RESUMO
There are few pathologic or molecular studies of penile precancerous lesions, and the majority refers to lesions associated with invasive carcinomas. Penile Intraepithelial Neoplasia (PeIN) is classified in two morphologically and distinctive molecular groups, non-HPV and HPV-related with special subtypes. The primary purpose of this international series was to classify PeIN morphologically, detect HPV genotypes and determine their distribution according to PeIN subtypes. A secondary aim was to evaluate the p16INK4a immunostaining as a possible HPV surrogate for high-risk HPV infection in penile precancerous lesions. Samples consisted of 84 PeIN cases, part of a retrospective cross-sectional analysis of 1095 penile carcinomas designed to estimate the HPV DNA prevalence in penile cancers using PCR and p16INK4a immunostaining. Penile Intraepithelial Neoplasia (PeIN) was classified in HPV-related (basaloid, warty-basaloid, warty, hybrid, and mixed subtypes) and non-HPV-related (differentiated), the former being the most frequent. PeIN subtypes were differentiated (non-HPV-related) and basaloid, warty-basaloid, warty, hybrid and mixed (HPV-related). Basaloid PeIN was the most commonly diagnosed subtype, and HPV16 was the most frequent HPV genotype detected. Warty-basaloid and warty PeIN showed a more heterogeneous genotypic composition. Most HPV genotypes were high-risk but low-risk HPV genotypes were also present in a few cases (4%). A single HPV genotype was detected in 82% of HPV positive cases. In contrast, multiple genotypes were detected in the remaining 18% of cases. The findings in this study support the paradigm that penile in situ neoplasia, like its invasive counterparts, is HPV dependent or independent and has distinctive morphological subtypes readily identified in routine practice. Considering that HPV16 is clearly the predominant type, and that the three available vaccines have HPV16, all of them will be suitable for vaccination programs; the price of the vaccines will be probably the main determinant to choose the vaccine.
Assuntos
Carcinoma in Situ , Carcinoma de Células Escamosas , Papiloma , Infecções por Papillomavirus , Neoplasias Penianas , Lesões Pré-Cancerosas , Neoplasias Cutâneas , Masculino , Humanos , Neoplasias Penianas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Carcinoma in Situ/patologia , Estudos Transversais , Estudos Retrospectivos , Carcinoma de Células Escamosas/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Neoplasias Cutâneas/complicações , Genótipo , Papillomaviridae/genéticaRESUMO
Penile intraepithelial neoplasia (PeIN) is currently classified in human papillomavirus (HPV)- and non-HPV-related subtypes with variable HPV genotypes. PeINs are frequently associated with other intraepithelial lesions in the same specimen. The aim of this study was to detect and compare HPV genotypes in PeINs and associated lesions using high-precision laser capture microdissection-polymerase chain reaction and p16INK4a immunostaining. We evaluated resected penile specimens from 8 patients and identified 33 PeINs and 54 associated lesions. The most common subtype was warty PeIN, followed by warty-basaloid and basaloid PeIN. Associated lesions were classical condylomas (17 cases), atypical classical condylomas (2 cases), flat condylomas (9 cases), atypical flat condylomas (6 cases), flat lesions with mild atypia (12 cases), and squamous hyperplasia (8 cases). After a comparison, identical HPV genotypes were found in PeIN and associated lesions in the majority of the patients (7 of 8 patients). HPV16 was the most common genotype present in both PeIN and corresponding associated lesion (50% of the patients). Nonspecific flat lesions with mild atypia, classical condylomas, and atypical condylomas were the type of associated lesions most commonly related to HPV16. Other high-risk HPV genotypes present in PeIN and associated nonspecific flat lesion with mild atypia were HPV35 and HPV39. In this study of HPV in the microenvironment of penile precancerous lesions, we identified identical high-risk HPV genotypes in PeIN and classical, flat, or atypical condylomas and, specially, in nonspecific flat lesions with mild atypia. It is possible that some of these lesions represent hitherto unrecognized precancerous lesions.
