Table 4.

Minimum HIV-1 RNA load needed to detect minor variants at a frequency (λ) of 0.1%, 1%, and 10%, assuming that the RNA is extracted from 1 mL of plasma.^a

Variant Frequency (λ)	Number of RNA molecules to be tested, according to the Poisson distribution (P>99%)	Fraction of elution volume used for cDNA synthesis (fe)	Minimum HIV-1 RNA load (copies/mL)
0.1%	5000	0.25	29 762
		0.5	14 881
		1	7 440
1%	500	0.25	2 976
		0.5	1 488
		1	744
10%	50	0.25	298
		0.5	148
		1	74

^aCalculations were derived from the formula: pVL= N_RNA(λ) / (V × f_e × E_RNAX× E_cDNA), where pVL is the plasma HIV-1 RNA copy number; N_RNA(λ) is the number of RNA copies that need to be tested according to the Poisson distribution to detect at least 1 variant with a probability > 99% if this variant is present at a frequency λ; V is the volume of plasma used for the RNA extraction in milliliters; f_e is the fraction of the RNA elution volume used for cDNA synthesis; E_RNAX is the efficiency of the RNA extraction process ; and E_cDNA is the efficiency of the cDNA synthesis.

HIV-1 RNA values were estimated using the following assumptions: V=1 mL, E_RNAX=0.96 and E_cDNA= 0.7, based on http://www1.qiagen.com/literature/qiagennews/0398/983hiv1.pdf and http://omrf.ouhsc.edu/~frank/CDNA.html. Importantly, E_RNAX and particularly E_cDNA are subject to significant variation in different conditions, and may need to be determined empirically. Therefore, HIV-1 RNA values would need to be adjusted if different plasma volumes were used for the RNA extraction or if E_RNAX or E_cDNA were different.

Note that the volume of plasma required for RNA extraction in order to detect a variant with frequency λ can be easily calculated as: V = N_RNA(λ) / (p VL × f_e × E_RNAX× E_cDNA).