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Alternatives to PCR

Alternatives to PCR


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PCR uses cycles of heating and cooling to denature the strands, calling for special thermostable DNA polymerases. In a cell, during replication, Helicase unwinds the DNA without the requirement of heat. Can Helicase be used instead of the heat cycling to achieve single stranded dna? Would that eliminate the need for heat cycling, and hence special thermostable polymerases?


Edited after clarifications in question,

Let's start with normal functions of both enzymes. Helicases separates DNA strands while polymerase synthesize DNA strands as shown in the following figure. (Image Source: Wikimedia Commons)

Watch this animation, it will clear your doubts about function. However, RNA polymerase does have both activities.

In cells, helicase action is required to separate DNA strands. After that only polymerase can act. During PCR, separation of strands is done by increasing the temperature. This process is called DNA melting. This temperature is generally very high, around $90^oC$ to $100^oC$ depending on your sequence. At this temperature many proteins will not function, hence you need thermostable polymarase. About 40 years ago, such an enzyme was discovered from extremophiles (Chien et al 1976). Now it is commercially available as Taq polymerase. These enzymes are recombinant proteins with a few mutations which have also increased its yield and stability (Villbrandt et al 1997).

Now coming to your main question (Which I think I understood correctly this time).

The concept you are asking about has already been successfully tested in RNA detection. Researchers have used "Isothermal reverse transcription thermophilic helicase-dependent amplification"(Goldmeyer et al 2007). They used not only helicase but also reverse transcriptase (because they were detecting RNA). The following image (taken from the paper) shows the main steps in this process. Circles indicate DNA polymerase, squares indicate reverse transcriptase, and triangles indicate helicase.

Quoting from the paper (bullet points are mine),

Arrows indicate specific primers, circles indicate DNA polymerase, squares indicate reverse transcriptase, and triangles indicate helicase. The first-strand cDNA is first synthesized by a reverse transcriptase

  • (steps 1 and 2). The RNA-DNA hybrids from the reverse transcription are then separated by UvrD helicases generating single stranded (ss) RNA and DNA templates
  • (steps 3 and 4). The ssRNA enters next round of RT reaction
  • (step 5-a) generating more first strand cDNA. The ssDNA was converted into double-stranded DNA by the DNA polymerase
  • (step 5-b) and amplified concurrently in the tHDA reaction
  • (steps 6 through 9). This process repeats itself to achieve exponential amplification of the RNA target sequence.

Isothermal DNA amplification technologies have been developed. You do not need thermal cycling to amplify DNA fragments by this method.

http://www.ncbi.nlm.nih.gov/pubmed/17720718


Microbiology: culture vs molecular

Microbiologist Dr Mark Wilks looks at the key themes and messages that emerged from this year’s British Society for Microbial Technology conference.

The recent 32nd Annual Scientific Conference of the British Society for Microbial Technology was entitled “Hot Topics in Microbiology”. It focused on areas in medical microbiology where change has been rapid and is likely to be even more so in the near future.

In diagnostic clinical virology, there has been a wholesale move from culture to polymerase chain reaction (PCR) and, more recently, sequencing. In contrast, diagnostic bacteriology has remained largely culture based. There are many reasons for this, including cost, the necessary skillset, and the fact that culture methods, whatever limitations, are generally adequate.

Opinions have been polarised, with some refusing to engage with the new molecular methods, while others look down upon those still using culture and insist that PCR is now outdated and next generation sequencing (NGS) is the only way to go. Some have suggested that the only obstacle to high-throughput sequencing is the “innate conservatism of the profession”.

The range of different topics covered at the meeting shows that there is, in fact, no conflict between the two different methods. Great gains are being made using molecular and cultural approaches to cope with different situations and, in some cases, a combined approach yields the best results. Let’s look at how the approaches are being used in some rapidly changing fields.

Improving sepsis diagnosis

Professor Paul Dark, University of Manchester and NIHR Clinical Research Network Critical Care Lead, gave the first presentation on “Moving towards delivering precision medicine in sepsis”.

Here we have the unusual situation where the gold standard – a positive blood culture – is actually not very good. Blood cultures are negative even in those in which sepsis is strongly suspected on clinical grounds. The most important reason for this is probably prior antibiotic treatment, rather than the inadequacy of culture itself. As well as being insensitive, culture methods are often too slow to be useful in cases of severe sepsis.

There are unlikely to be any significant improvements in culture methods, so what are the molecular alternatives? There are increasing efforts to develop molecular methods to rapidly detect bacterial and fungal DNA directly from blood without the need for blood culture. These are not affected by trial prior antibiotic treatment. Several CE-marked methods were reviewed and showed great potential, although the cost of each test was high.

A complimentary molecular approach is to look at host biomarkers, such as CRP and PCT, released into the circulation in response to acute pro-inflammatory stimuli. Bacterial stimuli are associated with rapid and high responses and, crucially, they fall rapidly with correct treatment for bacterial infection. So, when used quantitatively, they have the potential to aid antibiotic initiation and discontinuation decisions. Although of course they don’t give any direct information about causative pathogen or antibiotic susceptibility.

It’s likely that a combination of molecular methods to detect DNA and host biomarkers will give the best results and lead to major advances in the reliable and rapid diagnosis of sepsis.

Aetiology of community-acquired pneumonia (CAP)

One area in which the superiority of molecular methods has clearly been shown is in determining the aetiology of community-acquired pneumonia. In the pre-antibiotic era, Streptococcus pneumoniae could be isolated in up to 95% of cases, but in the majority of cases the credible pathogen is not isolated. It’s not clear whether this represents a genuine change in the aetiology of the disease, more widespread use of antibiotics or both.

Dr Kate Templeton, Consultant Clinical Scientist, Edinburgh, described a recent landmark study in which they performed quantitative multipathogen testing of sputum samples in adults hospitalised with CAP. They collected mucopurulent sputum samples (96%) and endotracheal aspirates (4%) from 323 adults with radiologically confirmed pneumonia admitted to two tertiary care hospitals in the UK. They performed quantitative culture and multiplex real-time PCR for 26 respiratory bacteria and viruses. With PCR, they identified a potential pathogen (bacterial or viral) in 87% of patients, compared with 39% using culture alone. Predictably, PCR detected bacteria more frequently than culture in patients who had received antibiotics (77.6% vs 32.1%).

Of course the isolation of a credible pathogen does not prove that it was responsible for the disease in every case, especially as many of the bacteria detected are carried in the upper respiratory tract without apparent harm in much of the population. Nevertheless, the results of the study are hugely encouraging.

Identification of Mycobacteria

NGS to detect Mycobacterium tuberculosis from sputum samples has been shown to be possible, however, at present the detection of mycobacteria relies on culture. Dr Pieter Jan Ceyssens, Head of the Antibiotics and Resistance Unit at the National Reference Centre for Mycobacteria and Tuberculosis in Belgium, described the use of MALDI-TOF for the rapid identification of mycobacteria. In this case, the molecules are proteins and not nucleic acids, as in the other examples described.

The identification of cultured bacteria by MALDI-TOF has revolutionised the way of working in most labs in the UK and Europe. The vast majority of bacteria and yeasts can now be identified in minutes with minimal preparation. However, mycobacteria and filamentous fungi have proved much harder to identify. Dr Ceyssens described some simple methods in which positive cultures are heat killed, extracted with ethanol, sonicated and then loaded onto the MALDI-TOF in the usual way. This has allowed the identification of approximately 90% of the different species of non-tuberculosis mycobacteria that are encountered in clinical laboratories in a matter of minutes, a huge step forward over existing techniques, which are complex, expensive and take several days. This cheap and rapid molecular method may turn out to be superior to NGS in the majority of cases.

Microbial dark matter

Professor William Wade, from Queen Mary University of London, showed how molecular and cultural methods can be used in conjunction to greatly increase our knowledge of microbiology in a field where the limitations of culture have perhaps been too easily accepted. His talk was worryingly entitled “Cultivating the Uncultured”. This turned out not to be a reference to the audience, but to an extremely ingenious and painstaking approach to growing new bacteria from the mouth.

Oral bacteria are typically fastidious and slow growing – requiring complex media and long incubation times. Many are strict anaerobes requiring extra care in sample collection, transport and incubation.

A comprehensive cultural analysis of samples is difficult, meaning that it is only possible to analyse small numbers of specimens and around half of oral bacteria detected by molecular methods are uncultivable.

Some of these belong to existing well characterised phyla, such as the Bacteroidetes, where there are many cultured representatives, such as Bacteroides fragilis, which have been known for over a century. Others constitute newly discovered-deep branching lineages with no cultivable representatives. The reasons for the lack of success could include under sampling – because culture is much more labour-intensive than molecular methods – and dependence on other bacteria in the community. This could be due to particular nutritional or signalling requirements, which are hard to reproduce in the laboratory or the bacteria may themselves be intracellular and parasitic and hence difficult to grow in pure culture.

His talk focused on his attempts to culture uncultivated members of the phylum Synergistetes. The underlying hypothesis was that some uncultivated oral bacteria required the presence of other bacteria, so it might be possible to grow them initially in mixed culture in vitro, with the aim of eventually weaning them off their dependence on other bacteria and thus get pure cultures.

The sample obtained from the periodontal pocket was cultured on blood agar and incubated anaerobically for 10 days. Plates were then photographed, replica plated and blotted onto nylon membranes. The membranes were hybridised with Synergistetes probe allowing the area of Synergistetes colonies to be rapidly located on the original plate. These colonies could then be subcultured on blood agar again and so the primary culture gradually enriched for Synergistetes. After eight passages, the mixture consisted of four well-described bacteria.

Molecular methods showed that there was not just the four species of bacteria, but the Synergistetes type W0 90. By passage 12, this organism was able to grow independently, although next to a culture of Parvimonas micra. The organism was described, named (Fretibacterium fastidiosum) and its whole genome sequenced.

Remember that each passage took 10 days and 12 passages were needed to get the isolate in pure culture, so nearly six months of painstaking work were needed to recover the organism in pure culture.

This could only have been done by combining traditional cultural methods with molecular methods and it really shows the absurdity of trying to pose cultural and molecular methods as mutually antagonistic.

