RESUMO
Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30-45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62-83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62-100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69-100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78-94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.
Assuntos
DNA Bacteriano , Mycobacterium tuberculosis , Tuberculose Pulmonar , Humanos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Adulto , Feminino , Peru/epidemiologia , Masculino , DNA Bacteriano/urina , DNA Bacteriano/genética , Tuberculose Pulmonar/urina , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Pessoa de Meia-Idade , Adulto Jovem , Reação em Cadeia da Polimerase em Tempo Real , Valor Preditivo dos Testes , Urinálise/métodos , Estudos de Casos e Controles , Reprodutibilidade dos Testes , IdosoRESUMO
BACKGROUND: Leptospirosis is an important anthropozoonosis. The study investigated the presence of anti-Leptospira antibodies and detection of Leptospira spp DNA in the urine as well as the biochemical profile in Neotropical wild primates living in a forest reserve from Southeast São Paulo State, Brazil. METHODS: Blood samples were obtained from 50 adult tufted capuchin monkeys (Cebus apella nigritus). Urine samples were obtained only from male primates. The screening for antibodies against Leptospira spp was evaluated by microscopic agglutination test (MAT). Leptospira DNA in the urine was evaluated by polymerase chain reaction (PCR) considering the target gene LipL32. Biochemical profile was evaluated by using a spectrophotometer. RESULTS: The MAT results included 39 (78%) serum reactive animals with the proportions of 28/39 males and 11/39 females. The most frequent reactive serogroups were Icterohemorrhagiae, Canicola, and Autumnalis. All urine samples were negative for leptospiral DNA. There were no significant differences between sexes for aspartate aminotransferase (AST) and alkaline phosphatase values, but alanine aminotransferase (ALT), creatinine, glucose, and urea were significantly higher in males. CONCLUSIONS: Tufted capuchin monkeys were sera reactive against leptospirosis. Prevalence was similar for the 2 sexes. Leptospiral DNA was not detected in the urine of sera reactive primates tested by the MAT method. ALT, creatinine, glucose, and urea values were higher in male animals.
Assuntos
Anticorpos Antibacterianos/sangue , Cebinae , DNA Bacteriano/urina , Leptospira/isolamento & purificação , Leptospirose/veterinária , Doenças dos Macacos/epidemiologia , Animais , Brasil/epidemiologia , Rim/microbiologia , Rim/patologia , Leptospirose/epidemiologia , Leptospirose/microbiologia , Fígado/microbiologia , Fígado/patologia , Masculino , Doenças dos Macacos/microbiologia , SapajusRESUMO
INTRODUCTION: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.
Assuntos
DNA Bacteriano , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , DNA Bacteriano/sangue , DNA Bacteriano/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , DNA Bacteriano , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , DNA Bacteriano/sangue , DNA Bacteriano/urina , Mycobacterium tuberculosis/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6 percent (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75 percent for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30 percent for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Adulto Jovem , DNA Bacteriano/urina , Hanseníase Dimorfa/diagnóstico , Hanseníase Virchowiana/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Biomarcadores/urina , Estudos de Casos e Controles , Hanseníase Dimorfa/urina , Hanseníase Virchowiana/urina , Mycobacterium leprae/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients' urine samples was successfully amplified by PCR-Pra in 46.6% (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.
Assuntos
DNA Bacteriano/urina , Hanseníase Dimorfa/diagnóstico , Hanseníase Virchowiana/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Biomarcadores/urina , Estudos de Casos e Controles , Feminino , Humanos , Hanseníase Dimorfa/urina , Hanseníase Virchowiana/urina , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Adulto JovemRESUMO
Genital mycoplasmas are natural inhabitants of the male urethra and are potentially pathogenic species playing an aetiological role in both genital infections and male infertility. This study aims to determine the presence of Mycoplasma genitalium DNA in urine samples of HIV-1-infected men in São Paulo city. Realtime polymerase chain reaction (PCR) was performed using the primers My-ins and Mgso-2 and the Taqman probe Mgen-P1 as described previously. A total of 223 HIV-1-infected men were tested with a mean age of 44 years. Thirteen (5.8%) presented M. genitalium in urine and the co-infection was more common among homosexual men (76.9% versus 51.9%, P < 0.26). In conclusion, realtime PCR was a useful and rapid method for detecting M. genitalium DNA in urine samples. Further studies should be conducted to assess the clinical significance of these results on HIV transmission and its impact on HIV viral load.
Assuntos
Infecções por HIV/epidemiologia , HIV-1 , Doenças Urogenitais Masculinas/epidemiologia , Infecções por Mycoplasma/epidemiologia , Mycoplasma genitalium/isolamento & purificação , Adulto , Brasil/epidemiologia , Comorbidade , DNA Bacteriano/urina , Homossexualidade Masculina , Humanos , Masculino , Doenças Urogenitais Masculinas/diagnóstico , Infecções por Mycoplasma/diagnóstico , Mycoplasma genitalium/genética , Reação em Cadeia da Polimerase , Prevalência , Fatores de RiscoRESUMO
Abstract Leptospirosis is a zoonotic disease that occurs worldwide and is caused by pathogenic bacteria of the genus Leptospira. Clinical manifestations of leptospirosis are similar to other febrile illnesses and this fact frequently retards the beginning of antibiotic therapy. Thus, early and accurate diagnosis is a prerequisite for proper treatment of leptospirosis. Antigen and DNA-based detection tests offer potential advantage over tests based on antibody detection for early diagnosis of leptospirosis since antibodies only reach detectable levels several days after the onset of the infection. This work describes a method for detection of pathogenic Leptospira that associates an immunoseparation step with a PCR assay and uses an internal amplification control (IAC) to ensure accuracy of the test. The immunoseparation was performed with protein A-magnetic beads in house coated with an MAb specific for LipL32, the major outer membrane protein of pathogenic Leptospira; PCR was performed using lipL32 specific primers. The IMS-PCR method enhanced detection of Leptospira in experimentally contaminated human sera and urine when compared to PCR performed alone. IMS-PCR was able to detect 10(2) Leptospira cells per mL of human sera and urine, corresponding to 25 genomic copies per PCR reaction.
Assuntos
DNA Bacteriano/sangue , Leptospira interrogans/isolamento & purificação , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Líquidos Corporais/microbiologia , DNA Bacteriano/urina , Humanos , Separação Imunomagnética/métodos , Leptospira interrogans/genéticaRESUMO
OBJECTIVE: The purpose of this study was to assess the utility and validity of pooling urine samples for molecular diagnosis of Chlamydia trachomatis infection. MATERIAL AND METHODS: Of 1,220 urine samples collected from Mexican female and male adolescents, 305 pools were composed of fourth individual samples each, based on a calculation of optimal pool size. These were processed by ligase chain reaction (LCR) for the detection of C. trachomatis. Positive and gray-zone pools were reanalyzed individually. Cost savings were calculated comparing actual costs of testing to the cost that would have been incurred testing all 1,220 samples individually. RESULTS: Pools results were: 56 positive, 19 gray-zones and 230 negative. Following individual retesting of positive and gray-zone pools, 59 cases of C. trachomatis infection were identified (4.8% prevalence). Thus, a total of 601 LCR tests were performed, for a 50.4% savings considering only the direct cost of the test. CONCLUSIONS: Our experience shows that sample pooling is both a reliable and convenient tool for CT surveillance in our setting. It should be considered in other similar settings where limited resources constraint surveillance of STIs.