Ch 11: Bacteria/Viruses

limit of detection (for viruses)

lowest viral concentration that can be detected over background or a negative control in the test assay

It's important to incorporate _________and ________ controls at multiple points in the workflow.

positive and negative

HCV is in the ___________ family

Flavi

What is the difference between genetic epidemiology and molecular epidemiology?

Genetic- based on population genetics. Not about environmental or genetic effects of chronic disease. Molecular- tracking evolution of pathogens and classifying new pathogen species.

What does HIV cause? What type of virus is it and what does that mean?

HIV causes AIDS which interferes with the body's ability to fight infections. It is an RNA, retrovirus. Retrovirus means it make a DNA copy of genomic RNA using encoded reverse transcriptase.

Name the two types of HIV. Which type is less pathogenic? Since it mutates and recombines rapidly, it forms different groups called "clades". Which group is responsible for 95% of HIV infections around the world?

HIV-1 HIV-2 - less pathogenic Group M causes 95% of infections

Insertion elements are segments of DNA that move independently throughout the genome and insert themselves in multiple locations. THE STRAINS ARE TYPED BASED ON what? Isolated from the same strain will have the same number and location of elements.

HOW MANY INSERTIONS ARE PRESENT AND WHERE THEY ARE LOCATED.

What is HAART?

High active antiretroviral therapy which consists of 2 RT inhibitors + protease/nonnucleoside RT inhibitor. Reduces viral loads (2 log 10) below detection limits for more than 3 years.

Why does the HCV virus mutate rapidly?

High error rate RNA-dependent RNA polymerase

What are Interspersed Repetitive Elements and why do they occur? How do they help you distinguish different species?

Identical or nearly identical DNA sequences that are scattered throughout the genome from transposition or retrotransposition events. The locations of these structures is specific to the species type. are related to species type.

How is RAPD/AP-PCR used to compare two organisms?

If two organisms have the same pattern, they are considered to be the same type but if the patterns differ, they are different types.

What are the main reasons for quality control when collecting/testing pathogen specimens?

-ensure integrity of specimens -ensure accuracy of patient results

Describe Bordetella pertusis and how it is tested

-extremely fastidious -PCR is lab-developed test based on specific regions of pertussis toxin gene promoter

List 2 disadvantages of using cell culture to diagnose viruses.

-the amount of time before viral growth is detectable resulting in a false negative report -the specimen must be collected in the acute phase of the disease (ex: within the first 5 days of illness)

Name the 8 steps of an HIV infection

1. Binding and fusion (HIV envelope glycoproteins gp120 and gp41 to host receptors CD4 and CCR5/CXCR4) 2. Reverse transcription (RT) - makes cDNA from viral DNA 3. Integration- cDNA integrates into the host DNA- can stay latent as a provirus or is replicated actively 4. Transcriptional regulation 5. Export 6. Post-translational regulation 7. Assembly 8. Maturation

Describe the process of identifying bacterial pathogens with molecular-based methods.

1. Extract total DNA 2. Perform: PFGE PCR Microarray Restriction Enzyme digest qPCR Sequencing

Describe the process of identifying bacterial pathogens with culture-based methods.

1. Grow and isolate known/unknown bacteria in selective/non-selective media 2. Staining & microscopy 3. Biochemical tests 4. Scoring and identification 5. Can be extracted for molecular methods

Linearity

A serial dilution of standard curve closely approximates a straight line

Describe BOX elements.

BOX are also repetitive subunits. -Contain boxA, boxB, boxC subunits which are 59, 45, 50 bp -Not related to ERIC/REP -Do form stem-loop structures like ERIC/REP -S. pneumoiae genome has 25 BOX elements

Sensitivity

Lowest level detected at least 95% of the time

_________ is used to characterize bacterial isolates using sequences of internal fragments of housekeeping genes.

MLST

Which genotypic method(s) used for molecular epidemiology has Good-High criteria across the board?

Plasmid analysis Ribotyping Repetitive elements

HIV is in the _______ family

Retro

What is ribotyping and how does it relate to RFLP?

Ribotyping = probing Ribosomal RNA genes in the RFLP procedure because they are highly informative. Probes target 16S and 23S ribosomal genes.

Describe the process of Amplified Fragment Length Polymorphism (AFLP)

Similar to RFLP - based on amplification of DNA fragments generated by cutting the test genome with restriction enzymes. Begins with restriction digestion of chromosomal DNA. The resulting fragments are ligated with adapters compatible with the restriction enzyme ends and complimentary to primers used to amplify them. The first amplification is performed with pre-selective primers that end in a 3' base (N) selected by the user. Selective primers with three added 3' bases are used for a 3' second round of PCR. This selection results in a characteristic pattern and only a fraction of the original fragments will be represented in the gel pattern.

What is the primary way to diagnose a virus in clinical virology labs?

detect growth of the virus in tissue or cell culture system

What are quantitative tests used for in HCV?

determine viral load and monitor replication in response to antiviral therapy

Give the order where the ITS sits between the rRNA subunits

18S [ITS] 5.8S [ITS] 28S

In PFGE, regardless of the current changing direction, there is a threshold fragment size that will run at the same rate and end up appearing as a single large diffuse band in the gel. What is this threshold size?

30-50kb

About how many deaths per year in the US due to HIV?

844 per 100,000 people

Compare AFLP to RAPD, PFGE, and REP-PCR.

-detect more polymorphisms than RAPD -faster than PFGE -more technically demanding than REP-PCR

Describe the process of identifying and diagnosing HIV using antibodies.

-Detect antibodies specific for HIV using ELISA (enzyme linked immunosorbent assay) -If positive, use Western Blot to confirm or use APTIMA HIV-1 RNA Qualitative Assay (GenProbe) to confirm. -Can also use non-invasive conjugated antigen test Rapidtest HIV Lateral Flow Test

● _______ sequences and ______ sequences occur in noncoding regions and contain inverted repeats ● ____ sequences are only 38bp long while _____ sequences are 126bp long. ● ____ sequences are more numerous in the genome ● ____sequences are more present in multiple copies at a single location. ● ____elements are not related to _____ and ______ sequences but do form stem-loop structures.

-ERIC, REP -REP, ERIC -REP -REP -BOX, ERIC, REP

Describe the HCV virus, which family it is in, and what symptoms it causes.

-Enveloped -Positive sense ssRNA virus -Flaviviridae family. It causes viral hepatitis, cirrhosis and some cancers of the liver

What are ITS used to identify and are these regions conserved?

-ITS used to identify and type yeast and mold. -They are conserved within species but polymorphic between species.

Describe how HCV infects and replicates

-Infection & Replication occur in hepatocytes of the liver on intracellular lipid membrane (ER) by RNA-dependent RNA polymerase. -Enters cell by cell-surface molecules and then takes over intracellular machinery. -HCV genome is translated to produce proteins which are processed by viral and cellular proteases to produce structural and nonstructural proteins. -RNA polymerase produces a RNA strand intermediate that serves as a template for the production of new positive strand viral genomes. -New viral particles are released at the cell surface. -Each infected cell produces about 50 virions/day.

What causes urethritis? How do you detect it? What is a common co-infection?

-Infection of the urethra by Neisseria gonorrhea. -Diagnose with microscopy/gram staining, culture, or nucleic acid amplification (NAA). -Coinfection with Chlamydia trachomatis. NAAT can detect both bacteria.

What is MRSA? How is it acquired? Why is it dangerous? What tests can detect it?

-Methicillin-resistant Staphylococcus aureus - Staph aureus that is resistant to beta-lactam antibiotics like methicillin and oxacillin. -community, hospital, live stock-acquired -Dangerous because it is multi-drug resistant and it is part of our normal flora but MRSA can invade human tissues easily -real-time PCR is FDA-approved -Can be cultured and visually identified as gram positive coccus chain. Or culture can be used for rapid latex agglutination test that detects the PBP2a protein that is responsible for oxacillin resistance.

Name 5 respiratory tract pathogens

-Mycoplasma pneumoniae -Chlamydophila pneumoniae -Streptococcus pneumoniae -Legionella -Bordetella pertusis

List 5 molecular based assays used to detect viruses

-PCR -Reverse transcriptase PCR (RT-PCR) -Quantitative/real-time PCR (qPCR) -Transcription mediated amplification (TMA) -Signal amplification assays (Branched DNA-bDNA and Hybrid Capture)

List 3 nucleic acid amplification assays for HCV diagnosis:

-RT-PCR (amplicor HCV, ROCHE) -Transcription-mediated amplification (Versant HCV RNA, bayer) -Branched DNA (Versant HCV RNA, Bayer)

Describe the chemical (non-molecular) tests used to diagnose HCV.

-Serology used to detect presence of antibodies against HCV. -If initial blood test shows positive for HCV, additional blood tests will measure the quantity of hepatitus C in your blood (viral load) and identify the genotype of the virus

Name 3 ways HIV is transmitted

-Sexual contact -HIV-infected blood or blood contaminated body fluids getting into broken skin, wounds, mucous membranes -Deep, open mouth kissing if both partners have sores or bleeding gums

What is C. diff? What are its symptoms and who does it affect? how do you get it? What are some ways you would diagnose it?

-Spore-forming, toxin-producing, gram-positive anaerobic bacterium -Causes diarrhea to life-threatening inflammation of colon. It can also be present without causing disease. -It affects older adults and those treated with antibiotics -Fecal and food borne transmission Diagnose with: -anaerobic culture form stool sample -enzyme immunoassay (EIA) screening for glutamate dehydrogenase (GDH_ antigen and toxins A and B (rapid detection) -Nucleic acid amplification testing (NAAT)

Name 7 urogenital track organisms

-Treponema palidum -Mycoplasma genitalium -Mycoplasma hominis -Ureaplasma urealyticum -Haemophilus ducreyi -Neisseria gonorrhoeae -Chlamydia trachomatis

Genetic methods used for strain typing are evaluated and compared based on these 5 criteria:

-Typing capacity -Reproducibility -Discriminatory Power -Ease of Interpretation -Ease of Test Performance

What is PFGE used for? How does PFGE work? What are the 4 types?

-Used for the separation of large DNA, RNA, protein, chromosomes. -Restriction digest that cuts infrequently -Electric field periodically changes direction to a gel matrix -Interpret PFGE results using "rule of 3" FIGE TAFE RGE CHEF

Name all of the molecular techniques used to detect antimicrobial resistant organisms.

-plasmid analysis -PFGE -RFLP -Typing capacity -Arbitrarily Primed PCR -Amplified Fragment Length Polymorphism (AFLP) -Interspersed Repetitive Elements -Internal Transcribed Spacer Elements -spa Typing -Multilocus Sequence Typing

t some considerations when collecting samples and/or choosing a test using molecular based testing.

-some molecular tests require high quality nucleic acid (especially RNA) -contamination could yield false positives -accurate sampling site is important to recover the maximum load of infectious agent -proper equipment and reagents should be used based on sample types and tests requested

List some considerations when collecting samples and/or choosing a test using culture based testing.

-viability of organisms -special collection systems needed for strict anaerobes or other fastidious organisms -require high load of infectious agent compared to molecular based -accurate sampling site is important to recover the maximum load of infectious agent -proper equipment and reagents should be used based on the sample types and test requested

Susceptibility testing is great and all, but it is a phenotypic method. What are the four reasons for using molecular-based methods?

1. When the MIC of an organism is at or near the breakpoint of resistance, detection of mutated genes contributing to resistance would be irrefutable evidence of the potential ineffectiveness of the agent. 2. You can detect the genes that are involved in the resistance of Antimicrobial agents in organisms closer to the time of collection. This saves the time required to isolate the organism and perform phenotypic MIC determinations on isolated colonies. 3. In epidemiological investigations, monitoring the spread of a resistance gene in multiple isolates of the same organism is more useful than following the trend in the MIC. 4. Molecular methods are considered the gold standard when new phenotypic assays are being developed.

As a lab personnel working with potentially infectious pathogens, what are 4 rules to live by?

1. assume sample is infectious 2. avoid contamination to sample and yourself 3. know the strengths and weaknesses of various methods because that chagnes the clinical significance of the findings 4. document everything

List 3 disadvantages of using antibody testing to diagnose viruses.

1. indirect method of diagnosis (the host immune response needs to be stimulated by the virus for the host to produce antibodies) 2. the "window" period: the patient is infected and infectious yet antibodies aren't yet detectable 3. to interpret antibody testing with confidence, paired sera should be collected (one collected during the acute phase of infection and the other collection as patient is convalescing).

Name 3 general (non-molecular) ways to diagnose viruses

1. test for antibodies agains the virus 2. measure the presence/absence of viral antigens (immunoassays or direct immunofluorescent assays) 3. detect growth of the virus in tissue or cell culture system

Assay determination for bacterial identification depend on these 5 things:

1. type of pathogen 2. stage of infection 3. sample source 4. titre/load of organism that can be recovered 5. treatment options available (Is an antibiotic sensitivity test required to determine treatment options? Does the presence of antibiotic resistance genes based on PCR tell us the resistance profile of the pathogen?)

Accuracy

Ability to determine the true value

Flexibility (in testing for viruses)

Accuracy of measurement of virus regardless of sequence variations

Describe Mycobacterium tuberculosis and describe the culture-based assay versus molecular-based assay used to detect it.

Airborne, causes TB -Direct fluorochrome stain of the specimen can be used to visualize but it requires a very high titre (10^4 organisms/mL) Culture-based: -Still Gold Standard -bacteria grow slowly -followed by biochemical tests or HPLC of mycolic acid (this takes a few weeks) Molecular-based: -PCR assay coupled with DNA probes for respiratory samples -highly sensitive -takes 3-8 hours

Give an example of an Interspersed Repetitive Element. Size, where its found.

Alu element. -300bp -in the short interspersed elements (SINE) family -found in primates and >1million copies in humans.

Why is it important to develop and use molecular assays to test for respiratory tract infections?

Because non-molecular methods lack sensitivity and are time consuming

How does restriction fragment length polymorphism (RFLP) analysis work?

Cut DNA with restriction enzymes, resolve the fragments by gel electrophoresis, then transfer the fragments to a membrane and probe with a specific probe that are radioactively labeled. Transfer membrane to x-ray paper and read with autoradiography (AKA southern blot). Exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence.

When reading a fluorescent AFLP, why is it that duplicate specimens do not produce the exact same pattern?

Due to band shifts and different band intensities.

Describe ERIC sequences

ERIC= enterobacterial repetitive intergenic consensus sequences -126bp genomic sequences -highly conserved in G- bacteria. -Located between genes in operons or upstream or downstream of a single open reading frame. -Flanked by inverted repeats that can form stem-loop or cruciform structures. G- bacteria: Bartonella, Shigella, Pseudomonas, Salmonella

What is ITS?

Internal Transcribed Spacer Element -Spacer DNA that sits between the small-subunit rRNA and large subunit rRNA genes in the chromosome -Can also sit in the transcribed region in the polycistronic rRNA precursor transcript

What is the problem with using NAAT for C. diff?

It can be too sensitive and cause overdiagnosis and overtreatment

Phenotypic methods like MALDI-TOF (mass spec) aren't always ideal. But why?

Lack of reproducibility and their Lack of ability to discriminate between isolates. Genotypic methods are used almost exclusively to type bacterial strains to determine the relatedness of multiple isolates.

What is Arbitrarily Primed PCR (AP-PCR or RAPD)?

Modified PCR using 10-base long oligonucleotides of random sequences to prime DNA amplification all over the genome. PCR products are generated without knowing the sequence of the target or targeting a specific gene. Similar band patterns obtained from performing PCR with the same arbitrary primers indicate that two organisms are the same or similar.

Describe MLST

Multilocus Sequence Typing -Used to characterize bacterial isolates using sequences of internal fragments of housekeeping genes. -Housekeeping genes are a type of amplification control =target that is always present. -6 or 7 genes are sequenced 450-500bps and the sequences are assigned as the alleles. -bacteria have enough variation so that there are multiple alleles of each housekeeping gene (about 30 different alleles/locus)

Specificity

Negative samples are always negative and positive results are true positives

Is RFLP still widely used?

No - almost obsolete but it was the first DNA profiling technique cheap enough for widespread application.

Should you use plasmid analysis for organisms with large genomes?

No-most organisms with larger genomes or multiple chromosomes can be identified with Pulsed Field Gel Electrophoresis

What are the 3 nucleic acid amplification (NAA) methods (with their targets) used for viral quantitation, disease prognosis, and treatment monitoring for HIV-1? Which is the most sensitive?

PCR/RT-PCR: gag gene (group M, subtypes A-H) NASBA: gag gene (groups M, O, N) bDNA: pol gene (group M, subtypes A-G) RT-PCR

Describe Plasmid Analysis aka DNA fingerprinting

Plasmids are autonomously replicating extrachromosomal DNA found within bacteria. Frequently, related strains contain the same number of plasmids with the same molecular weight and similar phenotypes. Involves the process of isolation and restriction mapping of bacterial plasmids. The simplest form of diagnostic digest is one in which you want to verify that the plasmid that you have is the expected size or that it is composed of a backbone and insert of expected sizes. Plasmid analysis has been shown to be useful in the characterization of specific bacterial strains.

What kind of testing has been the standard for monitoring drug therapy and HIV disease progression?

Quantitative HIV-1 RNA testing in plasma

Which genotypic method used for molecular epidemiology has poor reproducibility?

RAPD

Describe species identification using primed amplification of REP or ERIC.

Rep and ERIC sequences are present in different chromosomal location in bacterial subtypes. PCR extends outward oriented primers hybridized to the repetitive sequences and generates amplicons of different sizes based on the placement of the of the element.

Describe REP sequences

Repetitive Extragenic Palindromic sequences -occurs in noncoding regions (like ERIC) -contains inverted repeats -38bp long -more numerous in genome than ERIC -present in multiple copies in a single location (ERIC is not) -These elements can be primed in PCR and run on gel, electrophoresis, or microfluidics. -Used to type C. diff and fungal pathogens

Precision

Reproducibility of independently determined test results

What is spa typing?

Staphylococcus aureus Protein A (spa gene). MRSA has a VNTR (repetitive element) in the 3' coding region of the protein A gene. spa element can have 2-16 repeats. Analyzing these elements by PFGE or sequencing and comparing to known sequences is used to identify MRSA = spa typing. spa and VNTR typing has the same discriminatory power as PFGE but shorter TAT.

Discuss strengths and weaknesses of sequencing.

Strengths: -Can be targeted or non-targeted (good for diagnosis of unknown infection) -Metagenomics or WGS can help identify pathogenic genes like antibiotic resistance genes Weaknesses: -TAT can be long complicated/challenging workflow

Discuss strengths and weaknesses of PCR

Strengths: -speed -targeted Weaknesses: -Prone to false positives (contamination) -False negatives from inhibitors in the sample -Limited to KNOWN and specific sequences

Discuss strengths and weaknesses of microarrays.

Strengths: -speed -targeted Weaknesses: -complicated -prone to false positives -limited to KNOWN and specific sequences

Define molecular epidemiology

Study of causative genetic and environmental factors at the molecular level. Important for controlling and preventing the spread of diseases.

What is the purpose of susceptibility testing and what does this mean exactly? Is this a genotypic method or phenotypic?

Susceptibility testing measures the minimum inhibitory concentration (MIC) of an antimicrobial agent = the least amount of an antimicrobial agent that inhibits the growth of an organism. This is a phenotypic method.

Why is HIV viral load important and what is the purpose of antiretroviral therapy? What does viral load testing have to do with CD4?

The amount of HIV viral load is used as a marker for disease prognosis and to track the timing and efficacy of antiretroviral therapy. The goal of antiretroviral therapy is to get the viral load below 50 copies/mL of blood. If 100,000 copies/mL they are more likely to progress to AIDS than patients who maintain <10,000 copies/mL Viral load testing and CD4 counts should be performed in. conjugation.

What do the results of epidemiological studies conclude?

The origin, distribution, and best strategies for prevention of disease.

Why isn't AP-PCR/RAPD very easy to use?

The procedure conditions have to be followed strictly so that the differences are reproducible.

ITS sequences are amplified using primers and the amplicons are analyzed with sequencing, sscp, dgge, restriction enzyme analysis, and ssp. Why do we care about ITS sequences?

They are the most conserved region in the genome of ribosomal RNA genes in the 16S subunit. Barcode marker for fungi.

How is HCV transmitted and what are the symptoms?

Transmitted through exposures to infectious blood or body fluids that contain blood like: -injection drug use -receipt of donated -blood, blood products -needlestick injuries -birth to hcv-infected mother -tattoo equipment Most people have no symptoms. -fatigue -nausea -loss of appetite -yellow eyes/skin no vaccine for hep c

Describe the bacteria that causes syphilis. How is it transmitted to babies? What are common tests used to diagnose it?

Treponema pallidium - motile spirochaete. -Transmitted to a fetus by transplacental passage during the later stages of pregnancy which gives rise to syphilis. Treponemal tests -T. pallidum particle agglutination assay (TPPA) -T. pallidum hemagglutination assay (TPHA) Nontreponemal tests -Rapid plasmid reagin (RPR) -Venreal disease research laboratory (VDRL) test Quest has a LDT molecular test using RT-PCR

What does AMR stand for?

antimicrobial resistance

Name the 3 nucleic acid amplification methods used for HIV viral loads. List advantages/disadvantages for each.

bDNA: -high throughput, broad dynamic range, applicable for group M, subtypes A-G -no internal control, false positives Amplicor RT-PCR: -internal control, good specificity -limited dynamic range NASBA: -broad dynamic range, performed on many specimen types & volumes -doesn't detect all non-B subtypes

Why is it important to use the same method to determine viral loads when monitoring patients over time?

because results from different methods aren't always comparable

What are qualitative tests used for in HCV?

if antibody tests are negative but the person has symptoms, these can be used to confirm infection

What is the disadvantage of using PFGE to type strains?

time involved to perform the assay, which can take 2 to 3 days to complete ONE analysis