MODX LEC TRANSES 7Nr Mol Inherited Dis PDF

Molecular Detection of Microorganisms and Inherited Diseases LEC 13 2021 – 2022...

Molecular Detection of Microorganisms and Inherited Diseases LEC 13 2021 – 2022 2nd Semester MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Prof. Justin Kim C. Vergara, RMT, MPH TRANS 13 MODX311 LEC Date: June 3, 2022 Outline Mycobacterium is a fastidious organism and is difficult to At the end of the session, the student must be able to learn: grow and time-consuming 2. Hazardous to handle in the clinical laboratory (Histoplasma and I. Molecular Detection of microorganism Coccidioides) A. Application of Molecular Based Test\ B. Selection of Sequence Targets for Detection of Microorganisms Fungal infections, such as those not commonly isolated C. Selection of Target Sequences for a Nucleic Acid Test D. Bacteria fungal organisms are difficult and are part of bioterrorism E. Virus agents because their infectivity is high and is handled in a F. Parasite high-level facility. Molecular techniques are preferred for G. Mycology these organisms H. Resistance to Antimicrobial Agents We cannot culture Histoplasma and Coccidioides and other II. Molecular detection of inhereted diseases dimorphic. Most of the time we are just culturing then in A. Chromosomal Abnormalities reference laboratory. It requires higher lever of bio security III. Molecular Basis of single- gene disorder 3. When reliable laboratory tests were lacking (HCV and HIV)  Details Hepatitis C and HIV are confirmed using molecular  About the details above techniques. Side notes We used method for this is western blot technique and can also used ELISA which a serological test to screen them I. MOLECULAR DETECTION OF MICROORGANISMS but cannot confirm.  Molecular-based methods that have been used to detect and identify bacteria include nucleic acid-based hybridization and 4. Organisms that are received in clinical laboratories in high amplification procedures volumes (Streptococcus, Neisseria and Chlamydia)  Target detection is accomplished by a variety of methods: Genital specimens (Neisseria and Chlamydia), and Throat 1. Agarose Gel Electrophoresis specimens (Streptococcus) 2. Amplification Methods Since you have multiple samples you cannot process that (PCR, TMA, loop-mediated isothermal amplifi alone. cation [LAMP]), It test by batch Commonly used especially PCR 5. Genes that confer resistance to antimicrobial agents (mecA, Real-time PCR, or quantitative PCR (qPCR), is vanA/B/C, tonB, KatG, inhA) used frequently for the detection of infectious 6. Characterization of DNA, RNA, and protein was developed to agents because it provides a sensitive, safe find and identify new organisms and to further characterize or closed-tube assay with quantitative information classify known organisms (Influenza virus) not available from conventional PCR or other Molecular techniques are used to study SARS-CoV 2, “end-point” amplifi cation methods. differentiating it from SARS-CoV 1 The quantitative capability of qPCR allows distinction of subclinical levels of infection B. Selection of Sequence Targets for Detection of (qualitatively positive by conventional PCR) Microorganisms from higher levels with pathological  The primary nucleotide sequence of many clinically important consequences. Furthermore, qPCR programs microorganisms is available from the National Center for can be designed to provide closed-tube Biological Information (NCBI) or from published literature sequence or typing analysis by adding a melt-  The specificity of molecular methods targeting these sequences curve temperature program following the depends on the primers or probes that must hybridize amplifi cation of the target. Like conventional specifically to the chosen point in the genome of the PCR, qPCR is performed on nucleic acid microorganism extracted directly from clinical specimens, Many microorganisms shared the same sequences including viral, bacterial, and fungal pathogens especially when in the same genome or same species but 3. Sequencing different variant Identify new sequent variant of a particular This sequence would not be used for the detection of organism specificity strain. Ex.Ecoli. You have to choose a sequence 4. Immunoassays that specific for specific strain as well. You you will not Rapid testing for microorganisms choose the specific sequence the tendency it will cross They are frequently used as well especially in react over the other strain serology in detection of certain microorganism  Many microorganisms share the same sequences in 5. Western Blots evolutionarily conserved genes e.g HIV or other Retroviruses Hybridization technique Used for detection of protein particles of certain C. Selection of Target Sequences for a Nucleic Acid Test organism and the same time we can also used other hybridization technique such as soutern blot and northern blot for detection of nucleic acid 6. Mass Spectrometry Determination of amino acid sequence If you want to sequence particular protein coming from microorganisms or cells within a disease or tissues A. Application of Molecular Based Test 1. Microorganisms that are difficult and/ or time-consuming to isolate (Mycobacterium tuberculosis) Page 1 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH The diagnosis of tuberculosis (TB) may take prolonged periods, FIGURE 11.3 Selection of target sequences for a nucleic acid test. The during which time infections can spread. genomes of three organisms—the test target, a variant or different type If you will used culture it will take a long time that will grow for 2 of the test target, and another