Microbiological diagnosis of community-acquired respiratory tract infections by nucleic acid detection.

Background: Microbiological diagnostic procedures have changed significantly over the last decade. Initially the implementation of the polymerase chain reaction (PCR) resulted in improved detection tests for microbes that were difficult or even impossible to detect by conventional methods such as culture and serology, especially in community-acquired respiratory tract infections (CA-RTI). A further improvement was the development of real-time PCR, which allows end point detection and quantification, and many diagnostic laboratories have now implemented this powerful method. Objective: At present, new performant and convenient molecular tests have emerged targeting in parallel many viruses and bacteria responsible for lower and/or upper respiratory tract infections. The range of test formats and microbial agents detected is evolving very quickly and the added value of these new tests needs to be studied in terms of better use of antibiotics, better patient management, duration of hospitalization and overall costs. Conclusions: Molecular tools for a better microbial documentation of CA-RTI are now available. Controlled studies are now required to address the relevance issue of these new methods, such as, for example, the role of some newly detected respiratory viruses or of the microbial DNA load in a particular patient at a particular time. The future challenge for molecular diagnosis will be to become easy to handle, highly efficient and cost-effective, delivering rapid results with a direct impact on clinical management.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR) was applied for the simultaneous detection of hepatitis A virus (HAV), poliovirus (PV) and simian rotavirus (RV-SA11) and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp), PV (394 bp) and RV (278 bp) were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation.

Detection of Cryptosporidium - specific coproantigen in human immunodeficiency virus/acquired immunodeficiency syndrome patients by using a commercially available immunoenzymatic assay

The aim of this study was to verify the occurrence of Cryptosporidium infection in 52 human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients (group 1) and 38 clinically healthy individuals (group 2) by using enzyme immunoassay (EIA). All fecal samples collected were submitted to the Baermann, Lutz, and Ritchie methods, the Safranin/Methylene Blue, and Weber's chromotrope modified Trichrome staining techniques, and EIA. In group 1, parasitological staining techniques and EIA were both positive for Cryptosporidium sp. infection in 3/52 (5.8%) samples and both negative in 45/52 (86.5%) samples, while 4/52 (7.7%) samples were positive in EIA and negative in parasitological staining techniques. Concerning group 2, all samples were negative by EIA and microscopy for Cryptosporidium infection. In conclusion, EIA may be an alternative method for detecting Cryptosporidium-specific coproantigen in HIV/AIDS patients.

Viremic blood donor found by a rapid screening method in a season of high human parvovirus B19 activity in Niterói, Rio de Janeiro, Brazil

Erythrovirus B19 infection is usually benign but may have serious consequences in patients with hemolytic anemia (transient aplastic crisis), immunodeficiency (in whom persistent infection can lead to chronic bone marrow failure with anemia), or who are in the first or second trimester of gestation (spontaneous abortion, hydrops fetalis, and fetal death). Being non-enveloped, B19 resists most inactivation methods and can be transmitted by transfusion. B19 is difficult to cultivate and native virus is usually obtained from viremic blood. As specific antibodies may be absent, and there is no reliable immunological method for antigen detection, hybridization or polymerase chain reaction are needed for detecting viremia. A rapid method, gel hemagglutination (Diamed ID-Parvovirus B19 Antigen Test), can disclose highly viremic donations, whose elimination lessens the viral burden in pooled blood products and may even render them non-infectious. In order to obtain native antigen and to determine the frequency of viremic donors, we applied this test to blood donors in a period of high viral activity in our community. Positive or indeterminate results were re-tested by dot-blot hybridization. We tested 472 donors in 1998 and 831 ones in 1999. One viremic donor was found in 1999. We suggest that in periods of high community viral activity the gel hemagglutination test may be useful in avoiding highly viremic blood being added to plasma pools or directly transfused to patients under risk.

Meta-analysis based SVM classification enables accurate detection of Alzheimer's disease across different clinical centers using FDG-PET and MRI.

The application of support vector machine classification (SVM) to combined information from magnetic resonance imaging (MRI) and [F18]fluorodeoxyglucose positron emission tomography (FDG-PET) has been shown to improve detection and differentiation of Alzheimer's disease dementia (AD) and frontotemporal lobar degeneration. To validate this approach for the most frequent dementia syndrome AD, and to test its applicability to multicenter data, we randomly extracted FDG-PET and MRI data of 28 AD patients and 28 healthy control subjects from the database provided by the Alzheimer's Disease Neuroimaging Initiative (ADNI) and compared them to data of 21 patients with AD and 13 control subjects from our own Leipzig cohort. SVM classification using combined volume-of-interest information from FDG-PET and MRI based on comprehensive quantitative meta-analyses investigating dementia syndromes revealed a higher discrimination accuracy in comparison to single modality classification. For the ADNI dataset accuracy rates of up to 88% and for the Leipzig cohort of up to 100% were obtained. Classifiers trained on the ADNI data discriminated the Leipzig cohorts with an accuracy of 91%. In conclusion, our results suggest SVM classification based on quantitative meta-analyses of multicenter data as a valid method for individual AD diagnosis. Furthermore, combining imaging information from MRI and FDG-PET might substantially improve the accuracy of AD diagnosis.

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oral fluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAV IgM, 6 and 95% for anti-HBs, 13 and 100% for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV. The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 µg) and cefoxitin (30 µg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.