Comparison of conventional stool concentration and preserved-smear methods with Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay and ProSpecT Giardia EZ Microplate Assay for detection of Giardia lamblia.

Giardiasis is the most common parasitic infection in the United States. Variation in the numbers of cysts and/or trophozoites that are present along with the need for a skilled microscopist offer challenges in diagnosis. We compared the sediment wet preparation and permanent stained smear results (concentration in formalin-ethyl acetate and preparation of a smear prepared from a polyvinyl alcohol-preserved specimen) from 512 consecutive specimens with the results obtained by using the Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay (DFA; Meridian Diagnostics, Inc., Cincinnati, Ohio) and the ProSpecT Giardia EZ Microplate Assay (EIA; Alexon, Inc., Sunnyvale, Calif.). The Merifluor DFA detected 33 of 33 positive specimens, and the ProSpecT EIA detected 32 of 33 positive specimens. The diagnostic sensitivities of the Merifluor DFA and the ProSpecT EIA were 100 and 97%, respectively. The specificities of the assays were 99.8%. The Merifluor DFA and the ProSpecT EIA appear to be equally sensitive, and both are more sensitive than conventional microscopy.     

Evaluation of the ProSpecT Microplate Assay for detection of Campylobacter: a routine laboratory perspective

Objectives  To evaluate the use of the new enzyme-linked immunosorbent assay, the ProSpecT Campylobacter Microplate Assay (Alexon-Trend, Minneapolis, MN, USA), which allows 2-h detection of both Campylobacter jejuni and Campylobacter coli antigen directly in stool specimens.
Methods  Over 4 months, all stool samples preserved in Cary–Blair medium, or fresh specimens, from non-hospitalized children and HIV-infected patients (adults and children), submitted to our laboratory were evaluated with the ProSpecT Campylobacter Microplate Assay. Results were compared with those obtained by routine culture methods using both a specific medium and a filtration method for the recovery of Campylobacter spp.
Results  Of the 1205 stool specimens cultured, 101 were found to be positive for either C. jejuni or C. coli , giving an overall recovery rate of 8.38%. Ninety samples were positive by both culture and ProSpecT Campylobacter Microplate Assay, and 11 were positive by culture only, giving a sensitivity of 89.1%. In addition, of 1104 samples negative by culture, 25 were initially positive by ProSpecT Campylobacter Microplate Assay. We found no cross-reaction with other bacterial enteropathogens isolated from stool specimens. These results thus confirm a high specificity (97.7%) for both C. jejuni and C. coli. The positive and negative predictive values found were 78.3% and 99%, respectively. There was no statistically significant difference in sensitivity and specificity if the stool was fresh or preserved with Cary–Blair medium.
Conclusion  These data suggest that the ProSpecT Campylobacter Microplate Assay is a rapid and easy-to-use test for the detection of both C. jejuni and C. coli in stool specimens. It could be used for patients for whom early antibiotic therapy is needed or for epidemiologic studies.     

Multicenter Evaluation of the Xpert Norovirus Assay for Detection of Norovirus Genogroups I and II in Fecal Specimens

Norovirus is the most common cause of sporadic gastroenteritis and outbreaks worldwide. The rapid identification of norovirus has important implications for infection prevention measures and may reduce the need for additional diagnostic testing. The Xpert Norovirus assay recently received FDA clearance for the detection and differentiation of norovirus genogroups I and II (GI and GII), which account for the vast majority of infections. In this study, we evaluated the performance of the Xpert Norovirus assay with both fresh, prospectively collected (n = 914) and frozen, archived (n = 489) fecal specimens. A Centers for Disease Control and Prevention (CDC) composite reference method was used as the gold standard for comparison. For both prospective and frozen specimens, the Xpert Norovirus assay showed positive percent agreement (PPA) and negative percent agreement (NPA) values of 98.3% and 98.1% for GI and of 99.4% and 98.2% for GII, respectively. Norovirus prevalence in the prospective specimens (collected from March to May of 2014) was 9.9% (n = 90), with the majority of positives caused by genogroup II (82%, n = 74). The positive predictive value (PPV) of the Xpert Norovirus assay was 75% for GI-positive specimens, whereas it was 86.5% for GII-positive specimens. The negative predictive values (NPV) for GI and GII were 100% and 99.9%, respectively.     

Evaluation of a New Automated Homogeneous PCR Assay,GenomEra C. difficile,for Rapid Detection of Toxigenic Clostridium difficile in Fecal Specimens

We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.     

Comparison of Nine Commercially Available Enzyme-Linked Immunosorbent Assays for Detection of Giardia lamblia in Fecal Specimens

Overall performance, including ease of use, total hands-on time, incubation and processing times, sensitivity, and specificity, of each of nine enzyme-linked immunosorbent assays (ELISAs) were compared by using 222 individual fecal samples submitted for the detection of Giardia lamblia. The assays evaluated were manufactured by Alexon, Inc., Cambridge Biotech Corp., Meridian, Inc., and Trend Scientific, Inc. All assays used polyclonal antibodies except the “new and improved” Microplate (direct and diluted methods) by Alexon, which is a monoclonal antibody assay. Seventy specimens were positive for G. lamblia by ELISA, ova and parasite test, and/or direct fluorescent-antibody assay. One hundred fifty two were negative by all three methods. Sensitivities and specificities ranged from 88.6 to 100% and 99.3 to 100%, respectively. The total hands-on time needed to run one specimen ranged from 1 min to 2 min 17 s per specimen. All except one commercially available ELISA were found to be rapid, sensitive, and specific for the detection of G. lamblia in fecal specimens.     

Comparison of PCR and Microscopy for Detection of Cryptosporidium parvum in Human Fecal Specimens: Clinical Trial

PCR technology offers alternatives to conventional diagnosis of Cryptosporidium for both clinical and environmental samples. We compared microscopic examination by a conventional acid-fast staining procedure with a recently developed PCR test that can not only detect Cryptosporidium but is also able to differentiate between what appear to be host-adapted genotypes of the parasite. Examinations were performed on 511 stool specimens referred for screening on the basis of diarrhea. PCR detected a total of 36 positives out of the 511 samples, while routine microscopy detected 29 positives. Additional positives detected by PCR were eventually confirmed to be positive by microscopy. A total of five samples that were positive by routine microscopy at Western Diagnostic Pathology but negative by PCR and by microscopy in our laboratory were treated as false positives. Microscopy therefore exhibited 83.7% sensitivity and 98.9% specificity compared to PCR. PCR was more sensitive and easier to interpret but required more hands-on time to perform and was more expensive than microscopy. PCR, however, was very adaptable to batch analysis, reducing the costs considerably. Bulk buying of reagents and modifications to the procedure would decrease the cost of the PCR test even more. An important advantage of the PCR test, its ability to directly differentiate between different Cryptosporidium genotypes, will assist in determining the source of cryptosporidial outbreaks. Sensitivity, specificity, ability to genotype, ease of use, and adaptability to batch testing make PCR a useful tool for future diagnosis and studies on the molecular epidemiology of Cryptosporidium infections.     

Evaluation of a Screening Test for Detection of Giardia and Cryptosporidium Parasites

The Giardia/Cryptosporidium Chek test (TechLab, Inc.), a screening test for Giardia and Cryptosporidium, was evaluated with 136 fecal samples. Using the results of the Giardia II test and Cryptosporidium II test as gold standards, it was 98.4% sensitive and 100% specific and had positive and negative predictive values of 98.7% and 99.3%.     

Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay

The ColorPAC Giardia/Cryptosporidium (Becton Dickinson) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in human stool. Agreement between the Alexon-Trend ProSpecT Giardia Rapid EIA and the ColorPAC assay was 166 of 172 (96.5%). Agreement between the Alexon-Trend ProSpecT Cryptosporidium Rapid EIA and the ColorPAC assay was 169 of 171 (98.8%). No cross-reactions were seen with other parasites or human cells.     

Evaluation of Two Enzyme Immunoassays for Detection of Human Rotaviruses in Fecal Specimens

The two assays evaluated in this study (the Ridascreen rotavirus and the Pathfinder rotavirus) exhibited comparable sensitivities (100%) but highly divergent positive predictive values (93.74 and 57.7%, respectively) when compared on 393 specimens. This difference should be considered when using these tests on collectives with an unknown or low prevalence.     

Multiplex Assay Detection of Immunoglobulin G Antibodies That Recognize Giardia intestinalis and Cryptosporidium parvum Antigens

Giardiasis and cryptosporidiosis are common enteric parasitic diseases that have similar routes of transmission. In this work, we have identified epitopes within the Giardia variant-specific surface protein (VSP) sequences that are recognized by IgG antibodies from 13 of 14 (93%) sera from patients with stool-confirmed giardiasis. The conserved epitopes are shared among VSPs from both of the assemblages that commonly infect humans, and they are likely to be structural, as both sodium dodecyl sulfate treatment and dithiothreitol reduction decrease antibody recognition. In a multiplex bead assay (MBA), we used three VSP fragments from an assemblage A Giardia strain, three VSP fragments from assemblage B strains, and the α-1 giardin structural antigen to detect IgG antibodies to Giardia and used the recombinant 17- and 27-kDa antigens to simultaneously detect IgG antibodies to Cryptosporidium. The MBA differentiated between sera from Giardia and Cryptosporidium outbreaks and also identified a giardiasis outbreak that may have included cryptosporidiosis cases. Approximately 40% of cryptosporidiosis outbreak samples had high MBA responses for both the 27- and 17-kDa antigens, while <10% of nonoutbreak and giardiasis outbreak samples had high responses. At least 60% of giardiasis outbreak samples were positive for antibodies to multiple Giardia antigens, while ≤12% of nonoutbreak samples and samples from U.S. and British Columbia cryptosporidiosis outbreaks met our definition for Giardia seropositivity. A MBA using multiple parasite antigens may prove useful in the epidemiologic analysis of future waterborne or food-borne outbreaks of diarrheal disease.Giardia intestinalis (syn. Giardia lamblia and Giardia duodenalis) and Cryptosporidium spp. (e.g., Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium felis, and Cryptosporidium meleagridis) are enteric protozoan parasites with zoonotic potential that are commonly associated with diarrheal disease in humans (reviewed in reference 26). In the developing world where potential sources of fecal contamination are widespread, repeated and sometimes chronic infections occur at an early age (reviewed in reference 81). In the developed world, outbreaks are often associated with episodic events that result in the contamination of food, water, or recreational water with infectious organisms (15, 33; reviewed in reference 82). Because Giardia cysts and Cryptosporidium oocysts are resistant to commonly used disinfectants, such as chlorine, and have relatively low infectious doses (7, 25, 65), municipal water treatment failures in communities that draw from challenged raw water sources can result in widespread outbreaks of disease. The largest known community-wide, waterborne outbreak of cryptosporidiosis occurred in Milwaukee, WI, in 1993. Approximately 400,000 people (26% of residents) were symptomatic during the outbreak (42). A retrospective analysis of serum samples from Milwaukee children suggested that 37 to 70% of residents may actually have been infected (43). In addition to recognized outbreaks, low levels of community-acquired giardiasis and cryptosporidiosis have long been observed in the United States and Canada. Laboratory-based surveillance estimates (1999 to 2002) of the incidence of Giardia and Cryptosporidium infections in Calgary, Canada, were 19.6 and 6.0, respectively, per 100,000 residents per year (38). In the same general time frame, infection rates in the United States based upon case reports varied between 6.9 and 8.5 infections per 100,000 per year for Giardia and between 1.0 and 1.3 infections per 100,000 per year for Cryptosporidium (23, 24).Giardia and Cryptosporidium infection estimates based on case surveillance or the detection of organisms in stool are likely to significantly underestimate the actual values in a population, given that asymptomatic infection is documented, shedding of organisms by infected individuals can be intermittent and low level, and detection by microscopy can be challenging, especially in asymptomatic individuals (6, 14, 59, 64, 92). Several groups have shown that serologic IgG antibodies against parasite surface antigens can serve as a useful indicator of the levels of infection in a community (reviewed in references 12 and 17). Assays to detect antibodies to Cryptosporidium have focused on the 17- and 27-kDa antigens (reviewed in reference 79), two low-molecular-weight proteins that are associated with a detergent-extractable portion of the parasite membrane by way of posttranslational glycolipid or lipid modifications (71, 74, 76). Because C. parvum protein-based assays can be used to detect antibody responses among patients infected with non-C. parvum species, the immunodominant 17- and 27-kDa epitopes must be conserved between species (20, 73, 75, 86, 87). In previous work, we demonstrated that recombinant 17- and 27-kDa proteins, when used in the enzyme-linked immunosorbent assay (ELISA) format, detected IgG antibodies with good sensitivity and specificity relative to the “gold standard” Western blot assay in both nonoutbreak and outbreak populations (50, 70, 74).In contrast to the Cryptosporidium assays just described, most of the assays that detect antibodies to Giardia have used crude trophozoite or cyst antigens, and a sensitive and specific recombinant protein-based serologic assay has not yet been reported (12, 17). The immunodominant Giardia antigen is the variant-specific surface protein (VSP), a cysteine-rich (11 to 12% Cys) protein that covers the entire surface of the parasite (reviewed in reference 2). Although a trophozoite usually expresses only one VSP on its surface at a time, antigenic switching (perhaps using an RNA interference mechanism) occurs at a rate of one switch for every 6.5 to 13 generations (62, 77). Because of antigenic switching, the host immune system is exposed to many different VSP sequences during the course of an infection. The Giardia genome encodes a family of approximately 200 different VSPs, and the repertoire of genes found in the two main genotypes that infect humans (assemblages A and B) have been shown to be divergent (3, 18, 46, 47, 58, 61). Structurally, each VSP has a highly conserved, carboxy-terminal membrane anchor segment of 34 to 37 amino acids (part III), a moderately conserved segment of about 170 amino acids adjacent to the anchor (part II), and an amino-terminal region that varies greatly in both size and sequence (part I) (53). A diagram showing the positions and sizes of the three VSP regions for AS6 (CRP170) is shown in Fig. ​Fig.11 (4, 47). Because of differences in the hypervariable amino-terminal region, VSPs can range in size from approximately 20 kDa to 200 kDa. In addition to the highly conserved transmembrane region, VSPs contain CXXC, GGCY (indicated by an arrow in Fig. ​Fig.1),1), and Zn finger motifs as well as a CRGKY cytosolic tail that can be posttranslationally acylated on the cysteine with palmitate (3, 22, 69, 78, and reviewed in reference 55). VSP variation during infection and the antigenic divergence between the repertoires of the two Giardia assemblages may limit the effectiveness of the immune responses in animals and humans and may contribute to the frequent and often chronic nature of giardiasis (reviewed in reference 54).Open in a separate windowFIG. 1.Map showing the hypervariable (part I), semiconserved (part II), and highly conserved anchor (part III) regions of VSP AS6 (CRP170) (4, 47). The amino acid length of each region is indicated based on the conventions of Bienz et al. (8) and Muller et al. (53). The location of the 140-amino-acid VSP1 fragment targeted for amplification in this work is indicated beneath the map by a bracket. The location of the GGCY motif, 47 amino acids from the amino terminus of part II, is indicated by an arrow.Epitope mapping of the antibody responses that result from infection in a mouse model has shown that the VSP contains two antigenic regions (52, 53). The hypervariable amino terminus (part I) of VSP H7 stimulated a low-level antibody response early in infection, while the semiconserved region (part II) stimulated a more-pronounced antibody response later in infection. Some of the new VSPs that resulted from antigenic switching cross-reacted with antibodies to the VSP H7 semiconserved region, but none of new hypervariable regions were recognized by anti-H7 sera. Muller et al. (52, 53) were unable to demonstrate cross-reactivity between VSP H7 immune sera from the GS strain infection (assemblage B) and CRP170, a VSP from the WB (assemblage A) strain of Giardia (85). Fine-level mapping of VSP H7 by Bienz et al. (9) identified a 130-amino-acid sequence within the semiconserved part II region that was recognized by mouse immune serum. These results suggested that a serologic antibody assay using multiple VSP semiconserved regions from both assemblages might be possible.In this work, we used the multiplex bead assay (MBA) format to develop an assay that simultaneously detects specific human IgG antibodies to Cryptosporidium and Giardia surface antigens. Six VSP fragments, three from assemblage A and three from assemblage B, and the α-1 giardin (16, 67, 93) were used to detect antibodies to Giardia. In addition, the recombinant 17- and 27-kDa antigens were used to detect antibodies to Cryptosporidium. The assay provides new information about the serological response to two common waterborne protozoan parasites and offers a potential new tool for further study of the risks and prevalences of these infections at the community level.     

Evaluation of Four Enzyme Immunoassays for the Detection of Giardia lamblia Antigen in Stool Specimens

 Four enzyme immunoassays for the detection of Giardia lamblia antigen in stool specimens were evaluated: the ProSpecT Giardia Microplate Assay (Alexon, USA), the Giardia CELISA (Cellabs, Australia), the DSl-Giardia-ELISA (DSL, Germany), and the Melotest Giardiasis Ag (Melotec, Spain). Microscopic examination and enzyme immunoassays were performed on 168 stool specimens collected from 168 patients suspected to have giardiasis. All assays were easy to perform. The ProSpecT Giardia assay had the highest sensitivity of the assays evaluated (91%), and its interpretation was the easiest. The sensitivity of the three other assays ranged from 63 to 81%. The ProSpecT Giardia assay can be useful to detect Giardia lamblia and may replace microscopic examination in areas of high endemicity.     

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.     

Evaluation of an Immunochromatographic Dip-Strip Test for the Detection of Cryptosporidium Oocysts in Stool Specimens

The study presented here examined the efficacy of a commercially available qualitative immunochromatographic assay for detecting Cryptosporidium oocysts in stool samples. A total of 75 samples were tested, including 50 positive for Cryptosporidium spp. by acid-fast stain, 20 positive for other parasites (Blastocystis hominis, Endolimax nana, Entamoeba coli, Giardia lamblia, Ascaris lumbricoides, Strongyloides stercoralis and Trichuris trichiura), and five negative samples. The observed sensitivity was 98%, while specificity was 100%; the detection threshold was near 1,000 oocysts/ml. Correctly diagnosed positive samples included Cryptosporidium parvum genotypes 1 and 2, whereas the single false-negative sample corresponded to a Cryptosporidium meleagridis infection. Electronic Publication     

Site-Specific Clinical Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel for Detection of Infectious Gastroenteritis in Fecal Specimens

We evaluate the clinical performance of the Luminex xTAG gastrointestinal (GI) pathogen in vitro diagnostic (IVD) assay in a comparison between clinical and public health laboratories. The site reproducibility study showed 98.7% sensitivity with high positive and negative agreement values (96.2% and 99.8%, respectively), while assay performance against confirmatory methods resulted in 96.4% sensitivity with similar positive and negative agreement values (90.1% and 99.5%, respectively). High-throughput detection of multiple GI pathogens improved turnaround time, consolidated laboratory workflow, and simplified stool culture practices, thus reducing the overall cost and number of specimens processed.     

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid and Inexpensive Detection of Cytomegalovirus DNA in Vitreous Specimens from Suspected Cases of Viral Retinitis

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of cytomegalovirus (CMV) was developed and evaluated. The LAMP assay specifically amplified only CMV DNA, and no cross-reactivity with the DNA of herpes simplex virus type 1, varicella-zoster virus, adenovirus, Aspergillus flavus, or Staphylococcus aureus was observed. The sequences of the LAMP assay-positive CMV products were perfectly (100%) matched with the CMV sequence deposited in the GenBank database. The sensitivity of the LAMP assay was found to be 10 copies/μl of CMV DNA. Vitreous samples from 40 patients with suspected retinitis were subjected to LAMP and real-time PCR for the detection of CMV. Of 40 patients with suspected viral retinitis, 10 tested positive for CMV by the real-time PCR and LAMP assays. A 100% concordance was observed between the results of the two methods. The LAMP assay is a rapid, highly specific, and sensitive method for the diagnosis of retinitis caused by CMV.Viral retinitis is commonly caused by herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), and occasionally, Epstein-Barr virus (EBV) (7). In patients with atypical features and in the early stages of ocular manifestations, clinical differentiation between cases of retinitis associated with CMV and other herpesvirus infections is often difficult (6). The differentiation of CMV retinitis from HSV and VZV retinitis is very important early in the course of the disease, as the therapeutic agent to be used for treatment differs from virus to virus (7). Conventional methods for the diagnosis of viral retinitis include the detection of viral antigen and virus isolation from intraocular specimens (2). These tests have been shown to have low sensitivities for the detection of viruses and are not currently recommended for use for the diagnosis of viral retinitis (2).PCR has proved to be of great utility for the diagnosis of viral retinitis (3, 4, 5, 6, 8). However, owing to the expensive systems required, PCR is still not a very common diagnostic test. Notomi et al. have reported on a novel nucleic acid amplification assay termed the loop-mediated isothermal amplification (LAMP) assay (10). The assay amplifies the DNA under isothermal conditions (63 to 65°C) with high degrees of specificity, efficiency, and speed. The assay can be conducted in the laboratory in a water bath or heating block (10). Thus, the thermal cycling needs of a PCR are avoided. The assay can be used for the rapid detection of pathogens in peripheral health care settings in developing countries. The present study describes the development and evaluation of a simple and cost-effective LAMP assay for the rapid detection of CMV DNA in patients with viral retinitis.     

Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.     

Rapid Detection of Staphylococcus aureus Panton-Valentine Leukocidin in Clinical Specimens by Enzyme-Linked Immunosorbent Assay and Immunochromatographic Tests

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus (lukS-PV and lukF-PV) and the mecA gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL⁺); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL⁺ strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL⁺ strains ranged from 0 to 399 μg/ml by ELISA. By the use of 0.015 μg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.Staphylococcus aureus is an important human pathogen whose pathogenicity largely depends on extracellular virulence factors. One of these exoproteins, Panton-Valentine leukocidin (PVL), is produced by several community-acquired methicillin-resistant S. aureus and methicillin-sensitive S. aureus (CA-MRSA and CA-MSSA, respectively) clones currently spreading throughout the world (27).Historical publications show that PVL targets cells of the human immune system, such as polymorphonuclear neutrophils (PMNs), monocytes, and macrophages (26). In vitro, PVL forms pores on human and rabbit PMNs and monocytes that cause cytokine release and cell death by apoptosis or necrosis (13, 18). In rabbit models, PVL provokes a dose-dependent skin necrosis (7, 30); bacterial persistence in bone; and a rapid local extension of osteomyelitis (6), severe lung necrosis, pulmonary edema, alveolar hemorrhage, and death (10).Isolates of S. aureus harboring PVL genes have been epidemiologically linked to specific human S. aureus manifestations: not only to primary skin and soft tissue disease but also to severe necrotizing pneumonia and severe bone and joint infections (4, 11, 14, 19, 21, 24). In certain countries, such as the United Kingdom and France, PVL is now detected in clinical practice, and treatment regimens may be adjusted on the basis of the presence or the absence of PVL (12, 15, 17). The adjunctive use of antibiotics that suppress toxin production, such as clindamycin, linezolid, and rifampin, and intravenous immunoglobulin is advocated for the treatment of severe and invasive infections caused by PVL-producing strains.To date, the diagnosis of infection due to PVL-producing strains is mainly performed by PCR of colonies for the detection of PVL genes, while the latex agglutination assay and matrix-assisted laser desorption ionization-time of flight mass spectrometry method should be performed with colonies (3, 19, 23). However, the results of these methods of PVL detection may not correlate with in vivo production.In a previous study, we have shown that PVL can be detected in pus from a skin abscess caused mainly by PVL-producing methicillin-susceptible S. aureus (1). The objective of this study was to determine whether the PVL produced in clinical specimens from PVL gene-positive S. aureus infections could be detected by a specific enzyme-linked immunosorbent assay (ELISA) or immunochromatographic test (ICT), regardless of the genetic background and methicillin susceptibility of the PVL-producing strains.(This work was presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 12 to 15 September 2009.)     

Evaluation of Two Rapid Assays for Detection of Clostridium difficile Toxin A in Stool Specimens

Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.