College of American Pathologists
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  Clinical Abstracts





February 2010

Michael Bissell, MD, PhD, MPH

Characterization of human brain antigenic targets in neuropsychiatric SLE Characterization of human brain antigenic targets in neuropsychiatric SLE

Systemic lupus erythematosus is a chronic autoimmune disease characterized by multisystemic manifestations. Central nervous system involvement has been reported in six percent to 12 percent of systemic lupus erythematosus (SLE) patients. The features of neuropsychiatric SLE (NPSLE) are extremely diverse and include neurologic and psychiatric syndromes. The diagnosis of NPSLE is challenging because the drugs used to manage SLE, infections, or other non-SLE related pathologic conditions may be responsible for the neuropsychiatric manifestations and have to be excluded from the assessment. Moreover, there is no gold standard diagnostic test that can definitively confirm NPSLE. Because the treatment is dependent on the underlying cause, many authors have emphasized the need for new tools to diagnose NPSLE. Some evidence indicates that the immune system plays a role in NPSLE, since the disease typically occurs in the presence of serologically and clinically active SLE, and that concentrations of some chemokines, such as interferon-inducible protein 10/CXCL10 and fractalkine, are increased in the cerebrospinal fluid of NPSLE patients. However, while it is well established that autoantibodies can directly damage organs, especially the kidney, brain antigenic targets have not been fully identified in NPSLE. Several investigators have sought to identify autoantibodies that could bind directly to neurons and serve as diagnostic markers in NPSLE. Sera collected from patients with SLE have been tested for antibodies to microtubule-associated protein 2 (MAP-2) and were detected in NPSLE patients in particular. Nevertheless, further investigation is necessary to determine whether there is an alteration of the immune recognition of brain self-proteins in NPSLE. Moreover, most of the previous studies were performed with purified self-antigens or relevant peptides from preselected targets, or both. To avoid restricted analysis with preselected antigenic targets, the authors chose to assess global serum self-IgG reactivity against healthy or injured human brain tissue extracts. They used a Western blot method to compare serum self-IgG reactivities against human brain tissue extracts in NPSLE patients and in SLE patients without neuropsychiatric manifestations (non-NPSLE patients), Sjogren’s syndrome patients with and without central nervous system involvement, multiple sclerosis patients, and healthy subjects. The authors made no a priori assumptions. Using a proteomic approach, they then characterized the most discriminant brain antigenic targets in NPSLE patients. They confirmed previous results concerning the presence of anti-MAP-2B and anti-triose phosphate isomerase antibodies in NPSLE patients and characterized two other discriminant antigenic targets—Hsp70–71 and an unidentified p90 antigenic band. In contrast, one other protein band, which was characterized as septin 7, was never recognized by sera from the NPSLE patients but was recognized by a majority of sera from the non-NPSLE patients. The authors concluded that this characterization emphasizes the possible role of neuronal microtubules in the pathophysiology of NPSLE.

efranc D, Launay D, Dubucquoi S, et al. Characterization of discriminant human brain antigenic targets in neuropsychiatric systemic lupus erythematosus using an immunoproteomic approach. Arthritis Rheum. 2007;56:3420–3432.

Correspondence: Dr. Didier Lefranc at

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Automated antibiotic susceptibility testing for Streptococcus species Automated antibiotic susceptibility testing for Streptococcus species

