By G. Nasib. Cleary University.
These manual commercial extraction kits show good performance for nucleic acid extraction compared with in-house methods buy requip 0.25 mg free shipping. Given these differences buy requip with a visa, there are numerous choices available; the most appropriate method for a particular laboratory should be selected discount requip uk. Most of these kits use noncorrosive agents buy requip 0.25mg without prescription, so they are safe and easy to deal with. Although the entire extraction procedure is standardized by the manufacturer’s manual, the process is still complicated and is performed manually. To minimize such reproducibility problems, it is necessary to provide continuous training and quality control. The manual extraction method has been 11 Nucleic Acid Extraction Techniques 219 Table 11. Moreover, the number of speci- mens for molecular testing is increasing, which places more stress on the technolo- gists who are processing these specimens. This can affect the accuracy of tests as a result of a processing mistake or by contamination attributable to the complex pro- cessing procedure. Automatic Methods The introduction of commercial manual extraction kits was a valuable adjunct for molecular testing in the clinical microbiology laboratory. However, the manual extraction method is still labor-intensive and time-consuming and requires a well- trained technologist. There were also reports of outbreaks of cross contamination when the samples were treated at the same time [39, 75]. In recent years, many manufacturers developed and launched various automated extraction instruments; 220 J. It provides constant reproducibility for recovery of nucleic acid, avoiding person-to-person variations seen with manual extraction methods. It can also diminish cross contamination by reducing unnecessary han- dling steps and avoiding mistakes by personnel. It has an additional advantage for quality control monitoring, whereas the manual method needs intensive work for quality control monitoring [77 ]. Since many automated extraction instruments and kits have been developed, numerous evaluations have been reported [67, 78–81 ]. These studies included vari- ous kinds of extraction kits, clinical specimens, and pathogens. Even though some reports showed high detection rates with manual extraction method, the results of automated extraction methods were similar to or better than those of manual meth- ods in most of these studies [28, 29, 45, 64, 67, 78–81]. There were some variations of viral yields, which were only 50% compared with those of the manual kits. The differences of yields are not of great signiﬁcance if we consider the biologic range of viral loads in clinical practice as the manufacturer’s recommendation. We can face dif- ferent results for the evaluation of automated extraction instrument. The most important drawback we must consider is the economic aspect of the automated methods. To use such a system, an expensive instrument and extraction reagents, including disposables, are needed. However, the inﬂuenza A (H1N1) pandemic in 2009 demonstrated the value of an automated extraction system. At that time, requests for inﬂuenza A (H1N1) identiﬁcation were increasing rapidly, and many laboratories could not perform all the requested tests because of limited personnel. Although the detection rates and yield recovery are the most important factors in selecting com- mercial extraction methods, other factors, including ease of use and cost per extrac- tion, also must be considered . Conclusion In recent years, advanced molecular tests have come to occupy an important posi- tion in the diagnosis of infectious diseases because of their high sensitivity and speci ﬁ city [83, 84]. The optimal extrac- tion method should fulﬁll the following conditions: speed, short working time, cost- effectiveness, high sensitivity and speciﬁcity, good reproducibility, and safety [1 ]. However, at pres- ent, there is no one extraction method that satisﬁes all these conditions. On the contrary, there are signiﬁcant differences between extraction kits because nucleic acids can be different in speciﬁc clinical specimens. So, it is important to carefully evaluate the performance of any extraction method used in the clinical microbiol- ogy laboratory. Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M (2010) The era of molecular and other non-culture-based methods in diagnosis of sepsis. Gerna G, Lilleri D (2006) Monitoring transplant patients for human cytomegalovirus: diagnos- tic update. Klingspor L, Jalal S (2006) Molecular detection and identiﬁcation of Candida and Aspergillus spp. Stormer M, Kleesiek K, Dreier J (2007) High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components. Comp Immunol Microbiol Infect Dis 32:207–219 11 Nucleic Acid Extraction Techniques 225 66. Valentine-Thon E (2002) Quality control in nucleic acid testing—where do we stand? J Clin Microbiol 43:4616–4622 Chapter 12 Nonampli ﬁ ed Probe-Based Microbial Detection and Identi ﬁ cation Fann Wu , Tao Hong , and Phyllis Della-Latta Introduction Probe-based nonampliﬁed molecular assays were ﬁrst developed for detection of microorganisms decades ago. Over the years, the variety of molecular technologies for the laboratory diagnosis of infectious diseases has expanded greatly, largely due to the rapidly expanding ﬁeld of sequenced microbial genomes. Probe-based hybrid- ization assays remain a commonly used format in clinical microbiology laboratories due to its numerous advantages over routine culture-based methodologies. Conventional phenotypic methods of bacterial identiﬁcation which include the Gram stain, culture, and biochemical reactions, contain three major challenges. First, nonviable or nonculturable organisms simply cannot be identiﬁed due to growth restrictions. Second, some microbial strains may exhibit atypical biochemi- cal characteristics that do not match established patterns routinely used for identiﬁcation. Third, slow-growing or fastidious organisms require a prolonged time to identiﬁcation. In contrast, probe-based assays bypass many of the limita- tions of phenotypic methods and provide accurate pathogen identiﬁcation in a clini- cally relevant timeframe. A variety of commercial assays are available to identify pathogens from culture, and in addition there are several assays that can detect infectious agents directly from specimen. This chapter presents an overview of the design and clinical applications of prominent nonampliﬁed probe-based methods commonly used in clinical microbiology laboratories to identify pathogens. Della-Latta Clinical Microbiology Services, Department of Pathology , Columbia University Medical Center, New York-Presbyterian Hospital, 622 West 168th Street, C. The nucleic acid probe is labeled by a variety of reporter molecules that can be chemilu- minescent, ﬂuorescent, enzymatic, or antigenic in order to detect the double-stranded hybrids. There are a variety of probes and targets that are carefully selected when designing diagnostic assays. Ribosomes are highly conserved and essential organ- elles responsible for protein synthesis and are therefore present in all living cells in high quantity. The sequence variability allows the design of species speciﬁc probes for organism identiﬁcation. Probe Selection Probe selection and labeling have direct impact on hybridization assay efﬁciency. The ideal probe is single-stranded, lacks secondary structure, and does not self anneal. A critical feature of probe selection is the careful choice of probe sequence that is complementary to the sequence of the target of interest. Currently, nucleic acid probe hybrids are detected by incorporating pre-labeled probes in the methodology. Fluorescent-labeled probes offer the advantages of producing strong signals with less background, but have the disadvantages of poor ﬂuorescent signal stability and the purchase of a ﬂuorescence microscope with appropriate ﬁlters. Solid-phase hybridizations, such as line probe or dot blot assays, occur on a solid surface (nylon membranes) to which the nucleic acid probe is bound. In general, hybridizations assays on solid-support platforms are not as sensitive as those liquid-phase formats due to the lack of exposure to all target sequences. The controlled enzymatic digestion of cellular mem- branes and other proteins allow the probes to gain access to the target sequences. The labels for the nucleic acid probes, which can be biotin or digoxigenin, incorporate a signal compound, such as a colorimetric or a ﬂuorescent compound. Other formats have been designed to combine solution and solid- phased hybridization.
Careful descriptive notations about positions used during anesthesia and surgery buy requip, as well as brief comments about special protective measures such as eye care and pressure-point padding buy requip canada, are useful to include on the anesthesia record proven requip 0.5 mg. In potentially complicated or contentious circumstances order genuine requip online, a separate brief description of care documented in the patient’s record is advisable. Only in this manner can subsequent inquiries be properly answered on behalf of either the patient or the anesthesiologist. When credible expanded knowledge that further delineates mechanisms of positioning-related complications is available, these issues and the care of patients will be improved. General Principles Without doubt, direct compression of neural and soft tissue may result in ischemia and tissue damage. Many efforts have been directed at provider education over the years to reduce direct tissue trauma from compression. Most anesthesia providers are taught from the start of their training that various maneuvers, pads, and positioning devices are useful to reduce point 2006 pressure on neural and soft tissues. Is it a failure of education, the incorrect application of this information, or other issues that contribute to the continued presence of perioperative positioning injuries? Recent studies and editorials suggest that we do not yet fully understand the etiologic mechanisms of positioning issues. Surprisingly, the majority of these patients had widespread microvasculitic neuropathies, and many were responsive to immunologic modulation with high doses of corticosteroids. The inflammatory response may be dramatically altered in the perioperative period, and microvasculitic neuropathy appears to be a previously unrecognized cause of peripheral neuropathy. For example, anesthetic drugs and transfusion of blood products are known to promote systemic inflammation. In the meantime, these reports serve as evidence that a number of perioperative neuropathies may, in fact, have no relationship to intraoperative positioning or management of physiologic factors. As noted earlier, immunosuppression is present in a fairly significant proportion of patients undergoing major surgical procedures. This immunosuppression may provide opportunities for existing viruses or newly introduced viruses to activate, particularly in neural tissues. For example, the onset of shingles may be more frequent in surgical compared to general populations. Stretch of neural tissue may be an important factor in the development of peripheral and central neuropathies. Stretch of many mammalian nerves to 5% greater than their normal resting length has been shown repeatedly to lead to ischemia by reducing both arteriole and venule blood flow. The kinking of the arterioles and venules associated with neuronal stretch leads to ischemia. The impact of stretch on other soft tissues is less well documented and would be highly dependent on the type of tissue and amount of stretch. Cerebral circulation is slightly above heart level if the head is on a small pillow. B: Head-down tilt aids blood return from lower extremities but encourages reflex vasodilation, congests vessels in the poorly ventilated lung apices, and increases intracranial blood volume. C: Elevation of the head shifts abdominal viscera away from the diaphragm and improves ventilation of the lung bases. According to the gradient above the heart, pressure in arteries of the head and neck decreases; pressure in 2008 accompanying veins may become subatmospheric. There are many ways to reduce point pressure, but the most commonly used involve padding. Although there may be distinct differences in mechanical properties of various padding materials (e. The basic principle is to use any of these materials to protect nerves and soft tissues from point pressure. Supine Positions Variations of Supine Positions Horizontal In the traditional supine position, the patient lies on his or her back with a small pillow beneath the head (Fig. The arms are either comfortably padded and restrained alongside the trunk or abducted on well-padded arm boards. Either arm (or both) may be extended ventrally and the flexed forearm secured to an elevated frame in such a way that perfusion of the hand is not compromised, no skin-to-metal contact exists to cause electrical burns if cautery is used, and the brachial neurovascular bundle is neither stretched nor compressed at the axilla. The lumbar spine may need padded support to prevent a postoperative backache (see “Complications of Supine Positions”). Fortunately, most modern surgical tables have mattress pads that are sufficiently buoyant and thick to allow dispersion of point pressure. Although the horizontal supine posture has a long history of widespread use, it does not place hip and knee joints in neutral positions and is poorly tolerated for prolonged periods by an immobilized, awake patient. It is established by arranging the surface of the operating table so that the trunk–thigh hinge is angulated approximately 15 degrees and the thigh–knee hinge is angulated a similar amount in the opposite direction. Alternatively, a rolled towel, pillow, or blanket can be placed beneath the patient’s knees to keep them flexed. The patient of average height then lies comfortably with hips and knees flexed gently. A significant degree of perfusion can be restored if the compressive mass is rolled toward the left hemiabdomen by leftward tilt of the tabletop or by a wedge under the right hip. Each lower extremity is flexed at the hip and knee, and both limbs are simultaneously elevated and separated so that the perineum becomes accessible to the surgeon. For many gynecologic and urologic procedures, the patient’s thighs are flexed approximately 90 degrees on the trunk and the knees are bent sufficiently to maintain the lower legs nearly parallel to the floor. More acute flexion of the knees or hips can threaten to angulate and compress major vessels at either joint. In addition, hip flexion to greater than 90 degrees on the trunk has been shown to increase stretch of the inguinal ligaments. Branches of the lateral femoral cutaneous nerves8 often pass directly through these ligaments and can be impinged and become ischemic within the stretched ligament. Numerous devices are available to hold legs that are elevated during obstetric delivery or perineal operations. Care should be taken to ensure that angulations or edges of the padded holder do not compress the popliteal space or the upper dorsal thigh. Compartment syndromes of one or both lower extremities have resulted from prolonged use of the lithotomy position with various types of support devices. Thighs are flexed approximately 90 degrees on the abdomen; knees are flexed enough to bring the lower legs grossly parallel to the torso section of the tabletop. Arms are retained on the boards, crossed on the abdomen, or snugged at the sides of patient. Low For most urologic procedures and for many procedures that require simultaneous access to the abdomen and perineum, the degree of thigh elevation in the lithotomy position is only approximately 30 to 45 degrees (Fig. This reduces perfusion gradients to and from the lower extremities and improves access to a perineal surgical site for members of the 2011 operating team who may need to stand at the lateral aspect of either leg. Figure 29-4 Low lithotomy position for perineal access, transurethral instrumentation, or combined abdominoperineal procedures. High Some surgeons prefer to improve access to the perineum by suspending the patient’s feet from high poles. The effect is to have the patient’s legs almost fully extended on the thighs (Fig. The posture produces a significant uphill gradient for arterial perfusion into the feet, requiring careful avoidance of systemic hypotension. There is considerable variation in lower extremity perfusion pressure in volunteers placed in high lithotomy positions; they all tend to have low perfusion pressures, however. Exaggerated Transperineal access to the retropubic area requires that the patient’s pelvis be flexed ventrally on the spine, the thighs almost forcibly flexed on the trunk, and the lower legs aimed skyward so they are out of the way (Fig. The result places the long axis of the symphysis pubis almost parallel to the floor. This exaggerated lithotomy position stresses the lumbar spine, produces a significant uphill gradient for perfusion of the feet, and may restrict ventilation because of abdominal compression by bulky thighs. If pre- existing painful lumbar spine disease is present, an alternative surgical position may need to be chosen beforehand to avoid severely accentuating the lumbar distress after surgery. This position has been associated with a very high frequency of lower extremity compartment syndrome. Note potential for angulation and compression/obstruction of contents of femoral canal (A, inset) or stretch of sciatic nerve (B). Complications of Supine Positions Brachial Plexus Neuropathy Root Injuries Shoulder braces placed tight against the base of the neck can compress and injure the roots of the brachial plexus when steep head-down positions are used.
For example buy discount requip, in bloodstream infections purchase requip no prescription, peripheral blood samples from patients may have only 20 copies of the bacterial genome per milliliter of blood cheap requip 2mg amex, but will have millions copies of the host genome purchase requip 1mg without prescription. At that low signal-to-noise ratio, false positive rates will be extremely high and unacceptable for clinical applications. To further reduce the cost, a molecular tag (bar- code) system can be used during ampliﬁcation; then, after ampliﬁcation, hundreds of samples can be pooled together for one sequencing run, and software can be used to identify and differentiate the samples. If 10–20 samples are pooled in one run, the sequence analysis cost per sample will be under $50. The challenge then becomes providing an accurate measurement of the ampliﬁcation products in a rapid, high- throughput, and low-cost format. The simplest detection method is hybridization, which occurs without an enzy- matic reaction. Because of its ease of use, hybridization is the method of choice for many detection platforms. Currently, nucleic acids are arrayed on solid supports that are either glass slides or nylon membranes. The sequences on an array may represent entire genomes, including both known and unknown sequences, or they may be collections of sequences, such as apoptosis-related genes or cytokines. Many premade and custom arrays are available from commercial manufacturers, though many labs prepare their own arrays with the help of robotic arrayers. The methods of probe label- ing, hybridization, and detection depend on the solid support to which the sequences are bound. For each pathogen, target-speciﬁc capture probes are covalently linked to a speciﬁc set of color-coded microspheres. A microﬂuidics system delivers the suspension hybridization reaction mixture to a dual-laser detection device. A red laser identiﬁes each bead by its color-coding, while a green laser detects the hybrid- ization signal associated with each bead. Software is used to collect the data and report the results in a matter of seconds. This platform is speciﬁc, because only the probes that are captured by the beads are recognized by the green laser as signal. Any signal not associated with a speciﬁc set of color-coded beads is considered background. The green laser can detect the signal for as few as eight ﬂuorescent-labeled probes that are captured by a bead. Because everything occurs in a homogeneous solution (from bead manufacture, color-code staining, and capture probe coupling to product hybridiza- tion and data collection), highly repeatable results are obtained with this platform. Typically, there are 5,000 beads added per reaction for each color-coded bead set. Each bead set is speciﬁc for a particular disease marker, such as a mutation or a pathogen. Thus, the data represents 100 microbead- associated data points, not just one data point produced by a standard array. One more washing step is performed to remove unreacted signal ampliﬁcation reagents. Qualitative analysis of results (reading the array) can then be performed on the Verigen Reader. Integrated Solutions An integrated solution is one that incorporates different methodologies and instruments to allow sample-to-answer results. These companies are compared in the following categories: ampliﬁcation methods; detection platforms; multiplexing capability of more than ﬁve targets; fully integrating sample prep, ampliﬁcation, and detection steps to allow a maximum hands-on time of <3 min; and a closed reaction system so that amplicon contamina- tion can be eliminated. These steps are difﬁcult to automate and perform in an enclosed system, risking amplicon contamination that may lead to false-positive results or high background. More interestingly, they found that over 30 % of the patients had coinfections by detecting more than one pathogen in a sample . This system detected 18 molecular targets in a multiplex assay for Staph identiﬁcation and drug resistance gene detection. The StaphPlex system demonstrated 100 % sensitivity and speciﬁcity ranging from 95. However, the poten- tial risk of amplicon contamination still exists, because the ampliﬁcation and hybridization reactions are still set up in an open environment. This low-cost solution, however, has made their products more acceptable in emerging markets. The lack of automation and potential amplicon contamination may limit the ability of their products to penetrate the western market. At the core of the iCubate technology is a single-use cassette that comes preloaded with all the reagents necessary to perform extraction, ampliﬁcation, and detection steps. The closed design of the cassette guarantees that the high- concentration amplicons contained inside have no chance of contaminating the lab. The iCubate iC-Processor allows for the automated processing of iCubate cassettes. Each processor can run from 1 to 4 cassettes in a random access fashion; if more throughput is needed, up to 12 units can be linked together to run up to 48 samples simultaneously. The iCubate iC- Reader allows for automated data collection from iCubate cassettes. A high-speed rotating platter, laser, and photomultiplier tube allow the acquisition of data from each cassette in just seconds. The iCubate iC-Report software performs automated data analysis and generates individual reports for each cassette. Companies like Cepheid, Gentura Dx, and Idaho Technology have all developed sample-to-answer solutions that allow molecular assays to be performed in a contamination-free closed system. Nevertheless, the ease of use of these platforms has revolutionized the molecular diagnostics industry and beneﬁted millions of patients. Delivering Value Through Reducing Cost and Saving Lives The advances of genomic technology have changed the way we deﬁne diseases from a phenotypic, symptomatic description of clinical presentations to a genotypic, molecular classiﬁcation of underlying causes. Molecular differential diagnosis has become the hallmark of 21st century medical practice. Every infectious disease starts with an invasion by a microorganism’s genetic material into the human body. The expression of pathogen genes inside human cells can interrupt normal cellular function and induce systemic responses or clinical syndromes. To achieve this goal, we need a multiplex technology that uses one sample, one test, one technician, one machine, and a short period of time to obtain multiple answers. A difﬁcult cycle is often set into motion: a lack of rapid and accurate diagnostic tests combined with a lack of com- munication to the public and lack of scientiﬁc knowledge about the disease lead to panic and disruption of economic systems. Health-care practitioners therefore quickly identi ﬁ ed and properly treated those with pandemic ﬂu infection and those requiring regular care. Furthermore, these ﬁndings contradicted the conventional wisdom at the time, which was that anyone with ﬂu-like symptoms probably had the H1N1 virus and should be treated accordingly. The Koon study revealed a second critical point: among those with the H1N109 infection, 28 % were also infected with at least one other bacterial or viral pathogen [ 13]. Almost all genomes of human pathogens have been sequenced, and newly emerging resistant strain genomes are being sequenced as quickly as possible. This is a problem because, for a particular bacterial strain, drug resistance capability may come from many different genes and mutations. Instead of waiting days for culture results, a doctor can now act immediately based on a comprehensive molecular diagnosis. Instead of ordering the blood cultures to gain knowledge for future empirical treatment, a doctor can prescribe the test to seek immediate solutions. Instead of offering antibiotics to put families or parents (and sometimes the doctor) at ease, a doctor can now provide accurate treatment to actually improve a patient’s condition. Healthcare spending in the United States has grown rapidly over the past few decades—from $27 billion in 1960 to $900 billion in 1993 to $1. Depending on how you measure it, the healthcare industry represents between 15 and 16 % of the gross domestic product. Traditionally, these ﬁnancial activities occurred in three subcatego- ries: providers (such as hospitals, nursing homes, and diagnostic laboratories), payers (such as insurance companies), and life sciences (such as biotechnology and pharma- ceuticals). For example, the cost of developing a new drug can be as high as $800 million [ 17]. That cost is passed on from the life science sector to the payers and then to the providers.