increase in the amount of urinary PkDNA following anti-malarial treatment. This might have been due to the release of a large amount of PkDNA from the degraded parasites as a result of the anti-malarial treatment, leading to excretion of PkDNA in the urine. CONCLUSIONS: The cytb-PCR system using urine and faecal samples is of potential use in molecular epidemiological surveys of malaria. In particular, monkey faecal samples could be useful for the detection of zoonotic primate malaria in its natural hosts.
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ADN Protozoario/orina , Heces/química , Macaca/orina , Malaria/parasitología , Malaria/orina , Plasmodium knowlesi/genética , Animales , ADN Protozoario/análisis , Modelos Animales de Enfermedad , Femenino , Malaria/metabolismo , Malaria/fisiopatología , Microscopía , Técnicas de Diagnóstico Molecular , Parasitología , Reacción en Cadena de la PolimerasaRESUMEN
INTRODUCTION: Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. METHODS: A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. RESULTS: With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. CONCLUSION: Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.
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ADN Protozoario/orina , Leishmania infantum/genética , Leishmaniasis Visceral/parasitología , Manejo de Especímenes/métodos , Humanos , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/orina , Reacción en Cadena de la PolimerasaRESUMEN
Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost. .
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Humanos , ADN Protozoario/orina , Leishmania infantum/genética , Leishmaniasis Visceral/parasitología , Manejo de Especímenes/métodos , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/orina , Reacción en Cadena de la PolimerasaRESUMEN
The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.
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Enfermedad de Chagas/orina , ADN Protozoario/orina , Enfermedades Renales/orina , Trypanosoma cruzi , Animales , Antígenos de Protozoos , Enfermedad de Chagas/sangre , ADN Protozoario/sangre , Cobayas , Corazón/parasitología , Riñón/metabolismo , Riñón/parasitología , Enfermedades Renales/sangre , Enfermedades Renales/parasitología , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/orina , Reacción en Cadena de la Polimerasa/métodosRESUMEN
In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.
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ADN Protozoario/orina , Técnicas de Amplificación de Ácido Nucleico/métodos , Toxoplasma/aislamiento & purificación , Toxoplasmosis/parasitología , Animales , ADN Protozoario/genética , Femenino , Humanos , Ratones , Ratones Endogámicos ICR , Toxoplasma/genética , Toxoplasmosis/diagnósticoRESUMEN
A leishmaniose visceral ocorre em países dos cinco continentes e quando não tratada pode levar a óbito. Para se evitar esse desfecho, são essenciais o diagnóstico precoce e tratamento adequado. Com o objetivo de contribuir na pesquisa de novos diagnósticos para leishmaniose visceral, esse trabalho propôs desenvolver sistemas baseados em Nested-PCR convencional e em único tubo para o diagnóstico de leishmaniose visceral. A partir de uma revisão na literatura em busca de alvos moleculares utilizados no diagnóstico dessa parasitose, foram selecionados os alvos subunidade menor do RNA ribossômico (ssu rRNA) e espaçador transcrito interno 1 (ITS-1), que compõem o DNA do agente etiológico Leishmania infantum, para o desenvolvimento das nested-PCR. Foi também escolhido o alvo kDNA, o mais aplicado nas abordagens de PCR, para comparações com os sistemas desenvolvidos. Após otimizar todas as PCR com DNA genômico de L. infantum, esses sistemas foram avaliados em amostras de sangue, soro e urina de indivíduos com suspeita de leishmaniose visceral dos hospitais de referência da cidade do Recife - PE. Para utilização da urina, foram avaliados quatro protocolos de extração de DNA e identificou-se que a extração por fenol-clorofórmio, com modificações, foi a de melhor desempenho. Na avaliação com amostras biológicas, as PCR simples e nested-PCR com os alvos ssu rRNA e ITS-1 não tiveram boa sensibilidade ao se usar sangue, e não foram capaz de amplificar DNA do parasito em soro e urina. Esses sistemas desenvolvidos não podem ser usados para o diagnóstico da leishmaniose visceral. No entanto, a kDNAPCR apresentou bons resultados quando avaliada com urina. Mais estudos devem ser feitos para avalia-la como um diagnóstico seguro para esse tipo de amostra biológica. Esse trabalho representa um ponto de início para posteriores estudos que objetivem o aprimoramento e validação da nested-PCR único tubo para o diagnóstico da leishmaniose visceral.
Asunto(s)
Humanos , Técnicas y Procedimientos Diagnósticos , Leishmania infantum/genética , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Cartilla de ADN , ADN Ribosómico/genética , ADN de Cinetoplasto/análisis , ADN de Cinetoplasto/aislamiento & purificación , ADN Protozoario/inmunología , ADN Protozoario/sangre , ADN Protozoario/orina , Mycobacterium tuberculosis/aislamiento & purificación , Sensibilidad y Especificidad , Schistosoma mansoni/aislamiento & purificación , Trypanosoma cruzi/aislamiento & purificación , Wuchereria bancrofti/aislamiento & purificaciónRESUMEN
Saliva and urine from malaria-infected individuals contain trace amounts of Plasmodium DNA, and therefore, could be used as alternative specimens for diagnosis. A nested PCR targeting the mitochondrial cytochrome b gene (Cytb-PCR) of four human malaria species and Plasmodium knowlesi was developed and tested with 693 blood samples from febrile patients living in diverse malaria-endemic areas of Thailand, and compared with microscopy and nested PCR targeting small-subunit rRNA (18S-PCR). Cytb-PCR was 16% and 39.8% more sensitive than 18S-PCR and microscopy, respectively, in detecting all of these malarial species in blood samples. Importantly, 34% and 17% of Plasmodium falciparum and Plasmodium vivax mono-infections, respectively, detected by microscopy were, in fact, mixed P. falciparum and P. non-falciparum infections. Analysis of matched blood, saliva and urine from 157 individuals showed that microscopy and Cytb-PCR of saliva yielded no significant difference in detecting P. falciparum and P. vivax. However, Cytb-PCR of saliva was more sensitive than microscopy for diagnosis of mixed-species infections. A combination of Cytb-PCR of saliva and of urine significantly outperformed microscopy (p 0.0098 for P. falciparum, p 0.006 for P. vivax, and p 0.0002 for mixed infections). Furthermore, Plasmodium malariae and P. knowlesi could also be identified in saliva and urine with this method. Therefore, the Cytb-PCR developed herein offers a high potential for the use of both saliva and urine for malaria diagnosis, with a sensitivity comparable with or superior to that of microscopy.
Asunto(s)
ADN Protozoario/orina , Malaria/diagnóstico , Plasmodium/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Saliva/parasitología , Adolescente , Adulto , Anciano , Niño , Preescolar , Citocromos b/genética , ADN Mitocondrial/genética , ADN Protozoario/sangre , ADN Protozoario/genética , Femenino , Humanos , Malaria/sangre , Malaria/parasitología , Malaria/orina , Masculino , Microscopía , Persona de Mediana Edad , Mitocondrias/genética , Plasmodium/genética , Plasmodium/patogenicidad , ARN Ribosómico 18S/genética , Sensibilidad y Especificidad , Adulto JovenRESUMEN
BACKGROUND: Definite diagnosis of malaria relies on microscopy detection of blood stages of parasites in peripheral blood and requires blood sample collection. The nested PCR method has shown to be more sensitive and superior to microscopy in detecting co-infections of Plasmodium species in circulation while Plasmodium falciparum DNA can be identified in urine and saliva specimens of patients, albeit at a lower sensitivity. METHODS: Matched blood, saliva and urine samples were collected from 100 microscopy-positive and 20 microscopy-negative febrile patients who attended a malaria clinic in Tak Province, northwestern Thailand for nested PCR analysis targeting the small subunit ribosomal RNA gene of human malaria. Both P. falciparum and Plasmodium vivax have been known to circulate at a comparable rate in the study area. RESULTS: Comparing with microscopy results, nested PCR of saliva samples had a sensitivity of 74.1% for P. falciparum detection and 84% for P. vivax detection while 44.4% and 34.0% of the corresponding values were observed for urine samples. Both nested PCR results of saliva and urine samples had a specificity of 100% for identification of P. falciparum and P. vivax when compared with nested PCR results from blood. Co-infections of both species were found in four, 26 and 8 patients by microscopy and nested PCR of blood and saliva samples, respectively. Although the positive rates of nested PCR of saliva samples for P. falciparum increased with parasite density, no tendency occurred in results from nested PCR of saliva samples for P. vivax as well as those of urine samples. CONCLUSIONS: Saliva and urine samples could be alternative noninvasive sources of DNA for molecular detection of both P. falciparum and P. vivax. Further improvement of the detection method will offer an opportunity to use these samples for diagnosis of malaria.
Asunto(s)
ADN Protozoario/sangre , ADN Protozoario/orina , Genes de ARNr/genética , Malaria/diagnóstico , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/aislamiento & purificación , Saliva/parasitología , Adulto , Animales , Niño , Preescolar , Estudios Transversales , ADN Protozoario/genética , Femenino , Humanos , Malaria/sangre , Malaria/parasitología , Malaria/orina , Masculino , Microscopía/normas , Persona de Mediana Edad , Plasmodium falciparum/genética , Plasmodium vivax/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Tailandia , Adulto JovenRESUMEN
A pair of specific primers and a TaqMan probe were designed based on the sequence of Toxoplasma gondii B1 gene from GenBank database. Total DNA of T. gondii was extracted from fresh mice urine. DNA fragment of B1 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector. Following identification, the positive recombinant plasmid was used as reference template to generate standard curve and melt curve. Sensitivity, reproducibility, linear range and stability of reference plasmids were determined. The sensitivity of this method was 10(4) copies/ml. The coefficient of variation (cv) of intra-assay and inter-assay were 2.42% and 4.18%, respectively. Linear range was (10(3)-10(7)) copies/ml. The specificity was 100%. The reference materials were stable. Real-time FQ-PCR of T. gondii DNA in mice urine has been constructed, which is a convenient, sensitive and reliable method for quantifying T. gondii DNA in mice urine.