PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Comparison of the performance of polymerase chain reaction and pp65 antigenemia for the detection of human cytomegalovirus in immunosuppressed patients

INTRODUCTION: Human cytomegalovirus (HCMV) is often reactive in latently infected immunosuppressed patients. Accordingly, HCMV remains one of the most common infections following solid organ and hemopoietic stem cell transplantations, resulting in significant morbidity, graft loss and occasional mortality. The early diagnosis of HCMV disease is important in immunosuppressed patients, since in these individuals, preemptive treatment is useful. The objective of this study was to compare the performance of the in-house qualitative polymerase chain reaction (PCR) and pp65 antigenemia to HCMV infection in immunosuppressed patients in the Hospital de Clínicas of Porto Alegre (HCPA). METHODS: A total of 216 blood samples collected between August 2006 and January 2007 were investigated. RESULTS: Among the samples analyzed, 81 (37.5%) were HCMV-positive by PCR, while 48 (22.2%) were positive for antigenemia. Considering antigenemia as the gold standard, sensitivity, specificity, positive predictive values and negative predictive values for PCR were 87.5%, 76.8%, 51.8% and 95.5% respectively. CONCLUSIONS: These results demonstrated that qualitative PCR has high sensitivity and negative predictive value (NPV). Consequently PCR is especially indicated for the initial diagnosis of HCMV infection. In the case of preemptive treatment strategy, identification of patients at high-risk for HCMV disease is fundamental and PCR can be useful tool.

Development of cognitive abilities of children infected with helminths through health education

INTRODUCTION: The aim of this study was to evaluate the effect of health education in learning and cognitive development of children infected, previously treated in an endemic area for helminthiasis. METHODS: It is a longitudinal, experimental, with random allocation of participants. The study included 87 children of both sexes enrolled in the school hall of Maranhão, State of Minas Gerais, Brazil, and divided into two groups: intervention and control. Initially the children were submitted to the parasitological fecal examination for infection diagnosis and, when positive, they were treated. For the data collection, a structured questionnaire and the psychological tests Raven, Wisc-III and DAP III were applied, before and after the educational intervention. For the group comparison, the Mann Whitney test was used, and established significance level of 5%. RESULTS: It was found that previously infected children who received the educational intervention, children showed higher performance than the control group in strutured questionnaire (p<0.05). CONCLUSIONS: It is acceptable to suppose the positive influence and the importance in the use of educational interventions in the cognitive recovery and learning of children previously treated with anthelmintics.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Performance of a rapid diagnostic test for the detection of visceral leishmaniasis in a large urban setting

Introduction Rapid diagnostic tests (RDTs) may improve the early detection of visceral leishmaniasis (VL), but their real-world performance requires additional study. Therefore, we evaluated the performance of an rK39-based RDT (Kalazar Detect™) for the detection of VL in an endemic, large urban area. Methods Data were collected from a registry of rK39 RDT performed at 11 emergency care units in Belo Horizonte, Brazil, and from a national database of reportable communicable diseases of the Sistema de Informação de Agravos de Notificação (SINAN). Results The rapid rK39 test was performed in 476 patients, with 114 (23.9%) positive results. The analysis of rK39 RDT performance was based on 381 (80%) cases reported to the SINAN database, of which 145 (38.1%) were confirmed cases. Estimates for sensitivity and specificity were 72.4% (95% CI: 64.6-79%) and 99.6% (95%CI: 97.6-99.9%), respectively. Positive and negative predictive values were estimated at 99.1% (95%CI: 94.9-99.8%) and 85.5% (95%CI: 80.8-89.1%), respectively. In addition, close agreement between the rK39 RDT and indirect immunofluorescence was observed. Conclusions In summary, the rK39 RDT showed a high specificity but only moderate sensitivity. In endemic areas for VL, treatment may be considered in cases with clinical manifestations and a positive rK39 RDT, but those with a negative test should be subjected to further investigation.

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Introduction This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to define the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous finding in the histopathological examination, associated with an evident response to specific treatments to each clinical situation. Pleural fluid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplification. Results In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fluid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fluid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

Performance of the Vitek 2 system software version 5.03 in the bacterial identification and antimicrobial susceptibility test: evaluation study of clinical and reference strains of Gram-positive cocci

Introduction. The genera Enterococcus, Staphylococcus and Streptococcus are recognized as important Gram-positive human pathogens. The aim of this study was to evaluate the performance of Vitek 2 in identifying Gram-positive cocci and their antimicrobial susceptibilities. Methods. One hundred four isolates were analyzed to determine the accuracy of the automated system for identifying the bacteria and their susceptibility to oxacillin and vancomycin. Results. The system correctly identified 77.9% and 97.1% of the isolates at the species and genus levels, respectively. Additionally, 81.8% of the Vitek 2 results agreed with the known antimicrobial susceptibility profiles. Conclusion. Vitek 2 correctly identified the commonly isolated strains; however, the limitations of the method may lead to ambiguous findings.