Antimicrobial resistance among Streptococcus pneumoniae and other streptococci has been increasing since the early 1990s. Approximately 18 percent of the pneumococcal isolates in the United States are penicillin resistant (minimal inhibitory concentration, =2 µg/mL), and the overall rate of multi-drug resistance is 22 percent. Because of the emergence of antimicrobial resistance among streptococci, it is essential that clinical microbiology laboratories use accurate and convenient antimicrobial susceptibility testing (AST) methods. The authors conducted a study to evaluate the BD Phoenix Automated Microbiology System Strep AST panel (BD Diagnostics) in four clinical microbiology laboratories by comparing its performance to that of the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method for susceptibility testing of streptococci. The multi-center study, which used 13 agents, was performed on 2,013 streptococci (938 S. pneumoniae isolates; 396 group B streptococci; 369 viridans group streptococci; 290 β-hemolytic streptococcus groups A, C, and G; and 20 other streptococci) with the Phoenix system and the broth microdilution reference method. Clinical and challenge isolates were tested against cefepime, cefotaxime, ceftriaxone, clindamycin, erythromycin, gatifloxacin, levofloxacin, linezolid, meropenem, penicillin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. Clinical isolates with major errors or very major errors were retested in duplicate using both methods. The final results for the clinical isolates showed that for all of the organism-antimicrobial agent combinations tested, categorical agreement was 92 percent to 100 percent, with the exception of viridans group streptococci-penicillin (87 percent categorical agreement; all errors were minor). For S. pneumoniae, there was one major error with clindamycin (0.1 percent) and one or two very major errors with cefotaxime (4 percent), ceftriaxone (4.5 percent), erythromycin (0.9 percent), and tetracycline (0.7 percent). For groups A, C, and G, the categorical agreement was 97 percent to 100 percent, and the only very major errors were resolved through additional reference laboratory testing. For group B streptococci, there was only one very major error (tetracycline, 0.3 percent), and D-zone testing of 23 isolates with clindamycin major errors (one isolate unavailable) revealed inducible clindamycin resistance. For viridans group streptococci, the major error rates were zero to three percent, and very major errors occurred with seven agents (3.5 percent to 7.1 percent). The mean times required for organism groups to generate results ranged from 8.4 to 9.4 hours. The authors concluded that the Phoenix system provided reliable and rapid AST results for most of the organism-antimicrobial agent combinations tested.

Richter SS, Howard WJ, Weinstein MP, et al. Multicenter evaluation of the BD Phoenix Automated Microbiology System for antimicrobial susceptibility testing of Streptococcus species. J Clin Microbiol. 2007;45:2863-2871.

Correspondence: Sandra S. Richter at

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Speciating parasites with molecular methods Speciating parasites with molecular methods

Equine strongyles (Nematoda, Strongylida) belonging to the subfamilies Strongylinae and Cyathostominae are intestinal parasitic nematodes affecting equids worldwide. Since the morphological identification of adult cyathostomins can be challenging, and identification of larval stages is almost impossible, efforts have been underway to establish the molecular identification of horse strongyles. The use of oli­gonucleotide probes in reverse line blot (RLB) hybridization assays have proven to be useful for identifying a diverse range of clinically relevant human and veterinary pathogens, with high throughput. The RLB method enables the nonradioactive hybridization of polymerase chain reaction (PCR-generated) amplicons with different oligonucleo­tide probes in a single assay. In this method, oligonucleotide probes are covalently attached to a membrane in parallel lines using a miniblotter and then perpendicularly evaluated against biotin-labeled PCR products in the miniblotter. Hybridization is visualized using peroxidase-labeled streptavidin and chemiluminescence detection. Although species-specific oligonucleotide probes to identify some cyathostomes have been reported, it is still necessary to increase the number of species that can be identified by a molecular means using a simple approach adaptable to molecular parasitology laboratories. A preliminary RLB assay has been validated for the simultaneous molecular identification of eight species of cyathostomes. Building on this work, the authors conducted a study to develop an RLB hybridization assay that could simultaneously identify the most common large and small equine strongyles. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound, species-specific probes. All 13 cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles—Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris. The authors concluded that this assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. It could also represent a basic step toward developing a rapid and simple molecular test for early detection of drug-resistant genotypes of horse strongyle species.

Traversa D, Iorio R, Klei TR, et al. New method for simultaneous species-specific identification of equine strongyles (Nematoda, Strongylida) by reverse line blot hybridization. J Clin Microbiol. 2007;45: 2937–2942.

Correspondence: Donato Traversa at

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Bacteriology of diabetic foot infections Bacteriology of diabetic foot infections