nontarget organism—are depicted. weeks or 1month Sequence region A is not specific to the target organism and is, Nowadays we used genexpert therefore, not an acceptable area for probe or primer binding to detect the target. Sequences B and C are specifi c to the target. Sequence B D. Mycoplasma pneumoniae is variable and can be used to detect and type the target, although some variants may escape detection. Sequence C will detect variants  Amplification techniques and other characterizing methods such of the target organism but cannot be used for determining the type. as MALDI-TOF MS Three genes and there is a variant and other organism  Specimen of choice: BAL Sequence region s A, B and C MALDI libraries of 50 to over 300 Mycobacterium peptide spectra Sequence A is not specific to the organism, choosing B and C is are offered by at least one manufacturer. Due to the structure of good because it is specific to target but B is highly specific target their cell walls, mycobacteria require special preparation for to organism and can be used to detect and type the target, mass spectrometry testing. The bacteria are lysed by bead although some variants will not be detected. C will detect variants beating or in boiling water (also a safety measure), extracted with and target organism, but cannot be used for determining the type ethanol,dried, and resuspended in formic acid and acetonitrile for of variant. analysis. Several studies have shown favorable comparison of If you want your test to be highly specific, use sequence B, but if MALDI-TOF with conventional methods. you also want to check the variant, use sequence C Differentiation between type or target E. Chlamydophila pneumoniae Use of target specific primers and probes that will serve as a confirmatory test that is directed to the sequence desired. Intracellular pathogen  multiplex nucleic acid amplification tests D. Bacteria Specimen of choice: BAL, bronchial washings, throat swabs 1. Respiratory Tract Pathogens F. Streptococcus pneumoniae Ubiquitous in the environment  PCR, Sequencing and MALDI-TOF  Frequent testing targets include Bordetella, Legionella, Clinical significance of positive PCR assay is questionable, Mycobacteria, Chlamydia, and Streptococcus species because significant portion of population (children) is Laboratory identification and detection by non-molecular colonized by organism and PCR cannot discern between methods often lack sensitivity and time-consuming (biochemical colonization and infection; results are questionable tests) Colonization -doesn’t cause any harm on the host.  Molecular based assays that can detect and identify bacterial The bacteria multiply but it doesn’t have sign and pathogens directly in respiratory specimens have been symptoms. developed Infection - it can cause harm and it have sign ans  Individual IVD and analyte-specific reagent (ASR) systems have symptoms been marketed for individual testing from a variety of specimen For sequencing, 16S RNA is sequences but cannot sources differentiate alpha hemolytic Streptococcus species because they share more than 99% sequence homologies A. Bordetella pertussis  Primer and probe ASR for B. pertussis and Bordetella parapertussis detection by qPCR targeting IS481 and IS1001 A pathogen of the upper respiratory tract that is the causative agent of whooping cough. The organism is endemic worldwide and is transmitted via direct contact with infected respiratory secretions. B. Legionella pneumophila  PCR test targeted the macrophage infectivity potentiator (mip) gene and 16S and 5S rRNA genes The cause of Legionnaires’ disease, an infection of the lower respiratory tract that was first described in men attending an American Legion convention in Philadelphia in 1976. Since their first identification, Legionella species have been found in water, both in the environment as well as in air conditioners and hot water tanks in various types of buildings. Legionella species infections range from asymptomatic to fatal and are the third 2. Urogenital Tract Pathogens most common cause of community-acquired pneumonias. A. Neisseria gonorrhoeae and Chlamydia trachomatis C. Mycobacterium tuberculosis  nonamplification-based nucleic acid hybridization method that  PCR tests targeting the species-specific sequences, such as detected the rRNA and nucleic acid amplification assays such as IS6110 and 16S rRNA, allow detection of M. tuberculosis from target amplification assays, strand displacement amplification, fresh, frozen, or fixed tissue and transcription-mediated amplification  qPCR assays have also been developed for M. tuberculosis Sexually transmitted diseases detection with primers and probes targeting rRNA internal Considered as a good target for a development of molecular- transcribed spacer (ITS) elements in M. tuberculosis based method because the traditional laboratory method of M. tuberculosis is an important cause of respiratory tract detection and identification for these organism either lack infections causing significant levels of morbidity and mortality. sensitivity or are time consuming Page 2 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH It will detect through PCR Urine Among the first organisms to be targeted for determining clinical Chlamydia Urethral Culture MOMP specimens by molecular methods trachomatis Cervical Enzyme 16s RNA Cultures for N. gonorrhoeae and C. trachomatis have been Thin preparation immunoassay considered the gold standard, but nucleic acid amplifcation vials DFA assays have the advantages of being rapid, and testing can Conjunctiva be batched and automated, resulting in further savings for the laboratory. E. Viruses B. Treponema pallidum subspecies pallidum  The classical method for detection and identification of viruses in body fluids is tissue or cell culture as well as antigen and  Amplification of the T. pallidum DNA polymerase I gene (polA) is antibody detection highly accurate when tested on genital, anogenital, or oral ulcers Changes in the cell due to viral infection is called Serological tests are RPR (rapid plasma reagin) and VDRL cytopathic effect which is observed microscopically (venereal disease research laboratory) to detect. Evidence for virus infection has been detected by testing it accurate and precise when you used molecular-based method. for antibodies against the virus by measuring the presence T. pallidum cannot be grown in vitro. Laboratories have adopted of absence of viral antigens via serological techniques hemagglutination assays and enzyme immunoassays (EIAs) to → Serological tests can cross react with the other organisms screen patients for syphilis and fl uorescent antibody that is why molecular methods are used and is highly specific C. Mycoplasma hominis, Mycoplasma genitalium, and  Nucleic acid amplification assays have become indispensable in Ureaplasma urealyticum the clinical virology laboratory  Molecular methods are used to diagnose or monitor viral  PCR assays were developed targeting the adhesion gene (MgPa) infections or the rDNA gene of M. genitalium and laboratory-developed In the clinical virology laboratory PCR with hybridization is used for clinical detection of M.  Target amplification assays such as PCR, reverse transcriptase genitalium as well as M. hominis and U. urealyticum detection PCR (RT-PCR), quantitative PCR (qPCR), and transcription- Causative agents of non-gonococcal urethritis mediated amplification (TMA) as well as signal amplification assays such as branched DNA (bDNA) amplification and hybrid capture ORGANISM SPECIMEN TRADITIONAL GENE TARGET  MALDI analysis of PCR-amplified viral nucleic acid has been SOURCES DIAGNOSTIC successfully applied to clinical detection of HSV, HCV, HPV, METHOD enteroviruses, and respiratory viruses Geniltal ulcers Serologiccal TpN44.5a Multiplex PCR + MALDI (PCR Mass Assay) is shown to Blood (indirect and TpN19 identify multiple viruses/viral subtypes in a single test and Brain tissue indirect) TpN39 to differentiate them, MALDI is used Treponema Cerebrospinal fluid p01A pallidum Amniotic fluid Direct antigen TpN47 Placenta detection (dark 16S rRNA Umbilical cord field, direct p01A Fetal tissue fluorescent Serum antibody [DFA]) MgPa (adhesioon Mycoplasma Urine Culture gene genitalium Urethral Vaginal rDNA gene Cervical Mycoplasma Genital tract Culture 16S rRNA hominis Amniotic fluid Ureaplasma Genital tract Culture 16S rRNA genitalium Amniotic fluid Urease gene BOOK: Buckingham & Flaws – Molecular Diagnostics Gram stain 1.1kb target Nucleic acid amplifi cation assays have become indispensable in Haemophilus Culture groEL gene the clinical virology laboratory. Molecular methods are well suited ducreyi Serological to targeting the various configurations of nucleic acids found in human pathogenic viruses ( Table 11.4 ). Intergenic spacer Target amplifi cation assays such as PCR, reverse transcriptase between 16S and PCR (RT-PCR), quantitative PCR (qPCR), and transcription- 23S rDNA p27 mediated amplifi cation (TMA) as well as signal amplifi cation 16S rDNA gene assays such as branched DNA (bDNA) amplifi cation and hybrid capture are used in the clinical virology laboratory to diagnose or monitor viral infections. Urine Neisseria Urethral Culture amp III gene ssRNA check the if is positive sense or negative sense viruses gonorrhoeae Cervical opa gene Positive sense- positive RNA / directly translated RNA Thin preparation Cytosine DNA Negative sense - complementary to the translated strand vials methyltransferase or template strand cPPB gene In DNA have two stand which is sense and nonsense Site-specific Sense- non template strand. Can directly translate it recombinase gene In the picture the symbol * is positive sense. And for those cross symbol (+) are negative sense Page 3 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH C. CMV 1. Human immunodeficiency virus (HIV)  DNA is extracted and amplified by PCR using primers targeting the CMV polymerase (UL54) or glycoprotein B (gB) gene in  HIV infection is identified as antibodies specific for HIV in an EIA regions that do not share sequence homology with other and by confirming the specificity of detected antibodies for HIV herpes viruses products in a western blot or a qualitative RNA probe assay Cause cythopatic effect Rapidly mutates and recombines that is why it has Most people have been infected with CMV without obvious subtypes illness. Like other herpes viruses, CMV can go dormant Four HIV subtypes are subtype M (Major), O (Outlier), N and reactivate. (Non-M/O) and P (newly discovered) If symptoms occur, they are mild, except in  Quantitative nucleic acid amplification assays are performed after immunocompromised individuals. The virus is shed in an HIV diagnosis to determine how actively the virus is blood, urine, saliva, and other body fluids but dies quickly replicating (viral load), when to start antiretroviral therapy, and outside of the host. when to monitor the efficacy of treatment  HIV in patient samples can also be detected by PCR of D. VZV integrated DNA, nucleic acid sequence based amplification (NASBA), and bDNA  Qualitative PCR methods to detect VZV include PCR with Traditional test for HIV is the rapid test kit or enzyme immuno hybridization or direct gel electrophoresis. Quantitative PCR kits assay. Also ELISA is screening test if you wanted to know include internal controls to detect amplification inhibition whether the individual has a lower viral load you have to check No need to test using molecular based-method. Unless you CD4 count. need to differentiate from other skin lesion causing The confirmatory test is western blot technique. diseases. you have to detect the presence of any two bands either VZV is the causative agent of chickenpox and shingles (also p24, gp120 or 160 and the g41 called herpes zoster). The virus has a large genome containing over 70 open reading frames (ORFs). TEST ADVANTAGES DISADVANTAGES bDNA High throughput No internal control 3. Hepatitis C virus Broad dynamic range False-positive Applicable for group Results reported The initial approaches to HCV analysis are similar to those for M subtypes A-G HIV. Serology is used to detect the presence of antibodies against HCV. Amplicator RT-PCR Internal control Limited dynamic  Nucleic Acid Amplification assays for the qualitative detection Good specificity range and quantitation of HCV are RT- PCR, transcription-mediated amplification, and branched DNA NASBA Broad dynamic range Does not detect all  The qualitative HCV RNA assays are performed on patients with Performed on many non-B subtypes positive HCV antibody results to confirm active infection or on Specimen types and immunocompromised patients volumes  The quantitative HCV RNA assays are used to determine the viral load and to monitor viral replication in response to anti- viral Table 11.6 compares the advantages and disadvantages of each of therapy these assays for determining HIV viral load. Viral load should be  Laboratory methods available for HCV genotyping are PCR with determined before therapy is started, 2 to 8 weeks after therapy restriction fragment length polymorphism (RFLP) analysis and initiation to see the initial response, and then every 3 to 4 months to reverse hybridization, direct DNA sequencing and PCR with assess therapeutic effectiveness. MCA The viral load and the HCV genotype are used to determine the 2. Herpes viruses therapeutic protocol, both type of drug(s) as well as duration.  Herpes Simplex Virus (HSV), CMV, EBV, and Varicella-zoster 4. Human Papilloma Virus Virus (VZV)  Two approaches to molecular detection of HPV are hybridization A. HSV methods and amplification methods (target amplification, such as PCR, and signal amplification, such as hybrid capture)  detected by viral culture, antigen, and anti- body tests before the HPV is a double-stranded DNA virus recognized as oncogenic in application of PCR several human cancers. Able to reactivate and remain silent after many years of Oncogenic - it can induce cancer infections It is better to be vaccinated with HPV to prevent cervical cancer  PCR tests also offer the advantage of distinguishing HSV-1 from because it can trigger the formation of cervical cancer. HSV-2 directly without culturing Even males can have HPV vaccine because males also can  Methods include standard PCR and quantitative PCR targeting have cancer in genitalia because of HPV. type-specific HSV genes CERVARIX for female GARDASIL can vaccine in males B. EBV There are over 200 HPV types based on sequences of the viral genome compared with known HPV genomes. Five evolutionary  Latent infection is characterized by the presence of EBV-en- HPV genotype groups ( α , β , γ , μ , and ν ) have been defined. coded RNA (EBER) The largest, the α group, contains 64 types that mainly infect  Laboratory testing for EBV infection has been performed by mucosal epithelia in the anogenital tract and include the high risk immunohistochemistry, testing serum for antibodies to EBV- (HR) types (HPVs 16, 18,31, 33, 35, 39, 45, 51, 56, 58, 59, 68, associated antigens, Southern blot analysis of EBV DNA and 73, 82) that have been classifi ed as oncogenic and are found to Amplification methods cause anogenital cancers. Cause cythopatic effect The presence of high-risk HPV DNA in conjunction with an equivocal or ambiguous cytology result (atypical squamous cells of unknown significance [ASC-US]) indicates an increased risk for having an underlying cervical neoplasm. Page 4 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH Persistent infection with high-risk HPV may be the main risk factor for the development of high-grade cervical neoplasia and cancer. Women with normal cervical cytology who are negative for the high-risk HPV types are at low risk for having or developing cervical precancerous lesions. 5. Respiratory Syncytial Virus Detected using molecular-based method to differentiate from the other respiratory tract in detection  An ASR for RSV A + B RNA uses NASBA technology  A bead array technology can simultaneously detect RSV A and B, influenza A, nonspecific influenza A, H1, H3, influenza B, parainfluenza 1, parainfluenza 2, parainfluenza 3, In corona virus we used RT-PCR because most of the viruses metapneumovirus, rhinovirus, and adenovirus are single stranded RNA It hase same sign and symtoms. Even corona virus. If you wanted to detect the viral load of the microorganism or the Influenza - H1N1, H1N5, H1N3 SARSCov2 you wil used rRt-PCR or the qRt-PCR Seasonal and H1N1 flu viruses develop resistance to antiviral agents through sequence alterations.  Mutations can be detected by direct sequencing and digital PCR methods have also been developed to detect the frequently occurring alteration H275Y in the neuraminidase gene Hemagglutinin and neuraminidase - viral factor Neuraminidase mostly changed. It causes different strain oof the virus 6. BK and JC viruses are human polyomaviruses, the primate counterpart of which is SV40. BK, JC, and SV40 share sequence and antigenic homology. They are double-stranded DNA viruses with 5,000-bp genomes encoding 5 transcripts. Most polyomavirus infections are subclinical in adults. Infected children develop respiratory symptoms, and some have cystitis.  Several reagent sets are available for detection of BK or JC viruses in the clinical laboratory, including quantitative PCR tests F. Mycology Fungi are among the most ubiquitous microorganisms causing clinical infection. Traditional methods of identification by phenotype have become more difficult with the expanding diversity of organisms.  Molecular methods, particularly sequencing and PCR, have allowed for the detection and typing of fungi with greater sensitivity, specificity, and speed  Gene probes have been developed to confirm the identity of the organism growing in the culture For the HIV the amplified target it depends on what subtype are Fungi are important causes of human disease, especially in detecting immunocompromised patients. In addition, laboratory-acquired infections from fungi are a major risk for laboratory personnel. 1. Broad-range PCR and subsequent analysis are also used for clinical analysis of fungi :  Primers anneal to DNA sequences that are common to most of the clinically relevant fungi (Candida, Aspergillus, Rhizopus and other Zygomycetes, and Histoplasma and other dimorphic fungi)  Once the sequences are amplified, hybridization to species- specific probes or sequencing is used to identify the fungus to the genus or genus and species level PCR is much better to used because its faster and less hazardous Page 5 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH 2. Real-time PCR Common in health-care institutions are resistant Staphylococcus, Pseudomonas (very common) and  methods are used for direct identification of Aspergillus and Klebsiella Pneumocystis carinii MRSA can be isolated in tourniquets Fungal specimen causing in immunocompromised patient  Multidrug-resistant organisms may have one or more of these characteristics 3. Direct probe hybridization  A single- nucleotide variant in a drug target or transport protein can result in resistance Examples of this resistance mechanism are seen in S. aureus  assays detect Blastomycetes, Coccidioides immitis, and and N. gonorrhoeae that produce β -lactamase, an enzyme that Histoplasma capsulatum cleaves the β -lactam ring of the β -lactam antimicrobials such as the penicillins. 4. PFGE  is used in laboratory-developed methods for molecular typing of yeasts It has larger genome 5. Molds  can be typed by PCR and sequencing of 28S rRNA 6. MALDI-TOF Vancomycin resistant Staphylococcus aureus Has Tn1546 gene that resists Vancomycin  application to detection of fungi has been limited by their Instead of alanine, lactic acid is present in the cell wall biological complexity and different fungal growth phases organism is hard to disrupt Furthermore, their thick cell walls require chemical and physical Vancomycin targets cell walls of the organism and is used disruption methods. Cell walls are extracted with trifl uoroacetic if the organism is methicillin and oxacillin resistant acid, or formic acid and acetonitrile, possibly followed by physical disruption with beads. Mode of Examples Mode of Examples Examples of 7. Mass spectrometry Action Action Agents Affected  methods for identification of Saccharomyces cerevisiae and offer a reliable, rapid approach to testing for fungal sepsis Disrupts Beta-lactam Destruction/ B- B-lactams cell wall (penicillin and modification lactamases aminoglycosides G. Parasites synthesis cephalosporins) of agent (N.  Molecular-based testing for parasites has been limited mainly or integrity Glycopeptides gonorrhea because parasites are not a major cause of disease in developed and S. aureus) countries  Parasites found in complex matrices such as stool samples present a further difficulty due to inhibition of PCR and other Disrupts Polymyxins Elimination Multi-drug B-lactams enzymatic assays cell (Polymyxin B) of Agent efflux fluoroquinolones membrane Bacitracin systems macrolides 1. PCR assays structure chloramphenicol or function trimethoprim  have been developed for the detection of Trypanosomes, Plasmodia, Toxoplasma, Entamoebae, and Cryptosporidium in Inhibits Aminoglycoside Altered cell Thick cell hosts and water sources protein (gentamycin) wall walls that Vancomycin synthesis Tetracyclines structure exclude B-lactams 2. Real-time PCR methods Macrolides(eryt agent hromycin) Altered Lincosamides agent  are used for identification of Babesia, Trichomonas microti in (clindamycin) binding sites blood and from ticks, Encephalitozoon species microsporidia, and Trichomonas vaginalis Inhibits Quinolones Alternate Altered Sulfonamides 3. Multiplex PCR methods nucleic (ciprofloxacin) metabolic enzymes Trimethoprim acid Metronidazole pathways Integrity  have been designed to simultaneously detect multiple parasites  e.g a. Multiplex Real-time PCR assays for detection of Entamoeba histolytica, Giardia lamblia, and BOOK: Buckingham & Flaws – Molecular Diagnostics Cryptosporidium parvum simultaneously  There are several ways in which microorganisms develop  b. E. histolytica, Giardia intestinalis, and Cryptosporidium resistance ( Table 11.9 ). First, bacteria can produce enzymes spp. simultaneously in stool that inactivate the agent. Examples of this resistance mechanism The multiplex PCR methods also include an internal control to are seen in S. aureus and N. gonorrhoeae that produce β - determine the efficiency of the PCR and detect inhibition in the lactamase, an enzyme that cleaves the β -lactam ring of the β - sample, which is likely in stool samples. lactam antimicrobials such as the penicillins. Cleavage of the β - lactam ring destroys the activity of penicillin, rendering the H. Resistance to Antimicrobial Agents organism resistant to its antimicrobial action.  