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PubMed1995

PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs.

A PCR assay for the diagnosis of leishmaniosis was developed by using primers that were selected from the sequence of the small-subunit rRNA gene. The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (lysis of erythrocytes in Tris-EDTA buffer, digestion with proteinase K directly in PCR buffer, and no further purification steps). Furthermore, an internal control is included in every specimen in order to detect the presence of PCR inhibitors. The PCR was compared with diagnostic in vitro cultivation of promastigote stages for the detection of Leishmania spp. in clinical specimens from humans and dogs with a tentative diagnosis of leishmaniosis. PCR and cultivation gave identical results with all but 1 of the 95 specimens from humans. The PCR result in this case was false negative, possibly because of unequal apportionment of this sample. With 10 skin biopsies from six patients with cutaneous leishmaniosis, the sensitivity was 60%. For six human immunodeficiency virus-positive patients with visceral leishmaniosis, all bone marrow biopsies and 7 of 11 whole blood samples (after isolation of leukocytes by Ficoll-Paque) were positive in both tests. PCR detected one more case with the use of 500 microliters of whole blood with direct lysis of the erythrocytes in Tris-EDTA buffer. With dog lymph node aspirates, the sensitivity was 100% (16 of 16 samples) for both methods; furthermore, PCR was positive for 5 of 13 whole blood samples from dogs with leishmaniosis. The specificity of the PCR was 100% (70 specimens from patients without leishmaniosis). This PCR assay proved to be feasible as a routine diagnostic test, being reliable and faster than in vitro cultivation.

Animals↗

PubMed2003

Recombinant human thyrotropin-stimulated serum thyroglobulin combined with neck ultrasonography has the highest sensitivity in monitoring differentiated thyroid carcinoma.

Recombinant human TSH (rhTSH)-stimulated thyroglobulin (Tg) measurement and (131)I whole body scan (WBS) have been validated as informative tests in the postsurgical follow-up of differentiated thyroid carcinoma. We report the diagnostic accuracy of Tg measurement and diagnostic WBS, alone or in combination, after rhTSH stimulation in a retrospective, consecutive series of patients undergoing follow-up for differentiated thyroid cancer. Routine procedures also include neck ultrasound in every patient and post-therapy WBS when indicated. We studied 340 consecutive patients with differentiated thyroid carcinoma, previously treated with near-total thyroidectomy and (131)I thyroid ablation, scheduled for routine diagnostic tests. At baseline on L-T(4)-suppressive therapy, 294 patients had undetectable (<1 ng/ml) serum Tg and negative anti-Tg autoantibodies (TgAb), 25 patients had undetectable serum Tg and positive TgAb, and 21 patients had detectable serum Tg and negative TgAb. These patients were tested for the presence of active disease by rhTSH stimulation. The results of our study showed that rhTSH-stimulated Tg alone had a diagnostic sensitivity of 85% for detecting active disease and a negative predictive value (NPV) of 98.2%. After adding the results of neck ultrasound, sensitivity increased to 96.3%, and the NPV to 99.5%. rhTSH-stimulated WBS had a sensitivity of only 21% and a NPV of 89%. The combination of rhTSH-stimulated Tg and WBS had a sensitivity of 92.7% and a NPV of 99%. We conclude that the rhTSH-stimulated Tg test combined with neck ultrasonography has the highest diagnostic accuracy in detecting persistent disease in the follow-up of differentiated thyroid carcinoma. A detectable level of serum Tg on L-T(4), its conversion from undetectable to detectable after rhTSH, and/or a suspicious finding at ultrasound will allow the identification of patients requiring therapeutic procedures without the need for diagnostic WBS.

Adolescent↗

PubMed1986

Serial visual evoked potential recordings in Alzheimer's disease.