Assuntos
Carcinoma in Situ/virologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Neoplasias Penianas/virologia , Adolescente , Adulto , Idoso , Carcinoma in Situ/patologia , Condiloma Acuminado/patologia , Condiloma Acuminado/virologia , Genótipo , Humanos , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Papillomaviridae/genética , Neoplasias Penianas/patologia , Reação em Cadeia da Polimerase , Adulto JovemRESUMO
The International Society of Urological Pathology (ISUP) held an expert-driven penile cancer conference in Boston in March 2015, which focused on the new World Health Organisation (WHO) classification of penile cancer: human papillomavirus (HPV)-related tumours and histological grading. The conference was preceded by an online survey of the ISUP members, and the results were used to initiate discussions. Because of the rarity of penile tumours, this was not a consensus but an expert-driven conference aimed at assisting pathologists who do not see these tumours on a regular basis. After a justification for the novel separation of penile squamous cell carcinomas into HPV-related and non-HPV-related-carcinomas, the histological classification of penile carcinoma was proposed; this system was also accepted subsequently by the WHO for subtyping of penile carcinomas (2016). A description of HPV-related neoplasms, which may be recognised by their histological features, was presented, and p16 was recommended as a surrogate indicator of HPV. A three-tier grading system was recommended for penile squamous carcinomas; this was also adopted by the WHO (2016). Many of the distinctive histological subtypes of squamous cell carcinoma of the penis are associated with distinct grades, based on the squamous cell carcinoma subtype histological features.
Assuntos
Carcinoma de Células Escamosas/classificação , Neoplasias Penianas/classificação , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Humanos , Masculino , Gradação de Tumores , Infecções por Papillomavirus/complicações , Neoplasias Penianas/patologia , Neoplasias Penianas/virologia , Organização Mundial da SaúdeRESUMO
Chagas disease (ChD), caused by the protozoan Trypanosoma cruzi, is an endemic anthropozoonosis in Latin America, linked to deficients socio-economic and cultural aspects and is considered one of the neglected tropical diseases. We report a fatal case of Chagas disease reactivation with central nervous system involvement in a patient with HIV infection, whose diagnosis was confirmed by positive PCR (polymerase chain reaction) test of blood, with treatment response efficiency with benznidazol and management and etiologic treatment was difficult due to limited number of antitrypanosomal drugs and the occurrence of frequent and serious adverse effects.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Protozoárias do Sistema Nervoso Central/diagnóstico , Doença de Chagas/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Adulto , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Evolução Fatal , Feminino , Humanos , Imageamento por Ressonância MagnéticaRESUMO
Chagas disease (ChD), caused by the protozoan Trypanosoma cruzi, is an endemic anthropozoonosis in Latin America, linked to deficients socio-economic and cultural aspects and is considered one of the neglected tropical diseases. We report a fatal case of Chagas disease reactivation with central nervous system involvement in a patient with HIV infection, whose diagnosis was confirmed by positive PCR (polymerase chain reaction) test of blood, with treatment response efficiency with benznidazol and management and etiologic treatment was difficult due to limited number of antitrypanosomal drugs and the occurrence of frequent and serious adverse effects.
La enfermedad de Chagas, causada por el protozoo Trypanosoma cruzi, es una antropo-zoonosis endémica en Latinoamérica, vinculada con aspectos socio-económico-culturales deficitarios y considerada una de las enfermedades desatendidas. Presentamos un caso fatal de una reactivación de la enfermedad de Chagas con afectación del sistema nervioso central en un paciente con infección por VIH. El diagnóstico se confirmó por reacción de polimerasa en cadena (RPC) positiva en sangre. Tuvo una buena respuesta al tratamiento con benznidazol. Las dificultades en el manejo del tratamiento etiológico se debieron al número limitado de medicamentos antitripanosomiásicos y la aparición de efectos adversos graves.
Assuntos
Humanos , Feminino , Adulto , Doença de Chagas/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Protozoárias do Sistema Nervoso Central/diagnóstico , Imageamento por Ressonância Magnética , Infecções Oportunistas Relacionadas com a AIDS/parasitologia , Evolução Fatal , Infecções Protozoárias do Sistema Nervoso Central/parasitologiaRESUMO
Patients in treatment with allopurinol are in risk of having life threatening adverse reactions particularly at the beginning of the treatment. Two percent of the patients prescribed with this drug have associated severe cutaneous adverse reactions. We present two cases of allopurinol hypersensitivity syndrome in mexican patients in which asymptomatic hyperuricemia was the indication to its use. The general physician and the specialist must be alert of this syndrome that causes elevate morbidity and mortality.