Resistance can arise from alerted target binding, active extrusion,  Second, organisms produce altered targets for the antimicrobial or inaccessibility of the drug or microbial enzymes that inactivate agent. Mutations in the gene encoding a penicillin-binding the drug protein—for example, a change in the structure of the protein— render penicillin unable to bind to its target.  Finally, bacteria exhibit changes in the transport of the Page 6 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH antimicrobial agent either into or out of the cell. An example of this mechanism is seen in gram-negative bacteria that change A. Chromosomal Abnormalities their outer membrane proteins (porins) in order to decrease the infl ux of the antimicrobial agent. If the agent cannot get into the 1. Genome mutations (abnormalities in chromosome number) cell and bind to its target, then it is not effective in inhibiting or can be detected by Karyotyping, Ploidy Analysis by Flow killing the bacterium. Cytometry and Fluorescent In Situ Hybridization (FISH)  e.g. Polyploidy, Aneuploidy, Autosomal trisomy/monosomy, Organism Antimicrobial Gene(s) Conferring Mosaicism Agent Resistance BOOK: Buckingham & Flaws – Molecular Diagnostics Staphylococcus aureus Oxacillin mecA  Polyploidy (more than two of any autosome) in animals usually Streptococcus Penicillin pbp1a and pbp1b results in infertility and abnormal appearance. pneumoniae  Aneuploidy (gain or loss of any autosome) occurs with 0.5% Gram-negatives Beta lactams tem, shv, oxa, ctx-m frequency in term pregnancies and 50% in spontaneous Enterococcus Vancomycin vanA, vanB, vanC, abortions. Aneuploidy is caused by erroneous separation of vanD, vanE, vanG chromosomes during egg or sperm production (chromosomal Mycobacterium Isoniazid katG, inhA non-disjunction). Autosomal trisomy/monosomy (three tuberculosis copies/one copy of a chromosome instead of two) results from Rifampin rpoB fertilization of gametes containing an extra chromosome or missing a chromosome ( n + 1 or n – 1 gametes, respectively). BOOK: Buckingham & Flaws – Molecular Diagnostics  Autosomal monosomy is generally, but not always,  These genetic changes are most commonly brought about by incompatible with life. Sex chromosome aneuploidy is more mutation and selection processes. If a mutation results in a frequently tolerated, although it is associated with phenotypic survival or growth advantage, cells with the mutation will abnormalities. eventually take the place of those without the mutation, which are  Mosaicism, two or more genetically distinct populations of cells less able to survive and procreate. This process is stimulated by from one zygote in an individual (in contrast to chimerism: two or antibiotic exposure, especially if the levels of antibiotics are less more genetically distinct cell populations from different zygotes in than optimal. For example, S. aureus developed resistance to an individual), results from mutation events affecting somatic or antibiotics that target its penicillin-binding protein (PBP1) by germ cells. replacing PBP1 with PBP2a encoded by the mecA gene. PBP2a found in methicillin-resistant S. aureus (MRSA) has a low binding 2. Chromosome mutations (abnormalities in chromosome affi nity for methicillin. structure) larger than 4 million base pairs (bp) can be seen by karyotyping II. MOLECULAR DETECTION OF INHERITED DISEASES  Smaller irregularities can be seen with the higher resolution Mutations are changes in DNA nucleotide sequences. These of FISH or Microarray Technology changes range from single base pair or point mutations of  e.g. Translocations, Inversions, Deletions, Duplications, various types to chromosomal. Not all mutations lead to disease. Marker Chromosomes, Derivative Chromosomes  Polymorphisms are proportionately represented genotypes in a given population (Sequence polymorphism, Benign polymorphisms & Balanced polymorphisms) Sequence polymorphism means it can be located within or outside the gene because there are changes with the sequences Benign polymorphism is useful for mapping disease genes for identifying testing Balance polymorphism gives offsetting phenotypes  Epigenetic alterations do not change the primary DNA sequence Epigenicity means heritable change that does not affect DNA sequence but result in a change of gene expression  Epigenetic changes consist of three different forms: A. DNA Methylation Cytosine is methylated mostly down regulates RNA transcription B. Genomic Imprinting Selectively inactivates chromosomal regions e.g., X chromosome inactivation C. Chromatin Remodelling Sequesters large regions of chromosomal DNA through protein binding and histone modifi cation. Histone modification or changes with the protein binding III. MOLECULAR BASIS OF SINGLE-GENE DISORDERS Control the availability of DNA/RNA transcription  Penetrance is the frequency of expression of disease phenotype  Mutations in Germ Cells result in inherited disease. in individuals with a gene lesion Diseases that are in the germ line, which is passed from Complete penetrance is the expression of the disease generations phenotype in every individual with the mutated gene. Ex. Thus, a baby can have a heritable disease, such as Complete penetrance is common in homozygous recessive hemophilia, that can be passed on to future generations phenotypes. or a congenital condition, such as spina bifi da, that cannot be passed to offspring.  Variable expressivity is a range of phenotypes in individuals with the same gene lesion  Mutations in Somatic Cells result in cancer and some congenital also refl ects the interaction of other gene products and the malformations Genetic components are referred to as congenital (in-born); environment on the disease phenotype.  Single-gene disorders affect structural proteins, cell surface not necessarily heritable however congenital diseases (ex. receptor proteins, growth regulators, and enzymes Spina bifida) are present at birth Page 7 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH F5 gene. This DNA mutation destroys an Mnl I restriction enzyme site. An amplicon including the site of the mutation, when cut with Mnl I, will yield three fragments in normal DNA ( + / + ) and two products in homozygous mutant DNA (m/m). A heterozygous specimen ( + /m) will yield a combination of the normal and mutant pattern. 4. Methyltetrahydrofolate Reductase  Mutations Deficiency of the 5,10- methylenetetrahydrofolate reductase (MTHFR) causing hyperhomocysteinemia. These variants are detectable by standard or multiplex PCR with RFLP using restriction enzymes HinfI and MboII or sequencing 5. Hemochromatosis  caused by dysfunction of the hemochromatosis type I HFE or HLA-H gene product  The most frequently observed mutation in hemochromatosis is C282Y/ Cys282Tyr are detected using detectable using PCR- A. SINGLE-GENE DISORDERS RFLP autosomal-recessive condition that causes overabsorption of iron 1. Lysosomal Storage Diseases from food. Iron accumulation subsequently causes pancreas, liver, and skin damage; heart disease; and diabetes.  Mutations can be detected by direct sequencing, usually after an initial biochemical screening test for loss of enzyme activity 6. Cystic Fibrosis 2. Factor V Leiden  caused by loss of function of the CF transmembrane An example is the hypercoagulation phenotype resulting in conductance regulator, the CFTR gene (7q31.2) mutations in the factor V gene.  RFLP, PCR-RFLP, heteroduplex analysis, temporal temperature-  Leiden mutation (1691 A→G, R506Q) in the coagulation factor V gradient gel electrophoresis, single-strand conformation gene F5 (1q23) causes a hypercoagulable phenotype polymorphism (SSCP), SSP-PCR, Cleavase, bead array  The R506Q amino acid substitution is caused by a G to A technology, and direct sequencing change in exon 10 of the F5 gene which destroys an MnlI Cystic fi brosis (CF) is a life-threatening autosomal-recessive restriction enzyme site disorder that causes severe lung damage and nutritional defi ciencies. With earlier detection by genetic analysis and improved 3. Prothrombin treatment strategies, people with CF can live more comfortably surviving beyond the fourth decade of life. CF affects the cells that produce mucus, sweat, saliva, and digestive juices. Prothrombin is the precursor to thrombin in the coagulation Respiratory failure is the most dangerous consequence of CF. cascade and is required for the conversion of fibrinogen to fi brin.  Mutation in the 3ʹ untranslated region of the gene that codes for 7. Cytochrome P-450 prothrombin or factor II, F2 (11p11-q12), results in an autosomal- dominant increased risk of thrombosis  Multiplex PCR-RFLP method to simultaneously test for both F2  performed by allele-specific PCR for particular polymorphisms. or F5 mutations Multiple P-450 genetic variants may be screened by microarray, bead array, or sequencing BOOK: Buckingham & Flaws – Molecular Diagnostics FIGURE 12.7 PCR-RFLP for the factor V Leiden mutation. The R506Q amino acid substitution is caused by a G to A change in exon 10 of the Page 8 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH B. Single-Gene Disorders with Nonclassical Patterns of BOOK: Buckingham & Flaws – Molecular Diagnostics Inheritance Fig. 12.19 Interpretation of mutation analysis has long been complicated, however, by the extent of heteroplasmy (mutated  Mitochondrial mutations, genomic imprinting, and gonadal mitochondria and normal mitochondria in the same cell) and the mosaicism do not follow Mendelian rules of inheritance nature of the mutation. 19 A range of phenotypes may be present, even in the same family. 1. Mutations in Mitochondrial Genes Some Disorders Caused by Nuclear Gene Mutations Mitochondrial mutations are inherited maternally Mitochondria are cellular organelles responsible for energy production. Mitochondria contain their own genome, a circular  circular DNA molecule 16,569bp in length  Mutations in mitochondrial genes affect energy production and are therefore manifested as diseases in the most energy- demanding organs, the muscles and the nervous system  Mitochondrial mutations are easily detected by a variety of molecular methods: a. Southern blot is used for detecting large deletions b. PCR-RFLP is used for Point mutations 2. Nucleotide-Repeat Expansion Disorders  Nucleotide repeats include short tandem repeats (STRs) with 1 to 10-bp repeating units During DNA replication and meiosis, these STRs can expand (or contract) in length. Triplet-repeat mutations (expansions of STR with 3-bp repeating units) occur in coding and noncoding sequences of genes.  