Khatija Ahmed

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• Khatija Ahmed
• Setshaba Research Centre, Soshanguve
• Department of Microbiological Pathology,
  Microbicide Division
• University of Limpopo, Medunsa Campus
• Pretoria
• South Africa

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Laboratory Testing of Sexually Transmitted
Infections in Microbicide Clinical Trials
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Measurement of Endpoints

• Endpoints
• 1 endpoint
• 2 endpoints

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Primary Endpoints

• Measure the efficacy of the microbicide for the
  prevention of HIV
• Trial design - follow up visits and HIV testing

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Secondary Endpoints

• Effect of the Microbicide for the prevention of
  STIs
• Measuring of these raises concerns due to the
  following
• -MOA of Microbicides differ
• -asymptomatic infections - when to test?
• -sample collection devices (dacron or cotton
  swabs)
• -types of sample (swabs, urine, washings)
• - testing methods differ for STIs e.g. culture/
  serology/molecular
• - gel interference

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STIs- Testing Methods
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Limitations of Tests

• Stage of infection
• Site of specimen collection
• Type of specimen
• Transport of specimens
• All have different sensitivities and
  specificities
• Laboratory techniques and expertise

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Microbicides Impact on Laboratory Testing

• Microbicides-different compositions
• Residual Microbicides in women- quantity depends
  on
• -interval between insertion and specimen
  collection
• -volume inserted
• -viscosity of the microbicide
• -method of insertion
• -number of insertions- accumulative effect?
• -nature of specimen collection
• -type of test (culture and molecular)

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Phase 3 Clinical Trails
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Microbicides Impact on Laboratory Testing (1)

• Serological tests
• NO effect

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Microbicides Impact on Laboratory Testing (2)

• Culture
• - Young N et al (Abstract) Inhibitory effects
  of vaginal microbicides on Cobas Amplicor and
  Gen-Probe Chlamydia trachomatis and Neisseria
  gonorrhoeae, and InPouchTV Trichomonas vaginalis
  tests. International Journal of STD and AIDs
  200112 suppl 2193
• in vitro ( placebo, BufferGel, Carraguard)
• study showed
• - no inhibition of placebo (methyl cellulose)
• - the InPouch TV medium showed inhibition
• BufferGel 24 hrs was 10 48 hrs was
  41
• Carraguard 24 hrs was 4 48hrs was 78
• -No data on other Microbicides

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Nucleic Acid Amplification Tests ( NAATs)

• Usually used for NG and CT
• Also used for TV (not commercially available)
• Separate or combined
• Based on amplification of NA - PCR

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NAATs - CT and NG

• Commercial tests
• -Polymerase chain reaction PCR - (Roche)
• -Ligase chain reaction LCR- (Abbott)
• -Strand displacement Amplification SDA- (Becton
  Dickinson)
• -Aptima Combo (TMA) (Gen Probe)
• In-house tests (targets different genes)
• -cppB-gene based assay- NG
• -16S rRNA
• -Taqman Assay-opa genes

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NAATs Study 1

• Van Dyck E et al. Detection of Chlamydia
  trachomatis and Neisseria gonorrhoeae by Enzyme
  Immunoassay, Culture, and Three Nucleic Acid
  Amplification tests. Journal of Clinical
  Microbiology, May 2001, p. 1751-1756, Vol. 39,
  No. 5
• Drawbacks from the 3 commercial assays
• -incompatibility of collection and transport
  systems
• -handling instructions
• -extraction procedures (SDS, M4, STM etc)
• No significant difference in the sensitivities
  and specificities of
• -PCR
• -SDA N. gonorrhoeae and C. trachomatis
• -LCR

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NAATs Study 2

• Le Roux MC et al (Poster). Modification to
  Multiplex Amplification System for the Detection
  of Chlamydia trachomatis and Neisseria
  gonorrhoeae in Endocervical Swabs from
  Asymptomatic Women. Joint Congress of the
  Infectious Diseases and Sexually Transmitted
  Diseases Societies of Southern Africa,
  Stellenbosch.
• Tests done for CT/ NG
• Specimens collected via dacron swab- placed in
  transport medium (STM) (Amplicor - Roche)
• - initially inhibition experienced STM was
  replaced by M4 medium
• - inhibition decreased and a wash step was added
  on inhibited specimens only
• Carraguard Phase 2-same effectmodifications done

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NAATs Study 3 (1)

• Crucitti T et al (Poster, MP008). Inhibition of
  Nucleic Acid Amplification Tests by Residual
  Amounts of Microbicide 6 Cellulose Sulfate
  Vaginal Gel. 16th Biennial Meeting of the
  International Society for Sexually Transmitted
  Diseases Research, Amsterdam
• Assays used
• -Amplicor (Roche)
• -SDA CT/NG assay (BD)
• -In-house PCR (MOMP, cppB and ß2 microglobulin)

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NAATs Study 3 (2)

• Sub-study 1
• -single dose CS
• - specimens collected before gel use, 24 and
  96 hours later
• -inhibited specimens re-tested at ¼ and
  1/10 dilutions in buffer after spiking with
  CT and NG
• Sub-study 2
• -1 daily dose of CS for 4 days
• - specimens collected before gel use and daily
  after 24 hrs of gel use for 4 days
• -inhibited specimens re-tested at ¼ and
  1/10 dilutions in buffer after spiking with CT
  and NG

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NAATs Study 3 (3)

• Crucitti concluded
• Residual microbicide gel inhibits NAATs
• Did not recommend use of Amplicor and in-house
  PCR assays in CS trials
• SDA BD CT/NG assay was only assay not inhibited
  by 6 CS

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NAATs Study 4

• - Young N et al (Abstract) Inhibitory effects of
  vaginal microbicides on Cobas Amplicor and
  Gen-Probe Chlamydia trachomatis and Neisseria
  gonorrhoeae, and InPouchTV Trichomonas vaginalis
  tests. International Journal of STD and AIDs
  200112 suppl 2193
• in vitro ( placebo, BufferGel, Carraguard)
• Assays used
• -Amplicor and Gen Probe for CT and NG
• This study showed
• - no inhibition of placebo
• - For both BufferGel and Carraguard there
  was varying degrees of inhibition using both
  NAATs
• - after a modified procedure (transport
  medium and PBS wash),
  inhibition was removed from
  the Amplicor but not from the Gen-Probe

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NAATs Study 5 (1)

• Crucitti T et al. (Poster 02652). Inhibitory
  Effect of Vaginal Microbicides on Molecular
  Amplification Assays. Microbicides 2004, London.
• Gels evaluated
• 6 Cellulose Sulfate (CS)
• Acidform
• Polystyrene Sulfonate (PSS)
• K-Y Jelly
• PSS Placebo
• HPTN035 Placebo
• NAATs used
• Amplicor CT/NG PCR, Roche
• SDA BD ProbeTec ET CT/NG, Becton Dickinson
• MOMP PCR (CT amplification), primers according
  to reference
• cppB PCR (NG amplification) , primers according
  to reference
• ß2 microglobulin PCR, primers according to
  reference
• Extraction methods
• According to manufacturer

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NAATs Study 5 (2)
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NAATs Study 5 (3)

• Crucitti concluded
• The inhibitory effect of gels that will be used
  in clinical trials should be assessed on the
  current NAATs, prior to the start of the trials.
• The outcome of the interference study could
  influence the choice of the amplification assay,
  or require modifications to the test procedure.
• The inhibitory effect should be assessed not only
  in vitro but also in vivo.

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Conclusion

• Measurement of 2º endpoints in Microbicide
  clinical trials poses many challenges
• Prior to testing of STIs in Microbicide trials,
  in-vitro and in-vivo testing must be done on the
  individual Microbicide and the placebo to
  determine the optimal test and modifications
  required to the procedure
• If not done, the 2º endpoints data cannot be
  validated

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• THANK YOU