Los pacientes bajo tratamiento con alopurinol pueden presentar reacciones adversas potencialmente mortales, particularmente al inicio del tratamiento. Las reacciones cutáneas adversas por alopurinol tienen una prevalencia aproximada del 2 %. Presentamos dos casos de síndrome de hipersensibilidad por alopurinol en pacientes mexicanos en quienes la hiperuricemia asintomática fue la indicación para su uso. El médico general y el especialista deben estar alerta ante este síndrome que ocasiona alta morbilidad y mortalidad.
Assuntos
Alopurinol/efeitos adversos , Síndrome de Hipersensibilidade a Medicamentos/etiologia , Supressores da Gota/efeitos adversos , Adolescente , Alopurinol/uso terapêutico , Síndrome de Hipersensibilidade a Medicamentos/diagnóstico , Feminino , Supressores da Gota/uso terapêutico , Humanos , Hiperuricemia/tratamento farmacológico , Masculino , Adulto JovemRESUMO
Penile clear cell carcinoma originating in skin adnexal glands has been previously reported. Here, we present 3 morphologically distinctive penile tumors with prominent clear cell features originating not in the penile skin but in the mucosal tissues of the glans surface squamous epithelium. Clinical and pathologic features were evaluated. Immunohistochemical stains were GATA3 and p16. Human papilloma virus (HPV) detection by in situ hybridization was performed in 3 cases, and whole-tissue section-polymerase chain reaction was performed in 1 case. Patients' ages were 52, 88, and 95 years. Tumors were large and involved the glans and coronal sulcus in all cases. Microscopically, nonkeratinizing clear cells predominated. Growth was in solid nests with comedo-like or geographic necrosis. Focal areas of invasive warty or basaloid carcinomas showing in addition warty or basaloid penile intraepithelial neoplasia were present in 2 cases. There was invasion of corpora cavernosa, lymphatic vessels, veins, and perineural spaces in all cases. p16 was positive, and GATA3 stain was negative in the 3 cases. HPV was detected in 3 cases by in situ hybridization and in 1 case by polymerase chain reaction. Differential diagnoses included other HPV-related penile carcinomas, skin adnexal tumors, and metastatic renal cell carcinoma. Features that support primary penile carcinoma were tumor location, concomitant warty and/or basaloid penile intraepithelial neoplasia, and HPV positivity. Clinical groin metastases were present in all cases, pathologically confirmed in 1. Two patients died from tumor dissemination at 9 and 12 months after penectomy. Clear cell carcinoma, another morphologic variant related to HPV, originates in the penile mucosal surface and is probably related to warty carcinomas.
Assuntos
Adenocarcinoma de Células Claras/patologia , Infecções por Papillomavirus/complicações , Neoplasias Penianas/patologia , Adenocarcinoma de Células Claras/virologia , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Neoplasias Penianas/virologia , Reação em Cadeia da PolimeraseRESUMO
An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.
Assuntos
Doença de Chagas/sangue , DNA de Protozoário/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Doença de Chagas/diagnóstico , Doença de Chagas/genética , Doença de Chagas/parasitologia , DNA de Protozoário/isolamento & purificação , Humanos , Cooperação Internacional , Ensaio de Proficiência Laboratorial , Tipagem Molecular , Parasitemia/sangue , Parasitemia/diagnóstico , Parasitemia/genética , Sensibilidade e Especificidade , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Trypanosoma cruzi/isolamento & purificação , Adulto , Argentina , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Testes Sorológicos/métodos , Trypanosoma cruzi/genética , Adulto JovemRESUMO
BACKGROUND: According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10-12 months of age. METHODOLOGY AND PRINCIPAL FINDINGS: Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI. SIGNIFICANCE: This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/diagnóstico , DNA de Protozoário/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Parasitemia/congênito , Parasitemia/diagnóstico , Trypanosoma cruzi/isolamento & purificação , DNA de Protozoário/genética , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Carga Parasitária/métodos , GravidezRESUMO
This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.
Assuntos
Doença de Chagas/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática/métodos , Nifurtimox/uso terapêutico , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/imunologia , Adolescente , Adulto , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Estudos de Casos e Controles , Doença de Chagas/parasitologia , Doença Crônica , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.
