Clinical Abstracts
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December 2011 Editor:
Changes in tumor antigens, whether due to overexpression, mutation, or altered degradation, can lead to the development of autoantibodies. Tumor antigen-specific autoantibodies have been identified in the sera of patients with solid tumors, with antibody levels generally increasing with tumor burden. The long half-life and in vitro stability of these antibodies make them potential biomarkers for the early detection or prognosis of cancer, or both. As an autoantibody biomarker, p53-AAb are attractive because p53 is mutated in a variety of cancers. The development of p53-AAb is associated with tumor p53 mutations that lead to decreased protein degradation and reflect p53-dependent changes in tumor biology. P53-AAb are detected in the sera of six percent to seven percent of patients with limited-stage ovarian cancer and 19 percent to 30 percent of patients with late-stage ovarian cancer, suggesting that p53-AAb would have limited application as a diagnostic biomarker. Evidence supporting the utility of p53-AAb as a prognostic biomarker in ovarian cancer is mixed. These differences may reflect patient selection or differences in epitope detection in the assays. It is not known if cancer autoantibodies are involved in active immunologic surveillance or if they are byproducts of altered protein structure found in cancer cells. The authors investigated the utility of p53-AAb as biomarkers of diagnosis and prognosis for serous ovarian cancer. P53-AAb were detected by ELISA in sera obtained preoperatively from women undergoing surgery for a pelvic mass. This group included women subsequently diagnosed with invasive serous ovarian cancer (n=60) and nonserous ovarian cancers (n=30), as well as women with benign disease (n=30). Age-matched controls were selected from the general population (n=120). Receiver operating characteristic curves were constructed to compare the values of p53-AAb, CA 125, and HE4 as screening biomarkers. Kaplan-Meier curves and Cox proportional hazards modeling were used to assess the prognostic value of p53-AAb for survival. P53-AAb were detected in 25 of 60 (41.7 percent) serous cases, four of 30 (13.3 percent) nonserous cases, three of 30 (10 percent) benign disease cases, and 10 of 120 (8.3 percent) controls (combined, P=0.0002). P53-AAb did not significantly improve the detection of cases (area under the curve [AUC], 0.69) or the discrimination of benign versus malignant disease (AUC, 0.64) compared with CA 125 (AUC, 0.99) or HE4 (AUC, 0.98). In multivariate analysis among cases, p53-AAb correlated only with a family history of breast cancer (P=0.01). Detectable p53 antibodies in pretreatment sera were correlated with improved overall survival (P=0.04; hazard ratio, 0.57; 95 percent confidence interval, 0.33–0.97) in serous ovarian cancer. The authors concluded that antibodies to p53 are detected in the sera of 42 percent of patients with advanced serous ovarian cancer. Anderson KS, Wong J, Vitonis A, et al. p53 autoantibodies as potential detection and prognostic biomarkers in serous ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2010;19:859–868. Correspondence: Karen S. Anderson at kan derson@partners.org [ Top ]
Toxoplasma gondii infection is generally mild or subclinical in healthy people. However, when acquired during pregnancy, it exposes the fetus to a risk of congenital infection. The consequences of such infection range from severe fetal lesions diagnosed in utero or at birth, to the late development of retinal diseases in otherwise healthy children or adults. The early detection and treatment of congenital Toxoplasma infection are thought to reduce the risk of severe clinical lesions and neurological or ocular damage, although the precise benefits of such treatment remain unclear. Screening policies to prevent or diagnose congenital toxoplasmosis differ between developed countries and range from no prenatal screening, such as in the United States, to monthly prenatal testing, such as in France. Worldwide variations in the prevalence of past infection among pregnant women, such as 44 percent in France compared with 15 percent in the United States, help explain these differences. In several European countries, screening for Toxoplasma gondii infections in pregnancy is mandatory or recommended by national guidelines, but screening increasingly is being performed elsewhere at the request of pregnant women or on the initiative of their obstetricians. When acute infection is detected during pregnancy, the physician must, simultaneously, make decisions about the