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Chapter 16

With the coming of polymerase concatenation reaction technique, microbiologists were speedy to gain that this new diagnostic mode had possible to do microbiological diagnosing easier, cheaper and faster. Alternatively of conventional research lab methods that rely on phenotypic look of antigens or measuring of biochemicalproducts, usage of molecular methods helps in rapid designation of anumber of infective agents. Molecular methods have progressively been incorporated into the clinical microbiologylaboratory, peculiarly for the sensing and characterisationof viral infections, and for the diagnosing of diseases that occur due toinfection with fastidious bacteriums. The advantages are obvious and there is a rapid turn-around clip with high sensitiveness and specificity but it is hard to guarantee quality control and avoid taint during the usage of such methods.

Normally molecular methods involve the usage of PCR, which may be either straightforward PCR or the nested assortment. In future, it is expected that the usage of microarray would farther increase the public-service corporation of the molecular methods.

& lt ; H1 & gt ; MOLECULAR BIOLOGY IN VIROLOGY

Viruss possibly pose existent challenge for amicrobiologist, as they can neither be cultured nor can they be seen under an ordinary microscope. The responses that they generate in human organic structure are fickle and may non correlate with the type or badness of the infection. All these hindrances can make batch of jobs in diagnosing. That is where the function of molecular medical specialty comes in.

& lt ; H2 & gt ; CNS infections

The coming of polymerase concatenation reaction technique has revolutionised the diagnosing of CNS infections. Previously, the diagnosing of such infections wasa cumbersome and insensitive procedure. However, with the coming of the PCR technique, their diagnosing has become comparatively simple.

To understand this better, allow us hold a expression at the application of PCR in the diagnosing of HSV, which is the most common cause of acute sporadic focal phrenitis. The sensitiveness and specificity of CSF PCR for the diagnosing of HSV are 96 % and 99 % severally. About the same per centums characterise the molecular methods that are used to observe enteroviruses and Epstein-Barr virus and human CMV in the CSF. The sensitiveness and specificity rates are lesser for the VZV, HHV6, HIV and hydrophobias viruses. JCV is a slow-growing virus ( takes up to five hebdomads ) , so a diagnostic trial otherthan civilization is needed. Previously, the JCV virus used to be spotted by agencies of a nested PCR which had 92 % sensitiveness and 100 % specificity. However, in instances with attendant HIV infection, after the debut of HAART therapy, the PCR technique showed a strong lessening in the sensitiveness, because of the lower sum of the virus in the CSF, due to the Restoration of the immune system. However, with the aid of the existent clip PCR, sensitiveness and specificity has been restored to the old degrees.

& lt ; H2 & gt ; HPV infection

Early diagnosing of papillomavirus infection is a cardinal issue for the bar of HPV-related malignant neoplastic diseases. In some development states, these malignant neoplastic diseases represent the most prevailing neoplastic pathology. Screening programmes have been, and are, chiefly based on the scrutiny of cytologic vilifications from the cervical canal stained by Papanicolau staining technique ( Paptest ) .

Since antibody response is unfaithfully detected and does non needfully correlate with current viral presence, the diagnostic virologic checks for HPV infection are based on HPV DNA sensing and typewriting. Different PCR checks, largely utilizing general primers for the conserved L1 or E1 parts have been developed. In some instances, nested protocols utilizing combinations of the mentioned set of primers can be used. Typing is achieved by sequencing, limitation endonuclease digestion or investigation hybridisation, all performed on the amplified merchandise. In malice of these progresss, none of these methods can be considered a aureate criterion. Microarray based checks are presently being developed to better specificity and type scope.

Unlike cytologic showing, a vaginal swab is equal for obtaining a dependable consequence, since infected cells and virions withtheir high burden of viral DNA are spread throughoutthe venereal piece of land.

& lt ; H2 & gt ; Hepatitis viruses

Nucleic acerb sensing techniques are more sensitive than immunochemical assaies which detect viral antigen in samples of different originsfor diagnosing of HAV. HAV has been detected with techniques such as PCR – RFLP, SSCP, Southern blotting, sequencing, nucleic acid hybridisation and change by reversal transcription-PCR ( RT-PCR ) . Amplification of viral RNA by RT-PCR is presently the most sensitive and widely used method for sensing of HAV RNA.

Diagnosis of hepatitis C is based on serological and molecular checks which detect HCV-specific antibodies ( anti-HCV ) and HCV RNA severally. The molecular checks presently available are rearward RNA polymerase RT PCR. Third-generation anti-HCV enzyme-linked immunosorbent checks ( ELISAs ) are extremely sensitive every bit good as specific and stand for the primary diagnostic check. The recombinant immunoblot check ( RIBA ) is a auxiliary check that can be used to corroborate a positive ELISA, peculiarly in low-risk populations.

HCV infection can be diagnosed by RT-PCR every bit early as 1-2 hebdomads after infection or 4-6 hebdomads before anti-HCV seroconversion. The finding of HCV RNA is, in rule, of import for the choice of patients for antiviral therapy and for the appraisal of its efficaciousness. In instances with positive ELISA, RT PCR allows to know apart between patients with chronic hepatitis C and those with single-minded HCV infection that can stay anti-HCV positive for old ages or decennaries.

Discrimination of genotype 1 from genotypes 2 and 3 every bit good as quantitative finding of viraemia degrees has become of import for the choice of an optimum intervention regimen. In general, nevertheless, genotyping and quantitative RT PCR trials should be used merely in the context ofa defined therapy protocol and non for the initial diagnosing of HCV infection.

& lt ; H2 & gt ; Respiratory infections

Of the common viruses doing respiratory infections, the usage of molecular methods in the diagnosing of adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus ( RSV ) infections has non been clearly established. Although PCR techniques are available for the diagnosing of these viruses, other rapid conventional techniques are besides available: grippe virus and RSV can be detected in the clinical specimens by immunofluorescence ; parainfluenza virus and adenovirus can be detected by immunofluorscence after incubation for 48 H in shell vial civilizations. In these instances, nucleic acerb elaboration techniques have no added value in footings of sensitiveness or celerity.

Rhinoviruss and coronaviruses grow ill in cell civilization and rapid sensing techniques like immunofluorscence and civilization techniques are non available for these viruses. In such instances, usage of molecular methods presents a distinguishable advantage. The PCR technique is rapid and reasonably sensitive.Hantavirus pneumonic syndrome is characterized by febrility, myodynia, concern, andcough, followed quickly by respiratory failure. Antibodiesagainst heterologic hantavirus antigens were ab initio used toidentify the causative agent. After the coming of PCR technique, PCR was used to observe the hantavirus genomein necropsy specimens. The virus can non be cultured, therefore, PCR remains the bestdiagnostic possibility for this syndrome.

Besides these viruses, PCR based checks have besides been developed for the SARS and H5N1 strains of grippe.

& lt ; H2 & gt ; Gastrointestinal disease

Viruss are responsible for more instances of infective diarrhea as compared to bacteriums and other pathogens. The method of pick for microbiological diagnosing of rotavirus from stool samples is PCR. Norovirus, a calicivirus once known as Norwalk virus, canbe diagnosed by negatron microscopy, enzyme immunoassayand PCR ; but PCR is the most sensitive and rapid method.PCR is besides the most sensitive method for the diagnosing ofastroviruses and enteral adenoviruses ( serotypes 40 and41 ) .

& lt ; H2 & gt ; HIV

Although human immunodeficiency virus ( HIV ) infection is routinely diagnosed by serology, early HIV infection can be detected by HIV pro-viral DNA sensing much before HIV antibodies can be confirmed by Western Blot serology. Verticaltransmission of HIV infection can besides be detected in the neonateusing the HIV pro-viral DNA sensing technique. These methods are capable of cut downing thepotentially infective window period. The existent clip PCR can besides be used to quantitate the viral burden, molecular techniques can therefore be used to predict in instances with HIV infection. Finally, HIV genotyping for the sensing of drug opposition can steer antiretroviral therapy and complements viral burden appraisal.

& lt ; H1 & gt ; MOLECULAR BIOLOGY IN BACTERIOLOGY

Application of molecular biological science for diagnosing of bacterial infections should be considered in three scenarios, viz. ( a ) for the designation of an being already isolated in pure civilization, ( B ) for the rapid designation of an being in diagnostic scene from clinical specimens and ( degree Celsius ) for the designation of an being from non-culturable specimens, e.g. civilization negative endocarditis.

Most modern clinical microbiology diagnostic research labs rely on a combination of colonial morphology, physiology and designation of biochemical/serological markers for designation and successful categorization of the pathogen at the genus and species degrees. This is of import because it is necessary to right type bacteriums for epidemiological intents and for infection control.

The rapid designation of an being already isolated in pure civilization needs no amplification. Molecular methods can quickly insulate the nucleic acid from the civilized being and type it accurately and quickly. For rapid designation of beings in a diagnostic scene from clinical specimens, molecular biological techniques play an of import function. The civilization study may take a long clip to come and so, in many instances, patients possibly managed through empirical observation. In today ‘s universe, the importance of rapid diagnosing is besides apparent in the scene of a bioterrorist onslaught.

In instances where the being can non be cultured, molecular biological science has an of import function to play. Take a state of affairs where antibiotic therapy has already been instituted and the civilization is negative. Even in such instances, the being ‘s Deoxyribonucleic acid remains integral and this Deoxyribonucleic acid can be exploited in molecular nosologies.

By and big, molecular techniques in bacteriology usage DNA as the get downing stuff. Deoxyribonucleic acid has the advantage of being stable and bacteriums contain DNA s their familial material.Usually, molecular biological science is required to reply one inquiry ; whether or non the mark DNA is present. Quantitation has small significance unless some specific job like nutrient taint is being studied. Under these fortunes, DNA is equal as the get downing stuff.

At times, when there isno indicant sing the individuality of a bacterial being, elaboration of DNA encoding ribosomal RNA cistrons in concurrence with DNA sequencing of the ampliconcan be done. In bacteriums, thereare three cistrons which decide the rRNA functionality. These are 5S, 16S and 23S rRNA cistron. The 16S rRNA cistron has been most normally employed for identificationpurposes due to it being extremely conservedand holding a moderate transcript figure depending on thegenus. 16S rRNA cistrons are found in all bacteriums andaccumulate mutants at slow and changeless rate over clip, therefore, they may be used as “ molecular redstem storksbills ” . Highly variable parts of the 16S rRNA sequenceprovide alone signature to any bacteria and usefulinformation about relationships between different bacteriums.

More late, employment of the 16S-23S rRNAintergenic spacer part has become popular due to itslarge transcript figure, and more significantly, because of its high sequencevariability. The 23S rRNA fractional monetary unit has besides been used to place bacterial species.

In future, it is likely that the bacteriological diagnosing will look beyond merely the 16S rRNA sequence. Additionally, there are state of affairss where the 16S rRNA sequence may non be able to distinguish between species. Under these fortunes, sequences of indispensable cistrons such as that of heat daze proteins possibly employed. This is peculiarly utile in distinguishing between species such as Burkholderiacenocepaciaand B. multivorans.

& lt ; H2 & gt ; Fastidious Bacteria

The diagnosing of infections due tofastidious bacteriums has benefited greatly from moleculardetection methods. Many fastidious bacteriums, such as Mycobacterium TB, Chlamydiatrachomatis, Neisseria gonorrheaeand Bordetella whooping cough, have of import public wellness implications.Non-culture-based molecular testing has the advantage ofavoiding holds of yearss to hebdomads over conventional culturetechniques. This is of import for early acknowledgment and intervention as a public healthimperative.

Fastidious bacteriums normally transmit TB and sexually familial diseases. Collection of samples in instances of sexually transmitted diseases can be a beginning of intense embarrassment. Again, the opportunities of taint are rather high and most of the beings are extremely fastidious and require particular conditions for growing. Under these fortunes, molecular sensing is utile, since non invasive specimens that are unsuitable for traditional civilization, such as initial watercourse of urineand equanimous vaginal swabs can be used. These aremore convenient and acceptable and increase the patient ‘s compliancewith testing. Molecular proving methods provide sensitiveness and specificity which is tantamount to what is seen with standard civilization methods. In distant countries, molecular methods have the advantage of being performedon dry swabs with small debasement of DNA duringtransit compared to the troubles of transporting samples inspecialised transport medium to continue viability. In add-on, molecular methods can prove for multiple venereal pathogenssuch as C. trachomatis, N. gonorrhoeae, the Donovanosisagent and the venereal mycoplasmata from the same swab.

Mycobacteriology has been aided by the debut ofmolecular methods. However, it is of import to observe thatmolecular sensing of M. TB is one of the fewexamples where conventional civilization remains more sensitive.This is perchance due to the trouble in let go ofing the DNAfrom the bacterial cells during the extraction procedure. Yet, molecular methods are of import in sensing of M TB since it allows verification of acid-fast bacilliseen on microscopy with up to 98 % sensitiveness in pulmonarytuberculosis. Furthermore, the consequences are obtained within a twenty-four hours compared to two hebdomads or morefor civilization. Specimens that are smear-negative have a muchlower opportunity of molecular confirmation.Molecular biological science can besides differentiate the beings grown on a civilization home base in a twenty-four hours as compared to the four hebdomads it would usually take by standard methods.

& lt ; H2 & gt ; Antibiotic opposition markers

Detection of antibiotic opposition is of extreme importance in today ‘s scenario where bacteriums are developing opposition faster than the antibiotics can be discovered. Using rapid and dependable genotypic sensing to bacteriums with infection control deductions such as methicillin immune Staphylococcus aureus ( MRSA ) and vancomycin resistantenterococci ( VRE ) is of great possible benefit. Thedifferentiation of MRSA from other S. aureusis done bydetection of the mecAgene responsible for methicillin opposition. It is of import to observe MRSA early, non merely for epidemiological intents but besides for early induction of the patient ‘s intervention, which is more aggressive and specific in such instances. Similarly, sensing of VRE is rapid utilizing DNA-based elaboration techniques, which are extremely sensitive as compared to traditional methods.

Extended spectrum I?-lactamases ( ESBL ) are found inEscherichia coli and Klebsiellapneumoniaeand are readilytransmitted to plasmids and jumping genes. ESBL-containingbacteria can distribute quickly in wellness attention installations to causewound infections, urinary piece of land infections and blood poisoning. Molecular sensing of these point mutants at theactive site of the I?-lactamase cistron can corroborate the ESBL andallows for epidemiological typewriting.

Multi-drug immune TB ( defined as the presence ofboth rifampicin and isoniazid opposition ) is a serious problemin many parts of the universe. Rather than using traditional civilization methods which deliver consequences after several yearss, sensing of the rpoB and hsp65 cistron marks can observe the opposition cistrons in a individual twenty-four hours.

& lt ; H1 & gt ; MOLECULAR BIOLOGY IN MYCOLOGY AND PARASITOLOGY

Although non often applied for sensing of eucaryotic infections, molecular proving canbe helpfulin a figure of clinical fortunes. PCR can be used to observe Pneumocystis jiroveciinfection in HIV patients. The specificity of PCR is nevertheless limited in such instances, as this being is a omnipresent commensal and canbe detected even in the absence of pneumonia.Aspergillus spp. can be detected by PCR particularly in neutropenic patients. It is hard to civilization Aspergillus early in the disease and so molecular testing can be of great benefit.

Parasitological diagnosing is aided by molecular methods, sincemost parasites are non cultured in everyday research lab scenes, and hence, their diagnosing relies largely on the comparatively lesssensitive methods, such as microscopy and serology. Toxoplasma gondiican bedetected by PCR in amniocentesis fluid ( to corroborate foetalinfection ) and in CSF ( to name toxoplasma phrenitis ) .In instances of malaria, PCR can observe DNA of the malarial parasite diagnose malaria even after chemoprophylaxis and/ or intervention has been given. It can besides name assorted infections which are hard to name on microscopy.

& lt ; H1 & gt ; LIMITATIONS OF MOLECULAR METHODS

Despite so many advantages of molecular nosologies, conventional diagnostic methods can non be replaced, since many common trials performed in theclinical microbiology research lab are rapid and cheap. Culture methods have advanced well and the modern machine-controlled civilization systems allow rapid designation of bacteriums and susceptibleness testing. Bacterial civilization methods can observe a big figure of bacteriums and speciation can be done quickly. In contrast, PCR technique can observe merely thoseorganisms whose Deoxyribonucleic acid is complementary to the primers used.Therefore, covering a similar comprehensiveness of possible organismsas civilization would necessitate debut of cheap and simplemicroarray engineerings, that are non yet available.

& lt ; H2 & gt ; False Positive and False Negative Results

Contamination remains the bogeyman of molecular proving methods. The jobs of taint and research lab direction have been dealt with elsewhere in this book ( Chapter 13 – Establishing a Molecular Biology Laboratory ) , suffice to state that scrupulous attending to avoid taint demands to be given. To avoid false positive consequences due tolaboratory taint, comparatively big and separate research lab areasare required for reagent readying, specimen readying and merchandise sensing togetherwith high degree of staff preparation and accomplishment. Ampliconlaboratory taint can be reduced by ultravioletlight irradiation of reagents and chemical inactivation ofsurface taint with Na hypochlorite. Intersample taint can be reduced by the usage of disposable equipments, cotton filtertips, and disposable personal protective equipments such as caps, gowns and baseball mitts. Appropriate negative controls should be included in every PCR tally to observe any sort of taint.

Poor primer design can besides take to mistakenly positiveresults. Primers may be ill designed such that incidentalamplification of micro-organisms other than those soughtoccurs. Besides, primers are designed based on the knownsequences available through international databases, but planing a primer wrongly may ensue in non-specific elaboration. For farther elucidation on this topic, readers are requested to mention to primer design in the rudimentss of PCR ( Chapter 7 – The Polymerase Chain Reaction ) .

False negative consequences may besides be a job. It possibly hard to pull out DNA from beings like Mycobacterial spp. Substances in someclinical specimens such as phlegm and fecal matters can degrade DNA and RNA, while other specimens may incorporate substancessuch as polyoses, heme and curative drugs thatinhibit the PCR enzymes. It is, hence, of import to includeinhibitor cheques for each specimen to guarantee that the negative PCRreaction is non really an inhibited reaction. Applying internal control can look into for the presence of inhibitors every bit good as guarantee successful DNA extraction.

& lt ; H2 & gt ; Lack of Uniformity in Molecular Testing

Molecular diagnosing is besides complicated by the huge array ofin-house PCR trials used in different research labs. Many trials are non available merely because it is non commercially feasible to fabricate their kits. Research workers so develop their ain in-house trials which use different primers magnifying different cistrons and/ or different sequences within cistrons. The PCR format possibly different ( standard PCR, multiplex or nested PCR ) . These variables lead to a considerable deficiency of uniformity in proving.

& lt ; H2 & gt ; Differentiation between Infection and Disease

Since the presence of nucleic acerb sequence does non needfully meanthe presence of feasible beings, a job with interpretationof PCR consequences can emerge.For some infections, such as invasive meningococcal disease, the presence of meningococcal Deoxyribonucleic acid at a unfertile site has avery high positive prognostic value. However, the detectionof P. jiroveciin immunosuppressed patients may hold merely a 50 % positivepredictive value for PCP, since P. jirovecimay colonise every bit good as cause disease. In some instances, quantitative PCR possibly helpful because higher being tonss are more specific for infection. Besides, RNA can be used as a templet. Since RNA degrades easy, the presence of RNA would bespeak pathogen viability and reproduction.

& lt ; H1 & gt ; MOLECULAR BIOLOGY AND BIOTERRORISM

The earlier belief that biological terrorism is non a serious menace has been proved incorrectly. It is apparent from so many recent onslaughts since mid 1980 ‘s, that biological terrorism is non a myth but a existent job. Biotechnology can be used rather easy by committed terrorist groups to bring forth micro-organisms that are capable of big scale morbidity and mortality.

& lt ; H2 & gt ; Organism attributes

The five basic properties that characterize a perfect military biological warfare ( BW ) agent are as follows:

High virulency coupled with high host specificity ;

High grade of controllability ; the being should assail merely specific groups or populations of people and should non assail the people originating the bioterrorist onslaught.

High grade of opposition to adverse environmental forces ;

Lack of timely countermeasures to the attacked population ;

Ability to camouflage the BW agent with comparative easiness.

Some of these properties might non be so of import for BW agents that are applied for biological terrorism. For illustration, a terrorist group might non be concerned, whether the agents it uses can be controlled after release. However, these standards serve as utile considerations sing the type of micro-organisms which can perchance be used by the bioterrorists. In add-on, to develop perfect bioterrorist agents, modern biotechnology techniques may be applied to heighten any or all of eight features or traits of microorganisms i.e.- robustness, opposition, infectiousness, pathogenicity, specificity, sensing turning away, aging, and the feasible but non-culturable province.

& lt ; H2 & gt ; Use of molecular biological science in heightening bioterrorist arms

In 2001, Australian scientists manipulated the mousepox virus to stamp down the wild mouse population. The result was a modified virus that was far deadlier than the original 1. This modified strain was besides capable of killing mice of course immune to mousepox or those immunized against the mousepox virus. Since the variola and the mousepox viruses are correspondent to each other, it is wholly possible that the same experiment can be carried out in the variola virus. The smallpox virus is non readily available to terrorist organisations, nevertheless it is possible for them to modify other viruses to overthrow the human immune system. Again, it is non impossible to synthesise a new being. In 2002, scientists in USA were successful in synthesising infantile paralysis virus from abrasion utilizing chemicals available in the unfastened market.

It is non merely viruses that are prone to familial use. Bacteria and mycobacteriums are besides prone to familial alteration. Mycobacterias have been manipulated and a hypervirulent mutation strain of TB has been produced. Similar experiments have been carried out with Protozoas such as Leishmania major. It is, hence, possible to make deadly micro-organisms by easy available methods. It would be incorrect to presume that the entree to these methods would be limited to research research labs. Most of the techniques used are easy available and can be reproduced in an mean research lab.

& lt ; H2 & gt ; The molecular footing of sensing

It is easy for the bioterrorist to pull strings the microscopic universe for his benefits. However, it is every bit easy for the biotechnologist to observe the being and institute appropriate actions. Ideally, sensing platforms should be capable of quickly observing and corroborating biothreat agents, including modified or antecedently uncharacterized agents, straight from complex matrix samples, with no false consequences. Furthermore, the instrument should be portable, user-friendly, and capable of proving for multiple agents at the same time. Such an instrument is as yet unavailable.

The PCR can be used for the sensing of bioterrorist arms. However, it has built-in jobs which have been alluded to earlier. It besides requires a clean sample and is unable to observe protein toxins. It can besides give a false positive consequence because of its built-in sensitiveness. Q-PCR can be utilized to observe several marks at the same time utilizing different reporter dyes for different targets.However, accurate word picture or designation of bacteriums by Q-PCR is limited by the same prejudice and fluctuations that are built-in in many nucleic acid techniques.

Immunoassaies have progressively been used and developed for the sensing of infective diseases. Immunological sensing has been successfully employed for sensing of biothreat agents such as bacterial cells, spores, viruses, and toxins based on the construct that any compound capable of triping an immune response can be targeted as an antigen. Immunoassays by and large test for merely one analyte per check. The specificity of immunochemical assaies is limited by the antibody quality, and sensitiveness. The sensitiveness is typically lower than with PCR and other DNA-based checks. As betterments are made in antibody quality ( e.g. , production of antibodies from recombinant libraries ) and in assay parametric quantities, it may be possible to increase immunoassay sensitiveness and specificity. It is besides proposed to utilize antibody fragments for sensing of antigen fragments.

Aptamers are little DNA or RNA ligands that acknowledge a mark by form and non by sequence. RNA aptamers include the ribozymes that can be engineered to bring forth a signal after mark gaining control. Deoxyribonucleic acid aptamers bind to a mark after exposure to UV visible radiation. Aptamers can be used to observe the presence of full beings such as Bacillus anthracis or toxins like ricin.

Finally, the microarrays can be used to observing a bioterrorist attack.Microarrays can observe several beings at one spell, hence it potentially remains a tool for future in observing biological warfare agents.

To reason, it is possible for committed terrorists to pull strings micro-organisms utilizing available molecular techniques to do them more deadly without much trouble. However, watchfulness and entree to modern diagnostic methods can easy hold a bioterrorist onslaught. In decision, it can be stated that the cardinal constituents to the battle against a bioterrorist onslaught are readiness and consciousness.

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