Este trabalho comparou os tempos de soroconversão negativos obtidos pela sorologia convencional (CS) e teste ELISA-F29 em uma coorte de pacientes chagásicos crônicos tratados com nifurtimox ou benznidazol. Um estudo retrospectivo foi realizado com soro preservado de 66 adultos chagásicos assintomáticos com acompanhamento clínico e sorológico semestral ao longo de um seguimento médio de 23 anos. 29 pacientes receberam tratamento tripanossomicida e 37 outras permaneceram sem tratamento. O teste ELISA-F29 usou um antígeno recombinante obtido por expressão do gene de uma proteína flagelar de Trypanosoma cruzi de ligação de cálcio em Escherichia coli. Entre os pacientes não tratados, 36 mantiveram os títulos da CS. Um paciente apresentou sorologia duvidosa em alguns controles. ELISA-F29 apresentou reatividade constante em 35/37 e foi negativo no paciente com CS flutuante. Os pacientes tratados foram agrupados de acordo com os títulos da CS, em três grupos: 13 tornaram-se negativos, 12 diminuíram e quatro permaneceram inalterados. ELISA-F29 foi negativo nos dois primeiros grupos. O tempo de negativização foi significativamente menor para o teste ELISA-F29 do que para CS (14,5 ± 5,7 e 22 ± 4,9 anos, respectivamente). A soroconversão negativa foi observada somente nos pacientes tratados. Os resultados obtidos confirmam que o teste ELISA-F29 é útil como um indicador precoce de soronegativação em pacientes crônicos tratados.
Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Doença de Chagas/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática/métodos , Nifurtimox/uso terapêutico , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/imunologia , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Estudos de Casos e Controles , Doença Crônica , Estudos de Coortes , Doença de Chagas/parasitologia , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.
Assuntos
Doença de Chagas/parasitologia , DNA Satélite/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Carga Parasitária/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/genética , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase Multiplex/normas , Carga Parasitária/normas , Gravidez , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e EspecificidadeRESUMO
Quantitative real-time PCR (qPCR) is an accurate method to quantify Trypanosoma cruzi DNA and can be used to follow-up parasitemia in Chagas disease (CD) patients undergoing chemotherapy. The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) study is an international, multicenter, randomized, double-blinded and placebo-controlled clinical trial to evaluate the efficacy of benznidazole (BZ) treatment in patients with chronic Chagas cardiomyopathy (CCC). One important question to be addressed concerns the effectiveness of BZ in reducing overall parasite load in CCC patients, even in the absence of parasitological cure. This report describes the evaluation of multiple procedures for DNA extraction and qPCR-based protocols aiming to establish a standardized methodology for the absolute quantification of T. cruzi DNA in Guanidine-EDTA blood (GEB) samples. A panel of five primer sets directed to the T. cruzi nuclear satellite DNA repeats (Sat-DNA) and to the minicircle DNA conserved regions (kDNA) was compared in either SYBR Green or TaqMan systems. Standard curve parameters such as, amplification efficiency, coefficient of determination and intercept were evaluated, as well as different procedures to generate standard samples containing pre-established T. cruzi DNA concentration. Initially, each primer set was assayed in a SYBR Green qPCR to estimate parasite load in GEB samples from chronic Chagas disease patients. The results achieved from Bayesian transmutability analysis elected the primer sets Cruzi1/Cruzi2 (p=0.0031) and Diaz7/Diaz8 (p=0.0023) coupled to the QIAamp DNA Kit extraction protocol (silica gel column), as the most suitable for monitoring parasitemia in these patients. Comparison between the parasite burden of 150 GEB samples of BENEFIT patients from Argentina, Brazil and Colombia, prior to drug/placebo administration, was performed using Cruzi1/Cruzi2 primers in a SYBR Green approach. The median parasitemia found in patients from Argentina and Colombia (1.93 and 2.31 parasite equivalents/mL, respectively) was around 20 times higher than the one estimated for the Brazilian patients (0.1 parasite equivalents/mL). This difference could be in part due to the complexity of T. cruzi genetic diversity, which is a factor possibly implicated in different clinical presentations of the disease and/or influencing parasitemia levels in infected individuals from different regions of Latin America. The results of SYBR Green qPCR assays herein presented prove this methodology to be more cost efficient than the alternative use of internal fluorogenic probes. In addition, its sensitivity and reproducibility are shown to be adequate to detect low parasitemia burden in patients with chronic Chagas disease.