The most well-known examples of triplet-repeat expansion diseases are Fragile X Syndrome and Huntington Disease A. Fragile X Syndrome - associated with a triplet-repeat BOOK: Buckingham & Flaws – Molecular Diagnostics (CGG) expansion in the noncoding region 5ʹ to the fragile X  FIGURE 12.17 Mutations and deletions throughout the mental retardation gene, FMR-1 mitochondrial genome are associated with muscular and  In addition to the fragile X chromosome observed by neurological disorders. karyotyping, the state of the repeat expansion is also analyzed using PCR and by Southern blot BOOK: Buckingham & Flaws – Molecular Diagnostics  FIGURE 12.20 Triplet-repeat (CGG) expansions in sequences 5 ′ to the FMR - 1 gene are observed in fragile X carriers (up to 200 repeats) and fully affected individuals (more than 200 repeats). Normally there are fewer than 60 repeats. Expansion results from amplifi cation of the triplet sequences Page 9 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER  [MODX311] Molecular Detection of Microorganisms and Inherited Diseases | Prof. Justin Kim C. Vergara, RMT, MPH during meiotic recombination events. The very large repeats (more than 200 repeats) are methylated on the C residues. This methylation turns off FMR - 1 transcription.  FIGURE 12.21 The fragile X chromosome is characterized by a threadlike process just at the telomere of the long arm (arrow). This is the site of disorganization of chromatin structure by the GC-rich repeat expansions. B. Huntington Disease  Associated with expansion within the huntingtin structural gene (4p16.3)  In this repeat expansion, the sequence CAG Expansion is 6-35 is normal. If its over hundred that’s expands from 9 to 37 repeats to 38 to 86 in the symptomatic or abnormal huntingtin gene  The triplet expansion inserts multiple glutamine 3. Genomic Imprinting residues in the 5ʹ end of the huntingtin protein à causes the protein to aggregate in plaques, especially in nervous tissue à neurological symptoms  Genomic imprinting is transcriptional silencing through histone or seen in this disease DNA modification  Detectable by standard PCR methods and capillary Imprinting occurs during egg and sperm production and is electrophoresis different in DNA brought in by the egg or the sperm upon It occurs on the 3ʹ fertilization. The difference is exhibited in genetic disorders in C. Idiopathic Congenital Central Hypoventilation which one or the other allele of a gene is lost. Syndrome  The difference is exhibited in genetic disorders in which one or Is a rare pediatric disorder characterized by the other allele of a gene is lost inadequate breathing while asleep. More-affected  e.g Uniparental Disomy/Deletion- demonstrates the nature children may also experience hypoventilation while of imprinting on chromosome awake. CCHS occurs in association with an intestinal  A deletion in the paternal chromosome 15, del(15)(q11q13) → disorder called Hirschsprung disease and symptoms Prader–Willi syndrome of diffuse autonomic nervous system  A deletion in the maternal chromosome 15 → Angelman dysregulation/dysfunction. syndrome  A number of gene mutations have been observed in A disorder with very different symptoms, including ataxia, CCHS, including a polyalanine expansion of the seizures, and inappropriate laughter. paired- like homeobox (PHOX2b) gene (4p12)13  Both syndromes can occur in four ways:  In CCHS, a triplet-repeat expansion occurs inside of 1. A deletion on the paternal or maternal chromosome 15 the PHOX2b gene, resulting in the insertion of 2. A mutation on the paternal or maternal chromosome 15 multiple alanine residues into the protein 3. A translocation with loss of the critical region from one No treatment they should monitor while sleeping chromosome because it has tendency that they are no longer 4. Maternal or paternal uniparental disomy in which both breathing. chromosomes 15 are inherited from the mother and none from the father or vice versa C. Cytogenetic Methods 1. Standard Karyotyping  Translocations and some deletions Arrange chromosome 1 to the last 2. High-resolution karyotyping  smaller deletions 3. FISH with labeled probes BOOK: Buckingham & Flaws – Molecular Diagnostics FIGURE 12.25 The huntingtin repeat expansion occurs within the  can detect over 99% of cases coding region of the huntingtin gene. The expansion is detected 4. PCR of RFLP or STR analysis directly by PCR using primers fl anking the expanded region (top). A 32 P-labeled primer is used, and the bands are detected by autoradiography of the polyacrylamide gel (bottom). In this example,  used to demonstrate uniparental disomy PCR products from the patient (P) fall within the normal range with 5. Methylation-specific PCR and Southern blot using the negative control (–). The positive control ( + ) displays the band methylation-specific restriction enzymes sizes expected in Huntington disease. FIGURE 12.26 The triplet-repeat expansion of PHOX2b includes triplets that code for alanine (top). The expansion is detected by  Diagnosis of disorders in imprinting (DNA methylation) PCR with a 32 P-labeled primer and polyacrylamide gel 6. FISH, array-based comparative genomic hybridization (aCGH), electrophoresis (center) or by standard PCR and agarose gel and next-generation sequencing (NGS) electrophoresis (bottom). Normal specimens yield a single PCR product. CCHS specimens yield another larger product in addition to the normal product. The standard PCR test can rapidly show the  used for the detection of genome-wide uniparental disomy (UPD) presence of the expansion, and the PAGE test allows determination of the exact number of alanine codons that are present in the expansion. (Photos courtesy of Dr. Elizabeth Berry-Kravis, Rush References: University Medical Center. Justin Kim C. Vergara, RMT, MPH. MODX3111 Lecture. Our Lady of Fatima University, Valenzuela City. Page 10 of 10 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER