Use of Roche AMPLICOR Mycobacterium tuberculosis PCR in Early Diagnosis of Tuberculous Meningitis

Several nucleic acid-based amplification tests are available for the detection of Mycobacterium tuberculosis, but few data are available on their use in the diagnosis of tuberculous meningitis (TBM). We performed a prospective study to assess the Roche AMPLICOR Mycobacterium tuberculosis PCR test (TB AMPLICOR) for use in the diagnosis of TBM and compared it with direct Ziehl-Neelsen staining of smears, radiometric culture for M. tuberculosis, and clinical and cerebrospinal fluid (CSF) findings. Eighty-three CSF specimens collected from 69 patients with suspected meningitis in South Africa were tested by TB AMPLICOR. On the basis of clinical and laboratory findings, 40 of these patients were treated for TBM and 29 patients were not treated for TBM. Ten CSF samples from 10 patients were positive by TB AMPLICOR. Seven of these 10 patients were classified as having definite TBM, 2 were classified as having probable TBM, and 1 was classified as having possible TBM. The sensitivity of TB AMPLICOR for detecting cases of definite and probable TBM in patients from whom CSF specimens had been collected less than 10 days into antituberculosis treatment was 60.0%. Specimens from all 29 patients not treated for TBM were negative by the TB AMPLICOR, giving a 100% specificity. TB AMPLICOR is therefore more sensitive than the combination of Ziehl-Neelsen staining of smears and radiometric culture for M. tuberculosis and is a rapid and highly specific diagnostic test for TBM.In the past few years there has been a global increase in the incidence of tuberculosis (TB), with an increase in the number of notifications of TB in England and Wales (7), other European countries (20), and the United States (2). Tuberculous meningitis (TBM) is rare in developed countries, but Mycobacterium tuberculosis is an important cause of meningitis in many developing countries, where facilities to confirm it as the cause of meningitis are least available. Without treatment, death occurs in virtually all patients with TBM, while delay in treatment results in a considerable risk of irreversible neurological damage. Therefore, rapid diagnosis is important but difficult, since the spectrum of disease is wide and abnormalities of the cerebrospinal fluid (CSF), although usually present, are very variable. Direct smears of CSF for acid-fast bacilli (AFB), although virtually diagnostic, are usually positive in fewer than 10% of cases of TBM (11, 26), while culture for M. tuberculosis takes up to 8 weeks and is also often negative. A clinical response to antituberculosis treatment is usual, but patients with TBM may deteriorate on appropriate treatment. Hence, a rapid and sensitive test is required for the diagnosis of TBM.A number of nucleic acid-based amplification tests, most of them based on PCR, have been developed for the detection of M. tuberculosis in clinical specimens. Although considerable data are now available on their use with respiratory specimens in the diagnosis of pulmonary tuberculosis, little is known of their role in diagnosing TBM from specimens of CSF. Only six published studies (in the English-language literature) of M. tuberculosis PCRs for the diagnosis of TBM have included more than 20 patients with suspected or confirmed TBM (Table ​(Table1).1). Those studies used primers targeting either the MPB 64 protein (15, 22, 23) or the insertion sequence IS6110 (12, 16, 19). Overall sensitivities for the diagnosis of TBM ranged from 33% (19) to 90.5% (15), with specificities ranging from 88.2% (23) to 100% (15, 19). From these data it is clear that the role of PCR in the diagnosis of TBM is undefined.

TABLE 1

Studies of TB PCR for the diagnosis of TBM

Microevolution of a Standard Strain of Cryptococcus neoformans Resulting in Differences in Virulence and Other Phenotypes

Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.Cryptococcus neoformans is a frequent cause of life-threatening meningoencephalitis in immunocompromised patients, such as those with human immunodeficiency virus infection or lymphoproliferative disorders or patients who have undergone organ transplantation or immunosupressive therapies (30). One of the striking characteristics of this fungal pathogen is its ability to cause persistent infections. The virulence characteristics that allow C. neoformans to persist in the host are poorly understood, but there is accumulating evidence that cryptococcal strains can undergo genetic changes in vitro and in vivo (16, 45). This phenomenon may contribute to survival in the host by providing a means to evade host defenses.Previous studies have documented genetic and phenotypic instability among C. neoformans strains from clinical and environmental sources. Genetic instability has been demonstrated by electrophoretic karyotype changes after murine passage (16) and in serial isolates from individual patients (2, 44, 45). Another indication that C. neoformans can undergo rapid genetic change was provided by Block et al. (1), who reported that the development of 5-fluorocytosine resistance in vitro was associated with a high rate of mutation, i.e., 1.2 × 10⁻⁷ to 4.8 × 10⁻⁷ mutations per cell division. In addition, structural variation in the capsular polysaccharide has been observed among serial isolates from patients with recurrent meningitis and in one isolate of a serotype C strain in vitro (7, 8). Passage of environmental isolates in mice has been shown to alter the sterol content and composition and antifungal drug susceptibility (9). More recently, it was shown that one C. neoformans strain also displays differences in colony morphology suggestive of the phenomenon of phenotypic switching described for Candida albicans (17). Hence, there is strong circumstantial evidence from multiple studies that under certain conditions, C. neoformans strains can undergo genetic and phenotypic changes.During the course of studying the murine immune response to C. neoformans, we noted that one isolate of strain ATCC 24067 became attenuated with respect to virulence following continuous passage in vitro. This observation raised the question of whether strain ATCC 24067 could change with time under laboratory conditions. ATCC 24067 (also known as 52D) is one of the best characterized C. neoformans strains and has been the subject of intensive study for several years by various laboratories (Table ​(Table1).1). We hypothesized that the attenuation of our ATCC 24067 stock was the result of unknown selection pressures during in vitro maintenance, and we proceeded to investigate this phenomenon further. Our approach was to compare strains from multiple laboratories, with the premise that minor differences in laboratory handling could result in different selection pressures that may lead to emergence of new variants. Consistent with this hypothesis, we found that some isolates from the various laboratories had different phenotypes, including virulence, implying that strain ATCC 24067 can change with time. These results have important implications for pathogenesis, comparison of results obtained in different laboratories, and maintenance of C. neoformans strains.