Assuntos
Cardiomiopatia Chagásica/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Carga Parasitária/métodos , Parasitemia/parasitologia , Parasitologia/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Trypanosoma cruzi/isolamento & purificação , Antiprotozoários/administração & dosagem , Argentina , Brasil , Cardiomiopatia Chagásica/tratamento farmacológico , Colômbia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Método Duplo-Cego , Monitoramento de Medicamentos/métodos , Monitoramento de Medicamentos/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Nitroimidazóis/administração & dosagem , Carga Parasitária/normas , Parasitemia/tratamento farmacológico , Parasitologia/normas , Placebos/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
The relationship between parasite burden and vertical transmission of Trypanosoma cruzi was studied in pairs of chronically infected women and their children in a non-endemic area. Parasitemia was quantified by quantitative polymerase chain reaction (qPCR) in the peripheral blood amplifying a nuclear T. cruzi DNA and expressed as equivalent amounts of CL Brener parasites DNA per ml (eP/ml). Similar levels of parasitemia were found in non-transmitting pregnant women and in non-pregnant women: 1.8 ± 0.5 and 1.5 ± 0.7 eP/ml, respectively. In women pregnant with infected children parasitemia was 11.0 ± 2.7 eP/ml (n=20). In 12 of their neonates the infection was detected by microscopic observation of the parasites in peripheral blood in the 1(st) month of age. These children had variable levels of parasitemia (13,000 ± 7000 eP/ml), that were about 600-fold higher than that found in their mothers. To our knowledge, this is the first quantitative evaluation of parasitemia in these three groups of women and in their congenitally infected children. These parasite quantifications could be a basis to plan the control of mother-to-child transmission of T. cruzi.
Assuntos
Doença de Chagas/sangue , DNA de Protozoário/sangue , Sangue Fetal/parasitologia , Transmissão Vertical de Doenças Infecciosas , Técnicas de Amplificação de Ácido Nucleico , Complicações Parasitárias na Gravidez/sangue , Trypanosoma cruzi/isolamento & purificação , Adulto , Animais , Doença de Chagas/prevenção & controle , Doença de Chagas/transmissão , DNA de Protozoário/genética , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Centros de Saúde Materno-Infantil , Reação em Cadeia da Polimerase , Gravidez , Complicações Parasitárias na Gravidez/prevenção & controle , Trypanosoma cruzi/genéticaRESUMO
Penile squamous cell carcinoma shows an ample geographic variation in its prevalence with regions of low (North America, Europe, Japan, and Israel) and high (Africa, Asia, and South America) incidence. However, the geographic variation in the distribution of penile intraepithelial neoplasia is not well established. The aim of the present study was to compare the distribution of in situ and invasive lesions between geographic areas with low (France) and high (Paraguay) penile cancer incidence using a series of consecutive cases. The French series included 86 cases (57 in situ and 29 in situ + invasive squamous cell carcinoma), and the Paraguayan series, 117 cases (31 in situ and 86 in situ + invasive squamous cell carcinoma). Incidence of invasive squamous cell carcinoma in the overall samples was higher in the Paraguayan series (P < .00001). Comparing the Paraguayan and the French series, differentiated penile intraepithelial neoplasia was more prevalent in the former (65.0% versus 19.8%), whereas lesions showing warty and/or basaloid features predominated in the latter (35.0% versus 80.2%) to a significant level (P < .00001). This distinctive pattern of differential distribution was maintained when cases with associated invasive squamous cell carcinoma were excluded. The pattern of distribution of lichen sclerosus was also distinctive, with a significantly higher prevalence in the Paraguayan population when compared with the French series (32.5% versus 12.8%, P = .0015). In summary, there appears to be a distinctive distribution of penile precursor lesions depending on the geographic region in consideration. Penile intraepithelial neoplasia with warty and/or basaloid features predominated in low-incidence areas, whereas differentiated penile intraepithelial neoplasia was more prevalent in endemic regions for penile cancer. Further prospective studies in matched populations and from different geographic regions are needed to further clarify the reasons for this discrepancy.
Assuntos
Carcinoma in Situ/patologia , Carcinoma de Células Escamosas/patologia , Condiloma Acuminado/patologia , Doenças Endêmicas , Neoplasias Penianas/patologia , Lesões Pré-Cancerosas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma in Situ/epidemiologia , Carcinoma de Células Escamosas/epidemiologia , Condiloma Acuminado/epidemiologia , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus , Paraguai/epidemiologia , Neoplasias Penianas/epidemiologia , Lesões Pré-Cancerosas/epidemiologia , Prevalência , Adulto JovemRESUMO
INTRODUCCION: La detección precoz de Chagas congénito es sumamente importante, ya que el tratamiento inmediato aumenta la eficacia y disminuye las complicaciones en el neonato.OBJETIVO: Evaluar si la utilización de la reacción en cadena de la polimerasa (RCP) aumenta significativamente la sensibilidad y la precocidad diagnóstica con respecto al micrométodo para Chagas.METODOS: El diseño del ensayo fue prospectivo, observacional y longitudinal. Se estudió a 43 recién nacidos, hijos de madres con enfermedad de Chagas atendidas en tres hospitales de la provincia de Buenos Aires (Hospital Evita de Lanús, Hospital de Lomas de Zamora y Hospital de Ezeiza) desde enero de 2010 hasta abril de 2011. Antes del alta, se tomó una muestra de sangre a los neonatos con guanidina-EDTA para realizar la RCP y otra con heparina para realizar micrométodo, con un segundo y un tercer control dentro del mes de vida y serología al octavo mes.RESULTADOS: De las 43 muestras extraídas, 2 fueron eliminadas del estudio por dar negativa la RCP para B-actina. De las 41 muestras evaluadas, se obtuvieron los siguientes resultados: 1 con micrométodo y RCP positiva y 40 con micrométodo y RCP negativas. Se realizó serología a los 25 pacientes que llegaron al octavo mes de control, con micrométodo y RCP negativas, que resultaron no reactivas.CONCLUSIONES: Según el estudio, la RCP y el micrométodo tuvieron una sensibilidad y especificidad comparables. Por lo tanto, la RCP es un método alternativo para el control de la transmisión connatal.
INTRODUCTION: The early detection of congenital Chagas is extremely important, because the immediate treatment increases the efficiency and reduces complications in the newborn.OBJECTIVE: To assess whether the implementation of polymerase chain reaction (PCR) significantly improves the sensitivity and precocious diagnosis comparing to the micromethod for Chagas.METHODS: A prospective, observational and longitudinal test was performed. It analyzed 43 newborns from chagasic mothers assissted in 3 hospitals of Buenos Aires province (Lanús, Lomas de Zamora and Ezeiza) from January 2010 until April 2011. Before the discharge a newborn blood sample was taken and preserved in guanidine-EDTA for PCR, while another one was stored in heparin for micromethod, with a second and third control within the first month of life and serodiagnosis at the eighth month.RESULTS: Of the 43 blood samples collected, 2 were eliminated from the study because of the absence of band at PCR with b-actin. The 41 blood samples finally evaluated yielded following results: 1 with positive micromethod and PCR, and 40 with negative micromethod and PCR. Serodiagnosis was carried out to the 25 patients that reached the eighth month control, with negative micromethod and PCR, resultin non-reactive.CONCLUSIONS: According to this study, PCR and micromethod have a comparable sensitivity and specificity. Therefore, PCR is an alternative method to control the transmission of congenital Chagas.
Assuntos
Doença de Chagas , Doença de Chagas/diagnóstico , Reação em Cadeia da Polimerase , Argentina , Saúde PúblicaRESUMO
INTRODUCCION: La detección precoz de Chagas congénito es sumamente importante, ya que el tratamiento inmediato aumenta la eficacia y disminuye las complicaciones en el neonato.OBJETIVO: Evaluar si la utilización de la reacción en cadena de la polimerasa (RCP) aumenta significativamente la sensibilidad y la precocidad diagnóstica con respecto al micrométodo para Chagas.METODOS: El diseño del ensayo fue prospectivo, observacional y longitudinal. Se estudió a 43 recién nacidos, hijos de madres con enfermedad de Chagas atendidas en tres hospitales de la provincia de Buenos Aires (Hospital Evita de Lanús, Hospital de Lomas de Zamora y Hospital de Ezeiza) desde enero de 2010 hasta abril de 2011. Antes del alta, se tomó una muestra de sangre a los neonatos con guanidina-EDTA para realizar la RCP y otra con heparina para realizar micrométodo, con un segundo y un tercer control dentro del mes de vida y serología al octavo mes.RESULTADOS: De las 43 muestras extraídas, 2 fueron eliminadas del estudio por dar negativa la RCP para B-actina. De las 41 muestras evaluadas, se obtuvieron los siguientes resultados: 1 con micrométodo y RCP positiva y 40 con micrométodo y RCP negativas. Se realizó serología a los 25 pacientes que llegaron al octavo mes de control, con micrométodo y RCP negativas, que resultaron no reactivas.CONCLUSIONES: Según el estudio, la RCP y el micrométodo tuvieron una sensibilidad y especificidad comparables. Por lo tanto, la RCP es un método alternativo para el control de la transmisión connatal.
INTRODUCTION: The early detection of congenital Chagas is extremely important, because the immediate treatment increases the efficiency and reduces complications in the newborn.OBJECTIVE: To assess whether the implementation of polymerase chain reaction (PCR) significantly improves the sensitivity and precocious diagnosis comparing to the micromethod for Chagas.METHODS: A prospective, observational and longitudinal test was performed. It analyzed 43 newborns from chagasic mothers assissted in 3 hospitals of Buenos Aires province (Lanús, Lomas de Zamora and Ezeiza) from January 2010 until April 2011. Before the discharge a newborn blood sample was taken and preserved in guanidine-EDTA for PCR, while another one was stored in heparin for micromethod, with a second and third control within the first month of life and serodiagnosis at the eighth month.RESULTS: Of the 43 blood samples collected, 2 were eliminated from the study because of the absence of band at PCR with b-actin. The 41 blood samples finally evaluated yielded following results: 1 with positive micromethod and PCR, and 40 with negative micromethod and PCR. Serodiagnosis was carried out to the 25 patients that reached the eighth month control, with negative micromethod and PCR, resultin non-reactive.CONCLUSIONS: According to this study, PCR and micromethod have a comparable sensitivity and specificity. Therefore, PCR is an alternative method to control the transmission of congenital Chagas.
Assuntos
Doença de Chagas , Reação em Cadeia da Polimerase , Doença de Chagas/diagnóstico , Saúde Pública , ArgentinaRESUMO
AIMS: About 10-20% of all penile squamous cell carcinomas (SCCs) originate in the foreskin, but knowledge about preputial precursor and associated lesions is scant. The aims of the present study were to determine the prevalence of various precancerous and cancerous lesions exclusively affecting the foreskin, and to describe their pathological features. METHODS AND RESULTS: One hundred consecutive circumcision specimens from symptomatic patients living in a region of high penile cancer incidence were analysed. Clinical diagnoses included mostly phimosis and chronic balanoposthitis (40 and 35 cases, respectively), but also a tumour mass (11 cases). Histopathological lesions found included: squamous hyperplasia in 61 cases; lichen sclerosus in 53 cases; penile intraepithelial neoplasia (PeIN) in 30 cases (all differentiated PeIN, with two cases showing multicentric foci of basaloid and warty-basaloid PeIN); and invasive SCC in 11 cases (three usual, three pseudohyperplastic, two verrucous-pseudohyperplastic, and one case each of basaloid, papillary and mixed usual-basaloid carcinomas). Lichen sclerosus was present in all low-grade SCC cases. Patients with no lesions were younger (mean age 44 years) than those with precursor lesions (mean age 54 years) or with invasive SCC (mean age 68 years). Immunohistochemistry for p16(INK4a) was performed in 19 precancerous lesions. All differentiated PeINs (18 lesions) were negative, and one basaloid PeIN was positive. CONCLUSIONS: The frequent coexistence of lichen sclerosus, squamous hyperplasia, differentiated PeIN and low-grade SCC suggests a common non-human papillomavirus related pathogenic pathway for preputial lesions, and highlights the importance of circumcision in symptomatic patients for the prevention of penile cancer.
Assuntos
Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Prepúcio do Pênis/patologia , Neoplasias Penianas/epidemiologia , Lesões Pré-Cancerosas/patologia , Neoplasias Cutâneas/patologia , Adulto , Idoso , Carcinoma in Situ/epidemiologia , Carcinoma in Situ/patologia , Carcinoma de Células Escamosas/epidemiologia , Circuncisão Masculina , Comorbidade , Humanos , Hiperplasia/epidemiologia , Hiperplasia/patologia , Incidência , Líquen Escleroso e Atrófico/epidemiologia , Líquen Escleroso e Atrófico/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Penianas/prevenção & controle , Lesões Pré-Cancerosas/epidemiologia , Estudos Retrospectivos , Neoplasias Cutâneas/epidemiologiaRESUMO
BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3) par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85-95%, accuracy of 86.8-89.5% and kappa index of 0.7-0.8 compared to consensus PCR reports of the 16 good performing tests and 63-69%, 100%, 71.4-76.2% and 0.4-0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. CONCLUSION/SIGNIFICANCE: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.