Comparison of primary rhesus and cynomolgus monkey kidney cell cultures for viral isolation from clinical specimens.

Rhesus monkey kidney and cynomolgus monkey kidney cell cultures were compared for viral isolation by using clinical specimens that yielded 203 viral isolates. Cynomolgus and rhesus monkey kidney cells were comparable for the isolation of 22 adenoviruses, 12 coxsackieviruses, and one poliovirus. Four of 50 echoviruses and seven of ten herpesviruses were detected only in cynomolgus monkey kidney cells. Influenza virus was isolated in 84 instances, of which eight were detected only in rhesus and four only in cynomolgus monkey kidney cells. Rhesus monkey kidney cells yielded six more parainfluenza virus isolates. Except possibly for parainfluenza virus, cynomolgus monkey kidney cells appear to be as sensitive as rhesus monkey kidney cells for viral isolation from clinical specimens.     

Rapid detection of herpes simplex virus in clinical specimens with human embryonic lung fibroblast and primary rabbit kidney cell cultures.

The performance of a culture system for isolation of herpes simplex virus, consisting of one tube each of human embryonic lung fibroblasts and primary rabbit kidney cells, was evaluated. Cultures were incubated at 37 degrees C on a roller drum and observed daily for characteristic cytopathic effect for 5 days. During 1982, a positive isolation rate of 28.1% was seen among 3,154 specimens submitted. Cultures from genital sources were positive more frequently from males (43.8%) than from females (25.5%). Oral lesion cultures were positive as often from males (34.6%) as from females (38.4%). Although detection of herpes simplex virus occurred significantly earlier in rabbit kidney cells on days 1 and 2 of incubation, by day 3 the number of positive cultures was nearly the same in both cell types. By day 4 of incubation, 99.5% of the positive cultures were detected. These results demonstrate that cell culture can be a rapid and sensitive method for detecting herpes simplex virus.     

The value of including broth cultures as part of a routine culture protocol.

Three hundred seventeen clinical specimens from both superficially and deeply infected sites were prospectively examined to assess the true value of including liquid media as part of the routine culture procedure. All broth cultures were subcultured after overnight incubation onto plate media. The isolates obtained from the broth cultures were then compared with the isolates obtained on primary solid media. The isolates obtained from the broth cultures only were evaluated for clinical relevance by review of the patients' records. Twenty-two clinically relevant isolates were obtained from the broth cultures only, but the isolation of these additional organisms altered patient management for only two patients. It would appear from these results that the additional expense and time involved in culturing clinical specimens in fluid media is unwarranted.     

Failure of charcoal-horse-blood broth with cephalexin to significantly increase rate of Bordetella isolation from clinical specimens.

In a field trial conducted in cooperation with pediatricians in private practice, 198 nasopharyngeal swabs from children with suspected whooping cough were placed into charcoal-horse-blood transport medium with cephalexin (40 mg/liter). After preincubation at 36 degrees C for 1 to 2 days, the transport systems were mailed to the laboratory. There, the swabs were plated onto charcoal-horse-blood agar with cephalexin and were subsequently incubated for 48 h in cephalexin-containing charcoal-horse-blood broth which was then subcultured onto the agar. Forty-six Bordetella pertussis strains and seven Bordetella parapertussis strains were isolated (Bordetella isolation rate, 26.8%). Only three (5.7%) of the 53 Bordetella strains were detected exclusively by use of the broth. This low rate of additional isolates is probably explained by the fact that the swabs had been submitted in charcoal-horse-blood transport medium which itself acts as an enrichment medium.     

Characterization of Neisseria elongata subsp. glycolytica isolates obtained from human wound specimens and blood cultures.

Four slightly yellow-pigmented, alpha-hemolytic, gram-negative coccobacilli, three from wound specimens and one from multiple blood cultures of a patient with endocarditis, were identified as Neisseria elongata subsp. glycolytica on the basis of their overall biochemical and genetic similarities to this subspecies. These strains resembled N. elongata in their guanine-plus-cytosine contents (55.6 to 57.1 mol%) and in their overall cellular fatty acid profiles, which are characterized by large amounts of 16:0, 16:1 omega 7c, and 18:1 omega 7c fatty acids. Their identities were confirmed by species-level DNA relatedness (hydroxyapatite method) to the type strains of all three N. elongata subspecies. The biochemical profiles and cultural characteristics of these strains resembled those of the type strain of N. elongata subsp. glycolytica except for the production of a weak yellow growth pigment and alpha-hemolysis on sheep blood agar. They differed from N elongata subsp. elongata by the production of catalase, by the production of alpha-hemolysis on sheep blood agar, and by acid production from D-glucose. They differed from N. elongata subsp. nitroreducens by the production of catalase and an inability to reduce nitrate. These studies suggest a pathogenic potential for N. elongata subsp. glycolytica, usually considered to be a transient colonizer in humans.     

Characterization and identification of 95 diphtheroid (group JK) cultures isolated from clinical specimens.

Ninety-five cultures of group JK bacteria isolated from clinical specimens were characterized morphologically and biochemically. The microorganisms were isolated primarily from blood cultures. The bacterial cultures produced positive reactions when tested for catalase, Tween hydrolysis, and carbohydrate fermentation. Glucose and galactose were fermented by more than 90% of the organisms. Gas-liquid chromatography of trimethylsilyl derivatives of whole-cell hydrolysates of some of the group JK cultures yielded nearly identical elution profiles. The group JK microorganisms were susceptible to vancomycin but were resistant to most of the other 17 antimicrobial agents tested. A method is presented for differentiating the group JK microorganisms from other similar bacteria encountered in clinical specimens. Although these bacteria rarely occur in clinical specimens, they are capable of producing fatal infections (endocarditis and sepsis) in humans.     

Correlation of growth of aerobic blood cultures in hypertonic broth with antibiotic therapy.

The aim of this study was to elucidate the mechanisms by which sucrose improves growth in a hypertonic medium for isolating aerobes from blood. Clinical blood cultures were made routinely in duplicate in plain broth consisting of brain heart infusion broth with sodium polyanetholesulfonate, gelatin, and penicillinase and the same broth with 20% sucrose added. The growth patterns of Staphylococcus aureus and Enterobacteriaceae from plain and from hypertonic broth were correlated with the presence or absence of antimicrobial therapy in patients when the blood cultures were collected. In S. aureus bacteremias, 58.7% of the positive cultures collected during treatment of patients with beta-lactam antibiotics showed earlier growth or growth only in hypertonic broth, compared with 16.7% of the cultures taken during treatment with other antimicrobial agents (P less than 0.05) and 17.6% of the cultures made in antibiotic-free intervals (P less than 0.01). In the group of cultures yielding growth of Enterobacteriaceae, growth occurred earlier or solely in hypertonic broth in 28.9% of the cultures taken during treatment with beta-lactam antibiotics, compared with 15.7% of the cultures taken during treatment with other antimicrobial agents and 21.6% of the cultures collected in antibiotic-free intervals (differences not statistically significant). It is concluded that treatment with beta-lactam antibiotics is an important reason for the improved growth of S. aureus from hypertonic broth, but other factors are also involved.     

An experimental comparison of Thiol broth with Brewer''s thioglycollate for anaerobic blood cultures.

In a series of simulated blood culture experiments, small inocula of eight different strains of Bacteroides and five strains of anaerobic cocci were added to Difco Thiol broth and Southern Group Brewer's thioglycollate. Both methods enabled all of the strains to be isolated after one to three days' incubation, with the exception of Bacteroides melaninogenicus, and most strains to survive after one week. B. melaninogenicus grew more quickly in Difco Thiol broth than in Southern Group Brewer's whereas three strains of anaerobic cocci were isolated first from Southern Group Brewer's. Difco Thiol broth appears to be a satisfactory alternative to Southern Group Brewer's for the isolation of non-sporing anaerobes likely to be found in the blood.     

Comparison of rectal swabs and stool cultures in detecting Campylobacter fetus subsp. jejuni.

Rectal swabs and stool specimens were compared for the detection of Campylobacter fetus subsp. jejuni in marmosets. Rectal swabs were superior to stool specimens for detection of Campylobacter fetus subsp. jejuni (P = 0.016). Preliminary human data are also presented.     

Comparison of HeLa 229 and McCoy cell cultures for detection of Chlamydia trachomatis in clinical specimens.

Consecutive clinical specimens of Chlamydia trachomatis (1,048) were inoculated in parallel on DEAE-dextran- and cycloheximide-treated HeLa 229 cells and cycloheximide-treated McCoy cells. HeLa 229 cell culture detected 113 positive specimens, and McCoy cell culture detected 103 positive specimens. This difference is not significant. However, HeLa 229 cell culture yielded significantly more inclusions than McCoy cell culture in the 95 specimens positive in both cell types (P = 0.042). For routine diagnostic purposes, a choice for one of the cell types may be determined by local preferences.     

Enhanced primary isolation of Legionella pneumophila from clinical specimens by low-pH treatment.

During the 12-month study period, 919 clinical specimens were submitted to the laboratory for culture of Legionella pneumophila. All specimens were plated onto nonselective and semiselective media both directly and after treatment with a KCl-HCl solution, pH 2.2. Of the 51 specimens (5.5%) positive for L. pneumophila and Legionella-like organisms, 20 were recovered by acid pretreatment only, 5 were recovered by direct plating only, and 26 were recovered by both acid pretreatment and direct inoculation. These data demonstrated that acid pretreatment substantially improved the recovery of L. pneumophila and Legionella-like organisms from the clinical specimens examined.     

Comparison of throat swabs with sputum specimens for the detection of Chlamydia pneumoniae antigen by direct immunofluorescence.

AIM: To compare throat swabs with sputum specimens for Chlamydia pneumoniae antigen detection. METHODS: During a one year period, sputum and throat swabs from 50 patients over 15 years of age with acute or persisting lower respiratory tract infection were examined for C pneumoniae antigen by direct immunofluorescence. RESULTS: C pneumoniae antigen was detected in 18/50 patients (36.0%) from sputum, throat swab, or both. Paired sputum and throat swabs were received from 35/50 patients (70.0%). C pneumoniae antigen was detected in either or both specimens from 14/35 patients (40.0%). Of the 14 positive patients, both specimens were positive in nine (64.3%), throat swab only in four (28.6%), and sputum only in one (7.1%). Of the remaining 15 patients from whom only a single specimen was sent, a further three of eight throat swabs and one of seven sputum specimens were positive. There was no statistically significant difference between the results obtained from the two types of specimen. CONCLUSIONS: Throat swabs may be as good as sputum for the detection of C pneumoniae antigen.     

Evaluation of Abbott TESTPACK ROTAVIRUS with clinical specimens.

TESTPACK ROTAVIRUS was compared with the Pathfinder Rotavirus assay for the detection of rotaviral antigens in feces from 137 patients. The symptomatology and ages of the patients varied from no symptoms to watery diarrhea and from less than 3 months to greater than 60 years of age. The sensitivity and specificity, before resolution, were 89.2 and 90.0%, respectively. After resolution with a blocking reagent, the results were 100% sensitivity and 98.9% specificity. The performance was similar for all age groups and in both symptomatic and asymptomatic patients. Abbott TESTPACK ROTAVIRUS is a rapid (10-min), easy-to-perform assay for the detection of rotaviral antigens and has the sensitivity and specificity of enzyme-linked immunosorbent assays that require several hours to perform.     

Glucose-nonfermenting Gram-negative bacilli associated with clinical veterinary specimens.

Glucose-nonfermenting gram-negative bacilli (NFB) have been recognized recently as opportunistic pathogens of humans. With few exceptions, strains of NFB have not been considered important enough to be identified when isolated from animals. In this study, all NFB isolated during a 1-year period in a clinical veterinary microbiology laboratory were identified to determine their prevalence. Of the 347 strains of NFB obtained, the most common species were Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Bordetella bronchiseptica, and Pseudomonas pseudoalcaligenes. Of all clinical veterinary specimens submitted for cultures, 10% contained nonfermenters.     

Clinical comparison of glucose broth with nutrient broth blood cultures for the detection of "Streptococcus viridans" bacteraemia

Viridans streptococci were isolated from the blood stream of half of 50 patients undergoing dental extractions approx. 2 min before blood culture. Glucose and nutrient broths were tested in parallel. There was no significant difference between the isolation rates of streptococci by the two methods after incubation for 7 days, nor did the addition of glucose 0.1% to nutrient broth significantly increase the speed of detection of streptococci; the isolation rates of streptococci during the first 48 h of incubation were similar with both types of broth. Although there were a few more isolations of streptococci from the glucose broth than from the nutrient broth during the first 20 h, the difference was not statistically significant. No rapid lethal effect against streptococci was observed in glucose broth during incubation for 7 days. These results suggest that adding glucose 0.1% to nutrient broth for blood culture does not influence the recovery of organisms from patients with "Streptococcus viridans" bacteraemia after incubation of the broth for 2-7 days.     

Use of Gen-Probe AccuProbe Group B streptococcus test to detect group B streptococci in broth cultures of vaginal-anorectal specimens from pregnant women: comparison with traditional culture method.

Detection of vaginal-anorectal colonization with group B streptococci (GBS) is critical to the prevention of neonatal GBS disease. The recommended method for the detection of GBS is culture of the distal vagina and anorectum in a selective broth medium followed by subculture to solid media and identification of GBS on the solid media. The purpose of this study was to compare this standard culture method with the detection of GBS directly from an enrichment broth by utilizing the Gen-Probe AccuProbe Group B Streptococcus Culture Identification Test (GPGB). A total of 502 specimens were tested in this study. Both culture and the GPGB detected 90 of 95 positive specimens (sensitivity, 94.7%). There were two false-positive GPGB results (specificity, 99.5%). An analysis of 100 consecutive specimens was performed to compare the costs associated with the use of a primary tryptic soy agar plate with 5% sheep blood (BAP) and a 3-ml tube of Todd-Hewitt broth supplemented with 10 micrograms of nalidixic acid per ml and 15 micrograms of colistin per ml (LIM broth) with subculture to another BAP and the costs associated with the GPGB. Our estimated costs were $3.68 for a negative culture including 7.0 min of labor, $5.41 for a positive culture including 8.9 min of labor, and $5.16 for the GPGB including 3.6 min of labor (based upon a test run of 10 specimens and two controls and a cost of $70.00 for a 20-test GPGB kit). Accessioning and reporting of results are not included in these costs. In conclusion, we found that the GPGB was equivalent in sensitivity to our standard culture method. While overall costs were somewhat higher for the GPGB, the GPGB has lower labor costs and offers the potential for incremental savings with higher test volumes and computer interface capability.     

Genotyping of human papillomavirus DNA in anal biopsies and anal swabs collected from HIV-seropositive men with anal dysplasia

BACKGROUND: Human papillomavirus (HPV) causes anal intraepithelial neoplasia (AIN) in HIV-seropositive men. The detection of HPV genotypes in anal biopsies and swabs was compared. METHODS: HPV DNA was detected in anal swabs and biopsies obtained concurrently from 154 HIV-seropositive men [31 without AIN, 60 low-grade AIN (AIN-1), 62 high-grade AIN (AIN-2,3), and 1 indeterminate AIN] under or eligible to highly active antiretroviral therapy. RESULTS: HPV DNA was detected in 24.2% of normal biopsies compared with 93.5% with AIN-2,3 (P < 0.001) and 88.3% with AIN-1 (P < 0.001). The proportion of biopsies containing multiple genotypes was greater in AIN-1 (n = 21, 35.0%; P = 0.002) and AIN-2,3 (n = 38, 58%; P < 0.001) than in normal biopsies (n = 2, 6.5%). The most frequent genotypes in order of frequency were in AIN-2,3 biopsies HPV-16, 18, 58, and 45 and were in AIN-1 biopsies HPV-6, 11, 16, and 39. Controlling for age, CD4 count, and smoking, the presence of high-risk HPV DNA in biopsies [odds ratio (OR) = 50.8, 95% confidence interval (CI): 13.0 to 199.5] but not in swabs (OR = 2.0, 95% CI: 0.6 to 7.0) was associated with AIN-2,3. CONCLUSIONS: AIN-2,3 was associated with high-risk HPV infection detected in biopsies but not in swabs in men under or starting highly active antiretroviral therapy, possibly due to the presence of HPV foci outside of the neoplastic lesion.     

Effect of hyperlipidemic serum and irradiation on wound healing in primary quiescent cultures of vascular cells

After 8 weeks in culture, outgrowths from explants of aortic media of rhesus monkeys and New Zealand rabbits result in circular colonies of mature smooth muscle cells, quiescent in 10% serum. Such cultures were wounded by cutting out a 1.5-mm-wide strip. Migration of cells into the wound area was measured daily, and proliferation was assessed by [3H]thymidine incorporation. Migration began within 24 hr and at 7 days the defect was filled by proliferates of migrated cells. The cumulative labeling index was highest in the cells in the wound gap but was also increased in the remaining part of the culture. Wounding thus stimulated the uninjured portion of these primary cultures to proliferate, while in subcultures of these cells increase in [3H]thymidine incorporation was confined to the wound area. While hyperlipidemic serum has been shown to induce proliferation in unwounded cultures, it did not enhance cell replication elicited by wounding but reduced cell density and labeling index in the wound gap. Irradiation prior to wounding reduced cell proliferation to control values, while migration of cells was not significantly affected. In irradiated cultures, the inhibitory action of hyperlipidemic serum on cell migration became evident. Such quiescent cultures thus allow us to separate the effects of a specific injury on the proliferative and migratory responses of vascular smooth muscle.