Clinical Abstracts
Clinical Abstracts |
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November 2004 Editors:
New assay for factor V Leiden A new non-PCR-based nucleic acid diagnostic testing platform has been developed that is based on the use of DNA probes that are modified with photo-active cross-linking molecules. The sample is first processed to render the target nucleic acids available for hybridization. Capture and reporter probes are then added and hybridized to their complementary target sequences, at which time the sample is irradiated with ultraviolet light to induce the formation of a covalent cross-link between the probes and target. The potential benefit of this approach is that by forming an irreversible probe-target hybrid, the latter steps in the assay process, such as target capture and washing, are less constrained than those in a conventional hybridization-based assay. In the cross-linking assay format, capture and washing can be performed under conditions that may denature conventional probe-target hybrids. Consequently, the cross-linking approach can provide increased signal as a result of more efficient target capture and decreased background as a result of more effective removal of nonhybridized DNA probes and other nonspecific signal sources. The authors of this study aimed to improve the sensitivity and performance of the method as applied to the detection of factor V Leiden mutations. They developed new reporter probes with approximately 10-fold more fluorescein molecules than the original probes. The single cross-linker-modified capture probe was replaced by a three-probe system, separating the probe-target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/ flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing pretyped samples. The authors then tested 1,054 clinical samples using an automated sample processor. This results in a new assay with a 10-fold increase in signal-to-background ratio. Genotype results for 1,039 of 1,054 clinical samples (98.6 percent) agreed with those of a polymerase chain reaction-based method. Of the 15 remaining samples, 10 produced indeterminate results outside the defined genotype ranges, two yielded insufficient signal to be genotyped, and three gave a discordant result. All 15 samples were genotyped correctly after re-extraction of genomic DNA and retesting. French C, Li C, Strom C, et al. Detection of the factor V Leiden mutation by a modified photo-cross-linking oligonucleotide hybridization assay. Clin Chem. 2004;2: 296-305. Reprints: Michael Wood, NAXCOR, 320 Logue Ave., Suite 200, Mountain View, CA 94043; mikew@naxcor.com Detecting Mycobacterium tuberculosis using PCR The poor sensitivity of standard laboratory tests is a major obstacle to diagnosing tuberculosis in children because the organism load in children is generally much lower than in adults. Mycobacterium tuberculosis is isolated in less than half of clinically suspected pediatric cases. A rapid test to diagnose tuberculosis in children is needed. Rapid tests based on nucleic acid amplification techniques have been developed to detect mycobacteria in clinical specimens. It remains to be proven, however, whether they can fulfill the requirements of high sensitivity and specificity, simplicity, and reasonable cost. A wide range of nucleic acid amplification tests have recently been developed as alternatives to polymerase chain reaction (PCR), a few of which have been evaluated in the clinical laboratory and are commercially available, including PCR-based Amplicor (Roche), ligase chain reaction (LCX; Abbott), transcription-mediated amplification (TMA; Gen-Probe), and strand displacement amplification (BDProbe; Tec-SDA). The low sensitivity and high cost of these tests, however, have precluded their use in detecting pediatric tuberculosis. The authors evaluated a novel heminested IS6110 PCR assay as a tool for diagnosing tuberculosis in 222 children, compared the results of PCR with those of conventional acid-fast bacilli (AFB) smear microscopy and culture, and compared the diagnostic value of different specimen types obtained from children with suspected tuberculosis. In an analysis of 392 specimens—gastric aspirates, nasopharyngeal aspirates, and sputum samples—results of PCR were compared with those of three culture methods, AFB staining, and clinical assessment by the Stegen-Toledo score. The sensitivity of PCR (67 percent) was comparable to that of the three-culture method (71 percent) and was significantly higher than that of Löwenstein-Jensen culture (54 percent) or AFB stain (42 percent) for children with highly probable tuberculosis. PCR detection rates for culture-positive specimens were 100 percent for smear-positive samples and 76.7 percent for smear-negative samples. The specificity of PCR was 100 percent in control children. Compared with culture, PCR demonstrated a sensitivity of 90.4 percent, positive predictive value of 89 percent, specificity of 94 percent, and negative predictive value of 95 percent (κ=.85). With clinical assessment as the standard, PCR had a sensitivity of 71 percent, positive predictive value of 92 percent, specificity of 95 percent, and negative predictive value of 79 percent (κ=.67). The authors concluded that PCR is a rapid and sensitive method for the early diagnosis of pediatric tuberculosis. Montenegro SH, Gilman RH, Sheen P, et al. Improved detection of Mycobacterium
tuberculosis in Peruvian children by use of a heminested IS6110 polymerase chain
reaction assay. Reprints: Dr. Sonia H. Montenegro, Molecular Immunogenetics Laboratory, Division
of Research, Ochsner Clinic Foundation, 1516 Jefferson Hwy., Biomedical Research
Bldg., Room 1N408, New Orleans, LA 70121; smontenegro@ochsner.org
Aging is associated with increased circulating levels of inflammatory markers,
including proinflammatory and anti-inflammatory cytokines, cytokine antagonists,
and acute phase proteins. Low-grade inflammatory activity, in turn, has been
associated with morbidity, functional disability, and mortality in the elderly,
but it remains unclear whether low-grade inflammatory activity causes morbidity
and frailty or whether it results from underlying pathological processes. The
authors have reported that high plasma levels of tumor necrosis factor alpha
(TNF-α), a classic proinflammatory cytokine, are associated with Alzheimer’s
disease and atherosclerosis in people who are 100 years old. TNF-? has also
been correlated with levels of interleukin-6 (IL-6) and C-reactive protein (CRP)
in people aged 100 years, reflecting activation of the entire inflammatory cascade
in the very elderly. The authors hypothesized that TNF-α is a specific
risk factor in people who are 100 years old and aimed to assess whether it is
a prognostic marker of all-cause mortality in Danish centenarians, independent
of comorbid conditions and other inflammatory markers, including IL-6, IL-8,
and CRP. Enrolled in the study were 126 subjects who were at or near their 100th
birthday. Plasma levels of TNF-α, IL-6, IL-8, and CRP were measured at
baseline. The authors determined the associations between the markers of inflammation
and mortality during the subsequent five years. Only nine subjects were alive
after five years. Elevated levels of TNF-α were associated with mortality
in men and women (hazard ratio=1.34 per standard deviation of 2.81 pg/mL; 95
percent confidence interval: 1.12 to 1.60, P=0.001). Levels of IL-6
and IL-8 did not affect survival, and levels of C-reactive protein were not
associated with mortality when levels of TNF-α were included in the analysis.
Dementia and cardiovascular diseases represented the major causes of comorbid
conditions at baseline. TNF-α was still associated with mortality in multivariate
models that included these parameters as confounders. The authors concluded
that TNF-α is an independent prognostic marker for mortality in people
who are 100 years old, suggesting that it has specific biological effects and
is a marker of frailty in the very elderly.
Bruunsgaard H, Anderson-Ranberg K, Hjelmborg JvB, et al. Elevated levels of
tumor necrosis factor alpha and mortality in centenarians. Am
J Med. 2003;115:278-283.
Reprints: Dr. Helle Bruunsgaard, Dept. of Infectious Diseases, M7641, Rigshospitalet,
University of Copenhagen, Bledamsvej 9, DK-2100 Copenhagen East, Denmark; infdishb@rh.dk
Increased levels of sperm ubiquitin and sperm quality
Sperm with intrinsic defects may appear morphologically normal on light microscopic
evaluation. Therefore, additional objective techniques that are not based on
conventional measures of semen parameters are needed to evaluate semen. Ubiquitin,
an 8.5 kDa highly conserved protein, forms covalently linked polyubiquitin chains
on substrate proteins and targets such "ubiquitinated" substrates for endocytosis
or proteolytic degradation, or both, by the multi-subunit protease, the 26-S
proteasome. Surface ubiquitination of defective sperm occurs during epididymal
passage. Ubiquitin is also a major protein of human seminal plasma. The cofactors
required for the presentation of ubiquitinated proteins to proteasome are expressed
in mammalian sperm. The clearance of defective sperm and debris in the epididymis
is not complete, although the number of defective cells decreases appreciably
during epididymal passage. To examine the role of ubiquitin, the authors developed
an objective immunoassay (SUTI, or sperm-ubiquitin tag immunoassay) designed
to reveal defective sperm regardless of whether or not their defects are detectable
by light microscopic evaluation. The study aimed to further validate sperm ubiquitin
as a biomarker of human male infertility in a group of 43 fertile donors and
infertility patients with various etiologies. Anti-ubiquitin immunoreactivity
was measured by SUTI in the sperm samples of 28 infertility patients and 15
fertile donors. Semen analyses were performed by computer-assisted semen analysis
and World Health Organization morpohology. Median values of ubiquitin-induced
fluorescence had a strong negative correlation with sperm count (r=-0.63;
P=0.0003) and a positive correlation with percentage abnormal morphology
(r=0.55; P=0.01). Infertility patients (n=28) had
significantly higher levels of sperm ubiquitin. Of 28 patients, six reported
possible occupational exposures to solvents, three were smokers, and six were
ex-smokers. Within the patient group, men with known male factor infertility,
those with self-reported occupational exposure to solvents, and smokers had
the highest sperm ubiquitin levels. When men with jobs involving potential occupational
exposure to solvents were combined with smokers, the highest correlations were
found between sperm ubiquitin and motility (r=-0.74), sperm count (r=-0.82),
and percentage of sperm abnormalities (r=0.73). Increased sperm ubiquitin
was inversely associated with sperm count, motility, and percentage normal morphology,
supporting the use of ubiquitin as a biomarker of human semen quality. SUTI
confirmed poor semen quality in all men with poor clinical semen parameters
but was high in some patients with seemingly good clinical semen parameters.
Sutovsky P, Hauser R, Sutovsky M. Increased levels of sperm ubiquitin correlate
with semen quality in men from an andrology laboratory clinic population. Hum
Reprod. 2004;19:628-638.
Reprints: Peter Sutovsky, University of Missouri-Columbia, S141 ASRC, 920 E.
Campus Drive, Columbia, MO 65211-5300; sutovskyp@missouri.edu
Ethanol-drug interactions and platelet function
Ethanol inhibits platelet aggregation induced by collagen, in part by inhibiting
thromboxane A2 formation. However, ethanol also inhibits thrombin-induced responses
of aspirin-pretreated platelets, indicating that ethanol has other inhibitory
effects, possibly on the activation or activity of phospholipase C. It has also
been demonstrated that ethanol adds to the inhibitory effects on aggregation
of submaximal concentrations of prostacyclin or adenosine. Combinations of inhibitors
that act on different pathways have been found to be more effective than one
might predict by adding the effects of the inhibitors used singly. Because patients
at risk of a second myocardial infarction or other acute coronary syndromes
who would be candidates for oral GP IIb/IIIa inhibitor therapy often lead a
normal lifestyle, which may include consuming alcoholic beverages, the additional
inhibitory effect of ethanol on platelet function may result in a greater interference
with hemostasis and increase the antithrombotic effect of the GPIIb/IIIa blocker.
To test this hypothesis in vitro, the authors examined the effect of combinations
of the drug LY309562 (a novel 2,6-disubstituted isoquinolone RGD mimic that
competes for fibrinogen binding to GPIIb/IIIa), aspirin, and ethanol on platelet
aggregation and the secretion of granule contents by human platelets stimulated
by several platelet agonists. These in vitro studies were conducted with PRP
from blood anticoagulated with the thrombin inhibitor FPRCH2CI,
or PPACK, so that a physiological concentration of ionized calcium was maintained.
At inhibitory concentrations of LY309562, ethanol (2 or 4 mg/mL) further inhibited
platelet responses to various agents. Responses of aspirin-treated platelets
to U46619 were also inhibited, indicating that ethanol was not acting solely
by inhibiting thromboxane A2 formation. Since it is likely that the authors'
results with LY309562 are representative of results with other GPIIb/IIIa antagonists,
their in vitro data suggest that the concomitant use of GPIIb/IIIa antagonists
and consumption of alcoholic beverages may further impair the ability of platelets
to participate in hemostasis and thrombosis.
Rand ML, Jakubowski JA, Fisher JM, et al. Ethanol enhances the inhibitory effect
of an oral GPIIb/IIIa antagonist on human platelet function. J
Lab Clin Med. 2002;140: 391-397.
Reprints: Dr. Margaret L. Rand, Division of Haematology/Oncology, The Hospital
for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8, Canada; margaret.rand@sickkids.ca
Doxycycline as an apoptosis inducer
Tetracyclines have an anti-tumor effect; however, the cellular mechanisms
of these biological activities remain unknown. Inhibition of solid malignant
tumor proliferation, invasion, and metastasis by doxycycline or chemically modified
tetracyclines has been reported. Tetracyclines are also known to be well-tolerated
agents that can suppress matrix metalloproteinase (MMP) activation in many tissues,
including malignant tumors. The MMPs play a significant role in the growth,
invasion, and metastasis of many tumors. The inhibitory effect of doxycycline
on MMP expression is independent of its antibiotic properties. The authors'
data indicate that doxycycline suppresses the proliferation of human leukemic
CCRF-CEM cells in vitro and that it induces cell death by enhancing apoptosis.
Cells were incubated with doxycycline at concentrations ranging from zero to
50 μmol/L. Caspase-3 activity of cells grown in the presence of 10 μmol/L
and 50 μmol/L doxycycline increased dose-dependently after 24 hours in
culture. The demonstration that doxycycline induces APO 2.7 expression in CCRF-CEM
cells in vitro also supports its capacity for inducing apoptosis. The level
of matrix metalloproteinase-2 was significantly lower in the medium cultured
with 50 B5mol/L doxycycline than in the control medium. These phenomena suggest
that this well-tolerated oral agent has the potential to be of value in antileukemic
therapy.
Iwasaki H, Inoue H, Mitsuke Y, et al. Doxycycline induces apoptosis by way of
caspase-3 activation with inhibition of matrix metalloproteinase in human T-lymphoblastic
leukemia CCRF-CEM cells. J
Lab Clin Med. 2002;140:382-386.
Reprints: Dr. Hiromichi Iwasaki, Division of Transfusion Medicine, Fukui Medical
University, Shimoaizuki 23-3, Matsuoka, Fukui, Japan 910-1193;
hiwasaki@fmsrsa.fukui-med.ac.jp
Use of hydrogen peroxide in high-level disinfection
The heat-sensitive nature of many medical devices requires that they be chemically
disinfected between uses using manual or automated systems. The working solutions
of many germicides are meant to be used only once, but others are formulated
for reuse in manual systems for 14 to 30 days. During such reuse, the germicidal
activity of the solution can be compromised through incremental dilution, evaporation,
breakdown of active ingredients, or accumulation of soil from the devices being
processed. A reduction in the germicidal activity of such formulations during
reuse could increase the risk of spreading disease. Therefore, it is crucial
to ensure that products sold for reuse remain active against major classes of
pathogens at least for the time frame on their label. The only method available
for stressing high-level disinfectants during reuse was developed and endorsed
by the Environmental Protection Agency and the Food and Drug Administration.
This protocol challenges the solution being used with repeated stress in the
form of germ-loaded carriers as well as by soaking pieces of respiratory therapy
equipment on a prescribed schedule during the recommended period of reuse. Samples
of the test solution from the reuse bath are collected on a regular basis and
evaluated for their germicidal activity to demonstrate that the stressed solution
remains a high-level disinfectant even at the end of the recommended reuse period.
However, the carrier test protocols commonly used for this purpose are now recognized
as deficient in design, and their results are often difficult to reproduce.
Formulations derived from accelerated hydrogen peroxide have demonstrated relatively
rapid activity against all classes of human pathogens. The sporicidal, mycobactericidal,
fungicidal, and bactericidal activities of samples of stressed solutions can
be assessed using a quantitative carrier test. This method, which uses flat-bottomed
glass vials as carriers, is now a standard of ASTM International, which publishes
consensus-based standards for testing the germicidal activity of chemicals.
The virucidal activity of the test samples was determined using metal disks
as carriers, which has also recently been accepted as a standard of ASTM. The
authors of this study sought to determine if the EPA and FDA stress protocol
could be applied in combination with recently developed quantitative carrier
tests to assess the germicidal activity of an accelerated hydrogen peroxide-based,
high-level disinfectant under simulated reuse for 14 days. On alternate days,
baths with three lots of the test formulation were stressed by adding bacteria
(Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas
aeruginosa) on glass beads and spores (Bacillus subtilis
and Clostridium sporogenes) on metallic penicylinders. In addition,
one set of respiratory therapy equipment was subjected to three daily cycles
of disinfection in each bath. The pH and H2O2
levels in the test samples were measured, and the samples were subjected to
quantitative carrier tests for their sporicidal, bactericidal, virucidal, fungicidal,
and mycobactericidal activities. After 14 days of reuse, the pH of the test
solutions was unchanged. Although the level of H2O2
dropped from 7.66 percent to 6.40 percent, all lots showed the required level
of broad-spectrum germicidal activity after 14 days of stress. The authors concluded
that the stress test and quantitative carrier test were successfully combined
in demonstrating the broad-spectrum germicidal activity of a high-level disinfectant
subjected to 14 days of simulated reuse.
Sattar SA, Adegbunrin O, Ramirez J. Combined application of simulated reuse
and quantitative carrier tests to assess high-level disinfection: experiments
with an accelerated hydrogen peroxide-based formulation. Am
J Infect Control.
2002;30:449-457.
Reprints: Dr. Syed A. Sattar, University of Ottawa, Faculty of Medicine, Center for Research on Environmental Microbiology, 451 Smyth Rd., Ottawa, Ontario, K1H 8M5, Canada
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