Evaluation of the human immunodeficiency virus type 1 and 2 antibodies detection in dried whole blood spots (dbs) samples

Human Immunodeficiency Vírus Type 1 and 2 antibodies detection was performed in 457 dried whole blood spots samples (S&S 903). Q-Preven HIV 1+2 was the screening test used. The results were compared with the gold standard serum tests by ELISA (Cobas Core e Axsym HIV1/2 gO) and imunofluorescence was the definitive confirmatory test. The samples were obtained from the Hospital Nossa Senhora da Conceição in Porto Alegre, RS - Brazil, through whole blood transfer to filter paper card and sent to Caxias do Sul, RS - Brazil where the tests were performed. The dried whole blood spot stability was evaluated with two different panels. The first one was composed of five negative and five positive samples stored at room temperature, 4 ºC, -20 ºC and -70 ºC, while the second was composed of two negative and three positive samples stored at 37 ºC (humidity <50%). Each sample was screened every week for six weeks. These measurement results didn't show variation during the study period. The detected sensibility was 100%, specificity was 99.6%, the positive predictive value was 99.5% and negative predictive values were 100%. The results demonstrated high performance characteristics, opening a new perspective of dried whole blood spot utilization in HIV screening diagnosis.

Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

Fluids in which Mycobacterium tuberculosis are seldom found, such as pleural and cerebrospinal liquids, are good candidates to be studied using PCR techniques. We detail our experience with a PCR assay applied to pleural and cerebrospinal fluids using the primer MPB64. Seventy three specimens were analyzed: 30 pleural fluids (PF), 26 pleural biopsies (PB) and 17 cerebrospinal fluids (CSF). The gold standard for the diagnosis of tuberculous meningitis was the positive culture for M. tuberculosis in CSF. Tuberculous pleural effusion was diagnosed when cultures of PF and/or PB were positive for M. tuberculosis, or the PB histology showed granulomas. Our results, compared to the gold standards employed, showed a sensitivity of 70%, specificity of 88%, positive predictive value of 82% and negative predictive value of 80%. The high specificity of the MPB64 fragment while still retaining a good sensitivity makes it very well suited for pleural and cerebrospinal tuberculosis diagnosis.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

Validation of a case definition for leptospirosis diagnosis in patients with acute severe febrile disease admitted in reference hospitals at the State of Pernambuco, Brazil

INTRODUCTION: Leptospirosis is often mistaken for other acute febrile illnesses because of its nonspecific presentation. Bacteriologic, serologic, and molecular methods have several limitations for early diagnosis: technical complexity, low availability, low sensitivity in early disease, or high cost. This study aimed to validate a case definition, based on simple clinical and laboratory tests, that is intended for bedside diagnosis of leptospirosis among hospitalized patients. METHODS: Adult patients, admitted to two reference hospitals in Recife, Brazil, with a febrile illness of less than 21 days and with a clinical suspicion of leptospirosis, were included to test a case definition comprising ten clinical and laboratory criteria. Leptospirosis was confirmed or excluded by a composite reference standard (microscopic agglutination test, ELISA, and blood culture). Test properties were determined for each cutoff number of the criteria from the case definition. RESULTS: Ninety seven patients were included; 75 had confirmed leptospirosis and 22 did not. Mean number of criteria from the case definition that were fulfilled was 7.8±1.2 for confirmed leptospirosis and 5.9±1.5 for non-leptospirosis patients (p<0.0001). Best sensitivity (85.3%) and specificity (68.2%) combination was found with a cutoff of 7 or more criteria, reaching positive and negative predictive values of 90.1% and 57.7%, respectively; accuracy was 81.4%. CONCLUSIONS: The case definition, for a cutoff of at least 7 criteria, reached average sensitivity and specificity, but with a high positive predictive value. Its simplicity and low cost make it useful for rapid bedside leptospirosis diagnosis in Brazilian hospitalized patients with acute severe febrile disease.

Concordance between expected and observed bacilloscopy results of clinical forms of leprosy: a 6-year retrospective study in Recife, State of Pernambuco, Brazil

INTRODUCTION: Operational classification of leprosy based on the number of skin lesions was conceived to screen patients presenting severe forms of the disease to enable their reception of a more intense multidrug regimen without having to undergo lymph smear testing. We evaluated the concordance between operational classification and bacilloscopy to define multibacillary and paucibacillary leprosy. METHODS: We selected 1,213 records of individuals with leprosy, who were untreated (new cases) and admitted to a dermatology clinic in Recife, Brazil, from 2000 to 2005, and who underwent bacteriological examination at diagnosis for ratification of the operational classification. RESULTS: Compared to bacilloscopy, operational classification demonstrated 88.6% sensitivity, 76.9% specificity, a positive predictive value of 61.8%, and a negative predictive value of 94.1%, with 80% accuracy and a moderate kappa index. Among the bacilloscopy-negative cases, 23% had more than 5 skin lesions. Additionally, 11% of the bacilloscopy-positive cases had up to 5 lesions, which would have led to multibacillary cases being treated as paucibacillary leprosy if the operational classification had not been confirmed by bacilloscopy. CONCLUSIONS: Operational classification has limitations that are more obvious in borderline cases, suggesting that in these cases, lymph smear testing is advisable to enable the selection of true multibacillary cases for more intense treatment, thereby contributing to minimization of resistant strain selection and possible relapse.

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%).

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

Introduction: Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. Methods: In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. Results: For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. Conclusions: The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

Validity of the Brazilian Portuguese version of the bipolar spectrum diagnostic scale

OBJECTIVE: Bipolar spectrum disorders (BSDs) are prevalent and frequently unrecognized and undertreated. This report describes the development and validation of the Brazilian version of the bipolar spectrum diagnostic scale (B-BSDS), a screening instrument for bipolar disorders, in an adult psychiatric population. METHOD: 114 consecutive patients attending an outpatient psychiatric clinic completed the B-BSDS. A research psychiatrist, blind to the B-BSDS scores, interviewed patients by means of a modified version of the mood module of the Structured Clinical Interview for DSM-IV ("gold standard"). Subthreshold bipolar disorders were defined as recurrent hypomania without a major depressive episode or with fewer symptoms than those required for threshold hypomania. RESULTS: The internal consistency of the B-BSDS evaluated with Cronbach's alpha coefficient was 0.89 (95% CI; 0.86-0.91). On the basis of the modified SCID, 70 patients (61.4%) of the sample received a diagnosis of BSDs. A B-BSDS screening score of 16 or more items yielded: sensitivity of 0.79 (95% CI; 0.72-0.85), specificity of 0.77 (95% CI; 0.70-0.83), a positive predictive value of 0.85 (95% CI; 0.78-0.91) and a negative predictive value of 0.70 (95% CI; 0.63-0.75). CONCLUSION: The present data demonstrate that the B-BSDS is a valid instrument for the screening of BSDs.

Are apolipoproteins A and B better than lipoproteins for assessing risk of obstructive coronary heart disease?

OBJECTIVE: To evaluate whether apolipoproteins A-I (Apo A-I) and B (Apo B) have, higher ensitivity (SN), specificity (SP) and positive predictive value (PPV) than lipoproteins (LP), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), and triglycerides (TGL) in assessing the risk of coronary heart disease (CHD). METHODS: This is a transversal case-control study of 241 patients, who were divided into two groups: 1) 145 patients with CHD, and 2) 96 patients without coronary disease. A model of logistic regression to evaluate the relation between the LPs and CHD was developed in which variables with a p-alpha <0.1 were included. RESULTS: Apo A-I levels were higher in the patients without CHD, (OR 2.08, CI 1.20-3.57). There were no statistical differences between the values of Apo A-I and the remaining lipid fractions (Apo A-I: 67%; Apo B: 100%; PPV: TC= 71%; TGC=71%; HDL=71%; LDL=71%). The costs of the tests in Reais were as follows: Apo A-I: R$ 56.60; Apo B-100: R$ 56.60; TC: R$ 9.94; HDL: R$ 21.30; LDL: R$ 28.40; TGL: R$ 14.20. CONCLUSION: Levels of Apo A-I and Apo B have no advantage over conventional lipoproteins in predicting the risk of CHD, despite the statistical association between Apo A-I and CHD; in addition, their costs are higher than those of the conventional lipoproteins.

Chest pain in the emergency room. Importance of a systematic approach

OBJECTIVE: To evaluate the efficiency of a systematic diagnostic approach in patients with chest pain in the emergency room in relation to the diagnosis of acute coronary syndrome (ACS) and the rate of hospitalization in high-cost units. METHODS: One thousand and three consecutive patients with chest pain were screened according to a pre-established process of diagnostic investigation based on the pre-test probability of ACS determinate by chest pain type and ECG changes. RESULTS: Of the 1003 patients, 224 were immediately discharged home because of no suspicion of ACS (route 5) and 119 were immediately transferred to the coronary care united because of ST elevation or left bundle-branch block (LBBB) (route 1) (74% of these had a final diagnosis of acute myocardial infarction [AMI]). Of the 660 patients that remained in the emergency room under observation, 77 (12%) had AMI without ST segment elevation and 202 (31%) had unstable angina (UA). In route 2 (high probability of ACS) 17% of patients had AMI and 43% had UA, whereas in route 3 (low probability) 2% had AMI and 7 % had UA. The admission ECG has been confirmed as a poor sensitivity test for the diagnosis of AMI ( 49%), with a positive predictive value considered only satisfactory (79%). CONCLUSION: A systematic diagnostic strategy, as used in this study, is essential in managing patients with chest pain in the emergency room in order to obtain high diagnostic accuracy, lower cost, and optimization of the use of coronary care unit beds.

Echocardiographic Assessment of the Pulmonary Venous Flow: An Indicator of Increased Pulmonary Flow in Congenital Cardiac Malformations

OBJECTIVE: To identify the left inferior pulmonary vein as an indirect marker of increased pulmonary flow in congenital heart diseases.METHODS: We carried out a prospective consecutive study on 40 patients divided into 2 groups as follows: G1 - 20 patients diagnosed with congenital heart disease and increased pulmonary flow; G2 (control group) - 20 patients who were either healthy or had congenital heart disease with decreased or normal pulmonary flow. We obtained the velocity-time integral of the left inferior pulmonary vein flow, excluding the "reverse A" wave, with pulsed Doppler echocardiography.RESULTS: In G1, 19 out of the 20 patients had well-identified dilation of the left inferior pulmonary vein. No G2 patient had dilation of the left inferior pulmonary vein. Dilation of the left inferior pulmonary vein in conditions of increased pulmonary flow had sensitivity of 95%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 95% (1 false-negative case). The integral of time and velocity of the pulmonary venous flow obtained with pulsed Doppler echocardiography was greater in the G1 patients (G1=25.0±4.6 cm versus G2=14.8±2.1 cm, p=0.0001).CONCLUSION: The identification of dilation of the left inferior pulmonary vein suggests the presence of congenital heart disease with increased pulmonary flow. This may be used as an indirect sign of increased flow, mainly in malformations of difficult diagnosis, such as atrial septal defects of the venous sinus or coronary sinus type.

The Role of B-Type Natriuretic Peptide in the Diagnosis of Congestive Heart Failure in Patients Presenting to an Emergency Department with Dyspnea

OBJECTIVE: To determine the utility of B-type natriuretic peptide (BNP) in the diagnosis of congestive heart failure (CHF) in patients presenting with dyspnea to an emergency department (ED). METHODS: Seventy patients presenting with dyspnea to an ED from April to July 2001 were included in the study. Mean age was 72±16 years and 33 (47%) were male. BNP was measured in all patients at the moment of admission to the ED. Emergency-care physicians, blinded to BNP values, were required to assign a probable initial diagnosis. A cardiologist retrospectively reviewed the data (blinded to BNP measurements) and assigned a definite diagnosis, which was considered the gold standard for assessing the diagnostic performance of BNP. RESULTS: The mean BNP concentration was higher in patients with CHF (n=36) than in those with other diagnoses (990±550 vs 80±67 pg/mL, p<0.0001). Patients with systolic dysfunction had higher BNP levels than those with preserved systolic function (1,180±641 vs 753±437 pg/mL, p=0.03). At a blood concentration of 200 pg/mL, BNP showed a sensitivity of 100%, specificity of 97.1%, positive predictive value of 97.3%, and negative predictive value of 100%. The application of BNP could have potentially corrected all 16 cases in which the diagnosis was missed by the emergency department physician. CONCLUSION: BNP measurement is a useful tool in the diagnosis of CHF in patients presenting to the ED with dyspnea.

Standardization and evaluation of ELISA for the serodiagnosis of amoebic liver abscess

An ELISA test for the serological diagnosisof amoebic liver abscess (ALA) was standardized and evaluated in sera from three groups of patients: (1) three patients with diagnosis confirmed by isolation of the parasite,(2) thirty seven patients with diagnosis established by clinical findings and ultrasound studies and (3) seven patients whose diagnosis were established by clinical findings and a positive double immunodifusion test. Ninety one serum samples from healthy subjects and 22 from patients with other liver or parasitic diseases were also included in the study. the optimum concentration of Entamoeba histolytica antigen was 1.25 µg/ml and optimum dilutions of serum and anti-human IgG-alkaline phosphatase conjugate were 1:400 and 1:4000 respectively. The cut-off point of the ELISA test in this study was an absorbance value of 0.34. The test parameters were: sensitivity = 95.7 per cent, specificty = 100 per cent, positive predictive value = 100 per cent and negative predictive value = 98.2 per cent.The ELISA test was found to be of great use as a diagnostic tool for the establishment of amoebic etiology in patients with clinical supposition of ALA. The test could also be used for seroepidemiological surveys of the prevalence of invasive amoebiasis in a given population, since it allows the processing of a greater number of samples at a lower cost tahn other serological tests.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Brain natriuretic peptide and left ventricular dysfunction in chagasic cardiomyopathy

Global left ventricular (LV) systolic dysfunction is the strongest predictor of morbidity and mortality in Chagas disease. Echocardiography is considered the gold standard for the detection of LV dysfunction, but not always available in endemic areas where chagasic cardiomyopathy is most common. Brain natriuretic peptide (BNP) is a neurohormone that has been recently described as a simple and inexpensive diagnostic and prognostic marker for patients with congestive heart failure. Chagasic patients (n = 63) and non-infected healthy individuals (n = 18) were recruited prospectively and underwent complete clinical examination, echocardiography and 24-h Holter monitoring. BNP was measured from thawed plasma samples using the Triage BNP test. We observed high levels of BNP in association with depression of LV ejection fraction, with increase of LV end-diastolic diameter and with LV premature complexes. An elevated concentration of BNP, defined as a concentration of 60 pg/ml or more, had a sensitivity of 91.7%, specificity of 82.8%, positive predictive value of 52.4%, and negative predictive value of 98% for detecting LV dysfunction (LV ejection fraction < 40%).BNP measurement using a simple, relatively inexpensive and rapid test has a promising role in identifying LV dysfunction associated with chagasic cardiomyopathy. Equally important, patients with Trypanosoma cruzi infection who have low levels of BNP level in plasma have a very low likelihood of severe cardiac involvement, and echocardiography is probably not necessary.