Establishment of a particle-counting method for purified elementary bodies of chlamydiae and evaluation of sensitivities of the IDEIA Chlamydia kit and DNA probe by using the purified elementary bodies. To evaluate the sensitivity of commercially available test kits for detection of chlamydiae, we established a method of purifying Chlamydia trachomatis and Chlamydia pneumoniae elementary bodies (EBs). We then subjected the purified EBs, together with the purified EBs of Chlamydia psittaci, to the IDEIA Chlamydia (IDEIA) and DNA probe test kits to determine the EB numbers at the detection limits. The sensitivities of the test kits were thus compared. The results can be summarized as follows. (i) Intact EBs in the purified preparations were present at 100, 96.3, and 97% for the C. psittaci Cal 10, C. trachomatis L2/434/Bu (L2), and C. pneumoniae TW-183 strains, respectively. The preparations of the L2 and TW-183 EBs contained a few EB envelopes, which reacted with antilipopolysaccharide monoclonal antibodies, as did the intact EBs, indicating that elimination of EB envelopes is not required for testing of the IDEIA kit's sensitivity. (ii) We established a method of counting intact EBs and EB envelopes under a scanning electron microscope after sedimentation of EBs on a coverslip by centrifugation. (iii) The EB numbers per assay at the cutoff level, which is set up in the IDEIA kit, were 9.6 x 10(2), 6.5 x 10(3), and 2.5 x 10(4) for the L2, TW-183, and Cal 10 strains, respectively. When the same EB preparations were applied to the DNA probe kit, the EB number at the cutoff level was 7.5 x 10(3) per assay for the L2 strain, but no reaction occurred for the Cal 10 and TW-183 strains at any EB number, indicating that the DNA probe kit is highly specific for C. trachomatis. Although the IDEIA kit designed for detection of C. trachomatis showed a sensitivity superior to that of the DNA probe, the chlamydial species was not determined by the IDEIA kit.


Analysis of relationships among isolates of Citrobacter diversus by using DNA fingerprints generated by repetitive sequence-based primers in the polymerase chain reaction. Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.


Comparison of two culture approaches, blind passage and dual observation, for detecting Chlamydia trachomatis in various prevalence populations. Chlamydia trachomatis diagnosis in our laboratory consisted of dual inoculation of shell vials and detection of inclusions by using fluorescein-conjugated monoclonal antiserum; the second culture vial was conventionally used for blind passage when the first vial was negative. We compared the increase in positivity using blind passage with that of a strategy utilizing observation of two stained monolayers (dual observation) without blind passage, in an effort to reduce the reporting time and labor associated with the conventional approach. A total of 4,329 specimens were obtained from an obstetrics and gynecology (OB-GYN) clinic (2,563 specimens) and the sexually transmitted disease clinic (1,766 specimens). These specimens were used to compare the two strategies. Blind passage of 1,269 initially culture-negative specimens from the OB-GYN clinic resulted in an additional 6 positive chlamydial diagnoses. In comparison, a similar number of specimens (1,294) from the OB-GYN clinic collected subsequently to the first group were tested by dual observation. There were five additional positive findings. A similar evaluation of specimens from the sexually transmitted disease clinic was performed. Blind passage of 313 initially culture-negative specimens yielded 3 additional positive diagnoses, whereas dual observation of 1,435 similar specimens resulted in 9 positive diagnoses. On the basis of analysis of 4,332 specimens, sensitivity of dual observation is comparable to that of blind passage; labor, cost, and reporting time of dual observation are reduced in comparison to those of blind passage.


Urinary tract infection due to Rahnella aquatilis in a renal transplant patient. Rahnella aquatilis is an unusual gram-negative rod belonging to the family Enterobacteriaceae. It inhabits fresh water and is rarely isolated from clinical specimens. We report the case of a urinary tract infection caused by this organism in a renal transplant patient.


DNA probe for strain typing of Cryptococcus neoformans. A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.


Comparative study of antipneumocystis agents in rats by using a Pneumocystis carinii-specific DNA probe to quantitate infection. A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.


Colonization of the female genital tract with Staphylococcus saprophyticus. The prevalence of colonization by Staphylococcus saprophyticus of the urogenital tracts of 276 women from an outpatient gynecology practice was determined by using selective and enrichment culture techniques. Nineteen subjects (6.9%) were found to be colonized by S. saprophyticus. The rectum was the most frequent site of colonization and was responsible for 40% of the isolates; this was followed in decreasing order by the urethra, urine, and cervix. Women colonized by S. saprophyticus were more likely to have experienced a urinary tract infection in the previous 12 months (P = 0.058; odds ratio, 2.844; 95% confidence interval, 1.054 to 7.671). Patients colonized by S. saprophyticus tended to have had their menstrual periods more recently (P = 0.066), experienced sexual intercourse more recently (P = 0.168), and had a recent or concurrent diagnosis of vaginal candidiasis (P = 0.111; odds ratio, 2.393; 95% confidence interval, 0.877 to 6.528). A seasonal variation in colonization was observed, with colonization most likely occurring during the summer and fall. Follow-up for an average of 6.75 months failed to document any colonized woman progressing to symptomatic urinary tract infection. In addition, 21 women colonized by non-S. saprophyticus, novobiocin-resistant, coagulase-negative staphylococci were identified and characterized.


Immunoblot analysis of the humoral immune response to Pythium insidiosum in horses with pythiosis. Reactions to Pythium insidiosum by sera from horses with active pythiosis were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Five strains of P. insidiosum were grown in nutrient broth and then sonicated. After centrifugation, supernatant antigens were separated by SDS-PAGE. An exoantigen of Conidiobolus coronatus was also tested. Bands with molecular weights between 97,000 and 14,000 were identified by Coomassie blue and silver staining. After being transferred to nitrocellulose, the antigens were reacted against sera from six horses with pythiosis, sera from four horses cured a year earlier by vaccination, and sera from five healthy horses. The sera from horses with pythiosis recognized at least 20 antigens in all strains. Three antigens with molecular weights of 32,000, 30,000, and 28,000 appeared to be immunodominant and specific. Sera from horses cured by immunotherapy showed only five very weak bands, three of them the 32,000-molecular-weight (32K), 30K, and 28K antigens. No bands were observed with sera from healthy horses or sera from horses with a variety of other infections. Sera from horses with pythiosis cross-reacted with the 44K antigen of C. coronatus. The immunodominant antigens described here may be useful for diagnostic purposes and in immunotherapy for this oomycotic infection in horses.


Characterization and distribution of Pasteurella species recovered from infected humans. During a 3-year period, all Pasteurella strains recovered at the Clinical Microbiological Laboratory, Lund, Sweden, were studied biochemically with respect to their relationship to the recently described taxa of this genus. Of 159 strains recovered from 146 infected humans, 95 were identified as Pasteurella multocida subsp. multocida, 21 as Pasteurella multocida subsp. septica, 28 as Pasteurella canis, 10 as Pasteurella stomatis, and 5 as Pasteurella dagmatis. The homology within and between the Pasteurella species regarding cellular fatty acids and enzymatic activities was also studied. Strains of the different Pasteurella species were indistinguishable from each other regarding fatty acid composition; all strains contained major amounts of C14:0, C16:1, C16:0, and 3-OH-C14:0 acids and minor amounts of C18:2, C18:1, and C18:0 acids. Neither did the enzymatic activities distinguish between strains belonging to different species. In addition, of 56 strains examined, toxin production was demonstrated only in 1 strain each of P. multocida subsp. multocida and P. canis. Except for one severe case of necrotizing cellulitis involving P. dagmatis, P. multocida subsp. multocida or P. multocida subsp. septica was recovered in the more serious cases of infection. Except for P. canis, which in all cases was associated with dog bites, most Pasteurella strains were recovered in cases of infection associated with cat bites or scratches. Pasteurella strains occurred in four infected patients without evident connections with animals.


Comparison of cytobrushes with swabs for recovery of endocervical cells and for Chlamydiazyme detection of Chlamydia trachomatis. Endocervical swab and cytobrush specimens from 1,301 symptomatic women were microscopically analyzed for adequacy and tested using Chlamydiazyme (CZ) (Abbott Laboratories). When the swab specimen was collected first, blocking antibody-confirmed CZ-positive results were obtained from 48 (8.0%) of 599 patients, 42 (87.5%) from swabs and 46 (95.8%) from cytobrushes (not significant). When the swab specimen was collected second, confirmed CZ-positive results were obtained from 46 (6.6%) of 702 patients, 44 (95.6%) from swabs and 41 (89.1%) from cytobrushes (not significant). Cytobrushes offered no significant advantage over swabs for CZ detection of Chlamydia trachomatis.


Differential susceptibilities of Mycobacterium avium and Mycobacterium intracellulare to sodium nitrite. Sixty-seven of 72 strains of Mycobacterium avium (93.1%) were resistant to sodium nitrite at a concentration of 3 mg/ml in 7H11 agar medium, while 57 of 59 strains of Mycobacterium intracellulare (96.6%) were susceptible to the agent. The difference in the susceptibilities of M. avium and M. intracellulare to sodium nitrite is therefore useful for the differentiation of the two species.


Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by polymerase chain reaction. The polymerase chain reaction (PCR) was used to detect Toxoplasma gondii DNA in cerebrospinal fluid (CSF) from 14 patients with AIDS by amplification of the repetitive B1 gene. Positive PCRs were obtained in CSF from four of nine patients with toxoplasmic encephalitis. CSF samples from five control patients were negative for T. gondii DNA by PCR.


Evaluation of a chemiluminescent probe assay for identification of Histoplasma capsulatum isolates. Fifty-three of 54 isolates of fungi were correctly identified with an acridinium ester-labelled probe for Histoplasma capsulatum (Accuprobe; Gen-Probe, San Diego, Calif.). One isolate of Aspergillus niger was incorrectly identified as H. capsulatum. Age of the culture, medium for isolation, and morphologic state did not affect the results.


Experimental Borrelia burgdorferi infection of outbred mice. Infectivity of Borrelia burgdorferi strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 10(4) and 10(5) or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 micrograms of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi.


Fatal disseminated infection caused by Myceliophthora thermophila, a new agent of mycosis: case history and laboratory characteristics. We report a case of human infection caused by the hyphomycete Myceliophthora thermophila. A 7-year-old male with neurofibromatosis (type 1) was diagnosed in 1987 with acute myeloblastic leukemia associated with the chromosomal abnormality monosomy 7. The patient experienced multiple serious infections over a three-year period before expiring in 1990 while in the end stage of leukemia. Autopsy findings included fungal vegetations of the left atrium, ascending aorta, and pulmonary arteries and fungal invasion of both lungs. Cultures yielded M. thermophila. We believe that this is the first reported fatality caused by M. thermophila.


Uncommon Campylobacter species in infant Macaca nemestrina monkeys housed in a nursery. Studies were conducted to characterize 18 isolates of Campylobacter spp. that could not be identified as either Campylobacter jejuni or C. coli. The isolates were cultured from specimens from 13 of 18 infant nonhuman primates during a prospective epidemiologic study reported previously. Phenotypic tests, DNA hybridization, and analysis of DNA coding for rRNA identified the isolates as C. butzleri (seven isolates), C. hyointestinalis (seven isolates), and C. fetus subsp. fetus or C. fetus subsp. fetus-like organisms (four isolates). Ribotype and polyacrylamide gel electrophoresis patterns indicated that there was heterogeneity among the isolates of C. butzleri and C. fetus subsp. fetus-like organisms.


Detection of diacetyl (caramel odor) in presumptive identification of the "Streptococcus milleri" group. The caramel odor associated with the "Streptococcus milleri" group was shown to be attributable to the formation of the metabolite diacetyl. Levels of diacetyl in the 22- to 200-mg/liter range were produced by 68 strains of the "S. milleri" group; apart from one strain of Streptococcus mutans, all 92 other strains of streptococci belonging to 12 species produced < 13 mg of diacetyl per liter. Quantitation of diacetyl levels from cultures of streptococci is suggested as a rapid presumptive test for the "S. milleri" group.


Use of the E test to predict high-level resistance to aminoglycosides among enterococci. The E test and the reference agar dilution methods were compared for detecting high-level aminoglycoside resistance (HLAR) among 71 selected clinical isolates, including 62 Enterococcus faecalis and 9 Enterococcus faecium isolates. High-level gentamicin resistance alone was found in 11% (5 E. faecalis and 3 E. faecium strains) and high-level streptomycin resistance was found in 42% (28 E. faecalis, 2 E. faecium strains) of the strains tested, and 31% of the strains demonstrated high-level resistance to both antimicrobial agents (21 E. faecalis and 1 E. faecium strains). The E test detected all HLAR populations, but the streptomycin strip may require recalibration to achieve absolute MIC comparisons with the reference value (twofold less) or the use of an alternative interpretive resistance breakpoint, e.g., > 1,000 micrograms/ml. By the E test, MIC results indicate that ampicillin, imipenem, penicillin, piperacillin, and vancomycin remain active against the HLAR E. faecalis isolates; however, these tested drugs were less effective on the HLAR E. faecium isolates (< 50%).


Stability of dried blood spot specimens for detection of human immunodeficiency virus DNA by polymerase chain reaction. Blood sampling on filter paper has many advantages for the detection of perinatal human immunodeficiency virus (HIV) infection by the polymerase chain reaction (PCR). However, if the method is to be widely used, an assessment of its performance under field conditions is required. To simulate conditions in the field, 50-microliters aliquots of whole blood containing low levels of HIV proviral DNA (4 to 1,024 copies per 100,000 nucleated cells) were spotted onto filter paper; dried; and subjected to heat, humidity, and prolonged storage at room temperature. After exposure, the DNA was recovered and amplified with primers to human leukocyte antigen DQ alpha- and HIV-specific sequences. Treatment at 37 degrees C and 60% humidity for 7 days, storage for 12 weeks at 22 degrees C, and freeze-thawing twice had no adverse effect on PCR reactivity when compared with the results obtained with reference spots stored at -20 degrees C. The lower limits of HIV detection in all tests ranged from 4 to 16 HIV copies per 100,000 cells. Fixation in 70% ethanol improved the amplification of low levels of HIV DNA and reduced biohazard risks. These findings suggest that dried blood spots will provide a powerful new resource for testing for HIV by PCR, especially in remote areas where refrigeration and immediate sample processing are unavailable.


Western immunoblot analysis and serologic characterization of Blastomyces dermatitidis yeast form extracellular antigens. A major 98-kDa extracellular protein antigen of Blastomyces dermatitidis was shown in Western blot (immunoblot) analysis to be highly reactive with serum antibodies from patients with blastomycosis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of yeast form B. dermatitidis culture filtrates and cell extracts demonstrated over 50 proteins, only 24 of which were immunoreactive. Of these, a 98-kDa protein was found to be the most specific and was isolated. This protein was found in both broth culture filtrates and extracts of yeast forms. Western blot tests with this antigen detected serum antibodies in 91% of 32 patients with clinically proven blastomycosis, in contrast to an enzyme-linked immunosorbent assay (ELISA) with DEAE-purified antigen, which detected 88% of the cases. The Western blot test also exhibited lower reactivity with a panel of sera from patients with heterologous fungal infections that were cross-reactive in the ELISA with DEAE-purified B. dermatitidis antigen. The 98-kDa protein electroeluted from polyacrylamide gels was identical to the diagnostic A antigen used in the blastomycosis immunodiffusion test. Comparison of the 98-kDa antigen with a previously described 120-kDa yeast form cell wall protein antigen of B. dermatitidis showed that these two antigens are almost identical.


Salmonella serogroups C2 and C3 identified by agglutination using an immunoglobulin G3(kappa) monoclonal antibody (32-1-E3) reactive with a somatic factor 8-like polysaccharide antigen. An immunoglobulin G3(kappa) monoclonal antibody (MAb), MAb 32-1-E3, which was prepared in BALB/c mice by using a heated, alcohol-acetone-extracted Salmonella newport CDC 50 antigen, reacted with protein-free lipopolysaccharides from Salmonella groups C2 (O:6,8) and C3 (O:-,8) but not with those from any other serogroup tested. Sodium periodate did not inhibit antigen reactivity, which was consistent with its identity as the abequose-containing disaccharide O:8 antigen. Reactivity was inhibited by competition with serogroup C2 (O:6,8) and C3 (O:-,8) antigens but not with non-O:8 antigens. Reactivity was also inhibited by preincubation of the antigen with polyclonal rabbit antiserogroup C2 or C3 antibodies but not with antisera to serogroup C1 or other Salmonella serogroups. The MAb agglutinated with all strains of Salmonella serogroups C2 and C3 tested but not with other bacteria. Agglutination was inhibited by preabsorbing the MAb with either of two serogroup C3 Salmonella strains, S. virginia CDC 189 or S. haardt MDL 83A4545, which contain only O:8, but not by preabsortion with O:8-negative S. cholerasuis MDL 81A7623 (group C1; O:6,7), S. paratyphi type B CDC 157 (group B; O:1,[4],5,12), or Escherichia coli (O:157) (which contains no Salmonella serogroup antigens). The MAb reacted strongly (4+ agglutination) with all 140 wild-type strains of group C2 and C3 Salmonella spp. tested and showed no reaction with any of 1,324 wild-type strains of non-C2 or non-C3 Salmonella spp. tested. The MAb is useful as a replacement for absorbed, polyclonal, single-factor O:8 antiserum to discriminate Salmonella serogroups C2 and C3 from serogroup C1.


Comparison of epidemiological markers of Salmonella strains isolated from different sources in Spain. A comparative study of the phage types, antimicrobial susceptibility patterns, and plasmid profiles of 171 strains of Salmonella isolated from food, epidemic outbreaks, and water-contaminated environments as well as sporadic human isolates was carried out to determine the most adequate marker in epidemiological investigations. Typing based on the plasmid profiles appears to be the most effective method for grouping strains with the same serotype obtained from a single outbreak and from environmental sources. However, none of the three markers tested allow us total discrimination and identification of related strains from a common source for epidemiological tracing. Therefore, the combined use of the three methods is necessary for determining whether common source isolates are related.


Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates. A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen. MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M. bovis, and M. kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb. A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M. tuberculosis clinical isolates. Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots. This suggests differences in PGL-Tb antigen production among M. tuberculosis strains even when they are grown in the same culture media and conditions. This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens. This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M. tuberculosis clinical isolates.


Functional and serological analysis of type II immunoglobulin G-binding proteins expressed by pathogenic group A streptococci. Bacterial immunoglobulin-binding proteins expressed on the surface of group A streptococci represent a heterogeneous family of functionally related proteins. In this report, we describe efficient methods for extracting immunoglobulin-binding proteins and classifying them functionally and antigenically. A common characteristic of immunoglobulin-binding proteins expressed by group A streptococci appears to be the absence of internal methionine residues in the binding protein. This has enabled development of a rapid, efficient, cyanogen bromide-based extraction procedure for solubilizing these molecules from intact bacteria. Studies carried out with a series of monospecific polyclonal antibodies prepared in chickens have identified two major antigenic classes of immunoglobulin-binding proteins. The methods described in this report facilitate a rapid functional and serological screening of immunoglobulin-binding proteins that should now enable detailed epidemiological studies of the importance of these molecules in group A streptococcal infections and their relationship to other surface proteins, in particular, the antiphagocytic M protein.


Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. Current laboratory diagnosis of Lyme disease relies on tests for the detection of antibodies to Borrelia burgdorferi, the etiologic agent of the disease. These tests are often unreliable because of a lack of sensitivity and specificity and test-to-test variability. The purpose of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) amplification for detection of B. burgdorferi in skin biopsy specimens. Forty-six 2-mm skin biopsy samples were obtained from 44 patients with a clinical diagnosis of erythema migrans, 9 of whom were receiving antibiotic therapy at the time of biopsy. Specimens were ground in BSK medium with separate aliquots taken for culture and PCR. Of the specimens from the untreated group, 57% (21 of 37) were positive by culture and 22% (8 of 37) were culture negative; 22% (8 of 37) of the cultures were uninformative because of contamination. By comparison, 22 (59%) of 37 specimens were positive by PCR amplification. Of 21 culture-positive samples, 13 (62%) were also positive by PCR analysis. Thus, the sensitivity of the PCR was 59 to 62%, based on either a clinical or cultural diagnosis of untreated Lyme disease. None of the nine specimens from antibiotic-treated patients grew in culture, whereas two of the nine were positive by PCR analysis. Given the complexity and time required for culture, PCR is a promising technique for the diagnosis of early Lyme disease.


Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae. The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.


Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae. Rapid identification of Neisseria gonorrhoeae in clinical specimens is essential for effective control. Traditional culture requires a minimum of 24 h, and for some specimens harboring gonococci, the gonococci fail to grow or are misidentified. The recently described ligase chain reaction (LCR) is a highly specific and sensitive DNA amplification technique which was evaluated as an alternative to routine culture. Three LCR probe sets were used. Two of the probe sets were directed against the multi-copy Opa genes (Omp-II), while the third set was targeted against the multicopy Pilin genes. Each LCR probe set was evaluated with 260 microorganisms including 136 global isolates of N. gonorrhoeae, 41 isolates of N. meningitidis, and 10 isolates of N. lactamica; 26 nonpathogenic Neisseria strains; and 47 isolates of non-Neisseria species that may reside in clinical specimens. Amplification products were detected by using the IMx LCR format (Abbott Laboratories, Abbott Park, Ill.). Strains of N. gonorrhoeae were assayed at 270 cells per LCR (approximately 6.7 x 10(4) CFU/ml) with the Opa and Pilin probes, producing signals at least 21 and 15 times above background, respectively. In contrast, only background values were observed when testing the probe sets with 124 nongonococcal strains at 1.3 x 10(6) cells per LCR (approximately 3.2 x 10(8) CFU/ml). One hundred urogenital specimens were assayed by LCR, and compared with culture, the three probes were 100% sensitive (8 of 8) and 97.8% specific (90 of 92), resulting in an agreement of 98% (98 of 100). On the basis of the results of these preliminary studies, LCR has the potential to be an accurate and rapid DNA probe assay for the detection of N. gonorrhoeae in clinical specimens.


Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes. Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.


Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum. A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.


Evaluation of the RapID ANA II and API ZYM systems for identification of Actinomyces species from clinical specimens. Classification and identification of fermentative actinomycetes are labor-intensive and problematic. In this study, we evaluated the applicability and reliability of the RapID ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) and the discriminatory value of the API ZYM system (Societes Analytab Products Inc., La Balme Les Grottes, France) in the identification of Actinomyces-like bacteria by using conventional methods as a reference. Eighty-five strains, including 71 isolates from mixed anaerobic infections and 14 reference strains, were tested. The RapID ANA II system correctly identified all Actinomyces odontolyticus strains and 65% of Actinomyces israelii strains. All Arcanobacterium haemolyticum strains were misidentified as Actinomyces pyogenes. The most common isolates in the study were Actinomyces meyeri-like organisms, 84% of which, however, were aerotolerant. The identification of these aerotolerant strains thus remains unresolved and warrants further studies. New characteristics and changes to the conventional API ZYM enzyme profiles are suggested. The API ZYM enzyme profiles of A. odontolyticus and A. israelii were very similar, the only discriminating enzyme being alpha-fucosidase. In differentiation between A. pyogenes and Arcanobacterium haemolyticum, the production of beta-glucuronidase by the former and the production of acid phosphatase by the latter are suggested as new helpful characteristics for use in clinical laboratories. In summary, the RapID ANA II and API ZYM systems can be used as rapid preliminary methods in the identification of Actinomyces species but accurate identification requires supplementary conventional tests and gas-liquid chromatography.


Risk factors for sporadic Campylobacter infections: results of a case-control study in southeastern Norway. In 1989 and 1990, a case-control study designed to identify risk factors for sporadic infections with thermotolerant Campylobacter bacteria was conducted in three counties in southeastern Norway. The investigation was confined to infections which were acquired in Norway. A total of 52 bacteriologically confirmed cases and 103 controls matched by age, sex, and geographic region were interviewed. The following risk factors were found to be independently associated with illness in conditional logistic regression analysis: consumption of sausages at a barbecue (odds ratio [OR] = 7.64; P = 0.005), daily contact with a dog (OR = 4.26; P = 0.024), and eating of poultry which was brought into the house raw (frozen or refrigerated) (OR = 3.20; P = 0.024). The risk associated with consumption of sausages at a barbecue could not be attributed to cross-contamination from poultry products. By univariate analysis, consumption of poultry which was brought raw and frozen was associated with illness (OR = 2.42; P = 0.042), even though freezing substantially reduces the number of viable campylobacters. When poultry consumption was examined by country of origin, eating of poultry produced in Denmark or Sweden was strongly associated with illness (OR = 13.66; P = 0.014), whereas consumption of poultry produced in Norway was not (OR = 1.33; P = 0.41).


Streptococcal erythrogenic toxin genes: detection by polymerase chain reaction and association with disease in strains isolated in Canada from 1940 to 1991. The presence of genes encoding pyrogenic exotoxins type A (speA), B (speB), and C (speC) and streptolysin O (slo) was determined by the polymerase chain reaction (PCR) to target specific sequences in 152 strains of group A streptococci. These included reference strains, representative M and T type strains, and strains associated with scarlet fever and pharyngitis collected between 1940 to 1991 and included strains from patients with severe invasive streptococcal infections. PCR amplicons were detected by agarose gel electrophoresis, and specificity was established by restriction fragment analysis. The frequency of occurrence for each target gene among all strains tested was 33.6% for speA, 99.3% for speB, 28.9% for speC, and 100% for slo. Strains of non-group A streptococci, recognized toxigenic bacterial pathogens, and pneumolysin-producing Streptococcus pneumoniae strains were negative for all targeted gene sequences. Detection limits in the PCR were found to be 100 pg of total nucleic acids for the speB and speC genes and 1 ng for the speA and slo genes. Isolates associated with scarlet fever, pharyngitis, and severe invasive infections showed statistically significant differences in the presence of speA, with scarlet fever strains having the highest association (81.3%), severe infections the next highest association (42.9%), and pharyngitis the lowest association (18.4%). Although no significant differences were observed in speC frequencies in isolated associated with the three disease categories, a genotype of speB slo was significantly higher in isolates associated with pharyngitis (54.1%) than in strains associated with scarlet fever (18.8%) or severe invasive disease (23.8%). Streptolysin O targets were present in all the isolates tested, and only a single strain (T-11-M-11) was devoid of targeted speB sequences, thereby demonstrating that neither speB nor slo is associated with any particular clinical presentation.


Detection of serum Candida antigens by enzyme-linked immunosorbent assay and a latex agglutination test with anti-Candida albicans and anti-Candida krusei antibodies. Serum samples from 197 patients with and without candidiasis were assayed for Candida albicans mannan and Candida krusei mannan by an enzyme-linked immunosorbent assay (ELISA) and a latex agglutination test (LA) and for D-arabinitol by the enzymatic fluorometric method. Of the 43 patients positive for C. albicans mannan (> or = 0.2 ng/ml), 34 were infected with C. albicans and 9 were infected with Candida tropicalis. C. krusei mannan (> or = 0.3 ng/ml) was detected in 10 patients infected with Candida parapsilosis, 2 patients infected with Candida guilliermondii, and 1 patient infected with C. krusei. With both anti-C. albicans antibodies and anti-C. krusei antibodies, the sensitivities of ELISA and LA for detection of invasive candidiasis (58 patients) were 74 and 38%, respectively. No false-positive reactions were observed by the ELISA or the LA. The sensitivity and specificity of the D-arabinitol/creatinine ratio (> or = 1.5 mumol/mg) to invasive candidiasis were 50 and 91%, respectively. The ELISA with antibodies against both C. albicans and C. krusei may be useful in diagnosing invasive candidiasis caused by medically important Candida strains excluding Candida glabrata.


Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. A collaborative comparison of macro- and microdilution antifungal susceptibility tests was performed in five laboratories. MICs of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined in all five centers against 95 coded isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. A standard protocol with the following National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Testing recommendations was used: an inoculum standardized by spectrophotometer, buffered (RPMI 1640) medium (pH 7.0), incubation at 35 degrees C, and an additive drug dilution procedure. Two inoculum sizes were tested (1 x 10(4) to 5 x 10(3) to 2.5 x 10(3) CFU/ml) and three scoring criteria were evaluated for MIC endpoint determinations, which were scored as 0 (optically clear), < or = 1 (slightly hazy turbidity), and < or = 2 (prominent decrease in turbidity compared with that of the growth control). Overall intra- and interlaboratory reproducibility was optimal with the low-density inoculum, the second-day readings, and MICs scored as either 1 or 2. The microdilution MICs demonstrated interlaboratory agreement with most of the four drugs higher than or similar to that of the macrodilution MICs. In general, there was good interlaboratory agreement with amphotericin B, fluconazole, and flucytosine; ketoconazole gave more variable results.


Comparison of commercial serological tests for detection of Helicobacter pylori antibodies. Serological testing for Helicobacter pylori infection is one of the diagnostic methods of choice. Various commercial kits that use different antigens have been developed, but data on their diagnostic accuracy and direct comparisons between the tests are lacking. We aimed to evaluate the sensitivity, specificity, and predictive value of three immunoglobulin G enzyme-linked immunosorbent assay kits: Pylori stat, Helico-G, and Premier H. pylori. Serum samples and gastric biopsy findings from 76 patients were evaluated. We found by using a priori cutoff values that the Pylori stat, Helico-G, and Premier kits had overall sensitivities of 96, 96, and 88%, respectively, and specificities of 94, 86, and 96%, respectively, compared with gastric biopsy findings. For 232 serum samples, the Pylori stat test and a previously validated standard serological assay on which the test was based disagreed in 3% of the cases, while for 76 samples that were tested, Helico-G and a previously validated standard assay on which it was based disagreed in 8% of the cases. The intra- and interassay precision of each of the test kits was high. We conclude that serology based on any of these commercial tests represents a reliable and valid method for the diagnosis of H. pylori whether or not highly purified antigens are used.


Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica. A recombinantly expressed protein, recEh-P1, representing part of an immunodominant surface antigen of pathogenic Entamoeba histolytica, was used for serodiagnosis of invasive amebiasis. Expression was performed under the control of a T7-RNA promoter by using a modified procaryotic expression vector, designated pHisT7. This vector allowed high-yield expression of recEh-P1 fused to a stretch of sequence containing eight histidine residues, which facilitated purification by metal chelate affinity chromatography on Ni2+ columns under highly denatured conditions. Purified recEh-P1 was found to be water soluble after prolonged dialysis and was used as the antigen for the detection of antiamebic serum antibodies by immunoblotting and enzyme-linked immunosorbent assay. In both tests all sera of patients with invasive amebiasis reacted to recEh-P1 whereas none of those collected from healthy controls, including individuals with noninvasive amebiasis, or from patients suffering from bacterial or protozoan infections unrelated to E. histolytica did so.


Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies. Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA.


Detection of Toxoplasma gondii parasitemia by gene amplification, cell culture, and mouse inoculation. Diagnosis of Toxoplasma gondii infection is difficult, especially in immunosuppressed people. The AIDS epidemic has increased the number of people at risk and increased the need for better diagnostic methods. We have compared three methods for detection of T. gondii parasitemia. Rabbits were infected subcutaneously with 10(4) T. gondii tachyzoites. Blood samples were obtained, and buffy coat or leukocyte fractions were prepared. We sought the T. gondii B1 gene by gene amplification by the polymerase chain reaction, and we sought viable T. gondii cells by inoculating fibroblast cell cultures and by mouse inoculation. Thirty-two blood samples were obtained from seven infected rabbits, and 18 were obtained from four control, uninfected rabbits. Parasitemia was detected in 20 of 32 samples (62%) from infected samples by mouse inoculation, 12 of 32 samples (37%) by gene amplification and detection, and 8 of 32 samples (25%) by cell culture. Mouse inoculation requires use of live animals and has a long turnaround time. Currently, cell culture is the least sensitive but most practical and widely available method for the detection of T. gondii parasitemia. Gene amplification and detection was more sensitive than cell culture and may become available in clinical laboratories as techniques are developed further and automated.


The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.


Comparison of nine antigen detection kits for diagnosis of urogenital infections due to Chlamydia psittaci in koalas. Chlamydia psittaci is the major cause of infectious disease in the koala (Phascolarctos cinereus). It causes four disease syndromes in the koala, namely, conjunctivitis, rhinitis, cystitis, and infertility (females only). Diagnosis of chlamydial infections in koalas relies primarily on isolation of the organism in cell culture. Serology has generally not been useful, and little use has previously been made of the commercially available antigen detection kits. We examined the sensitivity, specificity, and usefulness of three direct fluorescent-antibody kits (Vet-IF [Cell Labs], IMAGEN [Celltech], Chlamydia-Direct IF [Bio Merieux]) and six antigen detection enzyme-linked immunosorbent assay (ELISA) kits (Clearview [Unipath], Surecell [Kodak], Pathfinder [Kallestad], Chlamydia-EIA [Pharmacia], Chlamydiazyme [Abbott], IDEIA [Celltech]) for the detection of urogenital infections in koalas. Laboratory studies showed that the direct fluorescent-antibody kits were the least sensitive in this case and did not detect fewer than 10(4) elementary bodies per ml, while most ELISA kits detected between 130 and 600 elementary bodies per ml. Field study results showed that the Clearview kit was the most sensitive (91%) compared with the IDEIA (88%) and the Surecell (73%) kits. All three kits were more sensitive than cell culture (36%), highlighting viability loss problems that occur during transport. This study showed that the Clearview kit is sensitive, specific, and easy to use for the detection of type II (urogenital) C. psittaci from koalas in the field and warrants further evaluation.


Application of rejection criteria for stool ovum and parasite examinations. We retrospectively determined the yield of 2,015 stool ovum and parasite (O&P) examinations performed over an 11-month period. Two aspects were evaluated: the yield of positive results from multiple specimens per patient versus the result of a single examination, and the yield of positive results from stools submitted after 3 days of hospitalization. There were 131 (6.7%) positive O&P examinations from 75 patients: for 35 patients the single examination was positive; for 18, two of two examinations were positive; and for 15, three of three examinations were positive. The remaining seven patients had at least one negative examination in a series, but pathogenic intestinal parasites were detected in only three of these patients. Seventeen patients with positive O&P examinations were inpatients: seven of nine patients examined for O&P within 3 days of admission had stool specimens that contained recognized pathogens, in contrast to only two of eight patients examined after 3 days of hospitalization. After reviewing the data and informing hospital clinics, wards, and physicians, we instituted the following policy for screening stool specimens submitted for O&P examination. Only one O&P examination was performed for each outpatient visit and for inpatients hospitalized for 3 days or less, and examinations were not performed on stools submitted after 3 days of hospitalization unless the clinician arranged for the examination on the basis of clinical need. Over the 6-month follow-up period, 29.9% of O&P requests were rejected, 22% for patients in hospital for longer than 3 days and 7.9% for multiple O&P requests. Of 265 initially rejected specimens, 22 (8.3%) were examined after the referring physician contacted the laboratory. None of these specimens was positive. We conclude that eliminating O&P examinations of patients hospitalized for more than 3 days and initially performing only one examination per patient significantly reduces the number of examinations performed and reduces laboratory charges without adversely affecting patient care.


Evaluation of methods for differentiation of coagulase-positive staphylococci. The purpose of this study was to determine the minimum number of tests that could be used to differentiate between the coagulase-positive strains of staphylococcus: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius. Eighty coagulase-positive strains of each of the three species were examined. The five tests conducted were growth on modified Baird-Parker agar, growth on P agar supplemented with acriflavin, production of acetoin, anaerobic fermentation of mannitol, and presence of beta-galactosidase. Positive test percentages for S. aureus were 100% for growth on modified Baird-Parker agar, 100% for growth on P agar supplemented with acriflavin, 94% for production of acetoin, 99% for anaerobic fermentation of mannitol, and 0% for presence of beta-galactosidase. Positive test percentages for S. intermedius were 0% for growth on modified Baird-Parker agar, 0% for growth on P agar supplemented with acriflavin, 1% for production of acetoin, 0% for anaerobic fermentation of mannitol, and 100% for presence of beta-galactosidase. S. hyicus isolates were negative in all five tests. Results from the 240 coagulase-positive staphylococcus strains tested would suggest correct identification of coagulase-positive staphylococci with P agar supplemented with acriflavin and the beta-galactosidase test. These two tests are simple to conduct and result in quick and easy differentiation of the three coagulase-positive staphylococcal species.


Cellular fatty acids in Fusobacterium species as a tool for identification. Identification of fusobacteria from clinical specimens currently requires analysis of metabolic end products by gas-liquid chromatography in addition to certain biochemical and enzymatic tests because of the relative biochemical inactivity of these bacteria. Even the finding of pointed, thin gram-negative cells on Gram-stained slides can no longer be relied on for identification of Fusobacterium nucleatum, since at least four other species of fusobacteria have been seen to exhibit similar morphology. We examined 46 clinical isolates and six American Type Culture Collection type strains of fusobacteria by conventional methods and by the Microbial ID Systems MIDI software package for analyzing cellular fatty acid patterns measured by capillary column gas-liquid chromatography. Distinctive patterns of major fatty acids could be used to reliably identify most clinical isolates to the species level. The MIDI system identified 89% of the isolates correctly and provides an alternative to conventional methods.


Evaluation of a commercially available complement fixation test for diagnosis of Helicobacter pylori infection and for follow-up after antimicrobial therapy. Commercially available complement fixation test reagents (Institute Virion Ltd., Rüschlikon, Zurich, Switzerland) available in package format were evaluated for the serodiagnosis of Helicobacter pylori infection. The assay was compared with bacterial culture and histological Giemsa stain of gastric biopsy specimens obtained from 930 patients of different ages and from different ethnic groups, with a variety of upper gastrointestinal tract symptoms. The prevalence, sensitivity, specificity, and positive and negative predictive values, respectively, were 35, 71, 90, 80, and 85% for Belgian patients aged 40 years or younger, 50, 81, 93, 92, and 83% for Belgian patients older than 40 years, and 83, 83, 79, 95, and 48% for Mediterranean patients. Using 645 serum specimens from 226 patients, we also evaluated the complement fixation test for its ability to monitor the eradication of H. pylori following antimicrobial therapy. Overall, H. pylori was eradicated from 122 patients while 104 patients remained infected with the organism. A significant decrease in antibody levels was observed 3 to 6 months after the end of therapy in the group of patients from whom H. pylori was eradicated.


Value of extended agitation and subculture of BACTEC NR 660 aerobic resin blood culture bottles for clinical yeast isolates. From 10,351 blood cultures, we prospectively studied 1,000 BACTEC NR 660 aerobic resin blood culture bottles (26+ and Peds Plus) for patients suspected of having yeast septicemia to determine whether extended agitation and subculturing would increase the recovery of yeasts. Aerobic bottles were agitated continuously for 144 h. On day 7, 1,000 culture-negative aerobic bottles which had fungal blood culture requests were agitated for an additional 14 days. During this time they were subcultured twice and read twice by BACTEC NR 660. ON days 1 to 7, 81 bottles were cultured positive for yeasts from 36 patients, which included 44 isolates of Candida albicans, averaging 1.4 days to detection, and 12 isolates of Cryptococcus neoformans, averaging 3.8 days to detection. The average detection time for all yeasts was 2.2 days. On days 7 to 21, no yeasts were detected by BACTEC or recovered from the subcultures. We conclude that when continuously agitated for at least 5 full days (120 h), the BACTEC NR 660 aerobic resin bottles reliably isolate yeasts, and it is unnecessary to subculture or hold these bottles beyond 5 days. It also eliminates the need for an additional blood culture system for yeast detection, thus saving (i) confusion in the collection process, (ii) patients' blood and money, and (iii) laboratory technologists' time.


Accuracy of the E test for determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial agents. We compared the results of the E test MIC method with the results of agar dilution susceptibility testing for 18 antimicrobial agents against 324 strains of gram-positive and gram-negative bacteria, including 99 strains of staphylococci, 101 strains of antimicrobial-resistant gram-negative bacteria, 40 strains of enterococci, and 84 isolates of Campylobacter jejuni. Overall agreement of MICs (+/- 1 log2 dilution) was 97.3% for staphylococci, 94.6% for gram-negative bacilli, and 100.0% for enterococci. The MIC results for C. jejuni showed an overall agreement of only 82.9%. This was due primarily to a number of offscale values that limited the number of comparisons with clindamycin, trimethoprim-sulfamethoxazole, and tetracycline. Interpretative criteria for the results of the two test methods, however, were similar. Overall, the E test produced MIC results comparable to those of agar dilution when multiresistant organisms were tested. However, it was necessary to add 2% NaCl to the agar when testing oxacillin against staphylococci for both the E test and agar dilution to obtain results comparable to those of the broth microdilution method.


Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA. Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.


Evaluation of a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens. Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly in the immunologically compromised. Monoclonal antibody reagents offer increased sensitivity and an excellent alternative to conventional staining methods. These reagents are helpful when screening large numbers of patients or those with minimal symptoms. Problems of false-positive and false-negative results with routine staining methods for stool parasites can be eliminated with monoclonal antibody reagents. Known positive formalinized specimens [Giardia sp. (n = 60), Cryptosporidium sp. (n = 55), and mixed Giardia-Cryptosporidium spp. (n = 10)] and negative formalinized specimens (n = 105), of which 46 contained other yeast or human cells or protozoa), were tested by the MERIFLUOR Cryptosporidium-Giardia direct immunofluorescence detection procedure. The MERIFLUOR reagent exhibited +/- to 4+ (majority, 2+ to 3+) on all Giardia cysts and 2+ to 4+ (majority, 3+ to 4+) on all Cryptosporidium oocysts. The cysts were generally oval (11 to 15 microns), while the oocysts were round (4 to 6 microns); both showed apple-green fluorescence against a background free of nonspecific fluorescence. All specimens positive for Giardia sp. and/or Cryptosporidium sp. showed fluorescence, and all specimens negative for the two organisms showed no fluorescence. There were eight specimens previously negative by the ova and parasite examination which were positive by the direct fluorescence method; four contained Giardia sp., and four contained Cryptosporidium sp. These positive results were confirmed after the examination of additional trichrome and modified acid-fast smears. The MERIFLUOR reagent was very easy to use, and even with a lower fluorescence intensity for Giardia sp. cysts, no false-negative or false-positive results among the specimens tested for either organism were found.


An enzyme-linked immunosorbent assay for enumeration of Pneumocystis carinii in vitro and in vivo. An enzyme-linked immunosorbent assay (ELISA) to quantitate Pneumocystis carinii organisms from culture supernatant and rat lung has been developed. A polyclonal antibody specific to P. carinii was produced in Sprague-Dawley rats by allowing P. carinii-infected animals to recover from infection. This antibody reacted strongly to P. carinii proteins of 50 to 55 kDa and weakly to those of 33 and 116 kDa. The ELISA used this convalescent-phase antibody to quantitate the number of P. carinii organisms in lung homogenates of infected rats and supernatants from infected tissue cultures which were used to screen drugs for P. carinii. The results of the ELISA were compared with those of direct microscopic counting of organisms, and the two methods were highly correlated (r > 0.9). Thus, the ELISA can be used as an alternative method for the quantitation of P. carinii organisms, and it is superior to the conventional microscopic method because it is easier to perform and less labor-intensive.


Novel coagglutination method for serotyping group B streptococci. A group G streptococcal strain was coated with antibody against six different serotypes (Ia, Ib, II, III, IV, and V) of group B streptococci. The coagglutination patterns of 114 strains of group B streptococci were compared with the serotypes determined after immunoprecipitation. The specificity of the method was 100% and the sensitivity 97%. It was used for the typing of 89 invasive and 101 colonizing isolates. The new method is swift, specific, and highly sensitive. It consumes only minute amounts of antibody.


Capsular polysaccharide serotyping scheme for Staphylococcus epidermidis. A scheme for the capsular typing of Staphylococcus epidermidis that is based on direct slide agglutination between proteinase-treated bacterial cells and specific antisera is described. Antisera were prepared from serum from rabbits immunized with two selected strains of encapsulated S. epidermidis isolated from bacteremic patients. Antisera were shown to be type specific and designated type 1 and type 2. Blood isolates of S. epidermidis from hospitals in different locations within the United States and Europe were serotyped, and it was found that over 90% of all strains were of type 1 or type 2. Type-specific antibodies mediated type-specific opsonophagocytosis and killing of S. epidermidis. The specificity was shown to be due to two distinct capsular polysaccharides. The data presented in this report may open a new window on the pathogenesis of S. epidermidis which could lead to the development of new vaccines and therapies.


Evaluation of the Wellcolex Colour Salmonella Test for detection of Salmonella spp. in enrichment broths. The Wellcolex Colour Salmonella Test was evaluated for detection of Salmonella spp. in enrichment broths of 1,010 stool samples. In 39 specimens, Salmonella spp. could be isolated from the selenite F broth (SF). Wellcolex agglutination indicative of the presence of Salmonella spp. was noted with the SF in 36 cases, 34 of which were in agreement with the subculture results. Therefore, relative to subculture, the sensitivity and specificity of the Wellcolex-selenite F procedure were 87 and 99%, respectively. Five false-negative results were noted. The gram-negative broth (GN) subculture revealed only 23 Salmonella spp. (59% sensitivity). The Wellcolex agglutination procedure applied to the GN indicated Salmonella spp. for 21 samples; its sensitivity was 70% and its specificity was 99% compared with GN subcultures. The Wellcolex agglutination procedure applied to the SF performed better than the agglutination of GN or direct plating procedures and detected 17 of the 39 Salmonella spp. at least 24 h earlier than did culture.


Bovine humoral immune response to Cryptosporidium parvum. Cryptosporidiosis is a diarrheal disease predominantly affecting cattle and humans. Sera from experimentally infected calves and calves of various ages with no histories of exposure were evaluated for immunoglobulin G to Cryptosporidium parvum. An age-associated increase in immunoglobulin G was present in experimental calves and in calves with no histories of infection from 1 to 3, but not > 3, months of age.


Rapid, sensitive detection of Mycoplasma pneumoniae in simulated clinical specimens by DNA amplification. The polymerase chain reaction (PCR) was investigated as a means of diagnosing Mycoplasma pneumoniae infections. The target DNA sequence was a 375-bp segment of the P1 virulence protein. This DNA segment was amplified in pure cultures of five different strains of M. pneumoniae but not in other species of Mycoplasma, Acholeplasma, or Ureaplasma that were tested. Simulated clinical specimens were used to compare PCR, culture, and the gene probe. The sensitivity of PCR was between 1 and 10 organisms. The sensitivity of culture was approximately 10(3) organisms, and the gene probe detected between 10(4) and 10(5) organisms. These results indicate that PCR has significant potential as a rapid, sensitive method for detecting M. pneumoniae in clinical specimens.


Peripheral intramonocytic leishmanias in an AIDS patient. Intramonocytic leishmanias were unexpectedly observed in blood smears of a Spanish AIDS patient. In immunodepressed patients from exposed countries, careful microscopic examination of blood smears should be requested by the clinician in cases of prolonged fever, and biologists must be informed that leishmanias may be fortuitously observed in the peripheral blood.


Early diagnosis of toxoplasmic encephalitis in AIDS patients by dot blot hybridization analysis. Central nervous system toxoplasmosis is a life-threatening infection with a mortality rate of higher than 60%. An early and rapid diagnosis is important for effective treatment of the disease. A new approach for detection of cerebral toxoplasmosis is described here. DNAs extracted from cells in cerebrospinal fluid samples (0.3 to 0.8 ml) of patients suspected of having cerebral toxoplasmosis were analyzed by a dot blot hybridization technique. A highly repetitive DNA sequence of Toxoplasma gondii (ABGTg4) was nonisotopically labelled with digoxigenin-dUTP and used as a specific DNA probe. Four of six patients analyzed gave positive signals in our hybridization assay. Two of them recovered with pyrimethamine-sulfadiazine, a drug recommended for treatment of toxoplasmosis. The other two patients with positive signals died soon after diagnosis. Patients with negative signals were found to suffer from mycobacterial infection (patient 1) or varicella-zoster virus infection (patient 6).


Immunoglobulin G antibody capture enzyme-linked immunosorbent assay: a versatile assay for detection of anti-human immunodeficiency virus type 1 and 2 antibodies in body fluids. In tests on specimens of dried blood, saliva, and urine from 55 human immunodeficiency virus (HIV)-seropositive and 55 HIV-seronegative patients, an immunoglobulin G capture enzyme immunoassay for the detection of antibodies to HIV types 1 and 2, GACELISA, gave 109 of 110, 109 of 109, and 109 of 110 correct results, respectively. This performance, achieved in a laboratory previously unfamiliar with the assay, suggests that GACELISA is a useful new epidemiological tool for the study of HIV infection, equally applicable to all three kinds of specimen.


Aspergillus quadrilineatus, a new causative agent of fungal sinusitis. Aspergillus quadrilineatus was found to be the etiologic agent of pansinusitis in a patient suffering from acute nonlymphoblastic leukemia and who had undergone allogeneic bone marrow transplantation. A. quadrilineatus was cultured from biopsy specimens of the maxillary sinus, and tissue sections with fungal stains showed a necrotic area containing dichotomously branching septate hyphae, which is morphologically consistent with Aspergillus species. The patient was successfully treated with a combination of surgical debridement, granulocyte transfusions, and intravenous administration of amphotericin B-cholesterol sulfate colloidal dispersion. This is the first report of an infection caused by A. quadrilineatus.


Treatment of severe spondylolisthesis in children by reduction and L4-S4 posterior segmental hyperextension fixation. Twenty children with severe lumbosacral spondylolisthesis underwent reduction, posterolateral fusion, and posterior fixation with an L4 to S2, 3, and 4 sublaminar wired rectangular rod to lessen lumbosacral kyphosis, allow early ambulation, and maintain correction. All patients had a postural deformity, 10 had preoperative neurologic findings, and 8 had severe pain. The average percentage of slip improved from 76% preoperatively to 55% postoperatively, and the slip angle improved from 25 degrees to 5 degrees (p < 0.0001). All patients had solid fusion by 6 months and no progression at 43 month follow-up on the average. We conclude that this technique reliably provides partial reduction, solid fixation, and fusion for patients with severe spondylolisthesis while allowing early ambulation. As with any spondylolisthesis reduction technique, neurologic risk should limit this procedure to well-selected patients.


Ambulation after transfer of adductors, external oblique, and tensor fascia lata in myelomeningocele. Forty-seven patients with myelomeningocele underwent triple (adductor, external oblique, and tensor fascia lata) transfers or double (adductor and external oblique) transfers and were retrospectively reviewed. Six patients lost significant neurologic function during the 4-years 6-month follow-up. Thirty-seven of the remaining 41 patients had improved gait pattern. Seventeen required less bracing after the muscle transfers. The need for assistive devices decreased in 21 patients. Twenty-seven were able to ambulate independently postoperatively as compared with seven preoperatively. The muscle transfers are indicated in midlumbar and lowlumbar spina bifida patients to improve hip stability, control, balance, and gait pattern.


Triple osteotomy of the innominate bone in treatment of developmental dysplasia of the hip. Ten children (11 hips) who underwent triple innominate osteotomy between the ages of 11 and 16 years for treatment of symptomatic acetabular dysplasia and who had > 10 years of follow-up since operation were reviewed to determine if satisfactory results reported in an earlier review were maintained. The mean length of follow-up was 12 years (range 10-16 years). All hips were examined roentgenographically, and functional assessment was made with the Iowa hip scoring system. Ten of the 11 hips improved roentgenographically and eight improved functionally after operation. One hip required replacement arthroplasty 16 years after triple innominate osteotomy.


Developmental hip dysplasia potentiated by inappropriate use of the Pavlik harness. Nineteen patients with 28 dislocated hips treated in a Pavlik harness for > 8 weeks with no evidence of hip reduction were identified. After discontinuation of the harness use, reduction was often difficult to obtain and maintain. Thirteen of these patients (17 hips) required operative intervention to obtain stable located hips. In this group of patients, we attempted to identify factors that led to an unusually high number of failures of closed management techniques. Prolonged positioning of the dislocated hip in flexion and abduction potentiates dysplasia, particularly of the posterolateral acetabulum, and increases the difficulty of obtaining a stable closed reduction. Confirmation of satisfactory reduction during treatment with the Pavlik harness is mandatory. Such treatment should be discontinued promptly if reduction is unobtainable.


Proximal femoral osteotomy using the AO fixed-angle blade plate. We describe the technique and results of proximal femoral osteotomy performed with the AO fixed-angle blade plate in 157 hips of 101 pediatric patients. Postoperative immobilization or restriction of activity was not prescribed. Fourteen complications occurred in 11 patients. Use of preoperative antibiotics decreased the rate of infection from 12% to 0%, which was significant. All osteotomies healed by 16 weeks postoperatively with no nonunions, malunions, device failures, or avascular necrosis. Using no postoperative immobilization in these children was successful and resulted in few complications when the appropriate-sized blade plate was used.


Single versus double screw fixation for treatment of slipped capital femoral epiphysis: a biomechanical analysis. Slipped capital femoral epiphyses (SCFE) were created in 10 pair of bovine femurs. Each pair was pinned with a single screw on one side and two screws contralaterally. The specimens were reloaded to failure. Double pin fixation yielded only a 33% increase in stiffness as compared with single pin fixation. Resistance to further slip was not proportional to screw number. The stiffness of neither double nor single screw fixation approximated that of the intact physis. Single screw fixation in slipped epiphyses is recommended because the small gains in stiffness with a second screw do not offset the risk of complications.


Computed tomography for early evaluation of developmental dysplasia of the hip. Computed tomography (CT) provides an axial image of the dysplastic hip. We reviewed 130 CT scans performed after closed reduction for developmental dysplasia of the hip (DDH) in 42 patients (52 hips). We evaluated methods of measuring CT scans and found no described measurement that was predictive of development of avascular necrosis (AVN) or persistent acetabular dysplasia. The CT scan provides an excellent image of the infant hip when the child is in a spica cast and is useful to confirm an adequate reduction. No patient in this series lost reduction after 2 weeks.


Primary epiphyseal transplants and bone overgrowth in childhood amputations. Seventeen children aged newborn to 14 years underwent major through-bone amputations or revision at our two institutions. Ten patients (group 1) had primary autogenous epiphyseal transplants taken from the amputated limb and used to cap the open medullary canal of the residual limb. Seven patients (group 2) did not have epiphyseal transplants. Nine of 10 patients in group 1 (90%) had no problems related to bone overgrowth or delay in prosthetic fitting. In group 2, six of seven patients (86%) had clinically symptomatic bony overgrowth of 20 months after the index amputation on the average. Four patients had surgical revisions. Therefore, provided healthy autogenous donor epiphyses are available, we recommend primary epiphyseal transplants to avoid the complications of bone overgrowth in childhood through-bone amputations.


Bone remodeling after leg lengthening: evaluation with plain radiographs, and computed tomography and magnetic resonance imaging scans. Ten patients who had undergone leg lengthening were monitored with plain radiographs and computed tomography (CT) scans at 3-month intervals. Four of them were examined with magnetic resonance imaging (MRI) scans. When the external fixator was removed, MRI scans could not demonstrate any continuous fat signals in the lengthened region, suggesting the lack of medullary cavity. CT scans disclosed double cortex in three patients and irregular new bone formation in seven. At 6 months, a medullary cavity could be detected. Other aberrations were less evident. Remodeling of new bone material required at least 1 year.


Changes of blood flow of the femoral head after subtrochanteric osteotomy in Legg-Perthes' disease: a serial scintigraphic study. Serial quantitative scintigraphic studies using 99mTc-methylene diphosphonate were performed on 25 hips with Legg-Perthes' disease to evaluate changes in local blood flow of the femoral head after subtrochanteric varizational and/or derotational osteotomy. Studies were made preoperatively and were repeated postoperatively at 2, 4, 6, and 8 weeks at 3 and 6 months, at 1 year, and finally at 2 years after osteotomy. The relative vascularity of the affected femoral head at 2 weeks after osteotomy was significantly decreased as compared with the preoperative value. Thereafter, it increased steadily, reaching the preoperative level by 6 months with no further statistically significant increase at < or = 2 years. Our scintigraphic study showed that local blood flow of the femoral head does not increase significantly after subtrochanteric femoral osteotomy in Legg-Perthes' disease.


Tetracycline labeling as an aid to complete excision of partial physeal arrest: a rabbit model. We determined whether ultraviolet (UV) visualization of tetracycline-labeled bone allows more complete excision of experimentally created physeal arrests while minimizing required bone resection. Lateral distal femoral growth arrests were surgically created bilaterally in eight 6-week-old New Zealand white rabbits, given 3 daily oxytetracycline doses (10 mg/kg); the animals were killed 6 weeks postoperatively. Femurs were excised and dissected free of soft tissue. Ten of 16 operated femurs randomized into two groups had consistent deformity. In group I, arrest was excised with a high-speed burr without, and, in group II, with ultraviolet visualization. In group II, metaphyseal bone and calcified arrest glowed brightly, contrasting well with the epiphyseal plate. All excised specimens were examined under x 9 magnification to assess remaining bone bridges. Excision defects were filled with modeling clay, which was then removed and weighed. Thorough excision was confirmed in each specimen. Resection was easier and less bone usually was resected in the UV group (difference not statistically significant). This technique would best be used in resection of arrests involving small physes such as the distal radius, in which maximal visualization and minimal resection are essential to preserve all possible growth potential. Tetracycline labeling and intraoperative UV visualization of fluorescence allows thorough excision of partial physeal arrest while minimizing required bone resection.


Meniscal cyst and magnetic resonance imaging in childhood and adolescence. We retrospectively reviewed 11 cases of meniscal cyst diagnosed at the Mayo Clinic from 1976 through 1990. Patient age ranged from 4 to 18 years (average 13 years). Two cases were diagnosed on clinical findings alone. Arthrograms were available in five cases. Magnetic resonance imaging (MRI) was used in four cases. These cases illustrate the difficulty in making the diagnosis of meniscal cyst. MRI is a useful tool for diagnosing meniscal cyst and aids in surgical planning. Because of the extensive differential diagnosis of the meniscal cyst, we recommend MRI in preoperative evaluation of soft tissue masses about the knee.


Physeal reconstruction with blocks of cartilage of varying developmental time. Reconstruction of physeal regions excised from the distal femoral chondroepiphysis was performed in a murine model. The excised regions were replaced with analogous blocks of syngeneic tissue. Vascularity, matrix formation, and cell division were assessed as well as growth. The blocks of tissue did not produce growth consistently, although individual specimens did show some growth. Tritiated thymidine incorporation by the proliferative zone of the transplanted block of tissue was not normal. The blocks of tissue did receive a nutritional supply from the host and did continue matrix formation after transplantation.


Neuropathic foot ulceration in patients with myelodysplasia. To determine if a significant relationship existed between type of operation and eventual development of pedal skin breakdown in a spina bifida patient population, 72 feet in 36 ambulatory patients with low lumbar or sacral myelomeningocele were followed for an average of 14 years 5 months. Using a clinical classification for foot suppleness and position, we determined that foot rigidity, nonplantigrade position, and performance of surgical arthrodesis were clinical indicators that had a strong statistical relationship with eventual development of neuropathic skin changes.


Post-traumatic stress disorder and coping in veterans who are seeking medical treatment. The present study examined psychological coping styles and mental health treatment histories in veterans with PTSD. This study also served as a replication and extension of an earlier investigation that assessed the prevalence of PTSD in World War II, Korea, and Vietnam combat veterans who were seeking medical treatment. Thirty-six combat veteran medical patients were compared to 38 war-era controls. Nearly a third of the combat veterans met psychometric criteria for PTSD; none of the controls met these criteria. Both PTSD-positive subjects and mental health treatment seekers showed a significantly greater use of emotion-focused coping. Results also showed that Vietnam combatants were more likely to have received individual mental health treatment. These findings and their treatment implications are discussed.


Supracondylar fractures of the humerus: a prospective study of percutaneous pinning. In 1981, we designed a protocol to treat displaced supracondylar fractures using a modified technique of closed reduction and percutaneous pinning. After the fracture was internally fixed, intraoperative anteroposterior (AP) radiographs of each distal humerus were compared. The reduction and pinning was accepted only if the radiographs demonstrated that Baumann's angle was < or = 4 degrees of that on the normal side. Seventy-one patients had clinical and radiographic evaluations at an average of 2 years 6 months after the operation. According to Flynn's criteria, the results were satisfactory in 70 patients and unsatisfactory in one. No patient had a cubitus varus deformity at follow-up evaluation.


Are the physical and TABP risk factors for heart disease unique to CHD? Scores of 40 hospitalized CHD patients on 11 Type A-related and 7 physical CHD risk factors were compared to those of 40 hospitalized non-CHD patients. Family history for CHD was the only physical risk factor for which a significant difference was found. CHD patients scored significantly higher on all seven interview-measured Type A and Type A subcomponent variables. Only two of the four Jenkins Activity Survey-measured Type A variables produced significant differences, with one higher for non-CHD subjects. It was concluded that some CHD risk scores also may be associated with other diseases, to the experience of being seriously ill, and/or to the experience of hospitalization.


Tibial physeal changes in renal osteodystrophy: lateral Blount's disease. In a group of 27 children with chronic renal failure and renal osteodystrophy, we identified eight patients with significant involvement of the proximal tibial physis (15 knees). The radiographic changes observed were analogous to those observed in tibia vara (Blount's disease) but involved the lateral physis rather than the medial physis. This observation is offered as further evidence that such changes are related to eccentric weight-bearing rather than alternative etiologies such as trauma, infection, or heredity.


A comparison of 16 systems to diagnose schizophrenia. Sixteen diagnostic classification systems were compared to determine the degree of agreement on the diagnosis of schizophrenia. Data were collected by a structured interview method with 284 patients, 52 first admissions, and 196 readmissions. The kappa coefficient was used to calculate interrater reliability and concordance of the systems. Adequate reliability was found for 12 of the systems. However, concordance was generally low; 11 of 120 combinations reached kappa of .40 or higher for the total sample, 16 of 120 for first admissions, and only 6 of 120 for readmissions. These results indicate the restriction of generalization and raise questions as to the empirical validity of the construct of schizophrenia.


Brachymetatarsia of the first metatarsal treated by surgical lengthening. The first metatarsal of six feet in four patients was surgically lengthened. Brachymetatarsia was caused by a congenital defect in two patients, nonunion after metatarsal osteotomy in one patient, and premature physeal closure associated with pin placement across the physis in one patient. In each patient, middiaphyseal osteotomy was performed, pins were placed into the proximal and distal metatarsal fragments, and an external distracting device was attached. After distraction, a fibular graft was inserted and the device was removed. The percentage of metatarsal length gained from the lengthening averaged 36% (range 12-68%). The fibular graft healed in the lengthened position in all patients.


Personality predictors of bulimic behavior and attitudes in males. This study was an investigation of personality factors associated with bulimic behaviors and attitudes in males. Various measures that have been noted as predictors of female bulimia were tested on 96 college students. Chronic dietary restraint and interoceptive awareness were the strongest predictors of more maladaptive eating patterns. The results suggest that maladaptive eating patterns in males and females may share a common set of features.


Preliminary results of orthotic treatment of pectus deformities in children and adolescents. Fifty-five children and adolescents with different types of pectus deformities and a minimum follow-up of 1 year were assessed to determine the effectiveness of nonoperative treatment with a dynamic chest compressor orthosis. Thirty-seven patients were treated and compared with 18 untreated patients. The treated patients had a better outcome. Reshaping of the contour of the chest wall was observed in 21 patients with inferior and lateral types of pectus carinatum. The dynamic chest compressor has minimal morbidity and is a valuable treatment option of pectus deformities in the growing period.


Differentiating symptom clusters of borderline personality disorder. This study is an attempt to delineate symptom clusters that may be considered most distinctive of patients diagnosed with borderline personality disorder (BPD). Medical records were examined to assess the extent to which each of the eight DSM-III-R BPD criteria was present in 89 psychiatric in-patients diagnosed with BPD. Structural analysis revealed three symptom clusters that can explain symptomatology for a majority of the sample. BPD patients can be identified initially by a core factor and separated subsequently into several BPD subtypes based on the patients' remaining symptomatology. A hierarchical diagnostic scheme for delineating BPD subtypes is proposed, and the implications of these findings for a theoretical separation of several BPD subtypes are discussed.


The Missouri Children's Behavior Checklist behavioral classification system: a construct validity study with nonreferred children. This study provides validity information about the Missouri Children's Behavior Checklist (MCBC) classifications system with nonreferred children. MCBC behavior patterns of 41 children were related to DSM-III symptomatology ascertained through a structured clinical interview, the Child Assessment Schedule, conducted with the mother. The findings indicated that considering the Undifferentiated Disturbance pattern as an indicator of poor adjustment may be unwarranted with nonreferred children.


Do computer-administered Minnesota Multiphasic Personality Inventories underestimate booklet-based scores? We ran meta-analyses that compared the Minnesota Multiphasic Personality Inventory scale scores produced in 770 booklet and 762 computer administrations described by nine studies. While most of the differences were small, the computer-based scores significantly underestimated their booklet counterparts on 8 of the 13 scales. The average underestimate was about three-quarters of a T-score point and accounted for around 4% of the reliable variance. These findings suggest that separate norms and profile sheets for computer-administered MMPIs might be helpful.


Osteoarticular complications of childhood brucellosis: a study of 57 cases in Saudi Arabia. Fifty-seven (36%) of a cohort of 157 children with brucellosis from Saudi Arabia had arthritis. Most gave a history of contact with farm animals or drinking unpasteurized milk. Associated features included pyrexia, arthralgia, hepatosplenomegaly, and lymphadenopathy. A subacute presentation with peripheral oligoarthritis predominantly affecting hips or knees was common. Specific chemotherapy resulted in rapid defervescence followed by slower resolution of the arthritis. Children with osteoarticular brucellosis had a higher relapse rate and a longer hospital stay. Supervised combination chemotherapy for at least 6 weeks was effective in preventing relapse. A brucellar etiology should be considered in any child from an endemic area who has osteoarticular manifestations.


Long-term effects of neonatal bone and joint infection on adjacent growth plates. Review of children with physeal damage from neonatal infection other than the hip at Winnipeg Children's Hospital showed that six patients had residual growth interference from adjacent infection in the bone or joint. Several of the infections involved multiple joints, with growth plate arrest occurring in the distal femoral growth plate in four, in the proximal humerus in four, in the proximal femur in two, in the distal radius in one, and in the distal humerus in one. Although the initial infection was frequently believed to be successfully treated in the neonate, the clinical effect of these infections on the growth plate was not fully appreciated in five of the infants until the children reached a mean age of 9 years. Because growth abnormalities in physeal bars may not be clinically evident for several years after the initial infection has been treated, we recommend that children with bone and joint infections occurring in the first month of life be followed to skeletal maturity, observing the adjacent physis for late tethering.


Depression, problem-solving ability, and problem-solving appraisals. Behavioral analytic methods were used to create a college student version of the Means Ends Problem Solving Procedure (MEPS). This instrument then was administered with measures of perceived problem-solving ability to depressed and nondepressed students to determine whether differences exist in both problem-solving ability and problem-solving appraisal. Analyses revealed that depressed subjects had more negative expectations and lower appraisals of their problem-solving ability. However, the groups did not differ in terms of the actual quality of their behavioral solutions to interpersonal, intrapersonal, and emotional problem situations. The results are interpreted as support for the role of cognitive factors in depression and problem-solving across a range of problem-solving situations.


Left cerebral dominance for bilateral simultaneous sensory stimulation? Hom and Reitan (1982) reported findings with respect to sensorimotor deficits in groups with lateralized cerebral lesions, but evaluated the results only within a framework that related cerebral dominance to deficits that resulted from lateralized lesions. Some of the variables they studied, however, depend upon evidence of retention of functions by one hemisphere under conditions of distracting input. While the right cerebral hemisphere appears to be dominant for tactile-perceptual abilities in terms of resulting deficits, the present study suggests that the left cerebral hemisphere is dominant for certain non-language functions that reflect maintenance of sensory-perceptual abilities under conditions of distracting stimuli.


Intralesional infiltration of corticosteroids in localized Langerhans' cell histiocytosis. The approaches to treatment of Langerhans' cell histiocytosis (LCH) have been as varied as the clinical presentation of the disease. The clinical course of localized LCH of bone is generally benign, and it tends to heal spontaneously in a period of months to years. If treatment is required, the disease can be controlled by local measures such as surgical curettement, low-dose irradiation, or intralesional infiltration with corticosteroids. We reviewed 15 children treated with intralesional infiltration of corticosteroids, either primarily for disease of bone (8 patients) or as adjunctive therapy for disseminated LCH (7 patients). Two patients developed complications as a result of this treatment method.


Anatomy of self-injurious, stereotypic, and aggressive movements: evidence for involuntary explanation. Self-injurious movements, common in persons diagnosed with Tourette syndrome, or mental retardation, are typically difficult to eliminate. The author considers the possibility that certain self-injurious movements are involuntary phenomena. An anatomical analysis of high-frequency movements in a patient with severe head slapping is presented by tracing the muscles and nerves involved. The median nerve innervates muscles that bring the hand/arm to the head and also muscles that control this patient's other frequent movements, viz., pill-rolling, thumb-gouging, wrist-flapping, and pinching the neck or cheek. Other patients underwent similar investigation: one who headbangs, one who hits out repetitively, and one with non-injurious stereotypic movements. An anatomical explanation suggests that certain self-injurious, aggressive, and stereotypic movements are involuntary muscle contractions that reflect abnormal innervation along specific nerves.