Identification of clinical isolates of anaerobic bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for the rapid identification of anaerobic bacteria that had been isolated from clinical specimens and previously identified by 16s rRNA sequencing. The Bruker Microflex MALDI-TOF instrument with the Biotyper Software was used. We tested 152 isolates of anaerobic bacteria from 24 different genera and 75 different species. A total of 125 isolates (82%) had Biotyper software scores greater than 2.0 and the correct identification to genus and species was made by MALDI-TOF for 120 (79%) of isolates. Of the 12 isolates with a score between 1.8 and 2.0, 2 (17%) organisms were incorrectly identified by MALDI-TOF. Only 15 (10%) isolates had a score less than 1.8 and MALDI-TOF gave the wrong genus and species for four isolates, the correct genus for two isolates, and the correct genus and species for nine isolates. Therefore, we found the Bruker MALDI-TOF MicroFlex LT with an expanded database and the use of bacteria extracts rather than whole organisms correctly identified 130 of 152 (86%) isolates to genus and species when the cut-off for an acceptable identification was a spectrum score ≥1.8.

Multidrug-resistant Corynebacterium striatum pneumonia in a heart transplant recipient.

Corynebacterium striatum is a rare, but likely underreported, cause of serious infections in immunocompromised hosts and generally is susceptible to multiple classes of antimicrobial agents. Here we report the first case of C. striatum infection in a solid organ transplant recipient. Three years after heart transplantation, a 58-year-old man developed bilateral pneumonia and pulmonary embolism. He did not improve with levofloxacin, piperacillin/tazobactam, and heparin treatment. A homogeneous population of abundant gram-positive rods was repeatedly demonstrated in sputum and bronchoalveolar lavage fluid, and C. striatum was grown in pure culture. The isolate was unusual for its multidrug-resistant (MDR) antimicrobial susceptibility pattern. The pneumonia resolved with 4 weeks of vancomycin therapy, in combination with rifampin given only during the first 2 weeks of treatment. The isolation of coryneforms ("diphtheroids") is often attributed to contamination. Their abundant presence on direct examination of specimens and/or their growth in pure culture suggest a pathogenic role, however, and indicate the need for accurate microbiological identification, particularly in immunocompromised hosts who have been hospitalized and previously treated with antibiotics. Combination therapy that includes vancomycin may be the most prudent treatment for MDR C. striatum infections.

Rapid detection of Clostridium difficile in stool using the VIDASR C. difficile Toxin A II assay.

A rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is important in patient management and in the administration of appropriate therapeutic modalities. The VIDAS(R) C. difficile Toxin A II (CDA 2) assay (bioMerieux, Inc., Hazelwood, MO) was compared with the cell culture cytotoxicity assay (CCA) for the rapid detection of C. difficile in stool from patients in whom C. difficile infection was suspected. Thirty-eight consecutively collected CCA-positive stool specimens, and 33 CCA-negative stool specimens were tested by the CDA 2 assay. Where appropriate, discordant specimens were repeated and/or tested by isolation utilizing cycloserine-cefoxitin-fructose agar (CCFA). Among 12 discordant stool specimens, 7 were VIDAS(R)-/cytotoxicity+, 2 were VIDAS(R) equivocal (E)/cytotoxicity+, 2 were VIDAS(R) E/cytotoxicity-, and 1 was VIDAS(R)+/cytotoxicity-. One VIDAS(R) E/cytotoxicity+ lacked sufficient stool to be repeated. From the single VIDAS(R)+/cytotoxicity- specimen, C. sordelli was isolated. Specimens that were equivocal by VIDAS(R), were omitted from incorporation into this study's test parameters. The sensitivity, specificity, positive and negative predictive values for the CDA 2 assay were 80.6, 96.8, 96.7, and 81.1%, respectively. The specimens which yielded false negative VIDAS(R) results had low levels of toxin based on endpoint titrations using the cytotoxicity assay. Although the CDA 2 assay displayed a reduced sensitivity compared with the CCA, the automated assay is rapid (results promulgated within 2 h), with computer generated readings obviating visual interpretations. Recognition of the CDA 2 assay's limitations is important to addressing this test's clinical utility.

Speciation of human microsporidia by polymerase chain reaction single-strand conformation polymorphism.

We describe the application of single-strand conformation polymorphism (SSCP) analysis to the speciation of human microsporidia after polymerase chain reaction (PCR) amplification with the panmicrosporidian primers PMP1 and PMP2. We compared the DNA extracted and amplified from different genotypes or isolates of Enterocytozoon bieneusi, Encephalitozoon cuniculi, E. hellem, and E. intestinalis plus an isolate of Vittaforma corneae. The PCR-SSCP, when performed at 20 degrees C, generated 2 bands in distinctive, reproducible patterns in polyacrylamide gels for each species of microsporidia tested, regardless of genotype or isolate. We found PCR-SSCP to be an easy and reproducible method for speciation of human microsporidia when the primer pair PMP1 and PMP2 is used.

Resistance to multiple fluoroquinolones in a clinical isolate of Streptococcus pyogenes: identification of gyrA and parC and specification of point mutations associated with resistance.

A strain of Streptococcus pyogenes resistant to multiple fluoroquinolones was isolated from the blood of an immunocompromised patient. Resistance to fluoroquinolones in S. pyogenes has not been previously studied. Compared to 10 sensitive strains of S. pyogenes, the fluoroquinolone-resistant clinical isolate of S. pyogenes presented point mutations in gyrA, predicting that serine-81 was changed to phenylalanine and that methionine-99 was changed to leucine, and in parC, predicting that serine-79 was changed to tyrosine. The mechanism of fluoroquinolone resistance in this isolate of S. pyogenes appears to be analogous to previously reported mechanisms for Streptococcus pneumoniae.

Performance of three enzyme immunoassays and two direct fluorescence assays for detection of Giardia lamblia in stool specimens preserved in ECOFIX.

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the "gold standard" for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.

Performance of para-Pak Ultra ECOFIX compared with Para-Pak Ultra formalin/mercuric chloride-based polyvinyl alcohol for concentration and permanent stained smears of stool parasites.

ECOFIX is a mercury and formalin-free fecal preservative that can be used for concentration of stool specimens and preparation of permanently-stained slides. In this study, the standard two-vial ParaPak Ultra system was compared with ECOFIX Ultra for the detection of intestinal parasites. A total of 261 specimens in 92 sets (77 with 3 specimens, 15 with 2 specimens) were collected in ECOFIX, formalin, and low viscosity polyvinyl alcohol (LV-PVA). Concentrations were performed from ECOFIX using Hemo-De and saline and from formalin using ethyl acetate and formalin. To prepare permanently-stained smears, ECOSTAIN (a modification of Wheatley's trichrome stain) was used on ECOFIX material and Wheatley's trichrome stain was used on specimens preserved in PVA. A total of 157 protozoa and helminths were detected; 132 (84.1%) were recovered in formalin/PVA and 129 (82.2%) in ECOFIX. In permanently-stained smears, 139 protozoa were observed, 116 (83.5%) in PVA-preserved material and 117 (84.2%) in ECOFIX. Fecal concentration yielded 111 parasites (103 protozoa and 8 helminths), of which 98 (88.3%) were detected in formalin-fixed stool and 48 (43.2%) in ECOFIX. Significantly fewer ECOFIX-preserved concentrates were positive for Blastocystis hominis (35 versus 15, p-value <0.001) and Endolimax nana (19 versus 2, p-value <0.001). In conclusion, use of the ECOFIX Ultra collection device in combination with ECOSTAIN resulted in largely comparable recovery of enteric parasites to the conventional two-vial ParaPak Ultra system when both sedimentation-concentration and permanently stained smears were performed, and 2-3 specimens per patient were evaluated.

Persistent diarrhea signals a critical period of increased diarrhea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil.

Persistent diarrhea (PD; duration >/=14 days) is a growing part of the global burden of diarrheal diseases. A 45-month prospective cohort study (with illness, nutritional, and microbiologic surveillance) was conducted in a shantytown in northeastern Brazil, to elucidate the epidemiology, nutritional impact, and causes of PD in early childhood (0-3 years of age). A nested case-control design was used to examine children's diarrhea burden and nutritional status before and after a first PD illness. PD illnesses accounted for 8% of episodes and 34% of days of diarrhea. First PD illnesses were preceded by a doubling of acute diarrhea burdens, were followed by further 2.6-3.5-fold increased diarrhea burdens for 18 months, and were associated with acute weight shortfalls. Exclusively breast-fed children had 8-fold lower diarrhea rates than did weaned children. PD-associated etiologic agents included Cryptosporidium, Giardia, enteric adenoviruses, and enterotoxigenic Escherichia coli. PD signals growth shortfalls and increased diarrhea burdens; children with PD merit extended support, and the illness warrants further study to elucidate its prevention, treatment, and impact.

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.

Blinded, externally controlled multicenter evaluation of light microscopy and PCR for detection of microsporidia in stool specimens. The Diagnostic Multicenter Study Group on Microsporidia.

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.

Mucosal injury and disruption of intestinal barrier function in HIV-infected individuals with and without diarrhea and cryptosporidiosis in northeast Brazil.

OBJECTIVES: To determine the relative effects of AIDS-related diarrhea with or without cryptosporidiosis and microsporidiosis on intestinal function and injury. METHODS: We studied 40 HIV-infected patients (20 with and 20 without diarrhea) and 13 healthy volunteers, using the differential urinary excretion of ingested lactulose and mannitol as respective markers of barrier disruption and overall villous surface area. We also examined them for fecal leukocytes, lactoferrin, and alpha 1-antitrypsin. Fasting subjects drank test solution containing lactulose (5 g) and mannitol (1 g). Urine was collected for 5 h and tested for sugars by high-performance liquid chromatography with pulsed amperometric detection. RESULTS: HIV-positive patients with diarrhea had a 2.8-fold higher lactulose:mannitol excretion ratio (L:M) than HIV-positive patients without diarrhea (p = 0.01) and 10.4-fold higher than healthy volunteers (p = 0.004). This was accounted for by a 1.5- to 3.1-fold higher rate of lactulose excretion by HIV patients with diarrhea than by those without diarrhea or by healthy volunteers. Mannitol excretion was 32-55% less in patients with diarrhea than in those without diarrhea or in healthy volunteers. Patients with cryptosporidial diarrhea had a nearly 6-fold higher L:M ratio than those without diarrhea (p < 0.001) and nearly 3-fold higher than those with non-cryptosporidial diarrhea (p = 0.02). One patient with microsporidial infection had a nearly 3-fold higher L:M ratio than controls without diarrhea. Alpha 1-Antitrypsin was positive in 40% of HIV-positive patients with cryptosporidial infections and none of 12 HIV-positive patients with non-cryptosporidial diarrhea. Fecal lactoferrin or leukocytes were increased in all HIV patients with diarrhea. CONCLUSION: HIV infection is associated with intestinal dysfunction and injury, even in patients who do not have diarrhea. However, those with diarrhea, especially with cryptosporidiosis or microsporidiosis, have even greater disruption of intestinal barrier function with potentially important nutritional consequences.

PIP: The effects of AIDS-related diarrhea--with and without cryptosporidiosis and microsporidiosis--on intestinal function and injury were studied in 40 AIDS patients and 13 healthy volunteers from Fortaleza, Brazil. The differential urinary excretion of ingested lactulose and mannitol was used as a marker of barrier disruption and overall villous surface area. HIV-infected patients with diarrhea had a 2.8-fold higher lactulose to mannitol excretion ratio than HIV-positive patients without diarrhea and a 10.4-fold higher ratio than healthy volunteers. Moreover, those with crypotosporidial infection had a lactulose to mannitol ratio almost 6-fold greater than those without diarrhea and nearly 3-fold higher than those with non-cryptosporidial diarrhea. This effect involved both decreased mannitol excretion (decreased intestinal absorptive area) and increased lactulose excretion (mucosal barrier disruption). The single patient with microsporidial infection had a nearly 3-fold higher ratio than healthy volunteers. Alpha1-antitrypsin tests were positive in two of five (40%) HIV-positive patients with cryptosporidial infections compared with none of 12 HIV-infected patients with non-cryptosporidial diarrhea. These findings confirm that HIV infection is associated with profound intestinal dysfunction and injury, even in those without diarrhea. Disruption of the intestinal barrier is even greater, however, in HIV-infected patients with cryptosporidial diarrhea, with potential nutritional consequences.

Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile.

The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA). Fifty stored isolates of C. difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C. difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive. Other Clostridium species that were PRO Disc positive could be differentiated from C. difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining.

Evaluation of two commercial microtiter cytotoxin assays for the detection of Clostridium difficile toxin B in stool specimens.

Two commercial microtiter cytotoxin assays using a fibroblast cell line (Bartels, Baxter Diagnostics, Inc., Deerfield, IL) and an epithelial cell line (Cytotoxi Test, Advanced Clinical Diagnostics, Toledo, OH) were evaluated for their ability to detect Clostridium difficile toxin B in stool specimens. After 48 hours, the assays had comparable sensitivity (90 versus 92%) and specificity (99 versus 98%). Although not statistically significant, the Bartels assay detected more toxin-positive specimens at 24 hours (84 versus 72%, P = 0.089, Fisher's Exact Test) and the Cytotoxi assay had fewer nonspecific reactions requiring repeat testing (2.2 versus 1.1%, P = 0.186, Fisher's Exact Test). Both cytotoxin assays had comparable analytic performance.

Application of molecular techniques to the diagnosis of microsporidial infection.

Microsporidia are now recognized as important pathogens of AIDS patients; the ability of these parasites to cause disease in immunocompetent persons is still being elucidated. Improved diagnostic tests for microsporidial infection are continually being sought for establishing diagnosis in order to avoid laborious electron microscopy studies that require invasively acquired biopsy specimens. Modified trichrome or chemofluorescent stains are useful for detecting microsporidia in bodily fluids and stool specimens, but they do not allow for speciation of microsporidia. Polymerase chain reaction with specific primers will allow the detection and speciation of microsporidia in biopsy tissue, bodily fluids, and stool specimens.

Evaluation of a rapid enzyme immunoassay system for serologic diagnosis of Mycoplasma pneumoniae infection.

A 5-min qualitative membrane enzyme-linked immunoassay (EIA) from Remel (Mycoplasma pneumoniae immunoglobulin G (IgG)/IgM Antibody Test System) was evaluated for its ability to detect IgM and IgG at levels indicating active or recent infection. Specimens from 131 patients were evaluated using an immunofluorescent antibody assay (IFA) to determine IgG and IgM titers and the membrane EIA. An enzyme-linked immunosorbent assay (ELISA) performed by a reference laboratory was used for discrepancy resolution. There were 34 IgM positive specimens (titer > or = 1:16), 19 IgG positive specimens (titer > or = 1:64), and 78 negative specimens. Compared with IFA and/or ELISA, the membrane EIA was 97% sensitive for the detection of IgM and 79% sensitive for the detection of IgG. Of the 78 specimens called negative, 17 specimens had IgG titers< or = 1:32) or an ELISA result indicating prior exposure, and the membrane EIA called seven of 17 (41%) positive. For the detection of both IgG and IgM, the membrane EIA had a sensitivity of 91%, specificity of 91%, and positive and negative predictive values of 87 and 93%, respectively. The Remel membrane EIA is a rapid and reliable assay for the diagnosis of active or recent M. pneumoniae respiratory tract infections.

Identification of microsporidia in stool specimens by using PCR and restriction endonucleases.

We report the development of a PCR-based assay for the detection of microsporidia in clinical specimens. A single primer pair complementary to conserved sequences of the small-subunit rRNA enabled amplification of DNA from the four major microsporidian pathogens of humans: Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, and Septata intestinalis. The extraction method allowed PCR amplification of E. bieneusi and S. intestinalis DNA from sodium hypochlorite-treated stool specimens. Differentiation of the microsporidian gastrointestinal pathogens E. bieneusi and S. intestinalis could be accomplished by restriction endonuclease digestion of PCR products using PstI and HaeIII.

Laboratory diagnosis of bacterial meningitis.

Bacterial meningitis is relatively common, can progress rapidly, and can result in death or permanent debilitation. This infection justifiably elicits strong emotional reactions and, hopefully, immediate medical intervention. This review is a brief presentation of the pathogenesis of bacterial meningitis and a review of current knowledge, literature, and recommendations on the subject of laboratory diagnosis of bacterial meningitis. Those who work in clinical microbiology laboratories should be familiar with the tests used in detecting bacteria and bacterial antigens in cerebrospinal fluid (CSF) and should always have the utmost appreciation for the fact that results of such tests must always be reported immediately. Academic and practical aspects of the laboratory diagnosis of bacterial meningitis presented in this review include the following: anatomy of the meninges; pathogenesis; changes in the composition of CSF; etiological agents; processing CSF; microscopic examination of CSF; culturing CSF; methods of detecting bacterial antigens and bacterial components in CSF (counter-immunoelectrophoresis, coagglutination, latex agglutination, enzyme-linked immunosorbent assay, Limulus amebocyte lysate assay, and gas-liquid chromatography); use of the polymerase chain reaction; and practical considerations for testing CSF for bacterial antigens.

Effect of treatment of shell vial cell cultures with dimethyl sulfoxide and dexamethasone and age of MRC-5 monolayers for detection of cytomegalovirus.

Pretreatment of MRC-5 cell monolayers in commercially prepared shell vials with 1% dimethyl sulfoxide (DMSO) and 10(-5) mol/L dexamethasone (DEX) was evaluated. Preliminary experiments indicated enhanced infectivity of AD-169 for pretreated MRC-5 cells in shell vials of ages 9 and 16 days. Compared with untreated shell vials, DMSO-DEX increased positivity (day 9, 19 vs. 26 shell vials, p less than 0.03; day 16, 13 vs. 29 shell vials, p less than 0.001) and increased the mean number of fluorescent foci (days 9 and 16, p less than 0.001). Pretreatment of 8-15-day-old monolayers was evaluated clinically using 146 urine specimens. Fifty specimens were positive for cytomegalovirus (CMV) in both treated and untreated shell vials with ten positive in untreated only and three positive in treated only (p = NS). The median number of fluorescent foci was not significantly higher in treated shell vials. Increased toxicity of MRC-5 cells was observed in treated monolayers (p less than 0.0001). Pretreatment with DMSO-DEX did not enhance CMV isolation from clinical specimens and can be toxic to MRC-5 monolayers.

Increased efficiency of stool culture for the detection of Salmonella and Shigella.

The Wampole Bactigen Salmonella-Shigella Latex Agglutination Test (SSLA) (Wampole Laboratories, Cranbury, New Jersey) was evaluated as a possible substitute for blind subculture of selenite broths from stool cultures. Recovery rates of Salmonella and Shigella from eosin-methylene blue (EMB) agar were reviewed to determine if this medium could be eliminated from primary stool culture. Salmonella was detected in 17 of 822 stools by both SSLA and culture. There were 52 false-positive SSLA for Salmonella (sensitivity 100%, specificity 93%). Of three Shigella isolated on culture, one was SSLA positive, one was SSLA negative, and one was negative by both SSLA and subculture of selenite broth. There were eight false-positive SSLA for Shigella (specificity 99%). Of 50 Salmonella and 11 Shigella isolated from 6200 stools in 1.5 years, two Shigella were isolated on EMB only. The SSLA test is a useful screening test for Salmonella. By eliminating unnecessary subcultures of selenite broth, it reduces turnaround time by 24 hr for negative stool cultures. The combination of primary culture with SSLA screening of enrichment broth should be adequate for the detection of Salmonella and Shigella from stool specimens. Our data suggest that EMB or other differential medium should be retained for primary culture to enhance detection of Shigella.

Effect of age of shell vial monolayers on detection of cytomegalovirus from urine specimens.

The effect of age of MRC-5 cell monolayers in shell vials on the detection of cytomegalovirus (CMV) from urine was evaluated. When the AD169 strain of CMV was used, 8-day-old monolayers had a higher mean count of fluorescent foci than 15-day-old monolayers did (5.78 versus 2.86) (P less than 0.02) and were more frequently positive (21 of 23 shell vials versus 14 of 22 shell vials) (P less than 0.04). Commercial shell vials used for clinical specimens were evaluated in groups of 8- to 11-, 12- to 15-, and 8- to 15-day-old monolayers. When compared with laboratory-prepared shell vials ranging in age from 3 to 9 days, commercial shell vials had a lower number of fluorescent foci in all groups (P less than 0.03, P less than 0.0001, and P less than 0.0001, respectively), the 12- to 15- and 8- to 15-day-old groups were less frequently positive (P less than or equal to 0.02 and P less than 0.02, respectively), and all three groups were more susceptible to the toxic effects of urine (P less than 0.0001, P less than 0.01, and P less than 0.0001, respectively). For all 191 specimens cultured (8- to 15-day-old group), one or both monolayers were destroyed in 60 (31.4%) specimens compared with 9 (4.7%) specimens toxic to laboratory-prepared shell vials (P less than 0.0001). Both the decreased sensitivity of older MRC-5 cells and the increased sensitivity to the toxic effects of urine made commercial shell vial less sensitive than laboratory-prepared shell vials for the detection of CMV.