Identification of bacteria recovered from clinical specimens by 16S rRNA gene sequencing.

The sequence of the 16S rRNA gene has been used extensively for phylogenetic classification, identification, and genotypic typing of bacteria. Identification of bacterial isolates by 16S rRNA gene sequencing, though generally performed in reference laboratories, has been recently introduced for routine use in clinical laboratories to identify isolates that cannot be identified by conventional methods. Described in this report is the use of 16S rRNA gene sequencing to identify uncommon bacteria, or bacteria with unusual phenotypic properties, with four brief case presentations to illustrate its clinical application. The feasibility, usefulness and limitations of performing this approach in the clinical laboratory are also discussed.

Comparison of six commercial DNA extraction kits for recovery of cytomegalovirus DNA from spiked human specimens.

We evaluated six commercially available DNA extraction kits for their ability to recover DNA from various dilutions of cytomegalovirus (CMV) added to four different specimens: bronchoalveolar lavage, cerebral spinal fluid, plasma, and whole blood. The kits evaluated included the Puregene DNA isolation kit (PG), Generation Capture Column kit, MasterPure DNA purification kit, IsoQuick nucleic acid extraction kit, QIAamp blood kit, and NucliSens isolation kit (NS). All six kits evaluated effectively removed PCR inhibitors from each of the four specimen types and produced consistently positive results down to a spiked concentration of 200 PFU of whole CMV per ml. However, the NS and PG resulted in the most consistently positive results at the lowest concentrations of spiked CMV (4 and 0.4 PFU/ml) and, in this evaluation, offered the most sensitive methods for extracting CMV DNA from the four different spiked specimens. Processing time and cost were also evaluated.

Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene.

Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.

Development of a PCR assay for diagnosis of Pneumocystis carinii pneumonia based on amplification of the multicopy major surface glycoprotein gene family.

We have evaluated a PCR technique using primers based on Pneumocystis carinii major surface glycoprotein (MSG) genes, a multicopy gene family, for utility in detection of P. carinii in BAL and oropharyngeal samples obtained from immunosuppressed patients. These primers were able to detect P. carinii DNA in as little as 16 fg of genomic DNA. PCR using MSG primers detected P. carinii DNA in 7 smear-positive BAL samples (100% sensitivity), and found no P. carinii DNA in 12 smear-negative BAL samples (100% specificity). Mitochondrial ribosomal RNA (mrRNA) primers, commonly used in PCR studies of PCP, detected P. carinii in six of seven positive samples (85.7% sensitivity) and none of 12 were negative samples (100% specificity). Diagnosis of PCP by amplification of 81 oropharyngeal samples using MSG primers had a 50% sensitivity (4/8) and 96% specificity (70/73). PCR with mrRNA primers was 37.5% sensitive (3/8) and 100% specific (73/73). All three false-positive MSG results showed a very low intensity on Southern hybridization. PCR using MSG gene primers should prove valuable in the diagnosis of PCP.

Long-term interferon alpha maintenance therapy for chronic hepatitis C infection in a patient with common variable immune deficiency.

A 46-year-old woman with common variable immune deficiency acquired acute non-A, non-B hepatitis from contaminated intravenous gamma globulin in 1983. For 6 years she had fluctuating elevations of her serum aminotransferase levels. In 1990 her serum was documented to be hepatitis C virusribonucleic acid positive by polymerase chain reaction, and her liver biopsy revealed chronic hepatitis with early cirrhosis (Knodell score, 15 points). Hepatitis C virus genotyping indicated that she had been infected with the type 3 genotype. She subsequently underwent treatment with interferon alpha (IFN-alpha) for 1 year and experienced biochemical, virologic, and histologic (Knodell score, 9) suppression. She was continued on maintenance therapy for an additional 7 years, with sustained biochemical and virologic suppression. During the sixth year of therapy, complications of portal hypertension were noted with mild ascites and eventually bleeding esophageal varices. This case report documents a favorable biochemical, virologic, and histologic response to IFN-alpha therapy in this setting; supports the notion that the natural progression of hepatitis C virus infection may be more aggressive in patients with common variable immune deficiency; and, although complications of portal hypertension eventually occurred, the suppressive maintenance IFN therapy may have delayed their onset. The future establishment of the long-term effects of IFN therapy on important clinical outcomes is necessary to understand better its therapeutic benefit in chronic hepatitis C infection.

An uncommon Helicobacter isolate from blood: evidence of a group of Helicobacter spp. pathogenic in AIDS patients.

An unusual Helicobacter sp. was isolated from the blood of a human immunodeficiency virus (HIV)-infected patient. This organism had spiral morphology, with single amphitrichous flagella, and was negative for hippurate hydrolysis, production of urease, and reduction of nitrate. 16S rRNA gene sequence analysis verified that the isolate was a species of Helicobacter, most closely related to an undescribed Helicobacter-like isolate from Vancouver, British Columbia, Canada, and to Helicobacter westmeadii, a recently described species from Australia. Both organisms had also been isolated from the blood of HIV-infected patients. These blood isolates, along with Helicobacter cinaedi, form a cluster of closely related Helicobacter spp. that may represent an emerging group of pathogens in immunocompromised patients.

Management of a Sabiá virus-infected patients in a US hospital.

OBJECTIVE: To describe the hospital precautions used to isolate a Sabiá virus (arenavirus: Arenaviridae)-infected patient in a US hospital and to protect hospital staff and visitors. DESIGN: Investigation of a single case of arenavirus laboratory-acquired infection and associated case-contacts. SETTING: A 900-bed, tertiary-care, university-affiliated medical center. PATIENTS OR OTHER PARTICIPANTS: The case-patient became ill with Sabiá virus infection. The case-contacts consisted of healthcare workers, coworkers, friends, and relatives of the case-patient. INTERVENTION: Enhanced isolation precautions for treatment of a viral hemorrhagic fever (VHF) patient were implemented in the clinical laboratory and patient-care setting to prevent nosocomial transmission. The enhanced precautions included preventing aerosol spread of the virus from the patient or his clinical specimens. All case-contacts were tested for Sabiá virus antibodies and monitored for signs and symptoms of early disease. RESULTS: No cases of secondary infection occurred among 142 case-contacts. CONCLUSIONS: With the frequency of worldwide travel, patients with VHF can be admitted to a local hospital at any time in the United States. The use of enhanced isolation precautions for VHF appeared to be effective in preventing secondary cases by limiting the number of contacts and promoting proper handling of laboratory specimens. Patients with VHF can be managed safely in a local hospital setting, provided that appropriate precautions are planned and implemented.

Optimal activation of isopsoralen to prevent amplicon carryover.

We compared the efficiencies of activation of the photochemical isopsoralen compound 10 and its resulting amplicon neutralizations under conditions with a UV transilluminator box at room temperature (RT) and a HRI-300 UV photothermal reaction chamber at RT and at 5 degrees C. Our data suggest that use of the HRI-300 reaction chamber at 5 degrees C results in a statistically significantly higher degree of amplicon neutralization.

Detection of Legionella by PCR in respiratory specimens using a commercially available kit.

Using polymerase chain reaction (PCR) for the detection of pathogens that are difficult to grow, such as Legionella species, may reduce difficulties encountered with culture and immunofluorescent staining. We evaluated a commercial PCR and hybridization kit, designed for environmental samples, for the detection of Legionella in respiratory specimens. Sixteen Legionella species cultures tested positive with the Perkin Elmer Legionella EnviroAmp Amplification and Detection kits (Perkin Elmer, Foster City, Calif). The assay detected as few as 100 colony-forming units per milliliter of spiked bronchoalveolar lavage (BAL) fluid, and no false-negative results were obtained. PCR inhibition by blood in the specimens was removed by washing pelleted specimens in sterile distilled water. Of 126 specimens screened with the kit, 1 induced sputum and 3 BAL specimens were positive by PCR. All 4 were validated as true-positive results by culture or serologic testing. The entire PCR and hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 12 days, and serologic conversion may not be demonstrated for weeks. Molecular techniques based on direct extraction and amplification of DNA from respiratory specimens nay be useful for the timely diagnosis of legionellosis.

Hepatitis C virus transmission by intravenous immunoglobulin.

The polymerase chain reaction was used to detect hepatitis C virus infection in patients who had previously been reported to have developed non-A, non-B hepatitis after intravenous immunoglobulin infusion. Of the 33 patients with intravenous immunoglobulin associated non-A, non-B hepatitis studied, HCV RNA could be detected in 15 out of 17 patients (88%) who were HCV RNA negative prior to the development of non-A, non-B hepatitis after implicated intravenous immunoglobulin batches. Similarly, eight out of nine patients (89%) in whom no sample was available for polymerase chain reaction testing prior to intravenous immunoglobulin therapy, had detectable HCV RNA after intravenous immunoglobulin therapy with intravenous immunoglobulin batches implicated in non-A, non-B hepatitis transmission. Two of the three intravenous immunoglobulin preparations implicated in non-A, non-B hepatitis transmissions that were available for polymerase chain reaction testing also had detectable HCV RNA, confirming that hepatitis C virus is the implicated virus in intravenous immunoglobulin-associated non-A, non-B hepatitis.

Immunoglobulin A antibodies directed against Campylobacter jejuni flagellin present in breast-milk.

We studied the relationship between IgA anti-campylobacter flagellin antibodies in breast milk samples and protection of breastfed infants living in a rural Mexican village from campylobacter infection. There were fewer episodes of campylobacter infection (symptomatic and asymptomatic combined) in infants breastfed with milk containing specific anti-flagellin antibodies (1.2/child/year, 95% CI 0.6-1.8) versus non-breastfed children (3.3/child/year, 95% CI 1.8-4.8; P < 0.01). Infants breastfed with milk that was anti-flagellin antibody negative by ELISA also had fewer episodes of infection compared with non-breastfed children, but the difference did not reach statistical significance (1.8/child/year, 95% CI 0.7-3.0 versus 3.3/child/year, 95% CI 1.8-4.8, P > 0.05). Breastfeeding has a protective effect against campylobacter infection and is associated with the presence of specific antibodies directed against campylobacter flagellin.

CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency.

Common variable immunodeficiency (CVI) is characterized by hypogammaglobulinemia and recurrent bacterial infections due to failure of CVI B cells to differentiate in vivo into immunoglobulin-secreting plasma cells. We hypothesized that T-cell dysfunction resulting in abnormal contact-mediated B-cell activation may play a prominent role in the failure of CVI B cells to produce specific antibody. We have previously shown that B-cell proliferation and IgE production after stimulation with anti-CD40 and interleukin (IL) 4 were normal in 22 CVI patients evaluated, indicating that CVI B cells respond to signals delivered via CD40. Here we report that CD40 ligand (gp39) mRNA expression by activated lymphocytes from CVI patients (n = 31) as a group was significantly depressed (P < 0.0001) compared with normal controls (n = 32). gp39 mRNA expression by activated lymphocytes from 13 CVI patients fell below the normal control range. T-cell surface expression of functional gp39 protein was correspondingly low in those patients with gp39 mRNA levels below normal control range and normal in patients with gp39 mRNA levels within normal control range. In CVI patients as a group, gp39 mRNA levels correlated with IL-2 mRNA levels (P < 0.002, r = 0.6) and production (P < 0.001, r = 0.7) but not with gene expression or production of other lymphokines evaluated, suggesting an as-yet-undetermined association between gp39 and IL-2 gene regulation. Of the 13 patients whose activated T cells exhibited gp39 mRNA expression below the normal control range, 2 had normal T-cell-derived lymphokine production, whereas the remaining 11 exhibited broader T-cell dysfunction, resulting in IL-2 deficiency, and in some patients deficient production of other lymphokines as well, reflecting a heterogeneity in the underlying mechanisms leading to depressed gp39 expression in these patients. The observation that both gene and surface expression of gp39 by activated T cells is depressed in a subgroup of CVI patients suggests that inefficient signaling via CD40 may be responsible, in part, for failure of B-cell differentiation in these patients.

The role of adhesion molecules in the regulation of antibody responses.

We used the T-cell-dependent antigen, bacteriophage (phage) phi X174, to study antibody synthesis in patients, guinea pigs, and dogs with complement component deficiencies (C2, C4, C3, C7); in patients with adhesion molecule deficiencies (CD11/CD18 or sialylated Lewisx); and in patients with the hyper IgM (HIM) syndrome (absence of functional gp39 expression by activated T cells). Patients and guinea pigs deficient in early complement components, patients deficient in CD11/CD18, and patients lacking functional gp39 on activated T cells responded to repeated phage immunizations with depressed antibody titers, lack of or inadequate amplification, and failure to switch from IgM to IgG, suggesting that defective T-cell-B-cell interaction is the cause of the antibody deficiency observed in these patients.

Interaction of group B streptococcal opacity variants with the host defense system.

Group B streptococci (GBS) demonstrate high-frequency phase variation of colony opacity. Colony opacity is a function of chain length, with opaque colonies consisting of GBS that form longer chains. Because opaque variants do not grow on standard streptococcal media, the role of opacity variation in GBS infection has not been studied. We have isolated stable variants from type III GBS that are either transparent (variants 1.2 and 1.3) or opaque (variants 1.1 and 1.5). In this study, we evaluated the interactions of these variants with different components of the host immune system both in vitro and in vivo. Opaque GBS were less immunogenic than transparent GBS. Opaque GBS were more susceptible to killing by polymorphonuclear neutrophils (PMNs) and could induce a chemiluminescent response of PMNs in the absence of antibody (Ab) or complement. Transparent GBS did not induce neutrophil chemiluminescence in the absence of Ab and complement. However, in the presence of Ab and complement, transparent GBS induced a stronger chemiluminescent response than did opaque GBS. Scanning electron micrographs of PMNs and GBS demonstrated differences in the attachment and engulfment of the different variants by the PMNs as well as different effects of the GBS on the PMNs themselves. Interactions with complement were affected by GBS opacity as well, with opaque variant 1.1 initiating complement activation in the absence of any Ab. The virulence of the GBS opacity variants was studied in vivo by inoculation of graded numbers of GBS into newborn mice. Transparent variants 1.2 and 1.3 were most virulent, with variant 1.1 intermediate and variant 1.5 minimally virulent. However, in mixed infections, variant 1.5 greatly enhanced the virulence of small numbers of transparent GBS. These results indicate that the opacity status of GBS can influence the interaction between the GBS and the host immune system.

Human neutrophil response to recombinant neisserial Opa proteins.

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.

Intravenous immunoglobulin therapy for active, extensive, and medically refractory idiopathic ulcerative or Crohn's colitis.

To determine whether intravenous immunoglobulin produces demonstrable clinical improvement in patients with refractory idiopathic inflammatory bowel disease, a pilot, open-label, nonrandomized, safety and therapeutic efficacy study was carried out at a tertiary care referral medical center. Twelve consecutive patients with refractory idiopathic colitis (nine ulcerative colitis, three Crohn's colitis) who were reluctant to receive immunosuppressive therapy or have surgical intervention were referred by physicians not participating as investigators in this study. Eleven patients were symptomatic for at least 6 months, with endoscopically moderate or severe mucosal inflammation despite medical therapy, including systemic corticosteroids in all cases, and one patient was dependent on oral prednisone to remain in clinical remission. Ten patients had extensive colitis, six of whom had pancolitis and four of whom had colitis extending to the hepatic flexure or transverse colon. Nine patients required hospitalization for treatment of colitis. Intravenous immunoglobulin was administered in one or two induction phases (2 g/kg over 2 or 5 days), followed by a maintenance phase (200-500 mg/kg every 2 wk for 12 or 24 wk). Tapering of systemic corticosteroid therapy was attempted, whereas other medications for idiopathic colitis were continued. Treatment response was assessed clinically and by colonoscopy with multiple biopsies whenever possible. Immunoglobulin therapy was well-tolerated and did not produce any biochemical abnormalities. In six patients who completed the treatment protocol, mean reductions +/- SE were achieved in subjective symptoms as quantified by a colitis activity score, 13.3 +/- 1.2 to 4.7 +/- 0.9 (p less than 0.001), and daily mg dose of prednisone, 41.7 +/- 8.0 to 1.9 +/- 1.2 (p less than 0.001). For all 12 patients, statistically significant reductions were achieved in the colitis activity score and daily prednisone dose. Of five patients who completed the treatment protocol and improved clinically, four underwent post-treatment colonoscopic and biopsy evaluations and had unequivocal reductions in the intensity of colonic mucosal inflammation. Three patients who had objective improvement with intravenous immunoglobulin experienced relapses of colitis after discontinuation of this therapy. Six patients did not complete the treatment protocol, two of whom required surgical intervention and four of whom withdrew to undergo colectomy electively. Intravenous immunoglobulin may be beneficial in subsets of patients with idiopathic colitis. The results of our pilot study justify the undertaking of a prospective, randomized controlled trial to determine the efficacy of intravenous immunoglobulin in carefully defined subsets of patients with idiopathic inflammatory bowel disease.