Foot infections in people with diabetes initially are treated empirically, but therapy directed at known causative organisms may improve outcome. Many studies have reported on the bacteriology of diabetic foot infections over the past 25 years, but the results have varied and often have been contradictory. To better define the bacteriology of such infections, the authors analyzed their data from a large prospective U.S. multicenter trial that investigated the outcome of treatment for diabetic patients who had moderate to severe lower extremity infections that required at least initial parenteral antibiotic therapy. As part of the trial, conducted from 2001 to 2004, the authors obtained 454 pretreatment specimens from 433 patients. After debridement, they collected wound specimens, mostly by curettage or biopsy, and sent them to the R. M. Alden Research Laboratory for aerobic and anaerobic culture. Among the 427 positive cultures, 83.8 percent were polymicrobial, 48 percent grew only aerobes, 43.7 percent had aerobes and anaerobes, and 1.3 percent had only anaerobes. Cultures yielded 1,145 aerobic strains and 462 anaerobic strains, with an average of 2.7 organisms per culture (range, one to eight) for aerobes and 2.3 organisms per culture (range, one to nine) for anaerobes. The predominant aerobic organisms were oxacillin-susceptible Staph­y­lococcus aureus (14.3 percent), oxacillinresistant S. aureus (4.4 percent), coagulase-negative Staphylococcus species (15.3 percent), Streptococcus species (15.5 percent), Enterococcus species (13.5 percent), Corynebacterium species (10.1 percent), members of the family Enterobacteriaceae (12.8 percent), and Pseudomonas aeruginosa (3.5 percent). The predominant anaerobes were gram-positive cocci (45.2 percent), Prevotella species (13.6 percent), Porphyromonas species (11.3 percent), and the Bacteroides fragilis group (10.2 percent). Pure cultures were noted for 20 percent of oxacillin-resistant S. aureus cultures, 9.2 percent of S. epidermidis cultures, and 2.5 percent of P. aeruginosa cultures. Two or more species of Staphylococcus were present in 13.1 percent of the patients. Ertapenem and piperacillin-tazobactam were each active against more than 98 percent of the enteric gram-negative rods, methicillin- sensitive S. aureus, and anaerobes. Among the fluoroquinolones, 24 percent of anaerobes, especially the gram-positive cocci, were resistant to moxifloxacin; 27 percent of the gram-positive aerobes but only six percent of the members of the family Enterobacteriaceae were resistant to levofloxacin. The authors concluded that moderate to severe diabetic foot infections typically are polymicrobial, and almost half include anaerobes. These antibiotic susceptibility results can help shape therapeutic choices.

Citron DM, Goldstein EJC, Merriam CV, et al. Bacteriology of moderate-to-severe diabetic foot infections and in vitro activity of antimicrobial agents. J Clin Microbiol. 2007;45:2819–2828.

Correspondence: Diane M. Citron at

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Inflammation in hemorrhagic shock Inflammation in hemorrhagic shock

Hypertonic solutions (7.5 percent saline ± 6.0 percent Dextran 70) have been investigated as alternative resuscitation strategies for critically injured people. Hypertonic resuscitation evokes an increase in serum osmolarity, which leads to redistribution of fluid from the interstitial and intracellular spaces to the intravascular space. This leads to rapid restoration of circulating intravascular volume, requiring a smaller volume of fluid compared with isotonic crystalloid solutions and leading to decreased accumulation of extravascular volume. The osmotic effects of hypertonic fluids also reduce intracranial pressure in patients with head injury. Therefore, this combination of increased systemic perfusion, which increases cerebral perfusion, along with a decrease in intracranial pressure, which reduces brain swelling, minimizes the progression of secondary brain injury. The authors hypothesized that administering hypertonic saline/dextran (HSD) would attenuate the initial neutrophil and monocyte proinflammatory response, which could lead to reduced inflammatory organ injury. In addition, modulation of the early innate immune response could result in a less dramatic counter inflammatory response and, therefore, a decrease in the risk of subsequent nosocomial infection and late organ failure. The authors conducted a study to determine the effect of pre-hospital resuscitation with HSD on the innate immune response after severe injury. They collected serial blood samples 12, 24, and 72 hours and seven days after injury from patients enrolled in a prospective, randomized, double-blind trial of traumatic hypovolemic shock, with HSD (250 mL) versus lactated Ringer’s solution (LR) as the initial resuscitation fluid. The authors assessed polymorphonuclear neutrophil (PMN) CD11b/CD18 expression via whole blood fluorescence­activated cell sorting analysis with and without stimulation (formyl-methionyl-leucyl-phenylalanine 5 µmol/L or phorbol-myristate-acetate 5 µmol/L). They assessed PMN respiratory burst using the nitro-blue tetrazolium assay. Monocytes stimulated with 100 ng lipopolysaccharide (LPS) for 18 hours were assessed for cytokine production (TNF-α, IL-1β, IL-6, IL-10, and IL-12). Enrolled in the study were 62 patients (36 HSD, 26 LR) and 20 healthy volunteers. The authors found that CD11b expression 12 hours after injury was increased 1.5 fold in patients resuscitated with LR compared with controls. Those resuscitated with HSD had a significant reduction in CD11b expression 12 hours after injury compared to those resuscitated with LR. No difference in respiratory burst was noted early after injury. Monocytes from injured patients expressed lower levels of all cytokines compared with those from normal controls. Patients given HSD showed a trend toward higher levels of IL-1β and IL-10 production in response to LPS 12 hours after injury. The authors concluded that HSD resuscitation results in transient inhibition of PMN CD11b expression and partial restoration of the normal monocyte phenotype early after injury.

Bulger EM, Cuschieri J, Warner K, et al. Hyper­tonic resuscitation modulates the inflammatory response in patients with traumatic hemorrhagic shock. Ann Surg. 2007;245(4):635–641.

Correspondence: Dr. Eileen M. Bulger at

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Relationship between human leukocyte antigen type and multiple sclerosis Relationship between human leukocyte antigen type and multiple sclerosis

Multiple sclerosis is the most common inflammatory demyel­inating disease of the central ner­vous system in young adults. The cause of multiple sclerosis (MS) is unknown, but it is widely believed to be an autoimmune disease occurring in genetically susceptible people after exposure to as-yet undefined environmental factors. Although the nature of the environmental triggers remains undetermined, progress has been made in characterizing the genetic factors of susceptibility to the disease. The first study focusing on the role of the human leukocyte antigen (HLA) system in MS susceptibility was carried out more than 20 years ago, and even today, the major histocompatibility complex (MHC) is frequently referred to as the only genetic region certain to contain MS susceptibility genes. A meta-analysis combining the raw genotyping data from nine populations with 719 families demonstrated strong evidence of linkage only in the MHC region on chromosome 6p21, confirming that this region represents the strongest MS susceptibility locus in the genome. The HLA genotype is estimated to account for between 10 percent and 40 percent of genetic risk. The development of molecular typing and polymerase chain reaction (PCR) techniques in particular has enabled a more detailed analysis of the associations between several different HLA class II ge­notypes and MS. The authors conducted a study to investigate the relationship between MS, the HLA-DRB1*15 allele, and other DRB1 alleles in a Portuguese population and their association with the clinical course of MS. The HLA-DRB1 alleles were analyzed by PCR using sequence-specific primers in 248 MS patients and 282 healthy controls. To relate HLA-DRB1 alleles to disease aggressiveness, patients with relapsing remitting MS and secondary progressive MS were subdivided into three groups: “benign” MS patients, who maintained an Extended Disability Status Scale (EDSS) score of three or less at least 10 years after disease onset; non-benign MS patients, who maintained an EDSS score of more than three after the same time period; and aggressive MS patients, who maintained an EDSS score of six or more within 15 years of disease onset. As expected, the authors found a higher frequency of HLA-DRB1*15 in MS patients (29.8 percent versus 19.9 percent; odds ratio [OR], 1.72; 95 percent confidence interval [CI], 1.15–2.56; P=.008). The HLA-DRB1*03 allele was positively associated with MS in the overall patient population (22.6 percent versus 15.6 percent; OR, 1.58; 95 percent CI, 1.02–2.45). In terms of disease aggressiveness, HLA-DRB1*15 occurred more frequently in the group with benign disease (42.6 percent versus 19.9 percent; OR, 2.99; 95 percent CI, 1.56–5.72) and the group with non-benign disease (34.1 percent versus 19.9 percent; OR, 2.09; 95 percent CI, 1.05–4.16) than in controls. When time to reach an EDSS score of three or six was considered as the end point, HLA-DRB1*15-negative patients were found to have a worse prognosis. The authors concluded that in this population of Portuguese MS patients, the HLA-DRB1*15 allele is established as a genetic marker for susceptibility to MS and is associated with a better outcome

Silva AM, Pereira C, Bettencourt A, et al. The role of HLA-DRB1 alleles on susceptibility and outcome of a Portuguese multiple sclerosis population. J Neurol Sci. 2007;258:69–74.

Correspondence: Ana Martins Silva at

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Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus.