Infectious Disease Flashcards
Risky travel activities with HIV-1/HIV-2 EIA positive. Western shows bands for p24 (HIV-1 capsid antigen) and gp41 (HIV-1 transmembrane glycoprotein). What are the four groups of HIV-1?
- M (main - A, B, C,D,F,G,H,J,K)
- O (outlier)
- N (non-M, non-N)
- P (primate *gorillas to humans)
Risky travel activities with HIV-1/HIV-2 EIA positive. Western shows bands for p24 (HIV-1 capsid antigen) and gp41 (HIV-1 transmembrane glycoprotein). What are the next two tests to order?
- HIV-1 RNA viral load
- HIV-1 genotyping
Which HIV genes are sequenced for resistance?
Protease
Reverse transcriptase
Ampliprep quantitative HIV-1 testing from 48-10,000 copies (1.68-7 log copies/mL) targets which HIV-1 M and N (and circulating recombinant forms -CRF) gene and which Group O gene?
gag (M and N)
long terminal repeat (O)
RealTime quantitative HIV-1 testing from 40-10,000 copies (1.68-7 log copies/mL) targets which HIV-1 gene?
integrase
None of the FDA-approved HIV viral load tests detect or quantify __
HIV-2
HIV-1 genotyping is limited by the need for _____ HIV-1 RNA copies/mL in plasma
500 copies/mL
HIV-1 resistance to _________, _______ and _____ inhibitors are not detected with the FDA-cleared platforms
fusion inhibitors
integrase inhibitors
CCR5 inhibitors
Beyond HIV sequencing, a ________ assay measuring IC50 and IC90 can be performed for resistance.
phenotypic
HIV-1 phenotyping is limited by the need for _____ HIV-1 RNA copies/mL in plasma
500
After RealTime HIV-1 testing below 40 copies/mL and additional testing by Ampliprep 2, Aptima RNA qualitative TMA, what conclusion may be made after 12 months about the patient and their quantitative testing?
HIV-1 elite controller (high CD4 T-cells and low viral copies)
Describe long-term non-progressors in HIV
CD4 > 500 without ART, detectable viral load under 5,000
Intra-assay variation for HIV-1 load is 0.1 to 0.2, there for a variation greater than ______ log copies/ml (3 fold) is required to represent biologically meaningful change in viral load
0.5
For positive 4th generation tests (HIV-1/2 antibodies and p24 capsid Ag) and negative Western, what are the two possibilities
Acute infection with p24 positive only
False positive
For positive 4th generation tests (HIV-1/2 antibodies and p24 capsid Ag) and negative Western, what testing could help distinguish whether there is an acute infection or a false positive?
HIV-1 RNA testing
HIV-1 groups (M-9 subtypes, N, O, P) are based on sequence diversity in ______ and ________ genes
gag, env
HIV-1 group M subtype __________ is the most common globally while subtype __________ is the most common in the US
C (globally)
B (US)
HIV binds _________on T-cells (using gp120 envelope protein) in syncytium inducing viruses and _______ on macrophage in non-syncytium inducing HIVs.
CXCR4 - CD4 Tcells
CCR5 - macrophage
A homozygous 32 bp ______ in CCR5 is associated with resistance to HIV-1
deletion
Beyond CCR5, __________ is associated with lower steady state viral levels and slow disease progression
HLA-I
Where is the predominant geographic location of HIV-2
West Africa
Same as group O (non-M, non-N)
What is the target for the Ampliprep HIV-1 version 1 test?
gag
Which of the following statements regarding HIV-1 genotyping is false?
A. Current FDA-cleared assays generally use sequencing technology to compare the patient’s sequence with the wild-type
B. Genotyping can be performed by the TRUGENE and ViroSeq assays
C. Genotyping reports provide information on antiretroviral drug resistance
D. Patients who are treatment naïve should have genotyping tests performed
E. Performing genotyping tests is the only way to determine a patient’s antiretroviral drug resistance profile
E.
phenotype assays also exist
Which of the following is not a characteristic of elite controllers?
A. They constitute <1% of the global HIV infected population
B. They have a normal CD4+ T-cell count
C. They maintain a viral load greater than 5000 copies/mL
D. They typically maintain a viral load less than 50 copies/mL
E. They will likely have a positive HIV-1 antibody screen
C.
Should be < 5000
CMV ganglciclovir resistance is based on which two genes?
UL97 - viral kinase
UL54 - DNA-dependent DNA polymerase
Which CMV gene more commonly has mutations?
UL97
1292-1998 - amino acids 430-666
Gangciclovir is an acyclic 2-deoxyguanosine that acts as a “suicide substrate” of the CMV DNA-dependent DNA polymerase encoded by the _____ (pol) gene.
UL54
Gangciclovir is initially phosphorylated by the ________-encoded viral kinase and then by cellular kinases
UL97 = viral kinase
CMV UL97 S/T kinase develops resistance through mutations at amino acids 460(________-_________ domain), 520 and 591-607 which is hypothesized to be the __________-_________ domain
460- phospho-transfer
591-607 gangciclovir-binding
Phenotypic assays in CMV define resistance by an IC50 change greater than ______ fold.
5-fold (IC50 change)
Given a major mutation in CMV (>5X IC50 change), what is the next step in therapy?
Foscarnet (herpes virus DNA polymerase) - does not require UL97 kinase
Cidofovir is another inhibitor of herpesvirus DNA polymerases but is not a recommended as a first line therapy. Why?
common UL54 (polymerase) mutations confer cross-resistance between cidofovir and gangciclovir.
Also nephrotoxic
What should be done clinically with a patient who has no resistance mutations and does not appear to be responding virologically?
Reduction of immunosuppression to augment the gangciclovir
In addition to reduction of immunosuppression in a patient that does not appear to be responding to gangciclovir (virologically), what are the treatment changes in low and high risk (lung, CMV negative patient in CMV positive allograft)patients
low- gangciclovir intensification
high- empiric foscarnet
CMV syndrome is a constellation of ______, ______ and _______
fever, anorexia and malaise
Due to the absence of a reference standard, patients should be followed for CMV titers using a _______________ assay
single (assay)
_______(pol) mutations that show resistance in gangciclovir show mutations in codons 300-1000
UL54
Gangciclovir inhibits CMV replication by disrupting
chain elongation
Gangciclovir resistance in solid organ transplant occurs most commonly due to:
Viremia after months of treatment
Gangciclovir resistance in solid organ transplanst most commonly occur in which pretransplant CMV serologic profile? (Donor____, recipient _____ )
Donor positive, recipient negative
Which HR-HPV type accounts for the highest percentage of disease of the cervix?
HPV 16
Transmission of HR-HPV which can lead to cervical cancer occurs by which route?
Contact by infected epithelium with mucous membranes (sexual activity)
The recommendations from the 2006 Consensus Guidelines suggest all of the following EXCEPT:
A. All women 30 years and older should be screened for high-risk (HR) HPV DNA testing from their cytology specimen
B. HPV-16 and HPV-18 genotyping is not recommended as the initial screening test for women 30 years and older
C. HPV-16 and HPV-18 genotyping should be used for women 30 years and older with HR HPV DNA to determine whether to perform colposcopy and biopsy of suspicious lesions or wait 12 months for repeat cytology testing
D. HR HPV DNA testing should be included in evaluating a patient with atypical glandular cells of undetermined significance (AGUS)
E. Women with atypical squamous cells of undetermined significance (ASC-US) cytologic results of any age should have HR HPV DNA testing on that cytology specimen
E. Women with atypical squamous cells of undetermined significance (ASC-US) cytologic results of any age should have HR HPV DNA testing on that cytology specimen
No ASCUS testing in less than 20 years
The only FDA-approved methods currently available for detection of HR HPV are based on which methods?
RT PCR and invader/cleavase signal amplification
Describe the Digene (Qiagen Inc) Hybrid Capture II assay?
It is a signal amplification method using RNA probes and antibodies specific for RNA:DNA duplex hybrids
What gold standard assay for cervical cancer screening?
biopsy
Pap smears suffer from low __________ (sensitivity/specifity) but have high_________ (sensitivity/specifity
Paps have:
low sensitivity
high specificity
FDA-approved Digene HPV Hybrid Capture2 (Qiagen) and Cervista HPV HR (Hologic) and Cervista HPV-16/18 genotyping are approved with the _________ liquid-based cytology media
Thin-prep (Hologic)
not Surepath (BD)
Cervista HPV HR test (Hologic) is a ____ ______ method of detection with _________ reactions
signal amplification
3 reactions
Cervista HPV HR test (Hologic) uses complementary oligonucleotides and a _________ which cleaves the FAM (6-carboxyfluorescein) fluorophore if the 3 targets are present.
cleavase
Cervista HPV HR test (Hologic) (Hologic) uses a RED (Redmond Red dye) tagged oligonucleotide to the human _____ gene as an internal control
H2AB
Cervista HPV HR test (Hologic) FAM FOZ (fold over zero) divides the ____ FAM FOZ value from any one of the 3 reactions by the ______ FAM FOZ value
highest/lowest
Cervista HPV HR Invader test (Hologic) uses _____ technology to generate a ______ sequence (when target is present) that is complementary to a secondary probe. The FLAP sequence binding on the secondary probe in a simultaneous reaction generates red fluorescence. Red means there ______ in the sample while FAM (green) fluorescence means there is _____.
invader
FLAP sequence
red = DNA
green = HPV
Digene HPV Hybrid Capture2 (Qiagen) is a _______ signal amplification technology based on RNA probes to detect ____ high-risk HPV types
chemiluminescent (RLU)
13
The Digene HPV Hybrid Capture2 (Qiagen) test has 93-96% ________ but the potential for ________ __________
93-96% sensitivity
false positives (cross reaction with LR HPV)
Gardasil contains HPV types ______ and _____ as well as LR HPV types ______ and _____
16, 18, 6, 11
The strain of lymphogranuloma venereum (LGV) round in European outbreak of rectal proctitis?
L2b
Which LGV gene is sequenced to determine the serovar?
Outer membrane protein A gene (ompA)
Performing an FDA-cleared test on a non-approved specimen requires a ___________?
validation
A Method Other Than Acceleration (MOTA) score provides a ___________ (quantitative/qualitative) result
qualitative
(does not indicate level of organism present in sample)
When should an internal control be used for a C. trachomatis molecular test s?
when testing a crude lysate
Differential for proctitis in HIV-1 positive MSM?
- Neisseria gonorrhoeae
- Chlamydia trachomatis serovars D-K and L1-L3
- HSV
- HPV
- Syphilis
Serology tests can/cannot? differentiate C. trachomatis A-K vs L1-3?
Cannot
______ __________ ____________ uses a bumper primer, a 5’ primer with restriction site, thiolated cytosine, exonuclease nicking, DNA polymerase and fluorescently tagged probe to achieve isothermal amplification
strand displacement amplification
Which enzymes are used in the isothermal Aptima CT assay - transcription mediated amplification (TMA)?
Reverse transcriptase (with RNaseH activity)
RNA polymerase (with T7 promoter included in primer)
LGV proctitis requires a 21 day course of __________________
doxycycline
The MRSA ___________ gene responsible for production of an altered Penicillin Binding Protein, PBP2a, which maintains staphylococcal cell wall integrity because of its low affinity for ?-lactam antibiotics.
mecA
What is the genetic target in the commercial MRSA PCR assays?
SCCmed/orfX insertion site
Which variants in S. aureus growth can cause discrepancies with PCR results?
anaerobic, blood requirements, low density growth and salt sensitivity
Healthcare facilities can use ____________ surveillance to support an overall infection control program
active surveillance
When the SCCmec cassette is inserted into the open reading frame (orfX) gene of S. aureus, it becomes the primary genetic basis for methicillin resistance.
staphylococcal cassette chromosome (SCCmec)
MRSA PCR as an “improved gold standard” has _____________ PPV than cultures but a _____________ NPV
lower PPV
higher NPV
MRSA PCR assay results can be MRSA positive, despite a culture negative status, for ______ to _____________% of specimens tested
5 to 10%
The BD GeneOhm and Cepheid Xpert both have ____________ performance for MRSA detection in Canada and Europe
diminished
Cepheid MRSA Expert has ____________ ____________ spores as a sample processing/assay control and PCR inhibition control
Bacillus globigii
Cepheid MRSA Expert measures the __________ ____________ control to ensure proper bead hydration, reaction-tube filling, probe integrity, and fluorophore stability.
probe check (control) -PCC
In addition, low bacterial densities in the nares samples can render the culture negative and the PCR positive, or vice versa, because low bacterial densities, typical with emerging subpopulations, may cause both methods to produce sporadic positive or negative results under the parameters described by the statistical phenomenon known as the ____________ effect.
Poisson (effect)
There is documented evidence that respiratory specimens, among others, are prone to false-________ results, which can be corrected by the use of selective agar to confirm the true presence of MRSA present in low density
negative (false-negative)
Microbial growth characteristics can also be responsible for PCR-positive, culture-negative results. Reasons for the discrepancy can include staphylococcal strains that _________ slowly, those that require the presence of _________ for growth, and those that grow only in _________ or __________ environments. In these cases, PCR would identify these fastidious species but the strains would not grow without extraordinary measures for cultivation. Furthermore,______ _________ variants of MRSA are a subset of fastidious strains and are becoming more commonly isolated, growing slowly on blood-based agar and sometimes on chromogenic agar, although they may not exhibit the typical colony color change on certain varieties of agar.
grow (slowly)
blood (for growth)
anaerobic or high salt
small colonogy
In addition to the microbial reasons for the discrepant results, an alternative scenario is possible. “______ __________ variants” of MRSA can exist, when the mecA gene is lost from the bacterial chromosome but the insertion site remains. Recurrent commercial PCR assays target genetic regions upstream from the mecA gene
empty cassette (variants)
Deletions within the SCCmec region of MRSA strains result in the absence of a functional _____________gene,
mecA
Deletions within the SCCmec region of MRSA strains result in the absence of a functional mecA gene, but these “empty cassette variants” cause PCR-positive, culture-negative results because current commercial PCR assays target genetic regions___________ from the mecA gene
upstream
The mecA and ____________ genes are targets for Cepheid’s second-generation MRSA assay, which also detects and confirms MSSA.
spa - (staphylococcus protein A)
The __________ gene is a recombinase gene responsible for insertion of the SCCmec cassette
ccr