Prevalence of neurocognitive disorders and depression in a Brazilian HIV population

AbstractINTRODUCTION:Combined antiretroviral therapy has enabled human immunodeficiency virus (HIV) carriers to live longer. This increased life expectancy is associated with the occurrence of degenerative diseases, including HIV-associated neurocognitive disorders (HAND), which are diagnosed via a complex neuropsychological assessment. The International HIV Dementia Scale (IHDS) is a screening instrument validated in Brazil for use in the absence of neuropsychological evaluation. HIV patients are frequently diagnosed with depression. We aimed to determine the prevalence of neurocognitive impairment using the IHDS and depressive disorders using the Hamilton Rating Scale for Depression (HAM-D17), compare the IHDS performance with the performances on the Timed Gait Test (TGT), the Digit Symbol Coding Test (DS) and the Brazilian version of the Scale of Instrumental Activities of Daily Living (IADL), and evaluate the association between the IHDS performance and clinical-demographic variables.METHODS:One hundred fourteen patients were evaluated in a cross-sectional study conducted in a public outpatient clinic for infectious diseases in Marília City, State of São Paulo, Brazil. Data were collected following consultation. Statistical analysis was performed in accordance with the nature and distribution of the data and hypotheses.RESULTS:According to the IHDS, 53.2% of the sampled patients were neuropsychologically impaired. According to the HAM-D17, 26.3% had depressive disorders. There were significant associations between the IHDS and the TGT and DS. Multiple regression analysis indicated that female gender, educational level, and cluster of differentiation 4 (CD4) levels were significantly and independently associated with neurocognitive impairment.CONCLUSIONS:The prevalence of neurocognitive impairment according to the IHDS is high and associated with female gender, education level, and low CD4 levels.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

Vancomycin monitoring in term newborns: comparison of peak and trough serum concentrations determined by high performance liquid chromatography and fluorescence polarization immunoassay

INTRODUCTION: Peak and trough serum concentrations of vancomycin were determined in term newborn infants with confirmed or suspected Staphylococcus sp sepsis by high performance liquid chromatography and flourescence polarization immunoassay. OBJECTIVE: To statistically compare the results of the high performance liquid chromatography and flourescence polarization immunoassay techniques for measuring serum vancomycin concentrations. METHODS: Eighteen peak and 20 trough serum samples were assayed for vancomycin concentrations using high performance liquid chromatography and flourescence polarization immunoassay from October 1995 to October 1997. RESULTS: The linear correlation coefficients for high performance liquid chromatography and flourescence polarization immunoassay were 0.27 (peak, P = 0.110) and 0.26 (trough, P = 0.1045) respectively, which were not statistically significant. CONCLUSION: There was wide variation in serum vancomycin concentrations determined by high performance liquid chromatography as compared with those determined by flourescence polarization immunoassay. There was no recognizable pattern in the variability; in an apparently random fashion, the high performance liquid chromatography measurement was sometimes substantially higher than the flourescence polarization immunoassay measurement, and at other times it was substantially lower.

Performance de populações de Milho (Zea Mays L.) em terras altas sob cerrado no Centro-sul do estado do Tocantins, Brasil1

No estado do Tocantins, o cultivo de milho em "terras altas sob cerrado" apresenta níveis de produtividade variáveis, cuja média não ultrapassa 2 t/ha. Fatores de solo, como acidez, baixos teores de Ca, Mg, Ρ e Κ e matéria orgânica, concorrem para reduzir as produtividades. O desenvolvimento de materiais genéticos melhorados busca contribuir na superação dessas limitações. Como atividade inicial em um programa de melhoramento genético, o presente estudo avaliou um total de 160 populações de milho, nas safras 1994/95, 1995/96 e 1998/99, nos municípios de Pedro Afonso e Gurupi, em termos de produtividade de grãos (PG), altura da planta (AP), altura da espiga (AE) e dias para florescimento feminino (FF). Para PG, os efeitos de populações foram significativos em todas as avaliações, ocorrendo produtividades entre 1,59 t/ ha a 7,46 t/ha. Destacaram-se as populações IAPAR 51, EGO 17, CMS 08, CMS 50, COMPOSTO TERRAS BAIXAS, TAIÚBA, TAITINGA, CMS 28, CMS 15 e SIKALQ, em Pedro Afonso, e K9201, CMS 39, BR 105 e CMS 03, em Gurupi, com produtividades acima de 5 t/ha. As diferenças nas performances produtivas e agronômicas em Pedro Afonso e Gurupi, juntamente com efeitos significativos das interações populações χ locais, alertam para a condução de programas de melhoramento específicos para cada local.