Staining and Labeling :: economics
Virol J. 2012 ;9 :27 22264275
Development of a SYBR green I based RT-PCR assay for yellow fever virus: application in assessment of YFV infection in Aedes aegypti.
Institut Pasteur, Insects and Infectious Disease Unit, CNRS URA 3012, 25 rue du Docteur Roux, 75724 Paris, France.
BACKGROUND Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases. RESULTS We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi). CONCLUSIONS The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.
Most cited papers:
Department of Medicine, University of Florida Health Science Center, Jacksonville.
Histopathologic interpretation of hematoxylin and eosin (H&E)-stained endoscopic biopsies is a common method for identifying Helicobacter pylori. Few studies report the accuracy of this method, and none have compared costs of other diagnostic methods. In the clinical setting of a community hospital using standard diagnostic techniques, the purpose of this study were to determine 1) the comparative sensitivities and specificities of the H&E stain, the Warthin-Starry silver stain, the Giemsa stain, and the CLOtest; 2) the sensitivity and specificity of an "experienced" pathologist in identifying H. pylori by H&E stains, compared with a rotating pathology faculty; and 3) the time to diagnosis (turnaround time) and current patient charges for each diagnostic method. Bacterial identification by the silver stain (or a combination of other tests which were likely to compensate for false-positive and false-negative silver stains) were used as the diagnostic standard in evaluating 94 consecutive cases with the following results: The H&E stain interpreted by the rotating pathology staff was the least sensitive method and one of the least specific tests that were studied. The silver and Giemsa stains were equally sensitive in identifying H. pylori; the silver stain was more specific. The CLOtest was less sensitive than the silver and Giemsa stains, but was equally specific. CLOtest was similar in sensitivity to the H&E stain examined by the "experienced" pathologist, but was more specific. An experienced pathologist was significantly more sensitive than the rotating pathologists in evaluating H&E-stained slides. Therefore, if H&E stains are used to identify H. pylori, which is a common practice, it may be advantageous to use an experienced pathologist. The CLOtest was a simple, rapid, and cost effective substitute for H&E stains in the identification of H. pylori.
Department of Laboratory Medicine, University of California, San Francisco.
Four stains for the detection of Pneumocystis carinii in respiratory specimens were compared for sensitivity, specificity, preparation time, and ease of interpretation. One hundred specimens were collected. Of these, 50 were induced sputum specimens and 50 were bronchoalveolar lavage fluid. All specimens were stained with Diff-Quik (DQ)(a modified Giemsa stain), a quick silver stain, and direct and indirect immunofluorescence stains. A positive specimen was defined as any smear positive by two or more of the methods. Fifty-eight percent of specimens were positive. Seventy-four percent of the sputum specimens and 42% of the bronchoalveolar lavages were positive. The sensitivities for detection of P. carinii in sputum were 92% with silver stain, 97% with direct immunofluorescence assay (DFA), 97% with indirect immunofluorescence assay (IFA), and 92% with DQ. The sensitivities for detection in bronchoalveolar lavage were 86% with silver stain, 90% with DFA, 86% with IFA, and 81% with DQ. Preparation times varied from 90 min for the silver stain and IFA to 3 min for DQ. Costs of the tests varied from $1.50 per slide for DQ to $10.00 per slide for the silver stain and DFA. Reading times varied from 10 to 30 min for the silver stain and DQ to less than 5 min for the immunofluorescence assays. We conclude that all of these tests are viable options for the clinical laboratory, and the choice will be influenced by factors such as clinical volume, ability to batch specimens, and expertise of technological support. A reasonable option may be to use the quick and inexpensive DQ as a screening test and to confirm negative smears with a more sensitive assay.
Gram stain method shows better sensitivity than clinical criteria for detection of bacterial vaginosis in surveillance of pregnant, low-income women in a clinical setting.
OBJECTIVE: The purpose of the study is to determine whether the Gram stain method is superior to the clinical criteria for the diagnosis of bacterial vaginosis in low-income pregnant women seen in a resident clinic setting. The clinical criteria is the current diagnostic method employed to diagnose bacterial vaginosis. STUDY DESIGN: In this study, 51 pregnant women with vaginal discharge were prospectively evaluated. All were screened using the clinical criteria, Gram stain method, and culture of the discharge. The modified scoring system instituted by Nugent et al.(J Clin Microbiol 29:297-301, 1991) was employed in reading the Gram stain smears. The clinical criteria were then compared with the Gram stain method. Isolation of moderate to many Gardnerella vaginalis growth by culture was used as the confirmatory finding. RESULTS: Sensitivity of the Gram stain method (91%) was significantly higher than that of the clinical criteria (46%),(sign test P = 0.0023,< 0.01). The Gram stain method also has both a low false-negative (4%) and high negative predictive value (96%), making it an ideal diagnostic test. CONCLUSION: The Gram stain method is a rapid and cost-effective test that is also highly reproducible and readily available in many laboratories. These features make the Gram stain method a more desirable screening procedure for bacterial vaginosis in a clinic population.
Department of Histopathology, Algernon Firth Institute of Pathology, General Infirmary at Leeds, UK.
AIM To determine whether two recently described staining methods (the modified McMullen's and the Helicobacter pylori silver stain HpSS methods) used for the histological identification of H pylori organisms are superior to two established techniques (the modified Giemsa and anti-H pylori antibody immunostain) in terms of availability, reproducibility, rapidity, sensitivity, and cost. METHODS Histological sections from 63 paired gastric biopsies from adult patients previously investigated for dyspepsia were stained with the four methods and these were assessed blindly and independently by two observers. Of the 63 patients, 30 were originally negative in all tests for H pylori infection, 30 were positive, and the remaining three cases had discordant results using a combination of five tests (rapid biopsy urease test, urea breath test, culture, serology, and histology). RESULTS Interobserver agreement was best with the antibody method (98%), followed by the McMullen's (90%), Giemsa (87%), and HpSS (85%). Of the 60 "gold standard" positive and negative cases, 30 were positive by the modified Giemsa stain, 29 by the McMullen's method, 29 by HpSS, and 30 by the antibody stain. However, there were two false positives with the HpSS method. The modified Giemsa is the cheapest and easiest to perform technically. CONCLUSIONS When H pylori are present, careful examination will almost always reveal them, whichever of these stains is used. However, the modified Giemsa stain is the method of choice because it is sensitive, cheap, easy to perform, and reproducible.
Endoscopic biopsy pathology of Helicobacter pylori gastritis. Comparison of bacterial detection by immunohistochemistry and Genta stain.
Department of Pathology, Baystate Medical Center, Springfield, MA 01199, USA.
OBJECTIVES To describe the endoscopic biopsy pathology of Helicobacter pylori gastritis, compare bacterial detection by immunohistochemistry using a specific antibody with the Genta stain, and to compare the relative costs of the 2 techniques. DESIGN One hundred cases of gastritis identified as positive for H pylori by Genta stain and 100 cases considered negative by the same technique were stained using an anti-H pylori-specific polyclonal antibody. Laboratory reagent and labor costs for the 2 methods were compared. RESULTS Chronic active gastritis with lymphoid follicles was significantly associated with H pylori infection (P <.0001). The immunohistochemical method had a sensitivity of 97% and a specificity of 98% compared with the Genta stain, with strong agreement for grading density of organisms (kappa = 0.85; P <.001). Reagent costs were similar for both methods, but immunohistochemistry using an autoimmunostainer required less dedicated technical time and hence was less expensive than the Genta stain. CONCLUSIONS Immunohistochemistry using a specific antibody is an accurate and cost-effective method for H pylori detection in gastric biopsies.
Department of Pathology, Queen Elizabeth Hospital, Charlottetown, Prince Edward Island, Canada.
Immunohistochemical staining of fresh frozen tissue to identify estrogen and progesterone receptors was tested as a means to supplant the dextran-coated charcoal assay in a small community hospital. Forty-three tumors were compared, yielding an overall concordance of 88% between immunohistochemical staining and the dextran-coated charcoal assay for determination of combined estrogen/progesterone receptor status. In addition, there was greater than 70% concordance for semiquantitative grades. Staining also revealed many other advantages, such as a 65% reduction in costs. For these reasons, the authors have abandoned the dextran-coated charcoal assay in favor of staining for estrogen/progesterone receptor status.
Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost.
Dept of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada. email@example.com
Control of Bordetella pertussis in the community is hampered by slow and insensitive diagnostic tests. We therefore examined the accuracy and cost of culture, direct fluorescent antibody (DFA) staining, and PCR in a routine clinical laboratory. Six hundred thirty seven nasopharyngeal swabs and aspirates in casamino acids transport medium were cultured, stained with polyclonal (Difco), and monoclonal (BL-5 and Accu-Mab) anti-B. pertussis reagents, and amplified by an IS481-specific PCR. PCR products were detected by a hybridization-enzyme immunoassay kit (Gen-eti-k DEIA, DiaSorin), with confirmation by a second PCR in a separate laboratory. Sensitivities and specificities of culture, polyclonal DFA, monoclonal DFA, and PCR were 36 and 100%, 11.4 and 94.6%, 8.3 and 98. 4%, and 95.0 and 99.3%, respectively, with a prevalence of 15.7%. The DFA tests were the most economical, and the PCR cost was 31% higher than culture. This study suggests that with minor improvements in economy, pertussis PCR can be implemented in a clinical laboratory with marked improvement in diagnostic accuracy.
Special histologic stains are rarely beneficial for the evaluation of HIV-related gastrointestinal infections.
Department of Medicine, University of Alabama at Birmingham, 35294-0007, USA
During a 28-month period, endoscopic mucosal biopsy specimens from all HIV-infected patients were submitted for routine histologic evaluation. Immunoperoxidase staining for cytomegalovirus and herpesvirus antigens (esophagus), mycobacterial and fungal staining, and Gram staining of mucosal biopsy specimens were done. Special fungal and acid-fast stains were selectively performed in patients with absolute CD4 cell counts of less than 200 cells per microliter (200 x 10(6)/L) and/or with diarrhea and or wasting syndrome. Treatment was based on the endoscopic and histologic findings, and long-term follow-up was performed. The 121 symptomatic HIV-infected patients underwent 221 upper and/or lower endoscopies with 285 biopsy sites. The sensitivity and specificity of H&E staining for the diagnosis of gastrointestinal cytomegalovirus were 97% and 100%, respectively. The results of fungal and mycobacterial stains neither altered therapy nor identified previously undiagnosed infections in any patient. Long-term follow-up revealed no patient in whom an infection was missed on routine H&E, which affected outcome. Routine H&E staining is accurate for the diagnosis of gastrointestinal opportunistic infections in HIV-infected patients. Special histologic stains for fungal, mycobacterial, and viral infections did not increase the diagnostic yield or alter medical therapy but doubled the costs.
Department of Pathology, University of Iowa, Iowa City.
Bronchoalveolar lavage (BAL) with Gomori methenamine silver (GMS) stain is commonly used to detect Pneumocystis carinii and fungal organisms as causes of infectious pulmonic disease in immunosuppressed patients. However, several reports have indicated that GMS stains are not any more sensitive than conventional cytologic stains in detecting Pneumocystis organisms in select patient populations, such as those with acquired immunodeficiency syndrome (AIDS). To examine the utility of GMS stains in our laboratory, we retrospectively reviewed 243 BALs from 188 patients. Sensitivity of the GMS stain for Pneumocystis and for fungi detection was 100%. Sensitivity for Pneumocystis and for fungi detection by Papanicolaou stain alone was 79% and 88%, respectively; by Diff-Quik stain alone it was 68% and 88%, respectively; and by combined Papanicolaou and Diff-Quik stains it was 79% and 100%, respectively. In four additional cases, fungi were detected by other methods (culture, biopsy) and not by BAL. The GMS stain result was correlated with a number of risk variables to determine which variables were associated with GMS positivity. Using stepwise logistic regression, Pneumocystis positivity by GMS stain correlated (P < 0.0001) only with the variable of history of AIDS or AIDS risk factors. Fungal organism positivity by GMS stain correlated (P = 0.02) only with the variable of history of BAL positivity for fungus. Cost savings analyses were performed, estimating the cost of the GMS stain at $45 (total cost of GMS in 243 BALs was $10,935).(ABSTRACT TRUNCATED AT 250 WORDS)