TABLE 1

Summary of studies that have employed C. neoformans ATCC 24067 (or 52D)

Primary Epitopes of Chicken Egg Yolk Antibodies to Peptidophosphogalactomannan

Egg yolks from hens immunized with peptidophosphogalactomannan (pPGalManⁱⁱ), which contains 10 phosphocholine diester residues and is secreted by Penicillium fellutanum, contain antibodies against 5-O-β-d-galactofuranosyl epitopes. These epitopes were the only significant determinants in pPGalManⁱⁱ. Approximately 60-fold less pPGalManⁱⁱ (1.6 μM galactofuran chains) was required for 50% inhibition than galactofurano-oligosaccharides or pPGalMan containing two galactofuranosyl residues per chain.Filamentous fungi produce soluble extracellular polysaccharides and glycopeptides (1, 9, 10, 14, 19, 21, 23). Many of these polymers have active antigenic determinants (3, 14, 17, 20, 27). Penicillium fellutanum (formerly Penicillium charlesii) peptidophosphogalactomannans (pPGalMan; M_w, 25,000 to 70,000) (9, 19, 21, 23, 25, 26, 32) contain a mannan with about 80 α-1,2- and α-1,6-mannopyranosyl residues and 12 small manno-oligosaccharidyl units, each attached to a 3-kDa peptide (Fig. ​(Fig.1).1). Eight to ten 5-O-β-d-galactofuranosyl-containing chains with 2 to 20 residues branch from the mannan. pPGalManⁱⁱ and pPGalManⁱⁱⁱ (26, 31) contain approximately 10 and 2 phosphocholine diester residues, respectively, and a variable number of galactofuranosyl-6-O-phosphodiester residues (5). Open in a separate windowFIG. 1Diagram of pPGalMan. The mannopyranosyl residues in each tetrasaccharide are attached by α-1,2 linkages and the tetrasaccharides are attached by α-1,6 linkages. The mannan is attached to the peptide by an O-glycosidic linkage to a seryl residue. Manno-oligosaccharides are attached to seryl and threonyl residues. An average of one galactan chain branches from each manno-octasaccharide and one phosphocholine phosphodiester is attached to C-6 of a mannopyranosyl residue.Sera from rabbits immunized with whole-cell preparations from P. fellutanum reacted with galactofuranosyl-containing heteropolysaccharide (20). Sera from guinea pigs injected with purified pPGalMan conjugated to bovine gamma globulin reacted weakly to manno-oligosaccharides of pPGalManⁱⁱ (11) and were unreactive to galactofuranosyl residues. Soluble pPGalMan did not elicit antibody in any of several species. This preparation, pP(Gal₂)Man, was later shown to contain an average of two galactofuranosyl residues per galactan chain (unpublished data).Antisera from rabbits immunized with extracellular polysaccharides of Penicillium sp. cell walls react with synthetic β-d-galactofuranosyl-containing oligosaccharides (17). The 5-O-β-d-galactofuranosaccharides resulted in the most inhibition.Antibodies that react specifically with furanosyl residues of parasites are of increasing clinical importance (4, 6–8, 14–16, 22, 28–30).The purpose of this investigation was to determine if stable antibody could be elicited from purified glycopeptides, such as pPGalManⁱⁱ in phosphate-buffered saline (PBS) without adjuvant, and to determine the polymers’ epitope(s). Laying hens challenged with immunogenic substances during the laying season produce eggs that contain immunoglobulin Y (IgY), which is similar but not identical to IgG, in their yolks. Antibody is selectively deposited in egg yolk and is obtained by noninvasive means (2, 18).

Preliminary experiments.

No immunological response was obtained in laying hens injected subcutaneously and in the footpad at weeks 1 and 3 with solutions of pPGalManⁱⁱ (200 μg/ml in PBS) and with whole P. fellutanum cells at weeks 6 and 9. A response to subcutaneous injections of rabbit IgG in PBS was obtained in these hens. In contrast, other chickens responded to a course of two subcutaneous and two intravenous injections of either pPGalManⁱⁱ or pPGalManⁱⁱⁱ in PBS. The immune responses to pPGalManⁱⁱ and pPGalManⁱⁱⁱ were similar. Yolks from eggs stored at 4°C for a year retained antibody with little loss of activity. In these experiments, anti-pPGalMan activity was tested routinely by an enzyme-linked immunosorbent assay (ELISA) procedure (24, 27) in microtiter plates (Dynatech Laboratories, Inc.) coated with 0.4 μg (0.057 nmol) of either pPGalManⁱⁱ or pPGalManⁱⁱⁱ (26) in 0.14 M NaCl–0.02% NaN₃. After incubation for 24 h at 4°C, the wells were washed with PBS containing 0.05% Tween 20. Unoccupied wells were blocked with 1 mg of bovine serum albumin in 0.1 ml of a solution of PBS, 0.01% NaN₃, and 0.05% Tween 20. Incubation at 24°C for 45 min followed. Plates were washed with PBS-NaN₃-Tween 20. Primary antibodies, prediluted with PBS, were added to all wells except those in the row that served as the secondary-antibody control. Plates were incubated for 60 min at 24°C. After the wells were washed, the quantity of chicken anti-pPGalMan antibody adsorbed to pPGalManⁱⁱ in each well was determined with rabbit anti-chicken IgG (whole molecule) alkaline phosphatase conjugate with p-nitrophenylphosphate as the substrate in 10% diethanolamine buffer (pH 9.8)–0.2% NaN₃. p-Nitrophenol released in each well was quantified with a Bio-Rad ELISA model 2550 enzyme immunoassay reader set at 405 nm.

Purification of chicken egg yolk anti-P. fellutanum antibody.

Antibodies were fractionated by polyethylene glycol precipitation, hydrophobic-interaction chromatography, and gel permeation chromatography (12). Anti-pPGalManⁱⁱ activity from permeation chromatography resulted in a 31-fold increase in ELISA units per microgram of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13) showed anti-pPGalManⁱⁱ activity at 28 and 62 kDa.

Immunochemical studies.

The reaction between pPGalManⁱⁱ or pPGalManⁱⁱⁱ and anti-P. fellutanum pPGalMan antibodies was quantified with 5 μg of protein/well. pPGalManⁱⁱ or pPGalManⁱⁱⁱ (0 to 1 μg/well) was used in an indirect ELISA system. Both pPGalMan species bound to Immulon wells in a hyperbolic concentration-dependent manner. Half saturation of the wells occurred with 26 nmol of either pPGalMan species (data not shown). Approximately 57 nmol (0.4 μg/well) of pPGalManⁱⁱ or pPGalManⁱⁱⁱ was used to coat the wells.Competitive inhibition experiments with a range of concentrations of soluble phosphogalactomannan (PGalManⁱⁱ) or pPGalManⁱⁱ as the inhibitor of antibody interaction with bound pPGalManⁱⁱ or pPGalManⁱⁱⁱ, respectively, showed 50% inhibition at 0.14 and 0.16 μM (1.4 and 1.6 μM galactofuran chains), respectively (Table ​(Table1).1). This suggests that phosphocholine phosphodiester is not a major epitope because pPGalManⁱⁱ, which contains at least fivefold more phosphocholine phosphodiester than pPGalManⁱⁱⁱ (26, 31), is not a better inhibitor than pPGalManⁱⁱⁱ. The epitope(s) on pPGalManⁱⁱ was determined with fragments derived by chemical or enzymatic degradation of pPGalManⁱⁱ. A range of concentrations of each fragment was tested as a hapten inhibitor of binding of anti-pPGalManⁱⁱ antibodies to pPGalManⁱⁱ in a competitive ELISA inhibition system. The concentration of inhibitor or galactofuran chains required to inhibit 50% of antibody binding to Immulon-bound pPGalManⁱⁱ (Table ​(Table1)1) was determined from plots of the percentages of inhibition versus log micromolar values of inhibition or chain.

TABLE 1

Inhibition of antibody binding to pPGalManⁱⁱ by modified pPGalManⁱⁱ and by oligosaccharide fragments^a

Differentiation of Corynebacterium amycolatum,C. minutissimum,and C. striatum by Carbon Substrate Assimilation Tests

We tested the carbon substrate assimilation patterns of 40 Corynebacterium amycolatum strains, 19 C. minutissimum strains, 50 C. striatum strains, and 1 C. xerosis strain with the Biotype 100 system (bioMérieux, Marcy-l’Étoile, France). Twelve carbon substrates of 99 allowed discrimination among the species tested. Additionally, assimilation of 3 of these 12 carbon substrates (maltose, N-acetyl-d-glucosamine, and phenylacetate) was tested with the API 20 NE identification system (bioMérieux). Since concordant results were observed with the two systems for these three carbon substrates, either identification system can be used as a supplementary tool to achieve phenotypic differential identification of C. amycolatum, C. minutissimum, and C. striatum in the clinical microbiology laboratory.Recent progress in molecular taxonomy (DNA-DNA hybridization and 16S rRNA sequencing) and in chemotaxonomy has profoundly modified the classification of coryneform bacteria. Since 1987, 24 former CDC groups have been assigned a new genus and/or species name (8). Corynebacterium amycolatum, C. minutissimum, and C. striatum are frequently encountered in the routine clinical microbiology laboratory (11, 15). Their normal habitat is the human skin and mucous membranes, and they are therefore sometimes isolated as contaminants in clinical samples. However, they have also been reported to be responsible for various types of infection such as pneumonia, endocarditis, and septicemia, especially in immunocompromised patients (8, 11). Consequently, they should not always be considered contaminants and should be identified to the species level. In published case reports, C. amycolatum (13) and C. striatum (3, 10, 12, 14, 16, 20) were all found to be responsible for infection. However, differential identification of these three species by biochemical tests remains difficult, and several misidentifications have been reported previously (7, 8, 21, 23). Furthermore, interpretation of the clinical importance of these species is still difficult. These species have been easily differentiated by methods that cannot easily be used in the routine laboratory, such as chromatography of mycolic acids (2), determination of propionic acid and lactic acid production by gas-liquid chromatography (5, 21), amplified ribosomal DNA restriction analysis (18), and amplification of the 16S-23S gene spacer regions (1). Identification schemes which simplify correct identification in the routine laboratory have recently been reported (8, 15, 19). Additionally, four new tests which allow convenient differentiation of C. amycolatum, C. minutissimum, and C. striatum have recently been established by Wauters et al. (22).We report here on a study of carbon substrate assimilation by 110 strains belonging to these three Corynebacterium species and conclude with a simple scheme allowing identification of C. amycolatum, C. minutissimum, and C. striatum in the routine microbiology laboratory.

Bacterial strains.

We tested 110 Corynebacterium strains isolated from various clinical samples from nonduplicate patients. They were obtained from the bacterial collections of the Département d’Étude et de Recherche en Bactériologie Médicale (Lyon, France), IUT A Lyon 1 (Lyon, France), bioMérieux Laboratories (La Balme-les-Grottes, France), and the Microbiology Laboratory, Faculty of Medicine (Strasbourg, France). In preparation for this study, these strains were identified to the species level: C. amycolatum (40 strains), C. minutissimum (19 strains), C. striatum (50 strains), and C. xerosis (1 strain) by recently described methods (1, 8, 15, 19).In brief, as a first step we inoculated API Coryne systems with these strains (bioMérieux, Marcy l’Étoile, France). The interpretations of the results were based on the second-generation database (9). In addition to the API Coryne system, we also determined the strains’ capability for growth under anaerobic conditions and in the absence of lipids and tested them for the presence of a tyrosinase by previously described protocols (15). In cases of ambiguity, the identification was confirmed by PCR-based amplification of the 16S-23S gene spacer region recently described by Aubel et al. (1). Identification of C. amycolatum strains was confirmed by the absence of mycolic acids according to the method described by Barreau et al. (2). Additionally, we tested the following reference strains: C. amycolatum CIP 103452^T (Collection de l’Institut Pasteur, Paris, France), C. minutissimum ATCC 23348^T, C. striatum ATCC 6940^T, and C. xerosis ATCC 373^T (American Type Culture Collection, Manassas, Va.).

Culture conditions.

Corynebacterium strains were grown at 37°C for 48 h on Columbia agar supplemented with 5% (vol/vol) sheep blood (bioMérieux) in an atmosphere containing 10% CO₂.

Carbon substrate assimilation tests. (i) Biotype 100 system (bioMérieux).

The system is composed of 99 test wells, each one containing a single dehydrated carbohydrate, organic acid, or amino acid, plus one control well without carbon substrate. As a minimal growth medium, we used Biotype Medium 2 (bioMérieux), which contains 31 growth factors and is therefore adapted to fastidious microorganisms. A bacterial suspension was prepared in 5 ml of distilled water and adjusted to the density of a 6.0 McFarland standard. Two milliliters of this suspension was transferred in 60 ml of Biotype Medium 2, and this final suspension was used to inoculate the system’s wells. The inoculated system was incubated at 30°C in a humid chamber. Growth was indicated by a higher turbidity observed in the test well than in the control well. In contrast to what is recommended for members of the family Enterobacteriaceae by the manufacturer, only growth (and not color development) was recorded in test wells 19 (esculin), 39 (hydroxyquinoline β-glucuronide), 59 (l-tryptophan), and 79 (l-histidine). Test results for the 99 wells were recorded with the Recognizer software package (P. A. D. Grimont, Taxolab, Institut Pasteur). The interstrain distances were calculated by using the complement of the Jaccard coefficient, which is not able to score double-negative characteristics. Clusters were formed with the unweighted pair group method with average (Taxotron package; Taxolab).

(ii) API 20 NE system (bioMérieux).

This system is commercialized for the identification of gram-negative bacilli. To adjust it to coryneform bacteria, we modified the manufacturer’s inoculation protocol by using a bacterial suspension adjusted to the density of a 6.0 McFarland standard. Ten drops of this suspension was then transferred in the AUX medium provided with the system, and this final suspension was used to inoculate the system’s auxanogram wells. The inoculated system was incubated at 30°C, and growth in the maltose, N-acetyl-d-glucosamine, and phenylacetate wells was observed after 2 and 4 days.

Results and discussion.

Use of the API Coryne system presents several problems, including difficulties in reading some enzymatic reactions, absence of C. amycolatum from the database, and often the need for supplementary tests to identify the two other species (6). The new API Coryne system database includes C. amycolatum, and C. xerosis is no longer included in this database (9). C. amycolatum, C. minutissimum, and C. striatum give the same code: (2-3)100(1-3)(0-2)(4-5). Certain C. striatum strains give a code such as 3100115; furthermore, a few C. amycolatum strains are urease positive (4).The results obtained with the Biotype 100 system are reported in Table ​Table1.1. Among the 99 carbon substrates tested, only 32 gave more than 20% positive results for at least one of the species tested (Table ​(Table1).1). C. amycolatum, C. minutissimum, and C. striatum used 13, 28, and 26 different substrates as sole carbon source, respectively. In general, we find that the metabolic activity of C. amycolatum is much lower than that of the two other species. The more discriminating carbon substrates were d-galactose, maltotriose, maltose, N-acetyl-d-glucosamine, phenylacetate, 4-aminobutyrate, 5-aminovalerate, l-glutamate, d-alanine, l-alanine, l-serine, and l-tyrosine.

TABLE 1

Percentages of positive carbon substrate assimilation reactions for C. amycolatum, C. minutissimum, and C. striatum^a

Iowa Variant of Familial Alzheimer’s Disease : Accumulation of Posttranslationally Modified AβD23N in Parenchymal and Cerebrovascular Amyloid Deposits

Mutations within the amyloid-β (Aβ) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer’s disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AβD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Aβ molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Aβ species—the Iowa brain Aβ peptidome—contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Aβ cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia.Amyloid β (Aβ) is the major constituent of the fibrils deposited in senile plaques and cerebral blood vessels of patients with Alzheimer’s disease (AD) and Down’s syndrome. It is an internal processing product of a larger type-I transmembrane precursor molecule APP, which is encoded by a single multiexonic gene located on chromosome 21.¹ Several mutations in the APP gene are associated with early onset familial AD (FAD) [reviewed in Refs. ² and ³ and AD Mutation Database (www.molgen.ua.ac.be/ADMutations/)]. Many of these mutations, located either 5′ or 3′ of the nucleotide sequence coding for the Aβ peptide, result in an overproduction of Aβ, particularly Aβ42, and are clinically associated with AD phenotype. In contrast, mutations located within the Aβ sequence (4^{,5,6,7,8,9,10,11,12,13,14,15,16}) are typically linked to early onset FAD and primarily associated with cerebral amyloid angiopathy (CAA), although they manifest with either cerebral hemorrhage or dementia. The bulk of the CAA-associated mutations are located in a short stretch—positions 21-23—of Aβ although more recently others have been reported.

Table 1

APP Mutations Located within the Aβ Sequence

Adherence to Guidelines for Hepatitis B,Pneumococcal, and Influenza Vaccination in Patients With Diabetes

IN BRIEF This single-center, cross-sectional study was designed to assess adherence to national guidelines for the immunization of patients with diabetes and to evaluate predictors of vaccination with the hepatitis B, influenza, and 23-valent pneumococcal polysaccharide vaccines. In patients considered to be at increased risk for infection and infectious disease complications because of their history of diabetes, extensive nonadherence to immunization recommendations for all three vaccines was found. Nonadherence to the 2011 Advisory Committee on Immunization Practices’ recommendation for hepatitis B vaccination was ubiquitous. Allocation of health care resources to increase vaccine coverage should remain a priority, with a focus on spreading awareness of the hepatitis B vaccine recommendation for people with diabetes.Diabetes has long been perceived to be associated with an increased risk of infection and worse health outcomes. The rate of infection arising from many common viral and bacterial etiologies has been shown to occur more frequently in patients with diabetes (1–4). The odds of developing acute hepatitis B are estimated to be more than double in patients with diabetes compared with those without (4). The incidence of hospitalization and odds of death are consistently elevated in people with diabetes compared with those without, during both influenza epidemic and nonepidemic years (5,6). Studies have suggested that patients with diabetes who develop pneumococcal pneumonia are more likely than those without diabetes to progress to systemic bacteremia (7–9).This apparent susceptibility to infection has been attributed to abnormalities in host defense mechanisms, including deficiencies in antibody response, cell-mediated immunity, leukocyte function, and colonization rates (9–11). The higher risk of infection may also be explained by the large burden of chronic disease in this population and associated organ dysfunction. Despite the potential for impaired immune function, most people with diabetes are capable of generating an adequate humoral response and sufficient antibody titers from vaccination (12–14).Morbidity and mortality associated with influenza and pneumonia are reduced in people who have received appropriate vaccination for each of these infectious diseases (9,15–17). The Advisory Committee on Immunization Practices (ACIP) and the American Diabetes Association both recommend annual vaccination with the influenza vaccine and at least one lifetime vaccination with the 23-valent pneumococcal polysaccharide vaccine (PPSV23) for individuals with diabetes or other conditions that increase the risk of complications from infection (18–22). As part of the Healthy People 2020 initiative, the U.S. Department of Health and Human Services has designated goal vaccination coverage rates for high-risk adults aged 18–64 years of 90% annually for the influenza vaccine and 60% for PPSV23 (22). Data from the 2011 National Health Interview Survey indicated that only 16.6% of these high-risk individuals had ever received the PPSV23 (23). Coverage with the influenza vaccine for the 2012–2013 season was estimated to be 47% for high-risk individuals (24). Substantial improvement will be required to meet coverage goals for these vaccines.

TABLE 1.

Adherence to ACIP Recommendations for Vaccination of People With Diabetes and Healthy People 2020 Coverage Goals for Each Vaccine

Human Antibody Response to Helicobacter pylori Lipopolysaccharide: Presence of an Immunodominant Epitope in the Polysaccharide Chain of Lipopolysaccharide

We have examined the antibody response to Helicobacter pylori lipopolysaccharides (LPS) in humans. We used sera from patients with gastroduodenal diseases and healthy adults infected or not infected with H. pylori. Data from the experiments for antibody binding to LPS suggested that the polysaccharide chains from many H. pylori strains showed high immunogenicity in humans. Sera from most (above 70%) H. pylori-infected individuals contained immunoglobulin G (IgG) antibodies against the polysaccharide region highly immunogenic H. pylori LPS. The IgG titers of individual serum samples that reacted strongly with highly immunogenic LPS were quite similar (r² = 0.84 to 0.98). The results suggest wide distribution among H. pylori strains of a highly antigenic epitope in the polysaccharide moieties of their LPS. Also, the similarity in the titers of individual serum samples against highly immunogenic LPS points to the existence of epitopes sharing a common structural motif. However, some strains showed low antigenicity, even those with polysaccharide-carrying LPS. The dominant subclass of IgG that reacted with the highly immunogenic LPS was IgG2, which was preferentially raised against polysaccharide antigens. Recently, a structure that mimics that of the Lewis antigens was identified in the O-polysaccharide fraction of H. pylori LPS; however, no correlation between antigenicity of the polysaccharide chain in humans and the presence of Lewis antigens was found. The IgA and IgM titers against H. pylori LPS seemed to be mostly nonspecific and directed against lipid A. In a few cases, however, sera from individuals infected with H. pylori gave strong IgA and IgM titers against the highly immunogenic polysaccharide. In conclusion, the LPS of many H. pylori strains possess an antigenic epitope in their polysaccharide regions that is immunogenic in humans. However, our results show that the antigenic epitope is unlikely to be immunologically related to structures mimicking Lewis antigens.Helicobacter pylori is an emerging candidate for the genesis of chronic gastritis and peptic ulcer (12, 14). Furthermore, H. pylori infection is thought to be one of the causative factors of gastric cancer (10, 15). Recently, extensive structural and immunological studies of H. pylori lipopolysaccharides (LPS) have been carried out. The O-polysaccharide region of H. pylori LPS has been found to be a major antigenic determinant (13), as are those of other typical bacterial LPS. Interestingly, many H. pylori strains have O-polysaccharide containing epitopes that mimic the structures of Lewis antigens, as shown by chemical (6, 7) and immunological studies (2, 3, 18, 20, 23). The Lewis antigens, which are made up of fucosylated lactosamine structures, are known as tumor antigens on cancer cells, and in normal cells they occur as blood-group antigens and a granulocyte marker antigen (CD15). Hence, the immunological response to the Lewis antigen-containing O-polysaccharides is considered to play a role in the pathogenicity of H. pylori through the establishment of an autoimmune response (3). We have been interested in the antigenicity of H. pylori LPS during natural infection in humans. We present data which suggest the existence of an antigenic epitope, immunologically unrelated to the Lewis antigen, in the polysaccharide moiety of the LPS of a wide range of H. pylori strains.

Bacterial strains.

Clinical strains of H. pylori were isolated from the biopsy specimens of lesions obtained from patients with chronic gastritis, gastric ulcer, duodenal ulcer, and gastric cancer (tumor sites and nontumor sites) in the Sapporo Medical University Hospital (Sapporo, Japan). After three to five laboratory subcultures, H. pylori cells were grown on brain heart infusion agar plates supplemented with 10% (vol/vol) horse blood at 37°C for 5 days under microaerophilic conditions by using the GasPak System without a catalyst (BBL, Cockeysville, Md.). The organisms were collected, washed with phosphate-buffered saline (PBS) three times, and lyophilized.

Human sera.

Sera of 25 patients with chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer and sera of 83 healthy adult volunteers were donated by the Hospitals of Sapporo Medical University and Akita University School of Medicine (Akita, Japan). The status of H. pylori infection was determined by using an enzyme immunoassay kit, Determiner H. pylori Antibody, originally distributed under the name HM-CAP by Enteric Products (Westbury, N.Y.) and purchased from Kyowa Medics (Tokyo, Japan). With this kit, the serum samples of 24 of 25 patients and 21 of 83 healthy adults were found to be positive for H. pylori infection.

IgG response to H. pylori LPS.

We examined the human antibody response to LPS isolated from H. pylori strains by enzyme-linked immunosorbent assay (ELISA). For most ELISA experiments, proteinase K-treated bacterial cells were used as an LPS antigen (2, 24). Briefly, H. pylori cells were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (11) at a concentration of 2 mg/ml and incubated at 100°C for 10 min. Two hundred microliters of proteinase K (2.5 mg/ml) was added, and the mixture was incubated at 37°C overnight and then at 65°C for 2 h. The resulting antigen was diluted 50-fold with 50 mM sodium carbonate buffer (pH 9.6), and aliquots were dispensed into a MicroTest III flexible assay plate (Becton Dickinson, Oxnard, Calif.). The plate was incubated at 4°C overnight, and after the reaction was blocked with 1% human serum albumin, the plate was used for ELISA (24). For purified LPS preparations used as coated antigens, an LPS preparation which was purified as described previously (1) was dissolved in 50 mM sodium carbonate buffer (pH 9.8) at a concentration of 5 μg/ml and then dispensed onto an assay plate. Human serum was diluted 3,000-fold with PBS containing 0.05% Tween 20 (PBST) and 2% human serum albumin. Horseradish peroxidase-conjugated goat F(ab′)₂ anti-human immunoglobulin G (IgG) antibodies (BioSource International, Camarillo, Calif.) and tetramethylbenzidine peroxidase substrate system (Kirkegaard & Perry Laboratories Inc.) were second antibody and substrate, respectively. Absorbance at 450 nm was measured after the termination of the reaction with 1 M phosphoric acid. The results of two ELISAs, with purified LPS and proteinase K-treated cells used as antigens, and immunoblotting analysis (24) were comparable.We found that H. pylori strains could be classified into three groups on the basis of the level of antigenicity of their LPS in humans (24). One group possessed high-antigenicity-polysaccharide-carrying smooth LPS. The second group had low-antigenicity-polysaccharide-carrying smooth LPS. Most H. pylori-infected individuals did not have an antibody titer against the low-immunogenicity LPS even though the LPS carried the polysaccharide chains detected by SDS-PAGE and silver staining (24; data not shown). The third group was characterized by the presence of rough LPS (e.g., CG10). We examined samples for correlation between the levels of IgG in individual serum samples for each type of LPS (Table ​(Table1).1). The levels of IgG for highly immunogenic H. pylori LPS derived from strains CG7, CG9, GU2, GU10, DU1, and DU2 were strongly (r² = 0.84 to 0.98) related. These results indicated that the antigenicities of all the highly immunogenic LPS were nearly the same. However, no significant correlation was observed between the titers of IgG for high-immunogenicity LPS, low-immunogenicity LPS (CA2, CA4, and CA6), and rough LPS (CG10). From the above results, we consider that the antigenicity of H. pylori LPS in humans can be expressed by the mean values of the binding activities to LPS in randomly selected serum samples from H. pylori-infected individuals. The results of ELISA using 10 randomly selected serum samples are shown in Fig. ​Fig.1.1. LPS of H. pylori clinical isolates showed various antigenicities. Among them, all the rough strains tested so far showed low antigenicity. The phenomenon is attributable to the antigenic epitope located in the polysaccharide chain. Interestingly, low-immunogenicity LPS, even those with the polysaccharide chain, were frequently found in strains derived from gastric cancer patients. In conclusion, there are high- and low-antigenicity-polysaccharide-carrying LPS among smooth strains of H. pylori.

TABLE 1

Correlation (r²) of titers of IgG antibody in serum samples tested to LPS fractions from H. pylori strains and S. minnesota Re mutant

Lab-on-Chip-Based Platform for Fast Molecular Diagnosis of Multidrug-Resistant Tuberculosis

We evaluated the performance of the molecular lab-on-chip-based VerePLEX Biosystem for detection of multidrug-resistant tuberculosis (MDR-TB), obtaining a diagnostic accuracy of more than 97.8% compared to sequencing and MTBDRplus assay for Mycobacterium tuberculosis complex and rifampin and isoniazid resistance detection on clinical isolates and smear-positive specimens. The speed, user-friendly interface, and versatility make it suitable for routine laboratory use.Multidrug-resistant tuberculosis (MDR-TB) requires long and expensive treatment and often results in poor clinical outcome in both low- and high-income countries (1, 2). The World Health Organization (WHO) has endorsed specific molecular diagnostics to improve fast diagnosis of MDR-TB (3,–5). However, the genotypic diversity and geographical distribution of Mycobacterium tuberculosis complex (MTBC), together with the inability to provide appropriate interpretation of silent mutations and the limited versatility are some of the restraints undermining the effectiveness of the current tools on a global scale (6,–13).In the present study, we evaluated a lab-on-chip (LoC) device, developed by STMicroelectronics (Geneva, Switzerland) and marketed by Veredus Laboratories (Republic of Singapore) as the VerePLEX Biosystem, for the diagnosis of MDR-TB and detection of common nontuberculous mycobacteria (NTM). The molecular assay was evaluated on both clinical isolates and direct specimens in low- and high-burden settings.We tested 91 MTBC isolates (see Table S1 in the supplemental material) harboring different patterns of mutations in rpoB, katG, and inhA genes to evaluate the probes on the array listed in 14). An additional 116 MTBC culture-negative specimens were included in the analysis. DNA from isolates and specimens was extracted by thermal lysis and sonication as described elsewhere (15). Phenotypic drug susceptibility testing (DST) for rifampin (RIF) and isoniazid (INH) was performed according to international recommendations (16). Some of the specimens were tested in a representative high-burden setting in Uganda (Nsambya Hospital, Kampala, Uganda), by trained staff.

TABLE 1

Probes spotted onto the array and targeted mycobacterial species and MDR-TB targets included in the assay

Enteroaggregative,Shiga Toxin-Producing Escherichia coli O111:H2 Associated with an Outbreak of Hemolytic-Uremic Syndrome

Shiga toxin-producing Escherichia coli O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative E. coli probe PCVD432. These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic E. coli.Enterohemorrhagic Escherichia coli (EHEC) is a well-known cause of severe disease, such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (10). The bacterium produces Shiga toxins (Stx; also known as verocytotoxins) (10), harbors large plasmids which code for production of enterohemolysin (EHEC-Hly) (24) and catalase-peroxidase (KatP) (4), and possesses the intimin-coding gene eaeA (27). The gene is part of a chromosomal gene cluster termed LEE, for locus of enterocyte effacement (16), which determines the production of attaching and effacing lesions on the intestinal mucosa and localized adhesion to HEp-2 cells (8, 12). Besides O157, by far the most important in human disease (10), EHEC strains belong to a restricted number of serogroups (10), among which O111 has been associated with both sporadic cases (9, 10) and outbreaks (3, 5, 20) of HUS.During a study aimed at characterizing Stx-producing E. coli (STEC) O111 strains from different countries (data not shown), we found that eight strains isolated in France during an outbreak of HUS (3) showed aggregative adhesion (AA), instead of the typical localized adhesion, to HEp-2 cells (18) and possessed the genetic markers of enteroaggregative E. coli (EAggEC). EAggEC, defined by its aggregating pattern of adherence to Hep-2 cells (18), has been associated with protracted diarrhea in children in developing countries (6) and with cases of childhood diarrhea in Europe (9, 11, 25). AA is associated with the presence of large plasmids carrying genes coding for bundle-forming fimbriae (17) and the production of EAggEC heat-stable enterotoxin 1 (EAST1) (23). Fragments from these plasmids have been used as DNA probes (2) or PCR targets (25) for identifying EAggEC. Since AA and Stx production have never been found to be associated in E. coli isolates, we describe here the molecular characteristics of these unusual strains.The E. coli O111:H2 strains, designated RD1 to RD8, gave negative results in the PCR analyses for the eaeA gene (26) and the EHEC plasmid markers ehec-hly (24) and katP (4). In addition, they did not produce hemolysin and did not hybridize with the eaeA (12) and EHEC (15) probes, even under low-stringency conditions. When tested in the HEp-2 cell assay (9), all these strains showed the aggregative pattern of adhesion typical of EAggEC (Fig. ​(Fig.1).1). Accordingly, they agglutinated rat erythrocytes in the presence of 0.5% mannose (17) and gave positive PCR amplification with the primer pairs which amplify a 630-bp region of the EAggEC probe (25) and the astA determinant of EAST1 (23). Moreover, they hybridized with the EAggEC (2) and astA (23) probes. The Vero cell assay (9) and the PCR analyses with Stx1- and Stx2-specific primers (21) confirmed that all the strains produced Stx2 alone. Taq cycle sequencing of the toxin B-subunit gene (21) showed 100% homology with the nucleotide sequence of the stx₂ B gene from the O157:H7 strain EDL933. Table ​Table11 summarizes the characteristics of the E. coli O111:H2 isolates in comparison with reference EAggEC and EHEC strains used as controls in all the experiments. As previously described by Savarino and coworkers (22), the O157:H7 strain EDL933 hybridized with a probe produced by PCR amplification of the astA gene present in strain 17-2 (23). However, it was negative in the astA PCR, thus suggesting the existence of a degree of variability in the astA nucleotide sequences present in the different groups of E. coli. Open in a separate windowFIG. 1Pattern of AA to HEp-2 cells of a representative STEC O111:H2 strain. Bar = 15 μm.

TABLE 1

Virulence properties of STEC O111:H2 strains and reference EHEC and EAggEC strains

Using Continuous Glucose Monitoring in Clinical Practice

Continuous glucose monitoring is poised to radically change the treatment of diabetes and patient engagement of those afflicted with this disease. This article will provide an overview of CGM and equip health care providers to begin integrating this technology into their clinical practice.

Continuous glucose monitoring (CGM) systems are more than just glucose monitors. Recent CGM systems have moved beyond mere blood glucose monitoring (BGM) by providing both real-time and predictive glycemic data. The robust data garnered from CGM can also be used for detection of trends, identification of asymptomatic events, and review of glycemic variability over a range of time.Increased frequency of glucose monitoring is associated with decreased hypoglycemia and increased glycemic time in range (TIR), which correlates with improved A1C (1). Moreover, glucose patterns captured via CGM data analysis can highlight areas in need of treatment intervention (e.g., to prevent hypoglycemia, improve glycemic control at specific times of day, and increase overall TIR). As is well known, the A1C test provides an indication of average glycemic control over the previous 2–3 months. However, it does not capture glycemic variability; thus, individuals who have the same A1C may have vastly different glucose ranges (2,3). CGM can be a good option for patients with inconsistent or confounding glycemic control, who desire engagement in their own disease management, or whose treatment plan puts them in danger of hypoglycemia.Health care providers (HCPs) can implement two different modalities of CGM. They may prescribe a personal CGM device, which a patient can use either continuously or intermittently, or they may purchase for their practices professional CGM systems that can be sent home with a patient for a brief period of time for diagnostic purposes. The Medtronic iPro2 and the FreeStyle Libre Pro are professional CGM systems for which data are blinded to the patient. The data are uploaded in the HCP’s office for retrospective review with the patient. The Dexcom G6 professional CGM system can be prescribed in blinded and unblinded modes. HCPs may use the unblinded option to help patients increase their awareness of their own glucose levels and make real-time treatment decisions.The data collected by these devices and either downloaded in the clinic or transmitted remotely allow for visualization of a patient’s true glycemic picture and the effects of current interventional treatments. CGM data also give HCPs insight into patients’ behaviors and glycemic patterns and may reveal previously undetected issues such as hypoglycemia (2,3). Retrospective review of CGM data can reveal therapeutic impacts on glucose management, aid in making treatment decisions, and provide opportunities for education.Professional CGM systems have been used clinically to measure the effects of variables over an intermittent or specific time interval, such as 3 days or 2 weeks. More specifically, such CGM has been used to evaluate the effects of various interventions, behaviors, and therapies, including the effects of foods or various types of exercise and medication titration (4–7).The abundance of data gathered via CGM can be reviewed and interpreted through the ambulatory glucose profile (AGP) report, a standardized CGM report that provides a graphical and quantitative display of glycemic activity. The AGP visually displays the dynamics of glycemic activity, including periods of hypoglycemia, glycemic excursions (both high and low), TIR, and recurring glucose patterns, all of which are meaningful metrics for guiding comprehensive diabetes management.From the patient perspective, CGM offers the benefit of real-time glycemic monitoring with glucose trend information indicated by directional arrows. These trend arrows are a visual display of the direction of glycemic activity (i.e., whether the current glucose level is rising, stable, or decreasing) (8). The visual display of CGM data allows patients to view their glycemic activity and monitor the effects of different types of food, timing of meals, activity levels, stress, and illness. This opportunity facilitates increased patient engagement with diabetes management. Having glucose data readily available is also relevant for loved ones and caregivers of people with diabetes, allowing them to better assist in care and offering them peace of mind with regard to hypoglycemia and hyperglycemia.Integrating CGM into clinical practice can be challenging for several reasons. Common issues reported include data overload, increased clinic staff time, and the need for HCP education on data interpretation (9,10). Orienting practice staff to the use of CGM technology and downloading reports to a standalone computer and printer that are separate from restrictive administrative firewalls can streamline analysis of CGM data.Although there can be some barriers to CGM use, there is also strong evidence for its utility in patients with either type 1 or type 2 diabetes and with either personal or professional CGM systems (11). Patient benefits include improvement in A1C, reductions in hypoglycemia and glycemic variability, and greater treatment satisfaction and improved sense of mental well-being (12–15).One solution to overcoming barriers is intermittent use, of personal or professional CGM, from every other week to perhaps every 6 months, followed by office review of the AGP report (16). This option permits an overview of the glycemic picture at important intervals, such as during lifestyle intervention or after medication changes. In addition, reviewing the AGP report with a patient offers an HCP the opportunity for patient education and a means of encouraging communication and shared decision-making.This article is intended to equip HCPs to effectively incorporate CGM into clinical practice by reviewing the overwhelming benefits of this technology and the strategies available to overcome therapeutic inertia with regard to its use. Practical tips and tools for streamlining the use of CGM are provided to maximize patients’ office visits through concise, proficient interpretation of CGM data. Topics include how to efficiently review and share the information displayed on the AGP report, how to interpret and act on that information, and how to bill for CGM use and data interpretation services.     

Correlation of Cecal Microflora of HLA-B27 Transgenic Rats with Inflammatory Bowel Disease

Transgenic rats with a high level of expression of the human major histocompatibility complex class I molecule HLA-B27 develop chronic inflammatory bowel disease (IBD) and arthritis. Assessment of the cecal microflora showed a rise in numbers of Escherichia coli and Enterococcus spp., corresponding to the presence and severity of IBD in these rats.The role of bacteria in Crohn’s disease and ulcerative colitis has been of intense interest for four decades, but no single bacterial pathogen has been identified. Current theory suggests that some aspect of the normal microflora may mediate the chronic inflammatory response characteristic of inflammatory bowel disease (IBD). Consistent with this, a number of occurrences of IBD that are seen in rodent models involving conventionally housed animals are prevented by raising the animals in a germfree environment (3). Among the models of IBD dependent upon the presence of intestinal bacteria is the enterocolitis that develops in HLA-B27 transgenic rats (8, 11). Here we report quantitative and qualitative characterization of changes in the bacterial populations of the cecum that correlate with the presence and severity of colitis in this animal model of IBD.Male and female rats from B27 transgenic disease-prone and disease-resistant lines from separate cages and animal housing rooms and nontransgenic littermates were used (4, 9, 10). Clinical disease was scored as described previously (1). Upon sacrifice, the cecum was excised and snap frozen, and the adjacent proximal colon was processed for histology (4). Previous studies have documented that the freezing process results in a modest decrease in total counts (0.5 log₁₀ CFU) and no qualitative differences in the major groups isolated. Frozen ceca were thawed in an anaerobic chamber. An aliquot was used to determine dry weight. Dilutions of 10⁻² to 10⁻⁸ in sterile saline were made from a second aliquot and plated onto both selective and nonselective microbiologic media (6, 7). Plates were incubated under anaerobic or aerobic conditions at 37°C for 48 h. Colonies were counted, and isolates of representative colony types were identified. All counts were reported as log₁₀CFU per gram (dry weight) for facultative and obligately anaerobic organisms. All microbiologic assessment was conducted without knowledge of clinical or histologic status of the animals. Correlations were sought between the microbiologic findings and the severity of clinical disease (severe, intermediate, and healthy), histologic score (proximal colon score range, 0 to 4 [8]), and quantitative histologic ulcer score (0 to 4).Representative Escherichia coli isolates were probed with pCVD434 for the presence of the toxin-associated eae gene and with pJPN16 for the presence of the EAF locus (5). Attachment of E. coli was evaluated using HEP-2 cells, and Vero cell cytotoxicity was assayed as described previously (2).The ceca from 29 rats were evaluated (6 healthy, with a mean age of 287 days; 12 with intermediate disease, with a mean age of 210 days; and 11 with severe disease, with a mean age of 158 days). Quantitative counts are presented in Table ​Table1.1. Rats with severe disease had somewhat higher numbers of aerobic and facultative organisms (mean log₁₀CFU, 9.51 ± 0.17) than either healthy rats or rats with intermediate disease. The total counts of anaerobic organisms (anaerobic counts) for healthy animals were approximately 0.5 log unit lower than for animals with either severe or intermediate disease (mean of 9.63 ± 0.27 versus 10.02 ± 0.16 and 10.23 ± 0.23, respectively). By one-way analysis of variance, neither the aerobic nor anaerobic total counts revealed significant differences among the three groups. While there was a significant correlation between age and colon score (P < 0.001), no correlation between age and total aerobic or anaerobic counts was observed. A significant correlation was observed between age and Enterococcus populations (P = 0.028); however, no correlation was observed between age of animals and the E. coli population. The total numbers of enterococci revealed a significant difference between rats with severe disease and healthy rats (mean log₁₀CFU, 9.37 ± 0.2 versus 8.13 ± 0.57, respectively [P < 0.045]). Those with intermediate disease (mean, 8.67 ± 0.26) were not significantly different from the other two groups. The most striking difference was seen in the counts for E. coli. Healthy animals had relatively modest numbers (mean, 5.89 ± 0.44; range, 4.6 to 7.14), whereas the rats with severe disease (mean, 8.44 ± 0.21; range, 7.5 to 9.7) and those with intermediate disease (mean, 7.85 ± 0.41; range, 5.49 to 10.31) had substantially higher counts (P <0.008 for all groups, P < 0.0001 for healthy versus severe disease, and P < 0.01 for healthy versus intermediate disease; r = 0.65 and P < 0.05 for E. coli count versus colon score). The increase in the total counts for facultative gram-negative organisms for animals with intermediate or severe disease was almost entirely due to the increase in the numbers of E. coli present (r = 0.94, P < 0.001). Assays of representative E. coli isolates from the three groups of rats for the presence of a cytopathic effect on Vero cells, the presence of the enterotoxin-associated eae gene and EAF locus, and attachment to the HEP-2 cell line were all negative.

TABLE 1

Quantitative bacterial counts in ceca of HLA-B27 transgenic rats

Anti-Neutrophil Cytoplasmic Antibody Pathogenesis in Small-Vessel Vasculitis : An Update

Vasculitides associated with serum positivity for anti-neutrophil cytoplasmic antibodies (ANCAs) that affect small- to medium-sized vessels are commonly known as ANCA-associated vasculitis (AAV) and include Wegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. Evidence derived from both in vitro studies and recent animal models points to a pathogenic role of ANCAs in AAV. In 2002, the first in vivo breakthrough in the pathogenesis of ANCAs showed that mouse ANCAs against myeloperoxidase (MPO) led to intrinsic pauci-immune renal vasculitis in mice. In 2004, a report using both in vitro and in vivo studies proposed that proteinase 3 (PR3)-directed autoimmunity involved the complementary peptide of PR3 (cPR3), which is encoded by the antisense strand of the PR3 gene. The last breakthrough came in October 2008 with a previously undescribed molecular explanation for the origin and development of injury in pauci-immune renal vasculitis, with potential clinical implications. This report showed that infection by fimbriated bacteria may trigger cross-reactive autoimmunity to a previously characterized ANCA antigen, lysosomal membrane protein-2, which is contained in the same vesicles that harbor MPO and PR3. Infection by fimbriated bacteria resulted in the production of autoantibodies, which activated neutrophils and killed human microvascular endothelium in vitro and caused renal vasculitis in rats. Although the evidence for a pathogenic role of ANCAs, mainly MPO-ANCAs, is striking, various questions remain unanswered. Understanding the key pathogenic mechanisms of AAV may provide a safer, more rational therapeutic approach than the traditional (ie, corticosteroids and immunosuppressants) treatment strategy.Anti-neutrophil cytoplasmic antibodies (ANCAs) were discovered by chance in 1982 when Davies et al¹ were studying antinuclear antibodies in serum samples from patients with segmental necrotizing glomerulonephritis. Using indirect immunofluorescence applied to neutrophils, a diffuse cytoplasmic, but not nuclear, staining pattern was observed. In 1985, van der Woude et al² found that cytoplasmic ANCAs occurred mainly in patients with Wegener’s granulomatosis (WG), and interest in ANCAs skyrocketed. In 1988,³ a distinct perinuclear pattern in serum samples from patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis was reported. Enzyme-linked immunosorbent assay showed that myeloperoxidase (MPO) was the chief antigenic target of perinuclear ANCAs. Two years later, proteinase 3 (PR3) was recognized as the major autoantigen accounting for the cytoplasmic ANCA pattern of WG.^4,5The vasculitides are often serious and sometimes fatal diseases that require prompt recognition and treatment. Symptomatic involvement of affected organs may occur in isolation or in combination with multiple organ involvement. Vasculitic syndromes are normally categorized by the type and predominant size of the blood vessels most commonly affected 6^,7 The distribution of affected organs may suggest a particular vasculitic disorder, but there is significant overlap.

Table 1

Classification of Vasculitis

Diversity of Hemagglutination Phenotypes among P-Fimbriated Wild-Type Strains of Escherichia coli in Relation to papG Allele Repertoire

Data regarding the hemagglutination (HA) patterns of the three variants (classes I, II, and III) of the Escherichia coli adhesin PapG are conflicting. These HA patterns usually have been assessed for each papG allele separately with recombinant strains in slide HA assays. We rigorously evaluated an alternative microtiter tray HA assay and then used it to assess the HA of four erythrocyte types (human A₁P₁ and OP₁, rabbit, and sheep erythrocytes) by multiple wild-type E. coli strains representing the four naturally occurring combinations of the papG alleles, i.e., class I plus III, class III only, class II plus III, and class II only. The microtiter tray HA assay displayed significantly better reproducibility of intraobserver (83%) and interobserver (86%) results than did slide HA assays (39 and 73%, respectively). Novel findings from the study of 32 wild-type P-fimbriated strains included reproducible determinations of phenotypic diversity among different papG categories, among strains within each papG category, and from day to day for individual strains. There was also substantial overlap of phenotypes between papG categories I plus III and III only and between II plus III and II only. A class III papG recombinant strain’s HA pattern differed significantly from that of the wild-type class III strains. These data demonstrate that HA phenotypes of wild-type P-fimbriated E. coli strains can be reproducibly assessed by a microtiter HA assay and that they correspond broadly to papG genotype but in a more complex and varied fashion than previously recognized.P fimbriae, the adhesins most clearly implicated in the pathogenesis of extraintestinal infection due to Escherichia coli (6, 15), mediate Gal(α1-4)Gal-specific binding to host epithelial surfaces (3, 26, 28, 31–33) via the tip adhesin molecule PapG (9, 39). A possible explanation for the subtle differences in binding preferences among P-fimbriated E. coli strains that were noted following the discovery of P fimbriae (5, 7) came with the later discovery that PapG occurs in three molecular variants (38, 40, 42). The PapG variants, sometimes categorized in classes I, II, and III, bind preferentially to different Gal(α1-4)Gal-containing compounds (25, 60, 61) and are encoded by distinct alleles of the adhesin gene papG (42) (Table ​(Table1).1).

TABLE 1

Characteristics of the three PapG variants, as reported in the literature^a