Mark Wilks is Lead Clinical Scientist, Microbiology at Barts Health NHS Trust and a Committee member of the British Society for Microbial Technology.


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Abstract

Objectifs Evaluer la concordance entre différentes méthodes de diagnostic et de confirmation des cas cliniques suspects d'infection àPlasmodium chez des enfants en Tanzanie et au Kenya.

Méthodes Des gouttes de sang digital ont été collectées chez 338 enfants avec une suspicion clinique de malaria non compliquée dans les cliniques de Tanzanie et du Kenya. La présence de parasites de Plasmodium a été détectée par la microscopie, par des tests de diagnostic rapide (TDR) et moléculaires quantitatives basées sur l'amplification des acides nucléiques (QT-NASBA) et par la PCR. Les résultats ont été comparés et analysés pour leurs concordances.

Résultats Le degré de concordance obtenu était élevé, allant de 88,6%à 100% entre les TDR ou les tests moléculaires et la microscopie. Dans les zones rurales du Kenya avec une incidence élevée des cas de malaria, le coefficient de corrélation allait de 0,94 pour les TDR à 0,76 pour la PCR. Dans les zones urbaines de la Tanzanie avec une incidence faible des cas, pour les TDR une valeur de R = 1,0 a été trouvée mais cette valeur était de 0,25 pour la PCR et de 0,33 pour NASBA.

Conclusions La malaria est surestimée lorsque le diagnostic est uniquement basé sur les signes cliniques. Dès lors, la confirmation de laboratoire est essentielle. La microscopie est une méthode fiable dans les zones rurales où la malaria est fréquemment rencontrée mais les TDR sont une bonne alternative avec l'avantage d’être simples et rapides. Les tests moléculaires sont plus sensibles mais plus difficiles à implémenter dans les zones rurales. Dans les zones avec une incidence plus faible, les tests moléculaires détectent un nombre significativement plus élevé d'infections àPlasmodium que les TDR ou la microscopie. Bien que l'implémentation des tests moléculaires soit difficile, leur avantage d'offrir un système de détection simple et moins cher les présentent comme outils prometteurs dans un futur proche.


DPCR: A Technology Review

Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

Keywords: absolute quantification arrays of microwells dPCR digital PCR droplet microfluidics microfluidic chambers microfluidic technologies microfluidics on-chip valves partitioning qPCR quantitative real-time PCR real-time PCR.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

( a ) Principles of the polymerase chain reaction (PCR). Each PCR cycle…

Real-time qPCR assay using a…

Real-time qPCR assay using a standard curve. ( a ) Amplification curves for…

Principles of digital PCR. The…

Principles of digital PCR. The sample is divided into many independent partitions such…

Comparison of PCR-based techniques. In…

Comparison of PCR-based techniques. In conventional PCR, the amplification products are analyzed at…

Quantification accuracy of dPCR. The…

Quantification accuracy of dPCR. The precision of dPCR is non-uniform and depends on…

Active partitioning platforms. ( a…

Active partitioning platforms. ( a ) Schematic of a push-up valve in a…

Passive partitioning platforms. ( a…

Passive partitioning platforms. ( a ) Megapixel digital PCR using planar emulsion arrays.…

Self-filling and partitioning platforms. (…

Self-filling and partitioning platforms. ( a ) Geometry for a staggered trap configuration…

Microfluidic droplet-based platforms. ( a…

Microfluidic droplet-based platforms. ( a ) Schematic illustrating the generation of droplets with…

Multiplex droplet dPCR assays. dPCR…

Multiplex droplet dPCR assays. dPCR assays can be multiplexed by coding the level…


Developing Norms for the Provision of Biological Laboratories in Low-Resource Contexts: Proceedings of a Workshop (2019)

Charles Chiu, MD, PhD, a member of the workshop planning committee, gave a presentation titled &ldquoMolecular Diagnostics in Low-Resource Settings.&rdquo He described the &ldquoclassical&rdquo (i.e., non-molecular) microbiological testing methods as including:

  • Culture
  • Serology
  • Microscopy
  • Biochemical profiling
  • Direct antigen testing: Lateral flow immunoassays and matrix assisted laser desorption/ionization (MALDI) for bacterial, viral, and fungal identification

Molecular diagnostics, or &ldquoDNA-Based detection,&rdquo include a variety of new, and even experimental, technologies, such as:

  • Hybridization (probes), for example, clustered regularly interspersed short palindromic repeats (CRISPR)-Cas based assays
  • Genotyping
  • Sequencing, including nanopore sequencing
  • Signal amplification
  • Target amplification (polymerase chain reaction [PCR]): Singleplex and multiplex

Molecular diagnostic tests offer some advantages for low-resource settings. The pathogens are inactivated for testing, so handling them is safer than in methods that require the use of infectious live organisms, decreasing the potential for occupational exposures. They do not rely on culture-based amplification, which is important because many pathogens

are not culturable. Because such molecular-based testing enables performance of diagnostics with noninfectious inactivated pathogens, the need for costly and complex BSL-3 or -4 containment is obviated for diagnostic work on very hazardous pathogens. Molecular methods also offer faster turnaround time and do not require large sample volumes.

However, Dr. Chiu continued, molecular testing also has significant disadvantages. First, these methods are more expensive than classical techniques. One participant noted, for example, that the cost of one PCR kit equals about 1 year&rsquos salary for a lab worker in low-resource settings. Second, performance assessment, validation, and regulatory approval of many of these methods are challenging, especially if the work is performed outside of highly controlled clinical laboratory environments, which may not be available in low-resource settings. Dr. Chiu reviewed some areas in which standards for molecular testing are lacking, particularly for environments that are not highly regulated: positive and negative controls, platforms, analytical performance, target pathogens, and reference databases. Because of this lack of standardization, the same assay run in two different labs may yield different results, and confirmatory testing is slow and costly.

In addition, the entities that normally certify and/or approve such tests (e.g., the U.S. Food and Drug Administration [FDA], the U.S. National Institute of Standards and Technology, the World Health Organization (WHO), and various nongovernmental organizations) have not yet done so for most of the new molecular technologies. In the United States, a regulatory framework, the Clinical Laboratory Improvement Amendments (CLIA), guides clinical laboratory testing. It sets minimum standards under which all clinical laboratories operate. CLIA laboratories are certified by inspection by an agency such as the College of American Pathology. Compliance with CLIA requires validation and quality assurance for all laboratory tests used in clinical care, including &ldquolaboratory-developed tests.&rdquo The Clinical and Laboratory Standards Institute (CLSI) also issues &ldquoGuidelines&mdashCLSI Molecular Diagnostic Methods for Infectious Diseases&rdquo (CLSI, 2015). But certification under these frameworks is not the same as FDA approval. Furthermore, many laboratories in low-resource settings may not meet CLIA or similar regulatory standards for proficiency testing, incorporation of standardized controls, etc.

Pre-analytical, analytical, and post-analytical concerns exist for molecular diagnostics, Dr. Chiu explained. Pre-analytical concerns include the need for proper sample collection methods, appropriate timing, proper storage conditions for both organisms and assay components (e.g.,

maintenance of a cold chain, which is especially important in low-resource settings and with labile ribonucleic acid [RNA]), and control of contamination. Analytical concerns focus on test performance evaluation&mdashsensitivity, specificity, precision, accuracy, linearity, matrix effect, interference, reproducibility, and limitations. Post-analytical concerns include proper reporting of results, copies/ml or IU/ml or Log IU/ml, positive and negative predictive values (PPV and NPV, respectively), and diagnostic value and clinical utility.

Based on these and other considerations, Dr. Chiu provided a list of relevant questions that should be addressed in the context of molecular testing in low-resource settings:

  • Who will pay for a test, and who is trained and certified to run it?
  • How often will the test be run? What volumes of material are required? Do the particular circumstances justify the costs?
  • Can clinically significant organisms be identified and quantitated in patient specimens or from culture?
  • If culture is not possible, molecular methods may be justified. But if they are to be used for prognosis, surveillance to guide public health interventions, or diagnosis to guide therapy for individual patients, will they provide the necessary accurate information?
  • Where will a test be run and in what settings? Are these settings appropriate for achieving accurate results?

Dr. Chiu then briefly described each technology category that he listed at the start of his presentation and commented on their states of development (see Box 4.1 at the end of this chapter).

Dr. Chiu explained that direct detection methods, such as sequencing, cannot fully replace serology, the branch of laboratory medicine that investigates blood serum to detect antibodies and antigens. 1 He views molecular testing as complementary to, but not a replacement for, classical testing methods. He also described the stage of development and use for each of the molecular technologies:

  • PCR is in place, but remains challenging because of lack of standardization.
  • Next-generation sequencing is still limited. Although not yet FDA approved, some nanopore sequencing is being used in the field.
  • CRISPR-Cas is very promising but remains in the research phase.
  • MALDI is generally too expensive for low-resource settings.
  • Multiplex PCR is available but is also expensive.
  • Host response-based assays are likely to evolve rapidly in the future.
  • Metagenomic sequencing is promising but also expensive and not yet widely available.

Dr. Chiu offered the following takeaway messages:

  1. A combination of traditional methods (e.g., immunofluorescent strips, real-time PCR) and state-of-the-art approaches (e.g., nanopore sequencing, CRISPR-Cas assays, multiplexed PCR) will likely be needed moving forward.
  2. Cost and other practical considerations favor true point-of-care molecular diagnostics (e.g., lateral flow immunoassays, CRISPR-Cas).
  3. It will be important to decide whether the focus should be on diagnostic testing or surveillance. Who (in loco, in country, international) should be doing what? Emerging infectious diseases do not respect borders.
  4. Sequencing has made the greatest impact in genomic surveillance, but not yet in molecular diagnostics.
  5. MALDI, multiplexed PCR platforms (e.g., BioFire, Luminex), and even single-plex PCR instruments (e.g., Cepheid GeneXPert) remain too expensive for use in diagnosis, but may be acceptable for targeted surveillance, such as during outbreaks.
  6. Inexpensive, field-ready multiplexed diagnostics are urgently needed but do not exist.
  7. Direct detection approaches likely will not replace serology (e.g., lateral flow assays) anytime soon.
  8. Complex data from genomic sequencing and other methods will require cloud computing resources to disseminate results quickly, which is critical in public health scenarios.

Dr. Chiu ended his presentation by stating that the effectiveness and accuracy of many of the molecular technologies must be demonstrated before they become widely usable in low-resource settings. Although some testing is occurring in low-resource settings, much work remains to be done.

During the discussion that followed, one participant said that his group is working to develop non-probe PCR techniques, trying to use multiplex immunoassays for serology, and providing Sanger sequencing using

remote analysis, where needed, as a backup to PCR field applications. He noted that the real questions are how to deliver the new tools to the field and train people in all these new skills, especially data handling, storage, and security. Some of the tools now available require no maintenance and have disposable cartridges. One approach for data is to use cloud-based bioinformatics to analyze data and return results so that no local bioinformatics talent is needed for this purpose, provided internet connectivity exists. However, another participant stated that communications are a real problem in the field because bandwidth is insufficient. Therefore, cloud approaches may not work in an outbreak situation.

A participant asked whether a case can be made for aiming for reagent self-sufficiency. Dr. Chiu replied that there is because the reagent market is not very competitive and competition would likely drive down costs. The same participant said that a cost-benefit analysis, which does not exist but is needed, could help donors to decide what type of support they should provide.

With Dr. Chiu&rsquos summary of the state of the art and his conclusions about technology readiness, the participants were ready to examine the practicalities of field deployment and use. Jonathan Towner, PhD, of the U.S. Centers for Disease Control and Prevention (CDC) gave a presentation on his field experiences during several hemorrhagic viral outbreaks, including the West African Ebola outbreak in 2014-2015. Dr. Towner&rsquos experiences illustrate the application of available techniques to real-world responses to major disease outbreaks. His first field experience was in Uganda in 2000-2001, where CDC used both ELISA-based and PCR-based testing. The work was performed in a hospital lab, and more than 1,000 samples were processed over a 3-month period, with more than 280 testing positive for the virus. In 2005, he participated in the response to an outbreak of Marburg virus in Angola. ELISA and PCR were again used, and this time the work was performed in an existing lab that was established for HIV diagnostics. This time 180 of 505 samples from blood or serum, breast milk, or swabs were found positive for the virus. From 2010 to 2016, Dr. Towner participated in a program of enhanced viral hemorrhagic fever (VHF) surveillance and diagnostics in Uganda to provide training for the local medical and other staff. This program placed CDC personnel in-country and helped to achieve a greatly reduced number of later VHF cases as well as reducing the time to diagnoses.

Then in 2014, the huge Ebola outbreak in West Africa occurred, seriously affecting Guinea, Sierra Leone, and Liberia. This event attracted, in Dr. Towner&rsquos words, a &ldquoUnited Nations&rdquo of field response with

Germany, France, Italy, Belgium, the Netherlands, England, Canada, the United States, Nigeria, South Africa, China, and Russia sending people, equipment, and other aid in an impressive response and collaboration effort. The U.S. agencies included CDC, the National Institutes of Health, and the Department of Defense. The aim was to provide rapid diagnostics in the field. In all, 27 field laboratories were set up across West Africa.

The scale of the response to the West African Ebola outbreak generated its own challenges. For example, many different real-time PCR assays were used in the large network of laboratories, creating a need for quality panels and attempts to standardize assays. For the panels distributed by CDC in Sierra Leone and Guinea, 2 of 6 laboratories were producing incorrect results at a rate of 10 percent, which required implementation of improvements. The need for a two-target Ebola assay, plus cell RNA PCR controls, emerged to reduce the rate of false positives or negatives when only one target was used. The lack of a cell RNA control also increased the risk of false-negative results.

There were also database, documentation, and reporting challenges. It was difficult to complete sample submission forms, so a considerable number of samples had little or no documentation. There was an absence of unique identifiers for samples and cases and difficulties with linking lab, clinical, and epidemiological data. Information on date of onset was often missing, as was knowledge of whether swabs were from corpses (appropriate) or live patients (inappropriate). Finally, there were problems with turnaround time for results, insufficient numbers of trained phlebotomists, and transport of samples.

Dr. Towner concluded that much was achieved, despite these problems. Peak testing occurred during the October-December 2014 period, with the highest number of samples tested at 180 per day in July 2015. On average, 71 percent of samples were tested on the day they arrived at the lab, and 99.9 percent were tested either the same or the next day. Samples were received for about 14 months from 12 of 14 districts and were mainly whole blood and cadaver oral swabs. Overall, more than 27,000 samples were tested. Dr. Towner&rsquos lab remained operational for 406 days, with no days off or disruptions in testing. A pilot study testing for viral persistence in male survivors began on May 23 and resulted in the testing of more than 500 semen samples. The Sierra Leone vaccine trial, or STRIVE, began on May 24, and 51 samples from 30 participants were tested. Twenty-eight teams of personnel from 17 different branches throughout CDC were trained in Atlanta on the Bo lab protocols and procedures and then deployed to help keep the lab operational.

After the crisis phase of the epidemic was over, Dr. Aiah Lebbie of Njala University in Sierra Leone was selected as the recipient of a 3-year CDC cooperative agreement to conduct ecological VHF surveillance on the region&rsquos bats. The University&rsquos laboratory facilities underwent major lab renovations from March through August 2017. There are now stable and properly maintained electricity, freezers, and other working equipment. Purchasing is operational, although delivery of perishables remains an issue. This arrangement with the University will provide educational as well as public health benefits for the region and exemplifies what partnering can accomplish. Approximately 5,000 bat specimens have been collected, all from forested areas. There are two field stations, one on Tiwai Island in Sierra Leone, which is starting renovations, and another in Gola Rainforest National Park in Liberia.

Following Dr. Towner&rsquos presentation, one participant noted that the new molecular technologies reduce risk, so minimal containment levels are needed if pathogens such as Ebola and Marburg are indigenous to a particular locality. In such a case, BSL-3 and -4 laboratories are probably not needed, but some recipients seek high-containment facilities for prestige rather than real needs. Another participant restated the need to distinguish between labs conducting surveillance and those conducting diagnostics, and noted that reference labs are necessary for identification of strains and for research. Perhaps low containment is adequate for the field while higher containment is required for reference labs.

Although Dr. Chiu said that the costs of the molecular technologies need to be reduced before they can be used in low-resource settings, one participant stated that acceptable cost may depend on the disease, the number of affected patients, the costs of care and treatment, and other factors. This translates into common diseases needing cheaper analytical capabilities. Some technology is already spreading. For example, 165 &ldquoGene Expert&rdquo machines have already been deployed throughout the Democratic Republic of Congo, although, as pointed out by one participant, their throughput is low. In addition, these machines only detect the Ebola Zaire strain, which, if it mutates, might be undetected like other strains. Another participant noted that funders should account for already-deployed capabilities when making support decisions.

A participant highlighted the different needs for normal operating situations vs. responses to epidemics. His organization uses Luminex testing, but what is appropriate depends on whether the researcher is looking for one specific pathogen or more. Another participant stated that anything new that is built should be linked to existing facilities that are

already part of a network so that reagents and other resources can be shared.

Dr. Chiu stated that direct detection of the organism of interest is the &ldquogold standard.&rdquo Clinicians are more conservative than researchers, so it might be 5 to 10 years before new test types are accepted as the basis for patient treatment. Because nasty incidents with drug and vaccine testing have already occurred in developing countries, there is a particular need to be conservative with molecular diagnostics as new tools for guiding medical treatment on these grounds as well.

A participant reiterated that the cost of reagents is an issue, especially because of the small budgets of low-resource countries. Another participant pointed out that the technologies themselves are very costly and donors should be advised to rely on proven technologies as a baseline. In light of cost factors, another participant said that it makes sense to use distributed sets of labs for basic testing and to reserve the expensive, high-throughput capabilities for central locations. Yet another participant noted that emergencies are special, but emergency response will improve if more laboratories are equipped to deal with normal business. Having a lab in place for normal operation also keeps personnel and supply chains trained and practiced. Donors should also inquire about quality management systems. In developing countries, reference labs and other resources may not be available to facilitate standardization, verification, and other necessary steps.

A participant said that the &ldquoWild West&rdquo can result from a lack of regulatory oversight&mdasheven in the United States, but more so in some developing countries. Another participant noted, however, that France provides oversight in Francophone countries. Another participant stated that the lack of reagents and of equipment maintenance and repair is a donor&rsquos nightmare and that training for these latter functions is greatly needed. However, another participant noted that the education levels in some localities are so low that the concept of maintenance does not exist and therefore providing such training for local personnel is very difficult. In addition, transportation poses a barrier to building reference capabilities. Partnerships are needed to provide fuel, dry ice, and other laboratory staples. Multinational corporations, such as Coca Cola, and oil companies, for example, have provided some of these supplies. The African Union, the Economic Community of West African States, and the South African Development Community have developed ways to address some of these problems,

A participant suggested that biobanks be sited in secure locations, such as military bases. Another participant noted that biobanks secure and

recognize the value of live samples, but may not have plans for what to do with them, raising questions about whether they should have collections without clearly defined purposes.

Participants called for a broader vision to ensure preparation for the next outbreak. They also posed the question of how to interest donors in enhancing what already exists instead of building new facilities. Finally, they wondered how donors could help with &ldquoleave behind&rdquo facilities after an outbreak, as was done in Sierra Leone after the 2014 Ebola outbreak.

BOX 4.1 The State of Development of Molecular Diagnostics: A Summary

Dr. Charles Chiu described the state of development of molecular diagnositcs. He began by explaining that in &ldquoprobe hybridization&rdquo or non-amplified nucleic acid probes, strands of DNA or RNA of less than 50 base pairs from a sample are probed for specific nucleic acid sequence &ldquotargets&rdquo that indicate the presence of particular pathogenic organisms. The DNA or RNA strands are labeled with enzymes, antigenic substrates, chemiluminescent molecular subunits, or radioisotopes. These bind with high specificity to complementary sequences of either DNA or RNA, which is referred to as &ldquohybridization.&rdquo The probes bind rapidly, under stringent conditions, and can detect even a single nucleotide change in a nucleic acid sequence. They can be used directly on patient specimens or on culture isolates. They are, however, 100-10,000 times less sensitive than amplification methods, and this level of sensitivity may not be sufficient for detecting organisms, such as Ebola virus, that have low copy number in tissue samples, such as blood. Probe hybridization is traditionally used when large numbers of organisms are present, although as noted the method is not very sensitive. It has been found particularly useful for confirming the presence of Mycobacteria species, systemic fungi, Campylobacter, Enterococci, Haemophilus influenzae, Group A and B Streptococci, Streptococcus pneumoniae, Neisseria gonorrhoeae, Staphylococcus aureus, and Listeria.

CRISPR-Cas based assays (CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and Cas for CRISPR-associated system) have received an enormous amount of recent publicity as a means of genome editing. These systems, derived from a natural bacterial defense mechanism against viruses that infect them, have made editing of the genome by adding, replacing, or removing DNA base pairs much more precise, efficient, flexible, and less expensive relative to previous strategies for changing gene sequences (National Academy of Sciences, National Academy of Medicine, 2017). They can be applied in non-human organisms, humans, and microorganisms. These advances have spurred an explosion of interest from around the globe in the potential ways in which genome editing can improve human health. Dr. Chiu stated that CRISPR-Cas-based molecular diagnostics could be a &ldquogame-changer&rdquo for the diagnosis of infectious diseases, surveillance of emerging pathogens, viral genotyping, detection of antibiotic resistance factors, cancer screening, and other applications.

CRISPR-Cas&minusbased assays have advantages and disadvantages according to Dr. Chiu. Among the advantages are that these assays are highly sensitive, have turnaround times of less than 2 hours, and have great design flexibility, requiring less than 1 week from design to implementation. They are highly portable, do not require electricity or expensive instrumentation, and can provide colorimetric readouts that are relatively easy to interpret. The University of California at Berkeley recently demonstrated that human papillomavirus can be detected directly from genital tissue without extraction in 1 hour using CRISPR-Cas. It is also currently being used to identify Zika and dengue viruses in clinical samples. The disadvantages, however, include several factors related to the fact that CRISPR-Cas remains in the phase. It has unclear multiplexing capability, it requires a target amplification step, which could introduce contamination issues, and its performance in most actual clinical uses is not yet proven. There are also important regulatory issues as well as concerns about test availability because many intended uses may be under patent.

Dr. Chiu described signal and target amplification technologies. The most widely used signal amplification method for diagnostics is the Branched Chain Technology. This technology has long been used to determine viral loads for HIV and hepatitis C (Tsongalis, 2006). It can be used to detect proteins and nucleic acids, both DNA and RNA. The concentration of the probe or target does not change, but sensitivity increases with increased concentration of labeled molecules attached to the target nucleic acid. Its advantage over target amplification methods, such as PCR, is that the detection &ldquosignal&rdquo is directly proportional to the amount of the target in a specimen, allowing for easier quantification. There is also a decreased risk of contamination, and inhibitors are not a problem.

Target amplification methods, such as PCR and Transcription-Mediated Amplification (TMA), use enzyme-mediated processes to make copies of a target nucleic acid. The result is that the analyst gets millions or billions of the target, which can lead to problems with contamination and false-positive results. The World Health Organization has issued standards for PCR (WHO, 2016).

PCR is a relatively simple technique for amplifying and detecting DNA and RNA sequences, such those associated with the genetic material of pathogens. Compared to traditional methods of DNA cloning and amplification, which can take days to complete, PCR requires only a few hours. Double-stranded DNA is first heat denatured to separate the strands. Primers then align to the separated DNA strands (annealing), and DNA polymerase then extends the primers. The result is two copies of the original DNA strand. The denaturation, annealing, and elongation process constitutes one cycle of amplification, which is repeated 20-40 times. The amplified product can then be analyzed.

The method is widely used to amplify DNA for experimental use, genetic testing, and detection of pathogenic material. PCR is highly sensitive and requires only small sample volumes. Dr. Chiu highlighted a 2010 paper (Boehme et al., 2010) that showed that a PCR-based technique could rapidly detect the presence of TB, including antibiotic-resistant forms, from sputum samples. Boehme noted that global control of TB is hampered by slow and insensitive diagnostic methods, particularly for the detection of drug-resistant TB. Early detection reduces the death rate and interrupts transmission, but the complexity and infrastructural needs of sensitive methods limits their accessibility and effect in low-resource settings. The rapid detection test developed by Boehme et al. was quickly deployed in Africa and elsewhere, although it is expensive.

There have also been numerous instances of inter-lab discrepancies in the results of PCR, indicating a performance variation among labs, although PCR assays are validated, accredited, and routinely used in some labs. In addition, &ldquogenomic drift&rdquo impacts these assays and PCR performance deteriorates over time as viruses mutate. Multiplex PCR, which is a desirable but not yet fully realized goal, would allow analyses of multiple targets in the same sample. Dr. Chiu cited a paper by Mahoney et al. (2007) on the development of a multiplex PCR panel test for the detection of 20 human respiratory viruses.

Dr. Chiu&rsquos particular area of expertise is DNA sequencing. Many devices and methods for DNA sequencing currently exist. For example, for bacteria, 16S RNA forms part of the bacterial ribosome. These RNA fragments contain regions of highly conserved and &ldquohypervariable&rdquo sequence that can be thought of as molecular &ldquofingerprints&rdquo that can be used to identify bacterial genera and species. These conserved regions can be targeted to amplify a broad range of bacteria from clinical samples. However, the technique is not quantitative because of copy number variation. The major advantage of this approach is that there is no need for culture, so it can be used on a very broad range of organisms. In this approach, DNA is extracted from a clinical sample, such as tissue or body fluid. The 16S RNA genes are amplified, sequenced, and compared with a reference database, such as GenBank, to look for a match. This type of testing is just becoming available in clinical settings. A similar approach using the 18S, 28S, and ITS genes is also available for eukaryotic pathogens (fungi and parasites).

Another approach is matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This technology has emerged as a tool for identifying and diagnosing unknown microbes from intact cells or cell extracts using the proteins translated from microbial genomes. It is rapid, sensitive, and economical. It has been adopted by microbiologists for microbial identification and strain typing, epidemiological studies, and detection of biological warfare agents, water- and food-borne pathogens,

antibiotic-resistance factors, and blood and urinary tract pathogens (Singhal et al., 2015).

Mass spectrometry (MS) involves ionizing chemical compounds into charged molecules and measuring their mass to charge (m/z) ratio. MS has been used since the early 1900s in the chemical sciences, but the development of electron spray ionization (ESI) and MALDI in the 1980s increased its applicability to large biological molecules, such as proteins.

Both ESI and MALDI are based on &ldquosoft ionization&rdquo methods that do not produce significant loss of sample integrity. In ionization by either, proteins are converted into ions by addition or loss of one or more protons. MALDI-TOF MS has certain advantages over ESI-MS because it produces singly charged ions, making data interpretation easy. In addition, prior separation by chromatography is required for ESI-MS, but not for MALDI-TOF MS, which is completely automated. The high throughput and speed associated with MALDI-TOF MS have made it superior for large-scale proteomics work.

The sample for analysis by MALDI-TOF MS is prepared by mixing or coating it with a solution of an energy-absorbent organic &ldquomatrix&rdquo compound. Both the matrix and the sample trapped in it are crystallized by drying, and the sample in the matrix is ionized with a laser beam. This generates protonated ions from the sample, which are then accelerated so that they separate from each other on the basis of their mass-to-charge (m/z) ratio. The ratio is measured by determining the time required for the ion to travel the length of the TOF tube (hence &ldquotime of flight&rdquo). Based on the TOF information, a characteristic spectrum called a peptide mass fingerprint (PMF) is generated for analytes in the sample. Microbes are identified by comparing the MS spectrum of an unknown microbial isolate to the spectra of known isolates in a reference database. Obviously, the limitation of the technology is that exact identification of new isolates is possible only if the reference database contains PMFs of the type strains of specific genera, species, and subspecies in the sample.

The topic of unknowns is very important in infectious disease. Dr. Chiu pointed out that of the top three common diseases&mdashpneumonia, meningitis/encephalitis, and fever/sepsis&mdash15-62 percent, 40-60 percent, and 20 percent, respectively, are caused by unknown organisms and are missed in hospital diagnostic labs even in developed countries. This makes the idea of being able to sequence everything in patient samples attractive, which can now be done using metagenomic next-generation sequencing. Metagenomics overcomes the twin problems of being unable to culture most microorganisms and dealing with the vast genomic diversity of microbes. These are the biggest roadblocks to advancement in clinical and environmental microbiology (National Research Council, 2007). Metagenomics seeks to understand

biology at the aggregate level, transcending individual organisms and focusing on the genes in an entire microbial community, whether this is from a soil sample or the human body. It also requires the development of advanced computational methods that maximize understanding of the genetic composition and activities of communities so complex that they can be sampled, but never completely characterized (National Research Council, 2007).

Although still a relatively new science, metagenomics has produced much new knowledge about the unculturable microbial world using radically new ways of doing microbiology. All metagenomics work starts with the same first step: DNA is extracted directly from all the microbes present in a particular sample. This mixed DNA sample can then be analyzed directly or cloned into a form maintainable in culturable laboratory bacteria. This enables researchers to create a library of genomes of all the microbes found in that sample. This library can, in turn, be studied either by analyzing the nucleotide sequences of the cloned DNA or by determining what proteins the cloned genes make when they are expressed. The technique lends itself to sorting out complex disease situations that can have many different causes. For example, tropical febrile illnesses can be caused by numerous species of bacteria, viruses, or parasites while presenting similar symptoms, and the use of metagenomics can help determine which specific organism is causing the fever in affected patients.

A metagenomic library is analogous to thousands of jigsaw puzzles jumbled into a single box, and putting the puzzles together again is one of the great challenges of metagenomics. The approach is now possible because of the availability of inexpensive, high-throughput DNA sequencing and the advanced computing capabilities needed to make sense of the millions of random sequences contained in genome libraries. The latter requires a robust bioinformatics pipeline, such as the Sequence-based Ultra-Rapid Pathogen Identification (SURPI) pipeline referenced by Dr. Chiu.

As an example of a metagenomics technology that might be suitable for use in low-resource settings, Dr. Chiu discussed nanopore sequencing, which allows for real-time metagenomic pathogen detection in patients with fever/sepsis. The advantages of nanopore sequencing include its ability to perform real-time sequence analysis, long reads, and direct sequencing of DNA, RNA, and protein from samples. It is portable using a pocket-sized device, does not require internet connectivity, and offers potentially fast turnaround times, which are key for infectious disease sequencing. Its disadvantages are that its use is costly ($500 per flow cell), it still has error rates of 8-12 percent, and the quality of its flow cells can be variable. Still, nanopore sequencing has been used successfully for real-time Ebola surveillance in West Africa (Quick et al., 2016). Quick, et al. show that they generated results in less than 24 hours after receiving an Ebola positive

sample, with the sequencing process taking as little as 15-60 minutes. This illustrates that real-time genomic surveillance is possible in low-resource settings and can be established rapidly to monitor outbreaks. Dr. Chiu&rsquos group at the University of California, San Francisco has also published a recent paper (Thézé et al., 2018) that describes the use of metagenomics to reconstruct the introduction and spread of Zika virus in Mexico and Central America.

In another paper cited by Dr. Chiu, Gardy and Loman (2017) state that:

&ldquoThe recent Ebola and Zika epidemics demonstrate the need for the continuous surveillance, rapid diagnosis and real-time tracking of emerging infectious diseases. Fast, affordable sequencing of pathogen genomes&mdashnow a staple of the public health microbiology laboratory in well-resourced settings&mdashcan affect each of these areas. Coupling genomic diagnostics and epidemiology to innovative digital disease detection platforms raises the possibility of an open, global, digital pathogen surveillance system. When informed by a One Health approach, in which human, animal and environmental health are considered together, such a genomics-based system has profound potential to improve public health in settings lacking robust laboratory capacity.&rdquo

There are, nevertheless, challenges to realizing this potential. Gardy and Loman describe some of these challenges for Zika virus, which exhibits low viral titers, a small genome (<11 kilobases), and transient viremia. Taken together, these factors complicate the detection of viral nucleic acid by a metagenomic approach. Gardy and Loman also report that obtaining a sufficient amount of viral nucleic acid for genome sequencing beyond simple diagnostics may also require PCR and an amplicon sequencing approach. Other challenges may include &ldquoaccess to reliable Internet connections, the ability to collect sample metadata, and translating genomic findings into real-time, actionable recommendations.&rdquo


Biology Virtual Labs and Simulations

Here are my favorite virtual labs and simulations for learning and exploring biology when hands-on options aren’t available.

Expandable Mind Software

I first found this gem of a site when I was looking for alternatives to animal dissections last year. While this is one resource that isn’t free, it’s well worth the very reasonable price tag.

The eFrog Virtual Dissection Lab

On this site you will find top-notch virtual animal dissections. Many dissections are available: fish (lamprey, shark, and perch), cat, fetal pig, earthworm, frog, and invertebrates (squid, starfish, and crayfish).

For each dissection, students can examine both the external and internal anatomy. As they use the virtual tweezers to remove organs, an explanation of each organ is provided. In addition, students can choose to watch a video clip relating to that particular organ. For example, when performing the virtual frog dissection, the student is able to watch video clips of intestinal peristalsis, the heart pumping, and the lungs inflating.

In addition to the virtual dissections, this site has other virtual labs.

In the Animacules lab, students can view a variety of cells through a virtual microscope.

I am just crazy about the eFly lab. Students explore genetic crosses as they virtually breed fruit flies. As they analyze the fruit fly progeny of their crosses, they become familiar with Punnett Squares, dominant and recessive gene alleles, and sex-linked traits.

This is by far one of my favorite resources for virtual labs! They have an option to try out the labs in demo mode before purchasing.

BioInteractive

In addition to some fantastic videos, lesson plans, and other activities, Biointeractive has some nice virtual labs for biology.

While completing the Bacterial Identification Lab, the student is guided through the process of using PCR to identify unknown bacteria isolated from a petri dish.

In the Bacterial Identification Lab, the student is guided through the process of using PCR to identify unknown bacteria isolated from a petri dish.

In the Cardiology Virtual Lab, the student examines 3 patients and uses the results to determine the diagnosis. This is a fantastic simulation for teens considering a career in medicine and is one I plan to use this year with my Anatomy and Physiology students.

Learn Genetics

The PCR virtual lab from Learn Genetics

For years, Learn Genetics has been one of my favorite sites for ideas for teaching about DNA and genetics. They have some virtual labs and simulations, too. Their interactive simulations help students understand the procedures scientists use to study cells and DNA, including PCR, gel electrophoresis, flow cytometry, DNA extraction, and DNA microarrays.

Bioman Biology

Bioman Biology offers games and virtual simulations on their website. These are a bit more like games than virtual labs, but they are done in ways that help students understand the material.

In the Respiratory Journey Simulation, students travel through all the parts of the respiratory system: first as a molecule of oxygen, and then as a molecule of carbon dioxide.

A simulation I plan to use in my Anatomy and Physiology class is called Respiratory Journey.

In it, you control the path of an oxygen molecule as it makes its way through the respiratory system: through the trachea, into the lungs, through the walls of the aveoli and into the bloodstream, through the heart and to the body cells. Students then drag the oxygen molecule into the cell’s mitochondria and watch as it is used to produce ATP. Then they take up a carbon dioxide molecule (waste from cellular respiration) and direct its journey back through the veins to the heart, back to the lungs, and back out the trachea.

It’s a really cool interactive experience and I anticipate that it will help students make multiple connections between gross anatomy and cellular processes.


A proposal of an alternative primer for the ARTIC Network’s multiplex PCR to improve coverage of SARS-CoV-2 genome sequencing

A group of biologists, ARTIC Network, has proposed a multiplexed PCR primer set for whole genome analysis of the novel corona virus, SARS-CoV-2, soon after the epidemics of this pathogen was revealed. The primer set seems to have been adapted already by many researchers worldwide and contributed for the high-quality and prompt genome epidemiology of this potential pandemic virus. We have also seen the great performance of their primer set and protocol the primer set was able to amplify all desired PCR products with fairy small amplification bias from clinical samples with relatively high viral load. However, we observed acute drop of reads derived from two particular PCR products, 18 and 76, out of the 98 designated products as sample’s viral load decreases. We suspected the reason for this low coverage issue was due to dimer formation between primers used to amplify those two PCR products. Here, we propose replacing just one of those primers, nCoV-2019_76_RIGHT(−), to a newly designed primer. The result of the replacement of primer showed improvement in coverage at both regions targeted by the products, 18 and 76. We expect this simple modification will extend the limit for whole SARS-CoV-2 genome analysis to samples with lower viral load and enhance genomic epidemiology of this pathogen.


Identification and Typing Methods for the Study of Bacterial Infections: a Brief Review and Mycobacterial as Case of Study

Graciela Castro-Escarpulli 1 , Nayelli Maribel Alonso- Aguilar 1 , Gildardo Rivera Sánchez 3 , Virgilio Bocanegra-Garcia 4 , Xianwu Guo 5 , Sara R Juárez-Enríquez 6 , Julieta Luna-Herrera 2 , Cristina Majalca Martínez 5 , Aguilera-Arreola Ma Guadalupe 1*

1 Laboratorio de Bacteriología Médica, Departamento de Microbiología, Mexico

2 Laboratorio de Inmunoquímica II, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, Mexico DF, 11340, Mexico

3 Laboratorio de Biotecnología Ambiental, Mexico

4 Laboratorio de Medicina de Conservación, Mexico

5 Lab. Biotecnología Genómica Centro de Biotecnología Genómica del Instituto Politécnico Nacional, Reynosa, 88710, Mexico

6 Laboratorio de pruebas especiales, Centro Médico Nacional 20 de Noviembre del Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Mexico DF, 03229, Mexico

*Corresponding Author: Dr. Ma. Guadalupe Aguilera Arreola
Laboratorio de Bacteriología Médica
Departamento de Microbiología
Escuela Nacional de Ciencias Biológicas
IPN, Esq. Prolongación de Carpio y Plan de Ayala s/n
Col. Casco de Santo Tomás, Del. Miguel Hidalgo
CP. 11340. México City, DF, Mexico
Tel: (+52-55) 57296300, extension 62374
Fax: (+52-55) 57296207
E-mail: [email protected]

Received date: October 25, 2015 Accepted date: December 05, 2015 Published date: December 15, 2015

Citation: Aguilera-Arreola MG. Identification and Typing Methods for the Study of Bacterial Infections: a Brief Review and Mycobacterial as Case of Study. Arch Clin Microbiol. 2015, 7:1.

Abstract

Several techniques based on molecular biology and analytical chemistry has been developed to reduce some of the bacterial characterization limitations. Molecular methods represent the best alternative to identify bacterial strains isolated from diverse origins and to improve research in the context of molecular epidemiology. However, these methodologies are laborious and costly compared to phenotypic or classical techniques, and there are no reliable routine laboratories. This review shall provide basic elements for the understanding of these methodologies and raise interest in their collaborative use among analytical laboratories where bacterial identification and typing are priorities, because molecular methods are not universally implemented but are available in research and reference laboratories.

Keywords

Identification Characterization Bacteria Infections

Introduction

One of the fundamental tasks of a microbiology laboratory is to fully identify the microorganisms involved in processes associated to infection or related to humans. This allows knowing their etiopathogenic implications, their clinical evolution, as well as applying an efficient antimicrobial therapy [1].

Identification and characterization of bacteria in the past were based on diverse phenotypic and genotypic methods (Table 1) however, in the last decades, it has been observed that the genotypic methods can represent a better alternative to establish the identity of bacteria and to enrich epidemiological research of infectious diseases [2].

Phenotypic methods Genotypic methods
Biochemical reactions Hybridization
Serological reactions Plasmids profile
Susceptibility to anti-microbial agents Analysis of plasmids polymorphism
Susceptibility to phages Restriction enzymes digestion
Susceptibility to bacteriocins Reaction and separation by Pulsed-Field Gel Electrophoresis (PFGE)
Profile of cell proteins Ribotyping
Polymerase Chain Reaction (PCR) and its variants
Ligase Chain Reaction (LCR)
Transcription-Based Amplification System (TAS)
Multilocus Sequence Typing (MLST)
Spoligotyping and MIRUS-VNTR

Table 1: Methods used in clinical laboratories for bacterial identification or typing.

Bacterial infections cause morbidity and mortality, and are responsible for the increase in costs and hospitalization times of patients. The time needed to identify a pathogen based on its phenotypic characteristics is the first challenge, as the sample has to be seeded and incubated for at least 24 hours and, then, conventional biochemical tests must be performed in at least another 24-hour period, conditions that delay results and compromise the patient&rsquos health.

Currently, in many microbiology laboratories, the use of automated or semi-automated commercial systems for bacterial identification is common practice, as for example: API ENTEROTUBE, VITEK, PHOENIX, MALDI-TOF MS and the GENOTYPE MYCOBACTERIUM CM system for mycobacteria. Some of the characteristics taken into account to choose the identification system are: the easiness to inoculate samples, characteristics to be determined, the required handling for the sample processing after incubation, and the availability and extent of databases [3].

Phenotypic methods are not always able to identify the microorganism to the species level, and much less to the strain level. Therefore, if a breakout, in which only one clone is responsible, is to be determined, more time and the use of genotypic (molecular) or more specific immunological techniques are required. Despite their limitation, phenotypic techniques provide an initial identification that allows taking decisions and is more available at clinical laboratories or hospitals due to their low costs and ample training of the personnel in this health area [4].

Methods for the isolation and identification of organisms from human samples, biological products, or of any other origin involve the isolation in a pure culture of the organism of interest, followed by the necessary tests to discern the microbial metabolism and/or by diverse immunological techniques that will facilitate identification. In many aspects, the culture methods and other techniques used for identification are limited in terms of sensitivity, specificity, or both. Improvements in sensitivity, specificity, and required time are based on progresses in molecular biology that have been integrated in commercial strategies for fast diagnoses. The use of molecular biology techniques for the identification and follow-up of pathogens is based on the characteristics of the genome of the particular organism to be detected or characterized. However, several aspects still complicate their application in the microbiology laboratory: the difficulty in the isolation, the slow growth, the costs of the tests, and their poor detection sensitivity for the identification of some bacterial species coming from complex samples, among others.

This review covers the different phenotypic, also called classical, methodologies, as well as different molecular biology methods that are applied to bacterial characterization. Likewise, it is aimed at raising the interest in the collaborative use of these methodologies among laboratories where bacterial identification and typing are priorities, since, although molecular methods are not yet universally implemented, they are available at research and reference laboratories that could provide the expertise to solve with first level methodologies the health problems of a country.

Phenotypic identification

For the identification of the causal agent of an infectious process, the following must be considered: 1) sample collection, 2) determination of microscopic and colonial morphotypes, and 3) identification based on the bacterial metabolism through conventional or automated tests [2]. The phenotypic study represents the classical point of view for identification, and most identification strategies are based on it [5].

In most cases, phenotypic identification is based not only on one method but rather on the combination of more than one. The sample must come from the site where the microorganism is causing the damage or must be representative of the site or product where it is multiplying. Some samples used in clinical microbiology are: feces, urine, pharyngeal exudate, cerebrospinal fluid, tears, semen, vaginal fluid, tissues, and/or biopsies. Some methods require a pure isolation of the microorganism from the sample, whereas others do not need it. Phenotypic bacterial identification is based fundamentally on the comparison of phenotypic characteristics of unknown bacteria to those of type culture. The reliability of the identification is in direct proportion to the number of similar characteristics. In medical bacteriology, the previous expertise of the analyzer and the association among the microorganism, the site, and type of infection are instrumental for the preliminary identification. Hence, in the traditional or classical bacterial identification process, three levels of processing have been established [1].

a) Primary tests are considered in the first level. These are fast and easy tests to perform, such as uptake of dyes and stains as Gram or Ziehl-Neelsen, microscopic determination of the bacterial morphotype revealed by the stains, growth characteristics at different incubation atmospheres, different temperatures, and in diverse culture media, production of oxidase and catalase enzymes, oxidation-fermentation, glucose fermentation, productions of spores, and mobility. Through these tests, it is generally possible to place the pathogen, provisionally, in some of the main groups of clinical relevance. Afterwards, other methods with greater discriminatory power can be used, to be able to discern among microorganisms that present a very similar aspect in the macro and microscopic analyses [6].

b) The second level of identification must specify the genus of the microorganism. In both this and the former level, the hypothesis on the probably identity of microorganisms is based on the characteristics of the culture and on the primary tests, which will allow determining the genus, group of genera, or, in some cases, the family of the isolate. Clinical data must also be taken into account. This will depend to a great extent on a stable pattern of phenotypic features and on the expertise of the microbiologist [7].

c) Finally, the third identification level is at the species level. Some biochemical tests allow identifying accurately most of the clinically significant bacteria. If this is not possible, a more ample battery of tests can be used, like those found in different commercial systems.

Numerous multi-test systems or equipments are available in the market to make bacterial identification fast and reliable. These techniques require a precise control of the inoculum, its purity, and way of inoculation, incubation, and reading of the tests, because not following these criteria may lead to errors. These systems can be manual, semi-automated, or automated. The result is compared to standardized tests or to the database of numerical profiles that the commercial methods have developed for this purpose. A limitation is the appearance of mutating strains and the acquisition of plasmids that can give origin to strains of different characteristics [5,8].

In contrast to the laboratories of clinical biochemistry or hematology that have benefitted from the technology to simplify sample processing and thus obtain results in a short time, automatization of the microbiology laboratory is more complex given the large variety of clinical samples to be analyzed and the inherent characteristics of different microorganisms. Recently, mass spectrophotometry (MS) has become part of the microbiology laboratory offering a fast and reliable alternative for the identification of microorganisms, including one of the most difficult identifiable bacterial groups, mycobacteria [9,10].

MS is an analytical technique that allows analyzing with great accuracy the composition of different chemical elements by permitting the measurement of ions derived from molecules and separating them in function of their mass/load (m/z) ratio [11]. The mass spectrum of each compound is named &ldquochemical trace&rdquo and is a graphical representation of the fragments obtained, by an increasing order of mass in terms of its relative abundance. Bacterial identification based on the proteins profile obtained by MALDI-TOF mass spectrometry was proposed several decades ago. However, it has been used only recently as a fast and reliable method for bacterial identification [9]. The currently commercialized platforms use MS for the identification of microorganisms through different approaches: identification based on the specific protein profile of each microorganism (proteomic approach) or on the analysis of its nucleic acids (genomic approach). Some of the commercial systems that use MS are: MALDI-TOF for microbial identification MicrobeLynxTM of Waters Corporation, MALDI BiotyperTM of Bruker Daltonics, and MS-ID of BioMérieux [12]. The last one allows the mycobacteria identification [13].

Genotypic (molecular) identification

In recent years and with the advent of new methodologies based on molecular methods great advances have been made in the diagnosis of clinically relevant bacteria. Among them, stand out the ribosomal RNA detection through hybridization with a DNA probe and that of nucleic acids amplification from clinical samples. These techniques improve the sensitivity and diagnostic specificity with respect to other detection techniques, including culture, and, in some cases, have allowed for the simultaneous detection of several microbial agents from the same sample [14].

The first step in the development of methodologies based on molecular biology techniques was supported by the detection of nucleic acids of microorganisms by means of a probe through hybridization. The genetic probe is a nucleic acid molecule, in a monocatenated state and marked, that can be used to detect a complementary DNA sequence. Oligonucleotide probes are obtained from natural DNA by cloning DNA fragment into appropriate plasmid vectors and then isolating the cloned DNA or through direct synthesis by means of combinatory chemistry. Probes can be labeled with substances that produce colorful reactions under adequate conditions [15].

DNA hybridization techniques are relatively easy to perform and interpret. Amplification techniques based on the detection of DNA using Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) or transcription-mediated specific rRNA amplification is already available both to be performed in house or commercially obtained. These techniques provide faster results with better sensitivity and specificity than conventional techniques. Depending on the type of sample these techniques detect from 15 to 20% more infectious agents than the conventional ones and 25 to 70% more than through immunofluorescence or Enzyme Immunoanalysis (EIA) [14,16].

Construction of probes to detect virulence markers, as those directed to genes encoding toxins, allows identifying those organisms that carry these genes in the clinical samples, without having to cultivate the samples. Examples of the later are the probes for Escherichia coli enterotoxins, for Vibrio cholerae toxin, or for toxins of Clostridium difficile, which can be applied directly to fecal samples [17].

Different target genes are used for the detection of microorganisms, for example those causing Sexual Transmission Infections (STI), which have been used in PCR assays among them are genes omp1 and omp2 of the Main Membrane Proteins (MOMP) to study the main etiological agents the cryptic plasmid pCT and genes 16S rRNA and 23S rRNA, for assays addressed at identifying C. trachomatis [14,18,19]. Focusing on genes 16S rRNA and 23S rRNA increases sensitivity of the assay, as normally there are multiple copies in microorganisms. However, some authors suggest that the crossed reactions with other bacteria could pose a problem whereas others have demonstrated that the use of conserved regions of gene 16S rRNA in the amplification reactions allows for species-specific differentiation [19, 20]. Use of genes and target regions for the detection of mycobacteria is a well studied area, particularly due to the difficulty posed for the isolation of these microorganisms from biological samples and furthermore because of the current hardships in handling these very virulent microorganisms. Several sequences, genes, and intergenic regions have been used for the identification of this bacterial genus, among them, the rRNA 16-23S region, genes 16S rRNA, gyrB and rpoB, the insertion element IS6110, and the eliminate differentiation regions RD1 and RD4 [21]. The study of these genes or genic sequences by means of PCR will eventually allow comparative sequence analysis of the obtained product with the sequences of reference isolates. Several commercial probes for the diagnosis of infectious diseases have been designed, but the capacity of detecting a small number of organisms or few copies of the gene in the clinical sample is still a limiting factor of this technique. However, combination of PCR with probes hybridization can become the method of choice, particularly, for microorganisms whose culture in the laboratory is slow and difficult [15].

PCR is an in vitro method of the DNA synthesis with which a particular segment of DNA is amplified by being delimited with a pair of flanking primers. Copying is achieved exponentially through repeated cycles of different incubation periods and temperatures in the presence of a thermostable DNA polymerase enzyme. In this way, millions of copies of the desired DNA sequence can be obtained in a couple of hours. This is a highly specific, fast, sensitive, and versatile molecular biology technique to detect the smallest amounts of a specific DNA, fostering its easy identification and avoiding the use of radioisotopes [22]. Despite the benefits that the PCR technique offers in comparison to culture for the detection of some microorganisms, the commercially available techniques are scarce and are limited to research laboratories or to reference laboratories specialized in molecular diagnoses, among other causes, due to their high cost. An alternative to make the use of molecular diagnoses feasible as routine techniques could be the acquisition of reagents in a separate manner and standardization of nucleic acids extraction and amplification protocols designed in each diagnostic laboratory this would lead to a significant reduction in technological dependence and to an increase in the sensitivity and specificity of the used diagnostic techniques [23].

The multiple amplification for the concomitant detections of some microorganisms enhances, in some cases, the sensitivity and specificity of those addressed at a single microorganism. This PCR variant is called multiple PCR (mPCR), in which more than one target sequence can be amplified simultaneously by the inclusion of more than a pair of primers in the reaction [24]. This technique has been applied successfully in many diagnostic areas, like the study of infectious diseases, species genotyping, diagnosis of hereditary diseases, identification of mutations, paleontology, anthropology, and forensic sciences, among others here, the technique has shown the potential to save considerable time, without compromising the usefulness and efficiency of the test [24]. On the other hand, plataforms for pathogen identification are becoming available like pyroseguencing and spectroscopy [25].

In the amplification-pyrosequencing platforms, bacterial identification is achieved by PCR of three variable regions of the 16S rRNA (V1-V3, or V1, V2 and V6). Lower amplicons of 500 bp are obtained, their nucleotides composition can be determined by means of the emission of light by the release of pyrophosphates (extension byproducts by polymerization of the DNA chain). This platform has been increasingly innovated based on the type of clinical sample and on the determination of different genic fragments corresponding to the different virulence factors and the resistance to antimicrobials, which has improved the versatility of this platform [2,25]

Another innovating platform for bacterial identification is the conjunction of amplification (by PCR) and mass spectrophotometry (PCR/ESI-TOF). The latter allows for the universal detection of one or more pathogens encountered in a wide variety of samples (environmental, clinical, foodborne, o in cultures). It consists in the extraction of nucleic acids and PCR amplification with primer pairs of ample range one or several PCR products are obtained that correspond to genomic identification regions of the different microbial domains in relation to the complexity of the problem sample. The products are desalted and then ionized and aerosolized towards the mass spectrometer, generating signals that are processed to determine their mass and composition. Results are interpreted with the TIGER (Triangulation Identification for the Genetic Evaluation of Risks) strategy, and accessing the information into a genomic database that assigns the species. The advantages of this platform are that it does not require culture, is efficient in polymicrobial samples, and, in the case of non-characterized new pathogens, it allows assigning them to bacterial genera or families. In addition, it also permits to detect some virulence and resistance genes, and typing of the identified microorganism [2,12].

Typing of microorganisms

After bacterial identification, microorganisms have to be typed for epidemiological studies hence, molecular typing systems constitute one of the molecular techniques contributions to microbiology widely used in the last years. These systems involve a large variety of techniques aimed at comparing the structure of genomes of highly inter-related organisms.

Typing methods (phenotypic and genotypic) allow differentiating one bacterial strain from another. Before using a typing technique it is important to ensure that the method can differentiate among non-related isolations, that it is able to detect the same strain in different samples, and that it reflects the gene relations among isolations with epidemiological relation [26].

From a practical point of view, a typing system should be reproducible, have a high discrimination capacity, and be easy to use and to interpret the results [26]. Notwithstanding, election of the molecular method depends also on other factors, such as the microorganism to be studied, the clinical sample, the target to be studied (a single gene or the whole genome), the area of application, the infrastructure available at the clinical laboratory, and the speed needed to reach a result. Once the microorganism has been identified, it is important to know whether it is responsible for a breakout therefore, the corresponding epidemiological research has to be performed. To accomplish this process diverse molecular techniques have been used these tools are aimed at determining the clonal relation that exists among several isolates of a given species. This information is useful in sporadic infections and even more during disease breakouts and epidemics because it allows determining the number of circulating clones, detecting the pathogens&rsquo transmission route, identifying the source of infection, recognizing particularly the virulent strains, and, thereby, leading to the most appropriate treatment [27,28].

The typing technologies based on the whole genome of the microorganism yield better results in establishing the clonal relation. However, for this analysis, digestion of the genome with restriction enzymes is needed to obtain DNA fragments of diverse sizes that provide patterns or profiles, once they have been separated by electrophoresis. On the other side, there is the inconvenient that the diverse fragments obtained from the restriction procedure are large-sized fragments that have to be analyzed by Pulsed-Fields Gel Electrophoresis (PFGE) [26]. PFGE is a technique widely used for typing clinically relevant bacteria. Its importance relies on that this type of electrophoresis is capable of separating fragments of a length higher than 50 kb up to 10 Mb, which is not possible with conventional electrophoresis, which can separate only fragments of 100 bp to 50 kb. This capacity of PFGE is due to its multidirectional feature, changing continuously the direction of the electrical field, thus, permitting the re-orientation of the direction of the DNA molecules, so that these can migrate through the agarose gel, in addition to this event, the applied electrical pulses are of different duration, fostering the reorientation of the molecules and the separation of the fragments of different sizes [29]. Along time, different types of PFGE equipment have been developed (Table 2), mainly to improve the resolution of gels and to diminish costs of reagents and electricity. The most used apparatus is the Contour Clamped Homogeneous Electric Fields (CHEF, BioRad), because it can separate molecules of 7000 kb, this characteristic is provided by its 24 electrodes that are hexagonally distributed and generate homogeneous electrical fields allowing for the samples to be run uniformly [29,30]. Some advantages of PFGE are: it possesses a high discrimination power, excellent reproducibility, easiness to measure the genome and it allows working with a large number of samples. Disadvantages include that most of the protocols require more than 4 days to get and analyze the pulse types,in comparison to other methods that can be less costly, but not appropriate to study clonally related strains (Tables 2 and 3) [27,29]. Application of PFGE to the study of mycobacterial infection has been limited by the need to count upon high mycobacterial DNA concentrations that are difficult to obtain, as effective breakage of the cell wall and disaggregation of the lipids covering the mycobacterium are complicated procedures additionally, mycobacterial growth tends to aggregate in clusters, which also hinders the assay [31].

Method Ease of the technique Results interpretation Duration of the test (days) Reproducibility among laboratories Intra-assay reproducibility Cost per test
PFGE Moderate Easy 3 Good Good Moderate
PCR-RFLP Easy Easy 1 Good Good Low
rep-PCR Easy Easy 1 Good Moderate Low
AP-PCR Easy Easy 1 Moderate Low Low
AFLP Moderate Moderate 2 Good Good Moderate
MLST Difficult Moderate 2 Good Good High

Table 2: Most relevant features of some typing methods based on PCR-amplification of nucleic acids in comparison to Pulsed-Field Gel Electrophoresis (PFGE).

Typing method Description No. Of markers Time scale Source of variation Discrimination power Reproducibility Application Database
MLST PCR amplification of housekeeping genes to create allele profiles 7 GE DNA sequence Moderate to high High Acinetobacter baumannii pubmlst.org
LE Clostidium difficile www.mlst.net
Coagulase negative staphylococci
Enterococci
Pseudomonas aeruginosa
Staphylococcus aureus
rep-PCR PCR amplification of repeated sequences in the genome NA LE Banding patterns Moderate to high Medium Staphylococcus aureus NA
Mycobacterium tuberculosis
Acinetobacter baumannii
PFGE Comparison of macro-restriction fragments NA LE Banding patterns Moderate to high High Acinetobacter baumannii NA
Staphylococcus aureus
Coagulase negative staphylococci
AFLP Enzyme restriction digestion of genomic DNA, binding of restriction fragments and selective amplification NA LE Banding patterns Moderate to high Low Acinetobacter baumannii NA
Klebsiella pneumoniae
Staphylococcus aureus
Mycobacterium tuberculosis
MLVA PCR amplification of loci VTR, visualizing the polymorphism to create an allele profile Oct-80 GE DNA sequence Moderate to high High Clostidium difficile minisatellites.upsud.fr
Enterococci www.mlva.net
Mycobacterium tuberculosis www.pasteur.fr/mlst
Staphylococcus aureus
RFLP Genomic DNA digestion or of an amplicon with restriction enzymes producing short restriction fragments NA LE Banding pattern Low High Staphylococcus aureus https://app.chuv .ch/prasite/index.htm
Pseudomonas aeruginosa
Mycobacterium tuberculosis

Table 3: Comparative analysis of some molecular biology techniques and bacteria of hospital relevance where they are applied.

Polymerase Chain Reaction-RFLP

Consists of a PCR for the amplification of a gene or parts of it, combined with the subsequent digestion of the PCR products using one or several restriction enzymes. The electrophoretic analysis of the restriction products reveals the polymorphisms of the gene or of the gene fragments (RFLP) and evidences the genetic changes among isolates. This technology is capable of revealing sequence polymorphisms rapidly it is technologically simple and highly reproducible. Besides, it compares well with other techniques like: Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), or single strand conformation polymorphism (SSCP), and Cut Fragments Length Polymorphism (CFLP), which also reveal sequence polymorphisms among strains without having to determine the whole sequence [32]. For mycobacteria, PCR-RLFP has been widely used, particularly for the study of the insertion element IS6110, where the Pvull enzyme is used to generate restriction fragments from genomic DNA [33]. Other sequences or genes used to identify and genotype Mycobacterium tuberculosis, as well as other non-tuberculous mycobacteria are 16S rDNA, and genes rpoB, recA, and hsp65, which have yielded variable results [34]. Of these sequences, the most consistent has been the gene that encodes the 65-kDa heat shock protein (hsp65), analyzed through a PCR-based assay and its posterior restriction with enzymes BstEII and HaeIII, an assay known as PRA (Polymerase Chain Reaction Restriction Enzyme Analysis&mdash PRA) [35]. Of the non-commercial molecular methods, the PRA method is one of the most used for the identification of nontuberculous mycobacteria of fast growth, due to its speediness, low cost, and above all, because the data base: https://app.chuv. ch/prasite/index.htm, is available and contains the restriction profiles of at least 113 species [36].

Rep-Polymerase Chain Reaction

Versalovic et al. [37] described a method to study the bacterial genome by examining the specific patterns of a given strain obtained through PCR amplification of repetitive DNA elements present in bacterial genomes. They used two main sets of repetitive elements for typing purposes, the REP with 38-bp sequences that consist of six degenerated positions and a variable loop of 5 bp between each side of a conserved palindromic portion. REP sequences have been described both for enteric bacteria and for Gram-positive [28,38] and, more recently, for mycobacteria including nontuberculous mycobacteria, in the latter with good results [39]. ERIC sequences are a second set of DNA sequences that have been used successfully for the typing of strains they are 126-pb elements that contain a highly conserved central inverted repetition and are located in extragenic regions of the bacterial genome. ERIC analysis has also been used for genotyping of mycobacteria [40]. REP or ERIC amplification can be performed with only one primer or with one set or multiple sets of primers. ERIC patterns are generally less complex than REP patterns, but both provide a good discrimination at the level of strains. The combined use of both methods (REP-PCR and ERIC-PCR) in the strains to be typed, increases their discrimination power [28]. Although REP and ERIC sequences are the most commonly used targets for DNA typing, BOX sequences are also used, the latter have been used to differentiate strains of Streptococcus pneumoniae. BOX elements are located in intergenic regions and they can also form stem-loop structures due to their even symmetry. They are a mosaic of repetitive elements composed of several combinations of three sequences known as boxA, boxB and boxC. The three-subunit sequences have molecular lengths of 59, 45 and 50 nucleotides, respectively. BOX elements have no sequence relation with REP and ERIC sequences [28].

Amplified Fragment Length Polymorphism

Amplified Fragment Length Polymorphism (AFLP) is a genomic fingerprinting technique based on the selective amplification of a subset of DNA fragments generated through restriction enzymes digestion. Originally, applied to the characterization of plant genomes, currently AFPL has been used for bacterial typing. Two variations of AFLP have been described: the first, with two different restriction enzymes and two primers for the amplification, and the second, with only one primer and one restriction enzyme. Bacterial DNA is extracted, purified, and then subjected to digestion with two different enzymes, such as EcoRI and MseI. Afterwards, the restriction fragments are bound to adaptors that contain each restriction site and a sequence that is homologous to a binding site of the PCR primer. The PCR primers used for the amplification contain DNA sequences that are homologous to the adaptor and contain one or two selective bases in their 3&rsquo ends [30,41]. The AFLP method has been adapted for the study of M. tuberculosis, however, it has been scarcely used due to its poor genotyping resolution power in M. tuberculosis [42].

Multilocus Sequence Typing

Multilocus Sequence Typing (MLST) is a genetic method with a high resolution power it is based on sequencing fragments of 7 genes of 450 to 500 bp (with a high degree of variability). The analysis detects variations in the different loci and permits the identification of identical microorganisms (clones) or of highly related ones (clonal lines or genotypes). Therefore, they are markers that have remained stable along evolution and are used for the comparison of strains in large time scales or from different geographical regions [43]. Sequencing allows detecting variants of just one change in the database of the analyzed gene. Hence, it has been calculated that if 30 different alleles are found per locus, and seven genes are studied, then up to 307 different genotypes could be distinguished [44]. Each allele is numbered considering its previous presentation in the database and each type of Sequence (ST) is defined by a bar code of seven digits that are unique to the seven loci [45].

Spoligotyping and MIRU-VNTR

Three methods are the most commonly used for mycobacterial genotyping: spoligotyping, MIRU-VNTR analysis, and analysis of restriction fragments obtained with the Pvull enzyme to detect the insertion element IS6110 by hybridization. Spoligotyping was the second method widely used for fast genotyping of mycobacteria, originally described for the typing of M. bovis isolates [46]. This technology combines a PCR amplification of the Direct Repeat (DR) region of each isolate and the hybridization of the spacer regions, the latter are unique sequences that separate DRs, there are 47 DRs for M. tuberculosis and 41 DRs for M. bovis these spacers depict a large polymorphism, which enables their use as variability indicators. The method was named &ldquospoligotyping&rdquo derived from &ldquospacer oligotyping&rdquo [47]. The hybridization pattern shown by each isolate is interpreted on a matrix that can be worked with the binary system or by means of the &ldquooctal code&rdquo to ease handling of the patterns [48]. Analysis of genomic loci of M. tuberculosis containing Variable Number Tandem Repeat Sequences (VNTR) is a fast genotyping method, similar to spoligotyping, and is based on the analysis of repeated sequences found in the mycobacterial genome (MIRUMycobacterial Interspersed Repetitive Units) that are different in the DR regions. The minimal set of MIRUS-VNTR to achieve a differentiation is of 12 loci, which yields a code that establishes the pertinence to the different genotypes through an informatics analysis [49]. The number of MIRUS loci has been increased to 24, granting a higher resolution to this protocol and has widened its application to evolutionary-type studies [50].

Conclusions

Isolation, identification, and analysis of isolates of a single sample are some of the main functions and objectives of a microbiology laboratory. However, it is fundamental to recall that the best bacteriological result is obtained when the sample received by the laboratory has been procured under the best conditions. In addition, it is a major task of the laboratory to differentiate a pathogenic microorganism per se or the potential pathogenicity of colonization and a contaminant, and to describe, if applicable, the possible resistance mechanisms to be able to propose the most efficacious treatment.

The medical bacteriology diagnostic laboratory currently counts upon diverse methodologies that constitute a fundamental cornerstone in the diagnostic process of bacterial-origin infections. Within its daily routine, the clinical laboratory applies phenotypic techniques to reach its goals. However, these techniques do present some limitations in terms of sensitivity, specificity, and time. These limitations are more evident for some types of bacteria of difficult or slow growth, of the so-called non-cultivable bacteria, or for the processing of samples from multi-treated patients. In the last decade, diverse techniques have been developed in the field of molecular biology and analytical chemistry with a great potential to diminish some of these limitations, and which have allowed for the search and identification of the causal agent, as well as the evaluation of clonality, for epidemiological research and objectives. Because these techniques are still laborious and of a higher cost than some phenotypic ones, they are usually not available at laboratories of public hospitals. Although their implementation is not universal they are available at research and reference laboratories.

To be clinically useful, the identification of a microorganism must be as fast as possible. Economic aspects and praxis propose the use of a minimal number of diagnostic tests. By necessity, identification in the clinical laboratory will always represent a compromise of accuracy and precision, on one side, and the speed and economy on the other side therefore, the collaborative efforts among public clinical laboratories and research and reference laboratories is undoubtly the correct course of action to better diagnoses.

Acknowledgements

This work was part of the research projects "Identification of etiological agents of intrahospital infection of the Centro Médico Nacional (CMN) 20 de Noviembre" funded by the Secretariat of Science and Technology and Innovation previously ICYT-DF under the ICyTDF/325/2011 agreement and "Basic and applied research in bacteriology" SIP 20150966 of the National Polytechnic Institute. MGAA, JLH, GCE, VB, GR, XG receives SNI, EDI and COFAA fellowships. NMAA was BEIFI fellowship.


Alternatives to PCR - Biology

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An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RLFP analysis

Claretta G. Losi, 1 Stefania Ferrari, 1 Enrico Sossi, 1 Riccardo Villa, 1 Maura Ferrari 1

1 Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy e-mail: [email protected]

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One of the major risks in cell culture laboratories is the misidentification and cross-contamination of cell lines. Several methods have been used to authenticate cell lines, including isoenzyme profiling, the test suggested by European Farmacopeia, which is performed at the Tissue Culture Centre in Brescia. However, this method displays several disadvantages, such as high variability and low reproducibility, and it is time consuming and requires high cell concentrations to be performed. Therefore, an alternative method has been developed to confirm the specie of origin of 27 different animal cell cultures. A polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) assay was optimized, based on the use of a pair of primers that anneal to a portion of the cytochrome b gene in all the species. The amplification product was digested with a panel of six restriction enzymes, and the pattern derived was resolved on 3% high-resolution agarose gel. For 23 species, this protocol produced a unique restriction pattern, and the origin of these animal cells resulted to be confirmed by this analysis. Furthermore, results indicate that cytochrome b PCR-RFLP was able to amplify target sequences using very low amounts of deoxyribonucleic acid (DNA). Its sensitivity in detecting interspecies, cross-contamination was comparable to that of isoenzyme analysis (contaminating DNA should represent at least 10% of the total DNA). For 4 of the 27 species (sheep, dog, Guinea pig, and Rhesus monkey) the observed pattern, even if highly reproducible, showed additional bands for these species, specific PCR was also performed.

Claretta G. Losi , Stefania Ferrari , Enrico Sossi , Riccardo Villa , and Maura Ferrari "An alternative method to isoenzyme profile for cell line identification and interspecies cross-contaminations: cytochrome b PCR-RLFP analysis," In Vitro Cellular & Developmental Biology - Animal 44(8), 321-329, (2 July 2008). https://doi.org/10.1007/s11626-008-9125-x

Received: 10 March 2008 Accepted: 16 May 2008 Published: 2 July 2008

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