Identification of Streptococcus pneumoniae revisited

The sensitivities and specificities of several different diagnostic assays for Streptococcus pneumoniae were assessed using 99 clinical isolates of S. pneumoniae and 101 viridans streptococci and were as follows: Pneumoslide, 99 and 87%, respectively; Directigen, 100 and 85%, respectively; Phadebact, 100 and 98%, respectively; deoxycholate drop test, 99 and 98%, respectively; deoxycholate tube test, 100 and 99%, respectively; optochin, 99 and 98%, respectively; and Gram Positive Identification Card, 90 and 96%, respectively. Identification of clinical isolates of S. pneumoniae should be confirmed using one or more diagnostic assays with well-documented high (e.g., > or =95%) sensitivities and specificities.     

Comparison of four enzyme immunoassays with a western blot assay for the determination of type-specific antibodies to herpes simplex virus

Most current enzyme immunoassays (EIAs) differentiate inadequately between types 1 and 2 herpes simplex virus (HSV) antibodies since significant cross-reactivity exists. We compared 4 IgG type-specific EIAs using a Western blot assay for resolution of discrepant results. The Diamedix had sensitivities of 100% for types 1 and 2 but specificities of only 71% and 61%, respectively. The cross-reactivity rate was 82% in positive samples tested. For HSV types 1 and 2, the Zeus sensitivities were 92% and 98%, respectively; specificities were 72% and 79%, respectively; the cross-reactivity rate was 54%. For HSV types 1 and 2, the Wampole sensitivities were 98% and 95%, respectively; specificities were 68% and 85%, respectively; the cross-reactivity rate was 47%. For HSV types 1 and 2, the Meridian sensitivities were 98% and 90%, respectively; specificities were 96% and 100%, respectively; no cross-reactivity was found between positive samples tested. While the Diamedix, Zeus, and Wampole assays showed good sensitivity, they lacked type specificity. The Meridian EIA offers the highest specificity along with no observed cross-reactivity. This EIA may be an easier, reliable alternative to Western blot for the determination of HSV type-specific antibodies.     

Comparison of eleven commercial tests for Chlamydia pneumoniae-specific immunoglobulin G in asymptomatic healthy individuals

The seroprevalence of anti-Chlamydia pneumoniae-specific immunoglobulin G (IgG) antibodies is high in the adult population. Experience is required to perform a microimmunofluorescence test (MIF), the current "gold standard" for serological diagnosis, and the assay still lacks standardization. Partially automated enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs), which are more standardized and for which the reading of results is less subjective, have been developed. The different commercially available serological tests differ in their sensitivities and specificities, depending primarily on the antigen used. Therefore, we evaluated 11 different tests (10 were species specific, 1 was genus specific) for IgG antibodies using serum samples of 80 apparently healthy volunteers. The interpretation of the results was based on the results of the gold standard, MIF: a sample was judged positive if it was positive by at least three of the four different MIFs. Based on this internal standard, we found that 71% of the samples were positive, while 8% were false positive by some tests. The correlations between the results of the different MIFs ranged from 83 to 99%, and the correlations between the results of the MIFs and the different ELISAs and EIAs ranged from 78 to 98%. Comparison of the IgG titers measured by MIF showed good agreement (r = 0.76 to 0.91). This analysis revealed that some ELISAs and EIAs fail to detect low IgG titers. The specificities of the species-specific tests varied from 95 to 100%, and the sensitivities varied from 58 to 100%. These results indicate that serological assays for the detection of anti-C. pneumoniae-specific IgG vary greatly in their sensitivities and specificities. MIF must still be considered the best method for the detection of IgG in apparently healthy subjects, but the sensitivities and specificities of new ELISAs approximate those of MIFs.     

Comparative Evaluation of the Nanosphere Verigene RV+ Assay and the Simplexa Flu A/B & RSV Kit for Detection of Influenza and Respiratory Syncytial Viruses

Using retrospective (n = 200) and prospective (n = 150) nasopharyngeal specimens, we evaluated the Nanosphere Verigene RV+ and the Focus Diagnostics Simplexa Flu A/B & RSV tests. Overall, RV+ demonstrated sensitivities and specificities of 96.6% and 100% for influenza A virus, 100% and 99.7% for influenza B virus, and 100% and 100% for respiratory syncytial virus (RSV), while the Simplexa test sensitivities and specificities were 82.8 and 99.7%, 76.2 and 100%, and 94.6 and 100%, respectively.     

Evaluation of an automated immunodiagnostic assay, VIDAS Rotavirus, for detection of rotavirus in fecal specimens.

Four hundred sixty-four fecal specimens were tested for rotavirus by three immunoassays, VIDAS Rotavirus (bioMérieux Vitek, Hazelwood, Mo.), Rotaclone (Cambridge Biotech Corporation, Worcester, Mass.), and Pathfinder Rotavirus (Sansfi Diagnostics Pasteur, Chaska, Minn.). Twenty-seven discordant specimens were evaluated by electron microscopy. The sensitivities, specificities, positive predictive values, and negative predictive values were 98, 99.3, 98.7, and 99%, respectively, for VIDAS Rotavirus; 100, 99, 98.1, and 100%, respectively, for Rotaclone; and 100, 92.4, 87.3, and 100%, respectively for Pathfinder Rotavirus.     

Evaluation of two colored latex kits, the Wellcolex Colour Salmonella Test and the Wellcolex Colour Shigella Test, for serological grouping of Salmonella and Shigella species.

Two colored latex kits (the Wellcolex Colour Salmonella Test [WCT-Salmonella] and the Wellcolex Colour Shigella Test [WCT-Shigella]; Division Diagnostics, Laboratories Wellcome S.A., Paris, France), which allow identification of the most frequently encountered Salmonella serogroups and Shigella species, respectively, were evaluated. WCT-Salmonella and WCT-Shigella yielded sensitivities of 98.4 and 98%, respectively, and a specificity of 100% when they were tested on pure cultures received at a reference laboratory.     

Accuracy of cefoxitin disk testing for characterization of oxacillin resistance mediated by penicillin-binding protein 2a in coagulase-negative staphylococci

The Clinical and Laboratory Standards Institute (CLSI) proposed, beginning in 2004, the use of cefoxitin disks to predict resistance mediated by the mecA gene in all species of coagulase-negative staphylococci (CoNS). The aim of this work was to evaluate the efficiency of the cefoxitin disk and of oxacillin-salt agar screening (MHOX) to characterize the oxacillin resistance mediated by the mecA gene in CoNS. One hundred seven CoNS isolates from different clinical samples were studied. Detection of the mecA gene by PCR was considered the "gold standard." The susceptibility to oxacillin and cefoxitin was detected by the disk diffusion and agar dilution tests, as described by the CLSI. MHOX was also performed with 6 microg/ml of oxacillin and 4% NaCl. The sensitivities of the oxacillin and cefoxitin disks for all CoNS species were 88% and 80%, respectively, whereas the specificities were 63% and 100%, respectively. The sensitivities of the agar dilution test for oxacillin and cefoxitin (for proposed breakpoints of > or =4 microg/ml for resistance and < or =2 microg/ml for susceptibility) were 90% and 85%, respectively, whereas the specificities were 76% and 98%, respectively. MHOX showed a sensitivity of 90% and a specificity of 95% for all CoNS species. Both the MHOX and the cefoxitin disk results indicate that these are appropriate methods for the evaluation of oxacillin resistance mediated by the mecA gene in all CoNS species.     

Evaluation of Toxoplasma gondii immunoglobulin G (IgG) and IgM assays incorporating the newVidia analyzer system

The new Vidia system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles, which allows a fast measurement of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM levels. The analytical performances of the Vidia Toxo IgG and IgM assays were compared with those of the automated Vidas, AxSYM, and Liaison Toxo IgG and IgM assays. The comparative evaluation was performed utilizing 204 frozen sera belonging to 166 subjects and 201 fresh sera collected from 198 subjects. For the Vidia Toxo IgG system, the sensitivities were 100% in both the retrospective and prospective studies, and specificities were 98.39% in the retrospective study and 100% in the prospective study, respectively. The sensitivities of the other three Toxo IgG assays were 100%, and the specificities ranged from 96.77% to 100%. For the Vidia Toxo IgM assay, the sensitivity and specificity were 100% in both the retrospective and prospective studies. The overall sensitivities and specificities of the other three Toxo IgM assays ranged from 80% to 100% and from 99.44% to 100%, respectively. In our study, the Vidia system revealed excellent sensitivity (100% for both IgG and IgM assays) and good specificity (99.25% for IgG and 100% for IgM assays).     

PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA

Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.     

Rapid detection of group A streptococci: comparative performance by nurses and laboratory technologists in pediatric satellite laboratories using three test kits.

Rapid tests for detecting group A streptococci in throat swabs are often performed outside hospitals or commercial laboratories by individuals with little or no technical training. We compared the abilities of nurses and technologists to perform and interpret three commercial kits (Directigen 1-2-3, ICON Strep A, and Culturette Brand 10-Minute Strep A ID) in three hospital satellite locations (the emergency department, a walk-in emergency clinic, and a general pediatric clinic). When the three tests were compared with culture, the sensitivities of the tests as performed by nurses and technologists, respectively, were 39 versus 44% for Directigen, 55 versus 51% for Culturette, and 72 versus 39% for ICON. A significant difference in sensitivity was found only with ICON tests. This result was largely explained by the tendency of technologists to test moist swabs, while nurses generally processed dry swabs; ICON test sensitivity was significantly greater with dry swabs. The specificities of Directigen and ICON tests performed by nurses and technologists were high (97 to 100%). The difference in the specificities of the Culturette test as determined from results obtained by nurses and technologists (80 versus 98%) was due to the tendency of one nurse to overinterpret the latex agglutination reaction. Analysis of the accuracies of the tests during practice periods compared with the accuracies of the tests during the study periods revealed statistically significant improvement in test performance. We conclude that these tests are specific but not sensitive when performed by nurses and technologists in satellite laboratories. With one exception, nurses and technologists performed the tests with comparable accuracy after brief training periods.     

Development and evaluation of serological assays for detection of Hantaanvirus-specific antibodies in human sera using yeast-expressed nucleocapsid protein

Indirect and capture enzyme-linked immunosorbent assays (ELISAs) for detection of Hantaan virus (HTNV)-specific immunoglobulins G (IgG) and M (IgM) in human serum samples were developed on the basis of recombinant yeast-expressed nucleocapsid (N) protein of HTNV. The sensitivities and specificities of the indirect and capture ELISAs were evaluated by comparing the reactivity of sera from patients with hemorrhagic fever with renal syndrome (HFRS) from China with that of a commercial IgG/IgM kit. The sensitivity of the indirect IgG and IgM ELISA tests was both 100% and the specificity of the indirect IgM and IgG ELISA test was 98% and 99%, respectively. The sensitivity and specificity of the capture IgM ELISA was 100% and 97%, respectively. The novel assays were found to detect HTNV-specific antibodies in acute phase sera from suspected HFRS patients in China. The results indicate that these novel ELISAs are suitable for the diagnosis of HTNV and for sero-epidemiological studies.     

Evaluation of rapid antigen point-of-care tests for detection of Giardia and Cryptosporidium species in human fecal specimens

In Bangladesh, a new parasite rapid antigen test was investigated demonstrating accuracy and feasibility. For Giardia species, it had a sensitivity and specificity of 94% and 100%, respectively. For Cryptosporidium species, it had a sensitivity and specificity of 100% and 100%, respectively. These are higher than or equal to the sensitivities and specificities of other tests on the market.     

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by enzyme immunoassay, culture, and three nucleic acid amplification tests

The purpose of this study was to evaluate and compare three commercially available nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis. Roche PCR and Becton Dickinson strand displacement amplification (SDA) were performed on 733 endocervical swab specimens from commercial sex workers. Abbott ligase chain reaction (LCR) was performed on a subset of 396 samples. Endocervical specimens from all women were also tested by culture for N. gonorrhoeae and by Syva MicroTrak enzyme immunoassay (EIA) for C. trachomatis. A positive N. gonorrhoeae result was defined as a positive result by culture or by two NAATs, and a positive C. trachomatis result was defined as a positive result by two tests. According to these definitions, the sensitivities and specificities for the subsample of 396 specimens of N. gonorrhoeae culture, PCR, SDA, and LCR were 69.8, 95.2, 88.9, and 88.9% and 100, 99.4, 100, and 99.1%, respectively; the sensitivities and specificities of C. trachomatis EIA, PCR, SDA, and LCR were 42.0, 98.0, 94.0, and 90.0% and 100, 98.0, 100, and 98.6%, respectively. The performance characteristics of N. gonorrhoeae culture, PCR, and SDA and C. trachomatis EIA, PCR, and SDA for all 733 specimens were defined without inclusion of LCR results and by discrepant analysis after resolution of discordant N. gonorrhoeae PCR results and of discordant C. trachomatis EIA and PCR results by LCR testing. The sensitivities of N. gonorrhoeae culture, PCR, and SDA before and after LCR resolution were 67.8, 95.7, and 93.9% and 65, 95.8, and 90.0%, respectively. The sensitivities of C. trachomatis EIA, PCR, and SDA decreased from 39.4, 100, and 100% to 38.7, 98.7, and 94.7%, respectively. All three NAATs proved to be superior to N. gonorrhoeae culture and to C. trachomatis EIA. The accuracies of the different NAATs were quite similar. SDA was the only amplification assay with 100% specificity for detection of both N. gonorrhoeae and C. trachomatis in endocervical specimens.     

International multicenter evaluation of latex agglutination tests for identification of Staphylococcus aureus

A newly marketed rapid agglutination kit for the identification of Staphylococcus aureus, Slidex Staph Plus (bioMérieux), was compared to Staphaurex Plus (Murex Diagnostics) and Pastorex Staph-Plus (Sanofi Diagnostics Pasteur). The study took place in three clinical microbiology laboratories in three different European countries. A total of 892 staphylococcal isolates, including 278 methicillin-sensitive S. aureus (MSSA) isolates, 171 methicillin-resistant S. aureus (MRSA) isolates, and 443 coagulase-negative staphylococcal isolates, were analyzed. The sensitivities (MSSA/MRSA) and specificities, respectively, were 98. 2% (98.9%/97.1%) and 98.9% for Slidex Staph Plus, 98.2% (98.2%/98. 2%) and 96.2% for Staphaurex Plus, and 98.7% (98.6%/98.8%) and 95.7% for Pastorex Staph Plus. The specificity of the Slidex Staph Plus kit was statistically significantly higher than the specificities of Staphaurex Plus and Pastorex Staph-Plus. The Slidex Staph Plus is a very reliable test for the identification of S. aureus.     

Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids

Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.     

Modified enzyme immunoassay to detect hepatitis C virus antibodies in oral fluid

Samples of oral fluid collected from 18 patients seropositive for hepatitis C virus (HCV) and 49 seronegative patients were tested for the presence of HCV antibodies with two modified serum-screening kits. The following sensitivities and specificities were obtained: HCV 3.0 assay, 72% and 98%, respectively, and Monolisa anti-HCV assay, 100% and 100%, respectively. The modified Monolisa assay demonstrated a striking concordance between serum and saliva samples. The use of oral fluids offers a convenient and noninvasive method applicable to HCV epidemiological studies and screening of high-risk groups.     

四种梅毒血清学实验检测方法的比较

目的比较四种梅毒血清学实验检测方法的准确性及在临床诊断中的价值。方法分别用甲苯氨红不加热实验（TRUST）、酶联免疫吸附法（ELISA）、梅毒螺旋体明胶颗粒凝胶实验（TPPA）、化学发光法检测180例梅毒血清样本。结果TRUST、ELISA、TPPA、化学发光法的敏感性分别为86．11％、97．78％、99．44％、98．89％，特异性分别为81．25％、98，75％、100％、98．75％。以TPPA作为标准．ELISA和化学发光法的敏感性高于TRUST，差异有统计学意义（P〈0．05），特异性ELISA和化学发光法间差异无统计学意义（P〉0．05）。结论ELISA和化学发光法可作为TPPA的替代实验，在临床中广泛应用。     

Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.     

Evaluation of two slide agglutination tests and a novel immunochromatographic assay for rapid diagnosis of infectious mononucleosis

The results of three tests used for the rapid diagnosis of infectious mononucleosis (IM) were compared with those of Epstein-Barr virus-specific serology. The sensitivities ranged from 15 to 33% in children under 13 years of age and from 59 to 81% in patients over 13 years. The specificities ranged from 86 to 100% in both age groups. These tests have a poor sensitivity for the diagnosis of IM, particularly in children.     

Development and evaluation of rapid urinary antigen detection tests for diagnosis of penicilliosis marneffei

Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of P. marneffei was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of P. marneffei urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis.