Efficient methodologies for sensitive HIV-1 RNA quantitation from plasma and vaginal secretions.
BACKGROUND
Quantifying HIV levels in mucosal secretions is essential to study compartmentalized expression of HIV and facilitate development of intervention strategies to prevent disease progression and transmission.


OBJECTIVE
To develop a sensitive, reliable, and cost-effective technique to quantify HIV from blood and vaginal secretions that is compatible with efficient implementation in clinical research environments.


METHODS
A sensitive, reliable, internally-controlled real-time reverse transcriptase (RT) PCR assay, which uses the HIV-1 pol gene as a target (Hpol assay) was developed to quantify HIV levels in plasma and genital secretions, and compared to the widely used Roche Amplicor HIV-1 Monitor assay. In addition, a simplified method of sample collection and processing of genital secretions (self-collection and use of RNAlater with batch processing) was compared to provider collection of samples and immediate processing.


RESULTS
The sensitivity and reliability of HIV levels detected by the assay described herein correlate well with measurements from Roche Amplicor HIV-1 Monitor assay for both plasma and vaginal secretions (R(2)=0.9179 and R(2)=0.942, respectively). The Hpol assay reproducibly quantifies a lower limit of 5 HIV-1 RNA copies per reaction, with low-levels of inter-assay and intra-assay variation. Additionally, vaginal viral levels and detection frequency did not differ significantly between the two the collection and processing methods.


CONCLUSIONS
The methodologies developed here provide sensitive, reliable, and cost-effective quantification of HIV levels in plasma and mucosal secretions, and are compatible with efficient use in clinical research studies.
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Tara Randolph
TR
Henning
Nedra
N
Lacour
Angela Martin
AM
Amedee
D005260:Female; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D015497:HIV-1; D006801:Humans; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D014621:Vagina / Q000737:chemistry; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics
D012367:RNA, Viral; D054302:pol Gene Products, Human Immunodeficiency Virus