Hemagglutination inhibition test can be used to confirm virus in brain-passaged material Haddow et al. Virus isolates from urine and saliva samples were recovered in Vero cell lines in one study, showing that Vero cell lines can be used to isolate ZIKV Bonaldo et al. Because live ZIKV sample handling requires specialized facilities to prevent its spread to handlers, not all laboratories can isolate the virus. Low-level viremias can also limit the opportunity for isolation of the virus Lanciotti et al.
Original Research ARTICLE
To test vaccines, several laboratory animal models of ZIKV infection have been evaluated. Two mouse models were studied, namely A type I interferon receptor knockout mice and AG type I and type II interferon receptor knockout mice.
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Three-week-old A and AG mice showed neurological signs and the presence of virus in their brains 7 days after challenge, whereas immunocompetent mice did not produce similar signs when challenged with ZIKV Brault and Bowen, In one study, 15 non-human and 18 human cell lines were used to determine the replication of ZIKV in various cells.
Replication in placental cell lines shows the ability of ZIKV to cross the placental barrier. Hemagglutination inhibition, serum neutralization, and complement fixation are the useful tests for the diagnosis of Zika fever Fagbami, ; Monath et al. Short viremias make it difficult to detect ZIKV; hence, serological diagnosis is a better way out to determine the status of an individual for a longer period of time Shan et al.
Seroconversion is confirmed by analyzing the antibodies titers in paired serum samples acute and convalescent phase during the course of infection. Authors suggest this assay can be a confirmatory test for the samples positive by commercial immunoassays for detection ZIKV antibodies Wilson et al. PRNT is a labor-intensive technique requiring a week for interpretation of results.
Hence, a reporter virus possessing the luciferase gene from Renilla was used in a neutralization assay, and it showed similar specificity as PRNT, but reduced the time of diagnosis to 2 days Shan et al. Similarly, this assay can be done on 96 well plates hence this assay minimizes the testing time and is a high throughput assay Shan et al.
The sensitivity was The sensitivity of the IgM ELISA differed among patients of Israeli origin and from European origin; hence, to minimize false negatives, further diagnostic tests are needed Lustig et al. ZIKV-specific epitope-based serological assays can prevent the problem of cross-reactivity with other flavivirus antibodies Landry and St.
The assay is robust, low cost strategy to surveillance program and seroprevalence studies and quickly adapted in ZIKV endemic areas. It possesses improved serological diagnostic capability for ZIKV. MIA allows incorporation of more than one antigen to enhance the diagnostic coverage. A MIA has been developed for detection of 6 flaviviruses, 6 alphaviruses, and 1 bunyavirus of human importance Basile et al.
A recently developed experimental murine model and ZIKV infection model in cynomolgus macaques may help to elucidate disease course, as well as would be useful in testing the anti-ZIKV drugs and vaccines Koide et al. In addition, a 3-[4,5-dimethylthiazolyl]-2,5-diphenyl-2H-tetrazolium bromide- MTT -based cell viability assay was developed to assess cell death in human and monkey cells caused by ZIKV.
Moreover, the easy preparation, smaller sample volume requirement, and short time to get results are significant advantages of the MIA. Therefore, the proposed MIA is a useful tool for assessing immune responses in vaccination trials and in clinical disease Wong et al. Electro-generated chemiluminescence was linked with polystyrene beads, which were also conjugated with monoclonal antibodies mAbs against ZIKV.
A pair of mAbs has been selected to construct a rapid diagnostic test in form of strip. The diagnostic strip was able to detect NS1 antigen is serum samples from various geographic areas in the Americas and India. The same test is linked with mobile phone camera and the images taken can be analyzed with ImageJ software, which allow objective analysis and eliminates user subjectivity in reading test results Bosch et al.source url
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Due to very short viremia time period during ZIKV disease, there exists a challenge in selecting the appropriate sample for diagnosis, as ZIKV RNA may not be detected in serum or plasma collected 10 days after the onset of disease. Urine samples were found to be more reliable than serum samples, as the viral load was higher, and results of this assay were more dependable than those of serology St.
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A study in Italy examined the whole blood, plasma and urine as samples for detection of RNA from 3rd day to 28 days post-ZIKV infection in 10 patients. Results showed that urine samples from all the individuals were ZIKV positive up to 21st day post-clinical sign while ZIKV could be detected in the whole blood up to 26 days Rossini et al. During early disease, viral genomes could be detected in serum Haug et al.
Another report showed that ZIKV persists for 81 days in whole blood, but 73 days in serum. Symptomatic pregnant women and affected persons display low-grade fever, maculopapular rashes, arthralgia, and non-purulent conjunctivitis for 2—7 days. Recently, in —, asymptomatic pregnant women in American Samoa were tested under a syndromic surveillance program, with results displayed in electronic health records Hancock et al. Similarly, the addition of nucleic acid stabilizers increased RNA recovery Tan et al.
Real time PCR offers several advantages over conventional RT-PCR including rapidity, low false positive, higher sensitivity and specificity with quantitative analysis. This is especially useful during an epidemic and for the testing of travelers Gourinat et al. The probes developed against NS5 region in a real-time PCR and the sensitivity was 32 genome-equivalents and 0. The assay was able to detect at least 37 ZIKV isolates representatives of wide geographic area in Africa and Asia over the period of last 36 years.
Also, it could differentiate ZIKV among 31 other flaviviruses. This assay alleviates the disadvantage of earlier developed real-time PCR as they could not detect all strains of ZIKV and, moreover, this assay also showed a detection limit of 5 RNA transcript copies Yang et al. A comparison of 7 published real-time RT—PCR assays to determine the analytical sensitivity revealed presence of up to 10 potential mismatches in oligos with the Asian lineage.
The two new assays developed by Corman et al. The continuous evolution in the viral genome and mismatch between oligos and genome may lead to reduced sensitivity. The study of ZIKV genomes, exhibited the presence of mutation in diagnostic regions, however these were present in lower frequencies Metsky et al. Hence, the diagnostic assay should be carefully chosen and need to be frequently evaluated and updated.
It is an assay into which the serum neutralization test and Real-time PCR tests are combined. The neutralization endpoint is measured by real -time PCR instead of counting plaques. The test takes 72 h to complete. Surface plasmon resonance-based technology for the sensitive detection of ZIKV RNA has been developed very recently wherein results revealed that bimetallic nanoparticle quantum dot-mediated fluorescent signals were stronger than those of single metal nanoparticles for the detection of ZIKV RNA Adegoke et al.
The technique is a reliable detection platform for rapid and ultra-sensitive detection of ZIKV genome. However, the higher cost of the test reduces its utility in low and middle-income group countries. Flow cytometry technique is also used for the detection of ZIKV NS3 antigen in the whole blood samples using polyclonal antibodies. It has been reported recently that most patients revealed a decrease in ZIKV antigen during later phases of the disease while some showed higher antigen level.
Immature dendritic cells present in human skin cells are permissive to ZIKV infection and 24 h post-infection express viral envelope when infected with 0. The intracellular presence of the viral envelope protein is detected by using a broadly neutralizing Ab 4G2 by flow cytometry.
The role of these genes as sensor of infection may also have a role in the detection of ZIKV Hamel et al. Viral genomes may be enriched by multiplex PCR from samples containing as low as 50 copies of viral genome. Clinical sample itself may be the starting material and within 1—2 days consensus viral sequence is obtained to do studies regarding evolution and spread of the virus Quick et al.
Deep sequencing can be employed to analyze the ZIKV genome, and a comparison with sequences available in a data bank can help to identify nucleotide changes in specific strains of the virus Buechler et al. This report also shows the suitability and importance of urine as a sample for ZIKV diagnosis Gu et al. It is a rapid assay which takes only 3—15 min and able to detect 21 RNA molecules. It produces RNA amplicons, opposite to other assays which produce DNA and have rapid kinetics, which result in higher amplitude amplification within 15—60 min. This assay is economic, specific, and provides a result in less than 8 min Bedin et al.
The LAMP assay has the disadvantage of product carry-over contamination; hence, closed-tube techniques are usually preferred Karthik et al. This assay used a portable LAMP box powered by a 5 V power source and a smartphone built using a chromaticity algorithm to scan for fluorescent signals. Use of this technology increased the sensitivity of this assay 5-folds compared to that of naked eye detection of colorimetric results Priye et al.
Finally, a portable device was developed that can extract nucleic acids using magnetic particles, then perform real-time RT-RPA or RT-PCR and interpret results by fluorescence detection. A 3D printer has also been used to process 8—12 samples for the detection of ZIKV, with fluorescence detection of amplified products by smart phone. This assay is cheap and portable Chan K. A higher sensitivity was obtained by Tian et al. The programmability of molecular sensors will help in addressing rapidly changing diagnostic requirements Pardee et al. RT strand invasion-based amplification by a battery-operated portable device for the diagnosis of ZIKV was reported to work well, even with incompletely purified RNA.
It relies on a recombinase coated single-stranded invasion oligonucleotide purposed to separate complementary target duplex. Resulting single-stranded target template is extended by DNA polymerase at a relative low and constant temperature. The method is simple and can run on low cost equipments. Also, it is able to reproducibly detect as low as 10 copies of RNA Eboigbodin et al. The technique offers and excellent stability of the BPD at room temperature and even at higher temperatures using metal—organic framework dependent biopreservation.
The technique eliminates the storage and transportation of device at low temperature Morrissey et al.
Competing interest statement
The same device may be adapted for diagnosis of other infectious agents also. Other recent technologies for ZIKV diagnosis include paper disk tools, which work on the principle of a color change from yellow to purple in the presence of ZIKV antigen Shukla et al. To concentrate ZIKV in samples with low viral loads, ultracentrifugation and polyethylene glycol precipitation can be performed, but these methods have their own limitations and can interfere with PCR assays Novotny et al. Therefore, recently, magnetic nanoparticle-based concentration has been preferred.
Nanoparticles like such as iron, nickel, and cobalt can be used, but their stability is poor; hence, they are encapsulated with silica, graphite, or a polymer Saraswati et al. A liposome-based biosensor that is cheap, portable, specific, and sensitive has already been developed for DENV Zaytseva et al. These platforms can also be utilized for the detection of ZIKV.
Different diagnostic platforms available for Zika virus detection are presented in Figure 2. An overview on different diagnostic platforms available for Zika virus detection. Extensive ZIKV epidemiological studies should be carried out to support advanced disease surveillance and monitoring approaches, and geographical information system GIS and appropriate networking programs should be employed to track the spread of the virus Dhama et al. Based on the surveillance data of the Zika epidemics in the Tolima department, Colombia, epidemiological mapping has been developed employing GIS.
Similarly other reports also show that GIS was employed to develop epidemiological mapping in Valle del Cauca and Pereira department of Colombia. Early reporting of ZIKV cases can allow the pattern of disease spread to be determined, as well as limit its further spread Nishiura et al. Efficient serological and molecular detection techniques are warranted for improved surveillance to prevent the spread of disease by adopting timely and appropriate control measures Waggoner and Pinsky, ; Sharma and Lal, When the recent ZIKV outbreak occurred in South and Central American states, several national and international organizations expressed their concern for the safety of athletes, players, coaches, and viewers during the Rio Olympic and Paralympic games.
Epidemiological studies confirmed that, after , approximately 1. Want to become a manufacturing leader and be promoted? What skills can you offer an employer? Learn powerful and practical keys to add greater value. From the Back Cover Soft Errors in Modern Electronic Systems describes the state-of-the-art developments and open issues in the field of soft errors. Frontiers in Electronic Testing Book 41 Hardcover: Springer; edition September 30, Language: Be the first to review this item Amazon Best Sellers Rank: Related Video Shorts 0 Upload your video.
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In a small clinical validation study, antibiotic susceptibility profiles of E. In conclusion, it is clearly established that rapid phenotypic susceptibility testing lowers the rate of incorrect empiric treatment choices, shortens the length of hospital stay and reduces patient mortality. Many novel options for rapid phenotypic AST will be available in the near future.
Before adopting one or more of these systems, clinical microbiologists will need to evaluate their benefit in the context of local requirements: Is there a need to bridge a particular diagnostic gap such as rapid AST in sepsis? What is the capacity of the system parallel processing? Is it cost-efficient under local circumstances? Is there sufficient peer-reviewed validation data? How is the flexibility of in-house solutions weighted against the ease-of-use of proprietary systems black box?
Finally, the benefits of rapid phenotypic AST will not translate into improved patient care unless extended staffing schedules and more rapid transmission of verified results can be provided. MALDI-TOF mass spectrometry fingerprinting has now been widely adopted by clinical microbiology laboratories for rapid identification of cultured microorganisms. Precise speciation can inform treatment decisions by facilitating better judgment of clinical relevance of microbial isolates e. Targeted modification of antimicrobial treatment can often be suggested upon identification of non-fermenting Gram-negative bacilli Acinetobacter spp.
Given the usually low rates of acquired resistance, species identification is exceptionally useful for the treatment of fungal infections. In observational studies, introduction of MALDI-TOF with antimicrobial stewardship intervention significantly reduced time to effective antimicrobial treatment in patients with bloodstream infection and Acinetobacter baumannii pneumonia 29 and shortened inappropriate use of vancomycin in patients with CoNS-contaminated blood cultures by more than 60 hours. Highest impact on turnaround times and prescription policies is expected for rapid identification from positive blood culture bottles.
Compared to conventional processing, direct identification of organisms from positive blood-cultures by mass spectrometry reduced turnaround times by at least one working day and provided species level identification results the day after sample collection in more than three fourths of cases.
Yet, in both studies, direct MALDI-TOF identification was only one aspect of an intervention bundle, which also comprised rapid susceptibility testing from positive blood cultures and intensified antimicrobial stewardship measurements. However, MALDI-TOF-based identification of bacterial pathogens directly from positive blood-culture bottles is comparably labor-intensive and currently few laboratories offer the service as part of their routine blood culture workup.
Beyond species identification, mass spectrometry has also been utilized for rapid susceptibility testing. The technique can be used to detect products of beta-lactam hydrolysis in bacterial cultures with unprecedented sensitivity and specificity. It has successfully been used to detect ESBL and carbapenemase production within 30 to minutes. Another promising approach involves direct identification of resistance determinants or biomarkers expressed by resistant bacteria by MALDI-ToF. In selected cases, MALDI-TOF mass spectrometry might also provide treatment relevant information via sub-species level differentiation of microbial pathogens.
While the achievable phylogenetic resolution varies considerably between species and is generally lower than with established typing tools, 45 the technique is much faster and cheaper than MLST or PFGE. The introduction of MALDI-TOF mass spectrometry into the clinical microbiology laboratory has considerably reduced time-to-result for species identification in culture based diagnostics. However, its impact on the rational use of antimicrobials critically depends on the timely translation of test results into clinical decision making via policies for empirical treatment based on local susceptibility data.
Application of MALDI-ToF mass spectrometry for rapid susceptibility testing or epidemiological problems is currently hampered by the lack of standardized protocols, test kits and software tools. While the analytical sensitivity of MALDI-ToF is insufficient for direct application to clinical samples, the low cost-per-sample and broad applicability make it an attractive bridging technology, which can be well complemented with nucleic acid based tests and conventional assays.
During the last two decades, amplification-based approaches towards pathogen detection have become irreplaceable in the clinical microbiology laboratory. More recently, the introduction of commercial multiplex PCR assays made rapid, sensitive and specific detection of both bacterial and viral pathogens from a single specimen broadly available. These assays can help to avoid unnecessary antibacterial treatment if viral pathogens are detected, which is of particular importance in infections of the respiratory system.
Some commercially available assays additionally detect a number of bacterial pathogens implicated in lower respiratory tract infections, e. Nevertheless, the published studies highlight some important problems. Most importantly, while one would assume that identification of a single viral pathogen in respiratory samples results in immediate discontinuation of antimicrobial treatment, several studies found that this is not generally the case. Interpretation of rapid molecular screening results becomes more complicated when bacterial pathogens are targeted by multiplex PCR.
In case of respiratory infections, typical patient samples include sputum and nasopharyngeal swabs. Not surprisingly, a study by Gilbert and co-workers using a combination of culture-based diagnostics and molecular tests to screen for viral or bacterial pathogens in community-acquired pneumonia, found bacteria as causative agents for respiratory infections at rates close to the reported colonization frequencies and often in conjunction with viral pathogens.
In consequence, false-positive rapid molecular test results may even trigger antimicrobial therapy when none is required and thus have a detrimental effect on antimicrobial stewardship initiatives. Bloodstream infections present another category of infections where rapid molecular diagnostics hold great promise to rationalize empiric antimicrobial therapy.
In fact, various commercial PCR assays from whole blood specimens remained negative while bacteria were recovered using conventional blood culture bottles, indicating a potential sensitivity issue with molecular sepsis assays. The interpretation of these results and the necessary differentiation from probable contaminations during sampling remains open. Most importantly, at present no data are available demonstrating the clinical impact of cost intensive PCR assays for direct pathogen identification in whole blood.
Although a recent study found a change in clinical management e. Thus, at present PCR assays are still waiting to find their place in sepsis diagnostics. Rapid, amplification-based methods could help to avoid unjustified broad-spectrum pathogen coverage and fast de-escalation of empiric antimicrobial therapy by immediate identification of molecular resistance mechanisms as soon as enough bacterial material becomes available during culture.
Obvious clinical need and the availability of abundant organisms have made positive blood cultures a primary target of tailored commercial assays. This already could have important stewardship implications, as knowledge on naturally occurring resistance phenotypes and availability of specific local resistance epidemiology could help to optimize antimicrobial therapies at an early stage of the diagnostic work up. Moreover, in certain scenarios a confirmed species identification could already help to discard bacteremia as a diagnosis and thus cease an antibacterial therapy e.
However, a major drawback of every approach that is restricted to rapid identification of bacterial pathogens is the lack of information on possible acquired resistance markers. Therefore, inclusion of primer sets for detection of specific resistance determinants is an obvious extension of PCR-based assays as long as there is an unambiguous association with a specific drug-susceptibility phenotype.
Here, due to the tremendous variability of resistance mechanisms, PCR-based methods as a basis to extrapolate a dedicated resistance phenotype are obviously of limited value in Gram-negative organisms. The almost monocausal reason for beta-lactam resistance in S. Most available, in house as well as commercial assays target positive blood cultures yielding growth of cluster forming Gram-positive cocci.
They allow to differentiate between coagulase-negative staphylococci and S. Turn-around times for PCR-based assays are between one to three hours and can thus significantly accelerate time to optimal targeted antimicrobial therapies or discontinuation of a running therapy, e. Moreover, data from the same study indicate that rapid MRSA detection is cost effective, e. If possible, the time to switch from empiric vancomycin therapy to a beta-lactam was 1.
Moreover, the mean length of hospital stay was 6. The diagnostic strategy of PCR-based differentiation of Gram-positive cocci directly from positive blood cultures including detection of mecA was reinforced by a later study from Australia investigating S. Emonet and co-workers recently analyzed the effect of an in-house multiplex real time PCR including specific primers for S. PCR was used to differentiate and preliminary deduce susceptibility of S. Introduction of the PCR assay significantly shortened the time-to-result to detect methicillin-susceptibility as compared to the standard workflow from More rapid availability of presumable beta-lactam susceptibility allowed for a quicker switch to an appropriate therapy in S.
Switching most often occurred in MSSA bacteremia, in which empiric glycopeptide usage was stopped and patients were treated with a beta-lactam instead. A drawback of these studies is that PCR was performed on all blood cultures yielding growth of Gram-positive cocci, resulting in significant costs especially when commercial systems are in use.
A way to lower these costs is to differentiate between coagulase-negative staphylococci and S. Ninety-six samples were assigned to the control group. Here, direct identification was followed by conventional susceptibility testing. Intriguingly, there was less unnecessary glycopeptide usage in patients with MSSA bacteremia in the intervention group 8.
Yet, despite the seemingly straightforward genotype-phenotype correlation for beta-lactam susceptibility in S. Nevertheless, the studies related to staphylococcal bacteremia highlighted above demonstrate the significant impact of direct bacterial species identification and detection of genetic resistance markers can have on the clinical management of septic patients. A similar strategy may also be applicable to other species, given that a reliable association between genotype and phenotype exists and that the respective genetic markers are of low variability.
For instance, this applies for vancomycin resistance in enterococci carrying vanA or vanB. In the past, optimal treatment ampicillin versus vancomycin of enterococcal bacteremia could be readily deduced from species identification, as resistance to aminopenicillins is low in E. However, due to the emergence of vancomycin-resistant enterococci VRE in Europe 82 and the high VRE prevalence in specific risk groups, 83 empiric administration of vancomycin may today be inappropriate even in E. Over the past couple of years several in-house as well as commercial systems have been developed, partially in integrated solutions in which Gram-negative and Gram-positive bacteria and some of their key resistance determinants are detected simultaneously from positive blood culture bottles [ e.
In addition, resistance phenotypes involving changes in gene expression levels or combined effects e. As a consequence, rapid reporting of molecular resistance results could potentially lead to wrong empiric treatment decisions by suggesting an all-clear to the clinician. Those reports should therefore generally include a comment on the limitation of the tests, and advise on considering the clinical context of the patient for example results from recent colonization screenings, epidemiological background, effectiveness of current antimicrobial treatment. As previously noted, rapid phenotypic methods will thus continue to be of significant importance in this context.
Although available commercial assays have been thoroughly validated in technical terms, the impact of using rapid amplification based methods on clinical decision-making and patient outcome is less well studied. However, such studies would be of significant importance in order to justify the increased cost and complexity of the diagnostic workflow.
The clinical impact of performing rapid identification and detection of resistance determinants in Gram-negative rods was tested in a retrospective study by Walker and co-workers. The amplification assay was performed immediately after the blood culture bottles were flagged positive, and results were directly reported. While the implementation had no effect on earlier appropriate antimicrobial coverage or de-escalation, length of ICU stay, day mortality and mortality associated with multidrug resistant organisms e.
ESBL-producing Enterobacteriaceae were lower in the group in which rapid molecular testing was applied. It should be noted that in this study, no additional stewardship measures were initiated to flank implementation of rapid diagnostics. A general drawback of most studies investigating the clinical impact of rapid pathogen identification in positive blood cultures is their observational study design and the use of historic controls.
In that respect, a recent publication by Banaerjee and co-workers is of special importance. While in one group standard work up was in place, in a second group the FilmArray BCID assay was used to differentiate organism immediately after blood culture bottles were flagged positive. Results in this group were communicated by a laboratory technician during 24 hours every day, accompanied by templated comments in the electronic medical record to guide antimicrobial therapy.
In a third group, FilmArray BCID results were communicated 24 hours every day by a member of the antimicrobial stewardship team. The authors found that in groups 2 and 3 clinicians were enabled to quickly initiate pathogen-directed antimicrobial therapy. In both groups an increased use of narrow spectrum antibiotics was observed, as was less usage of unnecessary vancomycin, decreased treatment of blood culture contaminants and more timely escalation if appropriate. The implementation of rapid molecular testing, however, had no effect on mortality, length of stay or costs.
The study not only provides evidence that implementation of rapid PCR testing of positive blood cultures can optimize patient treatment, but demonstrates that flanking stewardship measures are important clues to translate speed in diagnostic procedures into clinical action. The importance of structured communication and stewardship decision support, especially in Gram-negative bacteremia, was also reported by others.
In conclusion, PCR-based assays have a clear place in specific and fast, culture independent pathogen detection. The specific value in infections of the respiratory tract, especially hospital-acquired pneumonia, and the bloodstream is currently unclear. Certainly, PCR is of great value in rapid pathogen identification and resistance determinant detection in cultured bacteria.
This is especially true in the work-up of positive blood cultures — however, the investment in expensive diagnostic assays is only justified if results are communicated to the clinician in a way allowing for immediate clinical action, i. Techniques providing rapid information on bacterial pathogens and their antimicrobial susceptibility are of key importance for the management of infectious diseases patients.
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