most appropriate treatment, organize fetal ultrasound surveillance, and deal with the anxiety of the parents. Prenatal diagnosis using polymerase chain reaction (PCR)-based methods on amniotic fluid is widely used to detect fetal infection and guide decisions concerning the need to introduce or continue treatment with pyrimethamine and sulfonamides. Interpreting PCR results requires an understanding of the performance and limitations of the test. Most published data were obtained for conventional PCR assays, frequently based on the B1 gene, which have an overall sensitivity of only 65 percent. New techniques have been developed during the past 10 years to increase the performances of PCR tests through real-time amplification methods and by using more repetitive DNA targets. The information available to clinicians about the performance of these tests on amniotic fluid for the prenatal diagnosis of congenital toxoplasmosis remains scarce and is limited to retrospective samples. The authors conducted a prospective cohort study of women with Toxoplasma infection identified by prenatal screening at three medical centers that routinely perform real-time PCR for detecting Toxoplasma gondii in amniotic fluid. The data available were gestational age at maternal infection, types and dates of maternal treatment, results of amniocentesis and neonatal workup, and definitive infectious status of the child. The authors estimated sensitivity, specificity, and positive and negative predictive values overall and per trimester of pregnancy at the time of maternal infection. Polymerase chain reaction analysis was performed on amniotic fluid for 261 of the 377 patients included (69 percent). The analysis was accurate, with the exception of four negative results in children who were infected. Overall sensitivity and negative predictive value were 92.2 percent (95 percent confidence interval, 81 to 98 percent) and 98.1 percent (95 percent confidence interval, 95 to 99.5 percent), respectively. No significant association was noted relative to trimester of pregnancy during which maternal infection occurred. Specificity and positive predictive values of 100 percent were obtained for all trimesters. The authors concluded that real-time PCR analysis significantly improves the detection of T. gondii in amniotic fluid. It provides an accurate tool to predict fetal infection and to determine appropriate treatment and surveillance. However, postnatal followup remains necessary in the first year of life to fully exclude infection in children for whom PCR results are negative. Wallon M, Franck J, Thulliez P, et al. Accuracy of real-time polymerase chain reaction for Toxoplasma gondii in amniotic fluid. Obstet Gynecol. 2011;115:727–733. Correspondence: Martine Wallon at martine.wallon@chu-lyon.fr [ Top ]
Autoimmune bullous dermatoses are characterized by the production of autoantibodies against adhesive and anchoring proteins of the skin, such as proteins of the dermoepidermal junction or the desmosome complex. One group of diseases is characterized by autoantibodies directed against components of the basement membrane zone (BMZ) of stratified squamous epithelium that can be detected by indirect immunofluorescence (IIF). This group comprises the main entities of bullous pemphigoid (BP), a subepidermal blistering disease characterized by frequent tense blisters and erythema in which the main targets are BP180 and BP230; mucous membrane pemphigoid (MMP), a heterogeneous group of subepithelial bullous diseases with frequent oral and occasional conjunctival, genital, ear, nose, or throat mucosal involvement, as well as skin lesions; and epidermolysis bullosa acquisita (EBA) and bullous systemic lupus erythematosus (BSLE), two rare entities characterized by antibodies against type VII collagen, a component of anchoring fibers on the dermal side of the BMZ. A second group of diseases is composed of intraepidermal blistering diseases comprising pemphigus vulgaris (PV) and pemphigus foliaceus (PF). There is considerable clinical and histological overlap among these diseases, and differential diagnosis requires fine analysis of circulating autoantibodies. In addition to IIF and enzyme-linked immunosorbent assay (ELISA), a number of immunoblotting techniques have been developed for simultaneously identifying most antigen targets. The main substrates are epidermal and dermal extracts, keratinocyte culture, and placental amnion, which has the advantage of avoiding the technical problems of epidermal-dermal separation. The authors conducted a study to develop a simple, standardized, and reproducible immunoblotting technique using human amniotic membrane as the substrate and to compare the performance of this method with that of IIF (using various substrates) and ELISA (for anti-BP180, BP230, antidesmoglein 1 [Dsg1], and Dsg3 antibodies) in a large cohort of patients with various bullous dermatoses. Sera from 113 patients were tested by immunoblotting, rat and monkey oesophagus and salt-split skin IIF, and ELISA quantification of anti-BP180-NC16a and anti-BP230 or Dsg1 and Dsg3 antibodies. The study involved 56 cases of BP, 22 cases of MMP, eight cases of EBA, two cases of BSLE, 17 cases of PV, and four cases each of PF and paraneoplastic pemphigus (PNP). In BP, the three methods had similar sensitivity (84 to 89 percent) for anti-BP180-NC16a and anti-BP230 antibody detection. In MMP, autoantibodies (mainly directed against BP180 or laminin 332 subunits) were detected in 77 percent of patients by immunoblotting, compared with only nine percent by IIF on rat and monkey oesophagus and 36 percent on salt-split skin, and 14 percent by anti-BP180-NC16a and anti-BP230 ELISA. In patients with pemphigus, ELISA had 92 percent sensitivity for anti-Dsg1 and anti-Dsg3, but immunoblotting and rat bladder IIF were necessary to confirm PNP by revealing specific and rare patterns (antidesmoplakin I/II, antienvoplakin, and antiperiplakin antibodies). Immunoblotting also revealed anticollagen VII antibodies in 60 percent of patients with EBA and BSLE and antibodies to BP180, BP230, and Dsg3 in a few patients who were negative using the other two techniques. The authors concluded that amniotic membrane immunoblotting is an interesting diagnostic tool for bullous diseases as the entire panel of autoantibodies can be detected with a single extract. This method improves the identification of complex and heterogeneous autoimmune processes in conjunction with IIF and ELISA and is particularly useful for MMP characterization. Grootenboer-Mignot S, Descamps V, Picard-Dahan C. Place of human amniotic membrane immunoblotting in the diagnosis of autoimmune bullous dermatoses. Brit J Dermatol. 2010;162:743–750. Correspondence: Sabine Grootenboer-Mignot at sabine.grootenboer-mignot@bch.aphp.fr [ Top ]
The incidence of pregnancies involving twins has been increasing over the past three decades due to greater reliance on fertility treatments and rising maternal age secondary to delayed childbearing. Rates of twins from conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 26.9 percent and 26.02 percent, respectively. Rates of triplets are 2.8 percent and 2.9 percent, respectively. Together, these generate an estimated total of approximately 197,000 to 220,000 infants worldwide. Patients gestating multiples are at higher risk for fetal chromosomal anomalies than those gestating singletons. The overall probability that a multiples gestation contains an aneuploid fetus is directly related to its zygosity. If monozygotic twins most often have the same karyotype and the risk of an affected fetus approximates the maternal age risk of a singleton, then each fetus of a dizygotic twin pair has an independent risk of aneuploidy so that the pregnancy has approximately twice the singleton risk of an affected fetus. Therefore, invasive prenatal diagnosis should be considered an important aspect of the antepartum management of patients with multiples gestation. However, the recommendation to undergo this testing should be evaluated carefully, particularly for gestations resulting from fertility treatments, because of the possible increased risk of pregnancy loss resulting from invasive procedures. Previous studies of twin gestations have reported high success rates for chorionic villus sampling (CVS) and amniocentesis, whereas the rates of pregnancy loss vary widely. One study that evaluated the risk of amniocentesis in twins compared 101 sampled pregnancies with an unsampled control group scanned at a matching gestational age and detected no significant difference in total loss rates. The risk of fetal loss after using CVS in pregnancies involving twins varied widely. Those studies that have compared fetal loss rates in singletons and twins found that CVS was not associated with an increased risk of total pregnancy losses or single fetal losses. The authors found only two studies that compared CVS with amniocentesis in twin pregnancies in contemporaneously sampled groups. They found no difference in the pregnancy loss rate and a potential small benefit from CVS relative to the total fetal loss rate. No studies have compared second-trimester amniocentesis with first-trimester CVS according to sampling techniques: single versus double entry to the uterus. In some cases, one entry to the uterus may be made, sampling before the first placenta and then moving the needle to the other placenta in the case of CVS, or aspirating amniotic fluid from the sac of the first twin and then advancing through the intertwin membrane into the sac of the second one. The hypothesis is that by introducing a single-entry technique, the fetal loss rate in twins undergoing invasive prenatal diagnosis may be lower and similar to that of singletons. The authors conducted a study of 204 pregnancies involving twins in which the pregnant women underwent amniocentesis (100) or chorionic villus sampling (104) to assess the invasive prenatal diagnosis-related risk of pregnancy loss in twin gestations and in particular to compare second-trimester amniocentesis with first-trimester CVS according to sampling techniques. The authors found that the fetal loss rate at less than four weeks was 3.85 percent in the CVS group and 4.0 percent in the amniocentesis group (P value not significant). According to sampling technique, the fetal loss rate was 4.17 percent (CVS, one puncture), 2.7 percent (amniocentesis, one puncture), 3.75 percent (CVS, two punctures), and 4.76 percent (amniocentesis, two punctures). The rate of preterm premature rupture of the membranes at less than 34 weeks was 8.2 percent in the CVS group and 10 percent in the amniocentesis group (P value not significant). According to sampling technique, the rate of preterm premature rupture of the membranes was 12.5 percent (CVS, one puncture), 8.1 percent (amniocentesis, one puncture), 6.9 percent (CVS, two punctures), and 11.1 percent (amniocentesis, two punctures). The authors concluded that the double-entry technique does not significantly affect the outcomes evaluated for amniocentesis and chorionic villus sampling. Simonazzi G, Curti A, Farina A, et al. Amniocentesis and chorionic villus sampling in twin gestations: which is the best sampling technique? Am J Obstet Gynecol. 2010;202:365.e1–365.e5. Correspondence information not available. [ Top ]
Blood culture has played a major diagnostic role in medicine for decades, but it does generate false-positive results. Consequently, laboratories have employed numerous interventions with blood culture processes to reduce contamination. The authors introduced a new blood culture technique that significantly reduced the blood culture contamination rate in a study they conducted. The technique, the initial specimen diversion technique (ISDT), omits the first approximately 1-mL portion of venipuncture blood from the culture specimen without compromising or diminishing the volume of blood desired for culture. The authors developed ISDT based on the hypothesis that skin fragments incompletely sterilized by skin surface antisepsis and dislodged by venipuncture increase the blood culture contamination rate (R). The authors evaluated their hypothesis prospectively, first using ISDT versus control cultures for a nine-month period (group one; n=3,733 cultures). Next, they collected data for a second nine-month period with an all-cultures ISDT (group two; n=4,143) for comparison to the first group. The authors’ definition of a control culture (C): one venipuncture with two sequentially obtained specimens of 10 mL each, with the first specimen (M1) for aerobic media and the second (M2) for anaerobic media. The test ISDT culture (D) was identical, except that each was preceded by diverting a 1-mL sample (DS) from the same venipuncture. During the first of two sequential nine-month periods, the authors captured D versus C data (n=3,733), where DMXR and CMXR are R for D and C specimens. Their hypothesis predicted DS would divert soiled skin fragments from DM1 and therefore, CM1R would be significantly greater than DM1R. This was confirmed by CM1R (30 of 1,061 [2.8 percent]) less DM1R (37 of 2,672 [1.4 percent]; P=0.005), which equals 1.4 percent. For the second five-month followup period, data were compiled for all cultures (n=4,143), where ADMXR is R for all (A) diversion specimens, enabling comparison to test ISDT. The authors’ hypothesis predicted no significant differences for test ISDT versus all ISDT. This was confirmed by DM1R (37 of 2,672 [1.4 percent]) versus ADM1R (42 of 4,143 [1.0 percent]; P=0.17) and DM2R (21 of 2,672 [0.80 percent]) versus ADM2R (39 of 4,143 [0.94 percent]; P=0.50). The authors concluded that their hypothesis is valid: Venipuncture needles soil blood culture specimens with unsterilized skin fragments and increase R, and ISDT significantly reduces R from blood culture specimens obtained by venipuncture. Patton RG, Schmitt T. Innovation for reducing blood culture contamination: initial specimen diversion technique. J Clin Microbiol. 2010;48:4501–4503. Correspondence: Richard G. Patton at rpat ton@magnoliamt.com [ Top ]
Matrix metalloproteinases and their tissue inhibitors are important mediators of host-induced tissue destruction and are implicated in the pathogenesis of neuroinflammatory diseases. Matrix metalloproteinases (MMPs) are secreted by all cell types in the central nervous system and degrade constituents of the blood-brain barrier (BBB) basement membrane, such as type IV collagen, laminin, aggrin, and fibronectin. Models of central nervous system infections have shown that infiltrating leukocytes use MMPs to traverse the glia limitans. MMP knockout mice showed less immune pathology, BBB breakdown, and cellular infiltration, and MMP inhibitors provided better outcomes in experimental pneumococcal meningitis. However, the role of MMPs/tissue inhibitor of metalloproteinases (TIMPs) in the pathogenesis of central nervous system infection in humans is not well defined. The authors measured pretreatment cerebrospinal fluid MMP-1 to -10 and TIMP-1, -2, and -4 concentrations by ELISA in 96 HIV-negative, randomly chosen Vietnamese adults admitted to the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, with bacterial (n=31), tuberculous (n=11), fungal (n=13), parasitic (n=11), or viral (n=10) meningitis. Ten patients with cerebral malaria and 10 patients without infections served as controls. All were recruited between November 2006 and November 2008. The authors followed a predefined plan to explore relationships between CSF MMP/TIMP expression and prospectively recorded clinical features and outcome. Cerebrospinal fluid MMP and TIMP concentrations varied according to the etiology of meningitis. Patients with pyogenic bacterial meningitis had elevated MMP-1, -3, -7, -8, -9, and -10 concentrations (P<0.01). Cryptococcal and eosinophilic meningitis were associated with increased MMP-7, -10, and TIMP-2 concentrations (P<0.01). Tuberculous meningitis was characterized by a trend toward elevated MMP-1 concentrations. MMP-9 and CSF white cell count (WCC) were strongly correlated (R2=0.73; P<0.01), regardless of etiology. CSF MMP-3, -8, and -9 concentrations were significantly correlated with one another: MMP-8/-9 (R2=0.52; P<0.01), MMP-3/-8 (R2=0.47; P<0.01), and MMP-3/-9 (R2=0.13, P<0.01). TIMP-1 concentrations were correlated with CSF WCC (R2=0.35; P<0.01) and MMP-9 (R2=0.37; P<0.01). MMP-8 and -9 were also correlated with CSF lactate concentration (R2=0.62; P<0.01, and R2=0.33; P<0.01, respectively). The authors did not find correlations between CSF lymphocyte count and CSF MMP/TIMP concentrations or CSF total protein and MMP/TIMP concentrations. They examined the clinical features associated with severe coma (defined by Glasgow Coma Score of 11 or less) at presentation. On univariate analysis, low peripheral blood WCC, high CSF lactate concentration, and high CSF MMP-3 and -7 and TIMP-2 were associated with severe coma (P<0.1). On multivariate logistic regression analysis, high CSF TIMP-2 concentration was associated with a Glasgow Coma Score of more than 11 (odds ratio, 0.2; 95 percent confidence interval, 0.04–0.97; P<0.05) and high CSF lactate concentration remained predictive of severe coma (odds ratio, 6.4; 95 percent confidence interval, 1.02–39.8; P<0.05). In tuberculous, cryptococcal, and eosinophilic meningitis, coma severity was independently associated with high CSF MMP-8 concentration. In-hospital deaths occurred in those with cerebral malaria (5/10), as well as cryptococcal (4/13), eosinophilic (3/11), tuberculous (3/11), and bacterial meningitis (1/31). The authors did not find a significant relationship between death and CSF MMP/TIMP concentrations. Green JA, Chau TTH, Farrar JJ, et al. CNS infection, CSF matrix metalloproteinase concentrations, and clinical/laboratory features. Neurology. 2011;76:577–579. Correspondence: Dr. J. A. Green at justin.green@imperial.ac.uk [ Top ]
Neisseria gonorrhoeae, the etiological agent of gonorrhea, has developed resistance to multiple classes of antibiotics, including penicillins, tetracyclines, macrolides, and quinolones. The extended-spectrum cephalosporins are the mainstay of treatment in most settings. However, there are certain populations where, owing to factors ranging from limited use of antimicrobials to geographical and microbial isolation from populations harboring resistant strains, antimicrobial resistance is low and antibiotics such as penicillin can still be used successfully in accordance with World Health Organization (WHO) guidelines. An example of this is Australia’s Northern Territory, where, unlike the other Australian mainland states, in vitro resistance to penicillin is observed in less than five percent of the gonococci isolated from the local population. The authors conducted a study in which they developed and evaluated real-time polymerase chain reaction (PCR) for detecting penicillinase-producing N. gonorrhoeae (PPNG) in noncultured clinical samples. The assay (PPNG-PCR2) was designed to be an indirect marker of penicillinase activity by targeting a region of sequence predicted to be conserved across all N. gonorrhoeae plasmid types harboring the β-lactamase gene while not specifically targeting the β-lactamase-encoding sequence. The assay was evaluated using 118 N. gonorrhoeae clinical isolates and 1,194 clinical specimens, including 239 N. gonorrhoeae-positive clinical samples from which N. gonorrhoeae cells were isolated and for which phenotypic penicillinase results were available. Overall, the PPNG-PCR2 assay provided 100 percent sensitivity and 98.7 percent specificity, compared to bacterial culture results, for detecting PPNG in clinical specimens. PPNG-PCR2 false-positive results, presumably due to cross-reactions with unrelated bacterial species, were observed for up to 1.3 percent of clinical samples but could be distinguished on the basis of high cycle threshold values. The authors concluded that, in tandem with phenotypic surveillance, the PPNG-PCR2 assay has the potential to provide enhanced epidemiological surveillance of N. gonorrhoeae penicillin resistance and is particularly relevant in regions where penicillin is still used to treat gonorrhea. Goire N, Freeman K, Tapsall JW, et al. Enhancing gonococcal antimicrobial resistance surveillance: a real-time PCR assay for detection of penicillinase-producing Neisseria gonorrhoeae by use of noncultured clinical samples. J Clin Microbiol. 2011;49:513–518. Correspondence: David M. Whiley at d.whiley@uq.edu.au [ Top ]
Circulating tumor cells are a hallmark of invasive cancer cells, which are responsible for the development of metastases. It is imperative to develop new approaches for detecting and quantifying circulating tumor cells (CTCs), which will significantly contribute to clinical prognosis, diagnosis, individualization, and optimization of systemic therapy. Detecting rare CTCs in complex blood samples is a major challenge, requiring an exceptionally specific and sensitive assay for discerning and capturing CTCs with high efficiency. Some progress recently has been made in this area. The U.S. Food and Drug Administration approved the use of CellSearch (Veridex), which is based on immunomagnetic separation. A potential concern with this method is the impurity of leukocytes, leading to a false-positive rate. More recently, a CTC chip has been developed for monitoring therapeutic response, but the flow method is time-consuming and requires approximately six hours to process each sample. The majority of CTC techniques require an initial enrichment step because CTCs are rare events. After pre-separation, immunofluorescent labeling of captured cells is required to further validate the presence of CTCs. The authors reported on a direct assay based on highly specific and sensitive surface-enhanced Raman spectroscopy (SERS) technology to detect CTCs in peripheral blood, which requires no subsequent separation procedure. One of the key advantages of using SERS rather than a fluorescence technique is that the former gives a sharp fingerprint-like spectral pattern, which is distinct from other interferences within the complex biological milieu. In contrast, fluorescence spectra could be disguised by strong scattering signals from cells and protein clusters at low-signal intensities, such as rare CTC events. The distinctive SERS spectral pattern eliminates the need for tedious separation procedures. SERS nanoparticles with epidermal growth factor peptide as a targeting ligand have identified CTCs in the peripheral blood of 19 patients with squamous cell carcinoma of the head and neck, with a range of one to 720 CTCs/mL whole blood. The authors concluded that this technique may provide an important new clinical tool for managing patients with squamous cell carcinoma of the head and neck and other cancers. Wang X, Qian X, Beitler JJ, et al. Detection of circulating tumor cells in human peripheral blood using surface-enhanced Raman scattering nanoparticles. Cancer Res. 2011;71:1526–1532. Correspondence: Dong M. Shin at dmshin@emory.edu [ Top ]Clinical pathology abstracts editor: Michael Bissell, MD, PhD, MPH, professor, Department of Pathology, Ohio State University, Columbus. |