Primary presenile dementia slows the major positive component of the visual evoked potential to flash stimulation but does not affect the visual evoked potential to patterned stimulation. The progressive effect of Alzheimer's disease was followed in a 58 year old woman over three and a half years from the development of the earliest symptoms to complete mental incapacity. The pattern reversal visual evoked potential remained normal, but the flash visual evoked potential gradually slowed from 129 ms in 1981 to 153 ms in 1984. The severity of the abnormality of the flash visual evoked potential thus reflected the severity of the dementia. Electroencephalography, computed tomography, and psychometric tests indicated generalised cortical disease, but the results were not specific to dementia. The combination of a slowed flash and normal pattern visual evoked potential seems to be specific to Alzheimer's disease and supports the use of flash and pattern visual evoked potentials in routine diagnostic testing for this condition.

Alzheimer Disease↗

PubMed1998

Prognostic patch testing: the other kind of patch test.

Prognostic patch testing differs from diagnostic patch testing. Clinicians routinely perform diagnostic patch testing, but prognotic patch testing is the province of corporate research and development departments and, in the United States, contract laboratories. The three types of prognostic patch testing are described and referenced in the scientific and medical literature. A comparison among the three types of prognostic patch testing and diagnostic patch testing is provided. In addition, the variables in both prognostic and diagnostic patch testing are listed, as well as the types of ingredients and products routinely tested in prognostic patch testing.

Consumer Product Safety↗

PubMed2003

Development of a rapid polymerase chain reaction-ELISA assay using polystyrene beads for the detection of Toxoplasma gondii DNA.

AIMS: To develop a rapid colourimetric assay for the detection of Toxoplasma gondii DNA using polystyrene beads as solid support. METHODS AND RESULTS: A nested-polymerase chain reaction (PCR)-ELISA assay for the detection of T. gondii DNA was standardized by optimizing the hybridization time and probe concentration. Its detection threshold was then determined and compared with Southern blotting hybridization. These were found to be equivalent, but the PCR-ELISA-beads test is easier to perform and the turnaround time is much shorter than with Southern blot. CONCLUSIONS: The PCR-ELISA-beads assay is a valuable tool for the detection of T. gondii DNA. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Our results demonstrate that this PCR-ELISA assay, using polystyrene beads, can be used as a routine diagnostic test for the detection of T. gondii in clinical laboratories.

Animals↗

PubMed1995

Studies using immobilized platelet glycoproteins for detection of platelet alloantibodies.

The utility of an enzyme linked immunoassay for the detection of platelet alloantibodies to isolated platelet glycoproteins employing commercially available monoclonal antibodies was studied. The authors established the usefulness of the assay for antibodies to platelet alloantigens (APAA) by testing its sensitivity and specificity under different conditions and using a variety of alloantibodies from patients with immune mediated thrombocytopenia. The authors were able to specifically detect a variety of antibodies to platelet alloantigens on different platelet glycoproteins. These antibodies had specificity for HPA-1a (PIA1), HPA-3 (Bak) and HPA-5 (Br). These results show that the APAA is easily adaptable for routine diagnostic testing in hospital laboratories using commercially available monoclonal antibodies and for the diagnosis of alloimmune mediated thrombocytopenic conditions.

Antibodies, Monoclonal↗

PubMed1990

Evaluation of the indoxyl acetate hydrolysis test for rapid differentiation of Campylobacter, Helicobacter, and Wolinella species.

A total of 410 well-defined Campylobacter, Helicobacter, and Wolinella strains, comprising 26 named species, subspecies, and defined groups, were tested for indoxyl acetate hydrolysis by a disk method by using disks prepared at the Centers for Disease Control, Atlanta, Ga. All C. coli (43 strains), C. cryaerophila (34 strains), C. fennelliae (5 strains), C. fennelliae-Campylobacter-like organism 3 (2 strains), C. jejuni (66 strains), C. jejuni subsp. doylei (3 strains), hippurate-negative C. jejuni-C. coli (15 strains), "C. upsaliensis" (39 strains), H. mustelae (5 strains), W. curva (1 strain), and W. recta (1 strain) hydrolyzed indoxyl acetate. Four strains gave weak positive reactions, and the remaining 196 strains, which belonged to 15 species, subspecies, and defined groups, gave negative reactions. Of the 410 study strains, 246 and 125 strains were tested for indoxyl acetate hydrolysis by a disk method and a tube method, respectively, by using commercially produced disks. The disk method, regardless of source, required less time and interpretation than the tube method did. Better differentiation between Campylobacter spp. was obtained with the indoxyl acetate test than with the trimethylamine N-oxide test. The indoxyl acetate disk distinguished C. lari from C. jejuni and C. coli, C. cinaedi from C. fennelliae, and H. pylori from H. mustelae and suggested that W. succinogenes could be differentiated from W. recta and W. curva. The indoxyl acetate disk method could be performed in 5 to 30 min, was easy to read and interpret, and should be useful as a routine diagnostic test for identification of Campylobacter spp.

Bacteriological Techniques↗

PubMed1998

Comparison of Roche Cobas Amplicor and Abbott LCx for the rapid detection of Chlamydia trachomatis in specimens from high-risk patients.

OBJECTIVE: To evaluate two automated amplification systems for the detection of Chlamydia trachomatis in urogenital specimens, the Cobas Amplicor (Roche Diagnostic Systems, Branchburg, NJ) and the LCx (Abbott Laboratories, Abbott Park, IL). STUDY DESIGN: The two systems were compared testing specimens from 302 high-risk patients, including 98 female cervical swab specimens and 204 male urine specimens. The patients attended the state STD clinic in Reykjavik, Iceland, either because of symptoms or as a result of contract tracing. RESULTS: The prevalence of C. trachomatis infection was 15.3% in women and 13.2% in men. For the male urine specimens, the sensitivity and specificity were 100% and 99.4% for the Cobas Amplicor and 74.1% and 100% for the LCx. In the cervical swabs, both systems detected all 15 true-positive specimens. The internal control used with the Cobas Amplicor detected inhibition in 2% of the male urine and 20% female cervical swabs, respectively. CONCLUSION: The Cobas Amplicor demonstrated slightly better sensitivity than LCx in male urine specimens. Both systems offer the benefits of automation for routine diagnostic testing.

Chlamydia Infections↗

PubMed2002

Laboratory testing for suspected Lyme disease.

Laboratory testing for B. burgdorferi infection is intended to substantiate a physician's clinical judgment of whether a patient has Lyme disease or not. Cultivation of B. burgdorferi from a patient's skin or blood is the gold standard for demonstration of active infection, but it is expensive and lacks clinical sensitivity. Detection of spirochetal DNA in clinical samples by PCR has better sensitivity, but PCR for B. burgdorferi has not yet been standardized for more routine diagnostic testing. Detection of antibodies to B. burgdorferi is the most practical and common approach for laboratory work-up of a case of suspected Lyme disease. Serologic assays fall short of 100% sensitivity and specificity, however, and examination of a single specimen in time does not discriminate between previous and ongoing infection. Because of a background false positivity even among healthy populations of nonendemic regions, serologic testing is recommended only when there is at least a one in five chance, in the physician's estimation, that the patient has active Lyme disease. The pretest likelihood of the disease is determined by the physician in the context of epidemiologic and clinical facts of the case. This estimate can serve to reassure patients who are at low risk of B. burgdorferi infection but are seeking a Lyme test for complaints of a more nonspecific nature. Although new subunit serologic assays based on recombinant proteins are becoming available commercially, the longstanding two-test approach, in which a positive or indeterminate result with a standardized, sensitive ELISA test is followed by verification with a more specific Western blot assay, still provides the physician with a reasonably accurate and reliable assessment of the presence of antibodies to B. burgdorferi. More recent challenges for serologic testing are seropositivity in the population as the result of immunization with the Lyme disease vaccine and the emergence of new Borrelia species that cause Lyme disease-like illnesses.

Antibodies, Bacterial↗

PubMed2005

Reverse transcriptase-PCR for the detection of Astrovirus in children with nosocomial acute diarrhoea in Naples, Italy.

OBJECTIVE: This study was made to determine the incidence of nosocomial viral gastroenteritis in all children aged 0-4 years, admitted in the Pediatric Hospital over a 3-year period. METHODS: Astrovirus was detected by reverse transcriptase-PCR; routine diagnostic tests for Rotavirus, Adenovirus, and common bacterial pathogens were carried out on all samples. RESULTS: Of the 460 children with nosocomial diarrhea, 23 harbored Astrovirus (5%). Most cases occurred during the coldest months of the year. Children under 1 year of age were the most susceptible population. CONCLUSION: The collected data confirms the importance of viral etiology in nosocomial gastroenteritis. The reported rate of detection stresses the importance of Astrovirus in pediatric diarrhea. The authors recommend screening for this virus on a routine basis.

Astroviridae Infections↗

PubMed1992

The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma-interferon in whole blood culture.

The recently developed gamma-interferon (IFN-gamma) assay system for the diagnosis of bovine tuberculosis in cattle has been accredited by the Standing Committee on Agriculture for use in Australia. In this test system, whole blood is incubated with tuberculin purified protein derivative (PPD) antigens for 16 to 24 h. The plasma is then collected and assayed for IFN-gamma production using an enzyme immunoassay (EIA). The assay system has proven to be a rapid, sensitive and inexpensive method for measuring antigen specific cell-mediated reactivity when compared with the more traditional lymphocyte proliferation assay. The IFN-gamma assay is the first in-vitro cellular assay to be used as a routine diagnostic test in veterinary medicine. While the IFN-gamma EIA has been optimised, several conditions affecting the production of IFN-gamma in whole blood culture needed investigation. We determined that optimal IFN-gamma production required the use of heparinised blood, cultured with 20 micrograms/ml of PPD within 8 h of collection. The use of blood collected post mortem resulted in reduced sensitivity for the assay. The kinetics of IFN-gamma release were established as were the effects of intradermal tuberculin testing on the IFN-gamma assay.

Animals↗

PubMed1996

Myopathy in primary systemic amyloidosis.

OBJECTIVE: To define the natural history of primary systemic amyloidosis when muscle involvement is prominent at presentation. METHODS: A retrospective review was carried out of all patients seen at the tertiary referral practice of the Mayo Clinic between 1 January 1960 and 31 December 1994. All patients with primary systemic amyloidosis and proof of amyloid deposits by muscle biopsy were included for analysis. No patients were lost to follow up. RESULTS: Twelve patients were identified with amyloidosis in muscle. Muscle involvement was the most prominent symptom in patients who had widespread visceral involvement, which included the heart, peripheral nerve, and tongue. Of the 12, three had skeletal muscle pseudohypertrophy. All patients had a demonstrable dysproteinaemia by the finding of free light chain in the serum or urine, a discrete monoclonal peak on serum or urine protein electrophoresis, or a monoclonal population of plasma cells in the bone marrow. Measurement of creatine kinase was not a useful test. Of eight patients treated with chemotherapy based on alkylating agents, three responded. The median survival for the entire group was 12 months. CONCLUSIONS: The finding of a monoclonal protein in a patient with muscle weakness is an important clue to the diagnosis of primary systemic amyloidosis. Most patients have visceral involvement outside the musculoskeletal system. A subset of patients seems to respond to systemic chemotherapy. The overall survival, however, remains poor, with most patients dying of cardiac failure. Immunoelectrophoresis of serum and urine should be a routine diagnostic test during the evaluation of myopathy of unknown cause.

Aged↗

PubMed2001

Screening for inborn errors of metabolism among newborns with metabolic disturbance and/or neurological manifestations without determined cause.

CONTEXT: Inborn Errors of Metabolism are hereditary affections resulting from incompetence in enzymatic reactions of intermediary metabolism. At present, several hundred hereditary metabolic disturbances are known, many of which correspond to severe life-threatening disorders. OBJECTIVE: The early detection of carriers has motivated the screening for these disturbances among newborns at the Neonatal Unit of Hospital São Paulo, in an attempt to initiate support treatment, when available, before clinical manifestations become evident. DESIGN: Prospective study of risk patients. SETTING: Laboratory for Inborn Errors of Metabolism at the Center for Medical Genetics of the Departments of Pediatrics and Morphology of Universidade Federal de São Paulo/Escola Paulista de Medicina. Newborn care unit at a tertiary care hospital. PARTICIPANTS: 101 children admitted into the Neonatal Unit were included in this study by presenting hypoglycemia, metabolic acidosis, jaundice, difficulty in gaining weight, diarrhea, vomiting, hepato- and/or splenomegaly, cataracts, apnea, convulsions, hypo- or hypertonia. DIAGNOSTIC TESTS: Tests routinely utilized, performed for qualitative research of abnormal substances excreted in the urine in situations of metabolic disorder. RESULTS: Children were included in the study mainly because of presenting hypoglycemia, jaundice and metabolic acidosis. Sixty-four newborns presented at least one positive test result. Most of the positivity was due to transitory metabolic alterations of the newborn, such as the case of Transitory Neonatal Tyrosinemia, presented by 29 patients. Nine infants were referred to the Center for Medical Genetics of Universidade Federal de São Paulo for continuation of the diagnostic investigation. For three of them, the tests applied permitted us to formulate a diagnostic hypothesis of mucopolysaccharidosis, tyrosinemia type I and non-ketotic hyperglycinemia, respectively. CONCLUSIONS: The high positivity observed in the tests reflects the newborn's own metabolic immaturity. The selection of 9% of the studied cases for outpatient follow-up confirms that Inborn Errors of Metabolism must be suspected whenever a patient presents metabolic disturbances or neurological manifestations without a determined cause. They should be researched in parallel with the other diagnostic possibilities and not just taken to be exceptional diagnoses.

Female↗

PubMedDate not supplied

Methylene blue in the evaluation of gastrointestinal tract integrity: potential limitations.

INTRODUCTION: The integrity of the gastrointestinal tract can be evaluated by oral or rectal application of methylene blue. In the presence of anastomotic leaks or fistulas, methylene blue can be recovered in adjacent drains. However, parts of the dye can biochemically be reduced by intestinal bacteria to its colorless form leucomethylene blue, limiting the prediction of the test. MATERIALS AND METHODS: Diluted methylene blue was added to different concentrations of bacterial suspensions of Escherichia coli and Enterococcus faecalis. The time for discoloration of the suspension was measured. RESULTS: Reduction of methylene blue to leucomethylene blue was observed in both types of bacterial suspension. A 10(8) bacterial concentration discolorated the dye within 1 h in the E. faecalis suspension, respectively 2.5 h in the E. coli suspension. Longer bacterial interaction with methylene blue reduced the bacterial concentration required to achieve complete discoloration. DISCUSSION: Methylene blue can reliably be used as routine diagnostic test for the assessment of upper gastrointestinal integrity, where bacterial load is low. In the lower gastrointestinal tract, where bacterial load is generally higher, the dye can indicate leaks, only if extended intestinal passage after oral ingestion is avoided. In all other cases, the examiner has to be aware of false-negative results by bacterial discoloration of methylene blue.

Animals↗

PubMed2000

Methods for diagnosing onychomycosis: a comparative study and review of the literature.

OBJECTIVE: To identify the sensitivity of several readily available diagnostic tests for onychomycosis. DESIGN: Cross-sectional study. SETTING: Dermatology and podiatry departments at a teaching hospital. PATIENTS: Sixty-three adult men and women with a clinical examination highly suggestive of onychomycosis. MAIN OUTCOME MEASURES: Sensitivity of each test and of several test combinations. RESULTS: Nail samples underwent 6 diagnostic tests. Routine histopathologic examination with periodic acid-Schiff stain (PAS) (PATHPAS) was 85% sensitive. Sensitivities for potassium hydroxide dissolution and centrifugation combined with PAS, fluorescent brightener, or chlorazol black E were 57%, 53%, and 53%, respectively. Culture on Sabouraud agar withchloramphenicol and cycloheximide (Mycosel agar) was 32% sensitive; on Littman-oxgall agar, 23% sensitive. The most sensitive combination of tests, both culture methods plus PATHPAS, was 94% sensitive (not statistically different from the sensitivity of PATHPAS alone [P = .26]). CONCLUSIONS: When onychomycosis is suspected clinically, PATHPAS of the nail is the single most sensitive of the diagnostic tests we evaluated. Because it is quickly performed and relatively operator independent, PATHPAS is practical for clinical and research purposes. Further study is needed to determine if sensitivity may be enhanced by combining PATHPAS with cultures obtained by several collection methods (clipping, curettage, and shaving). Such combinations may serve as sensitive and efficient strategies for diagnosing onychomycosis.

Adult↗

PubMed1999

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification.

When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody. For stool samples, RNAzol, PEG-CETAB, and magnetic beads with antibody allowed detection of the virus in 11/12 and 12/12 of samples. For shellfish samples, three protocols allowed RNA to be extracted in 90% of cases, RNAzol, PEG-CETAB, and GTC-silica. Their rapidity and low cost make RNAzol and GTC-silica the most suitable for routine diagnostic testing. reserved.

Feces↗

PubMed2001

Blood levels of toxic and essential metals in motor neuron disease.

Toxic and essential metals have been implicated in the pathogenesis of sporadic motor neuron disease (SMND), but attempts to measure blood levels of these metals have led to contradictory results. We, therefore, measured blood levels of various metals using paired SMND/controls. In 20 subjects with SMND (15 males, five females, mean age 56.8 years) and 20 partner controls (15 females, five males, mean age 55.0 years) cadmium, lead, mercury, copper, zinc and selenium levels were measured in blood, plasma and red cells with inductively coupled plasma mass spectrometry and manganese levels with atomic absorption spectrophotometry. Results were analysed using non-parametric tests. Hypo-osmotic red blood cellfragility was estimated in six SMND/control pairs to see if hemolysis could account for increased metal levels. The plasma cadmium level was significantly raised in SMND cases (P = 0.005), but with considerable overlap between SMND and controls. No other metal levels were significantly different, though plasma lead in SMND had a tendency to be higher than controls. No difference in red cell fragility was found between groups. In conclusion, plasma levels of cadmium were raised in this SMND group, but the biological significance of this is uncertain. The measurement of metals in the blood of SMND cases seems unwarrranted for routine diagnostic testing.

Adult↗

PubMed2006

SMAD4 mutations found in unselected HHT patients.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease exhibiting multifocal vascular telangiectases and arteriovenous malformations. The majority of cases are caused by mutations in either the endoglin (ENG) or activin receptor-like kinase 1 (ALK1, ACVRL1) genes; both members of the transforming growth factor (TGF)-beta pathway. Mutations in SMAD4, another TGF-beta pathway member, are seen in patients with the combined syndrome of juvenile polyposis (JP) and HHT (JP-HHT). METHODS: We sought to determine if HHT patients without any apparent history of JP, who were undergoing routine diagnostic testing, would have mutations in SMAD4. We tested 30 unrelated HHT patients, all of whom had been referred for DNA based testing for HHT and were found to be negative for mutations in ENG and ALK1. RESULTS: Three of these people harboured mutations in SMAD4, a rate of 10% (3/30). The SMAD4 mutations were similar to those found in other patients with the JP-HHT syndrome. CONCLUSIONS: The identification of SMAD4 mutations in HHT patients without prior diagnosis of JP has significant and immediate clinical implications, as these people are likely to be at risk of having JP-HHT with the associated increased risk of gastrointestinal cancer. We propose that routine DNA based testing for HHT should include SMAD4 for samples in which mutations in neither ENG nor ALK1 are identified. HHT patients with SMAD4 mutations should be screened for colonic and gastric polyps associated with JP.

Activin Receptors, Type II↗