Stability of HIV-1 in human plasma samples at 37°C for up to 6 days.

Sir,

Viral load monitoring is an important factor to access the effectiveness of antiviral therapy in HIV-1 patients. This is directly dependent on reliable procedure followed for processing of samples prior to quantification. In most of the countries, the laboratories which carry out viral quantification are stationed at distant places from the rural areas. Preprocedure and logistic factors such as storage and transport conditions can affect the quality of results. It is recommended that samples should be tested within 24 h of their collection. However, it is not feasible in resource-limited areas and samples may take more than 24 h to reach the testing laboratory. During this time period, samples are stored in cooled gel packs. Viral RNA decay has been reported in simulation studies.[1] The degree of decay due to these preprocedural factors is not known. The whole-blood samples collected in ethylenediaminetetraacetic acid did not reveal any change in viral load at room temperature up to 7 days. However, whole-blood samples spiked in culture medium showed 1 log viral load decay after 120 days at room temperature.[2] The present study was conducted to find out decay in HIV-1 load in human plasma samples at 37°C for up to 6 days. Five HIV-1 RNA-positive human plasma samples were taken for this study. Eight aliquots of each sample were prepared. Two aliquots of each sample were separately incubated at 37°C in a water bath for 2 days, 4 days, and 6 days and two aliquots of each sample were reserved for controls. After incubation, isolation of HIV-1 RNA and viral load determination was carried out using Cobas TaqMan HIV-1 test, version 2.0 and Cobas TaqMan 48 (m/s Roche molecular systems, Inc., South Branchburg, New Jersey, USA), respectively. The values of viral load determined are summarized in Table 1. Two samples, HI-21 and HI-24, showed 0.58 log10 copies/ml and 0.55 log10 copies/ml decrease in viral load, respectively, after incubation for 6 days. However, the difference was less than clinical cutoff after incubating HI-21 and HI-24 for 2 and 4 days. In all other cases, viral load decrease or difference was less than the clinical cutoff of ±0.5 log10 copies/ml. The differences of less than ±0.5 log10 copies/ml are clinically insignificant[3] which indicates that HIV-1 is quite stable in human plasma samples at 37°C for up to 6 days. In other studies, the stability of HIV RNA in whole blood reported is up to 7 days at 4°C and 3 days at 25°C.[4] Less than 0.5 log10 copies/ml decrease in HIV RNA at 25°C in 1 day has been shown by a study conducted in the year 1997.[5] Similar results were obtained in our study. In three different studies, the decrease in HIV RNA at 4°C was 0.04 log copies/ml after 7 days and 0.01 log copies/mL after 14 days. At 30°C, it showed insignificant decline of 0.12 log after 2 days and after 7 days the decline was 0.74 log copies/ml. It further declined to 1.6 log copies/ml after 14 days.[136] The reported decrease in viral load at 37°C after 7 days was 0.92 log copies/ml. However, it was negligible (0.03 log copies/ml) at 22°C after 7 days.[6] In our study, most of the samples showed <0.5 log copies/ml decrease after incubation at 37°C for 6 days. Two samples showed a decrease little over clinical cutoff after 6 days.

Table 1

Viral load of human immunodeficiency virus-1 samples

Sample ID	Incubated for (days)	Replicate-1 viral load (copies/ml)	Replicate-2 viral load (copies/ml)	Mean viral load (copies/ml)	Mean viral load (log10 copies/ml)
HI-11	0	915	1100	1007.5	3
HI-11	2	810	876	843	2.92
HI-11	4	471	682	576.5	2.76
HI-11	6	885	667	776	2.88
HI-12	0	1110	913	1011.5	3
HI-12	2	551	582	566.5	2.75
HI-12	4	632	453	542.5	2.73
HI-12	6	438	324	381	2.58
HI-21	0	1650	1400	1525	3.18
HI-21	2	818	818	818	2.91
HI-21	4	1430	1500	1465	3.16
HI-21	6	95.6	718	406.8	2.6
HI-24	0	5090	7070	6080	3.78
HI-24	2	3680	3600	3640	3.56
HI-24	4	2370	2920	2645	3.42
HI-24	6	1680	1770	1725	3.23
HI-31	0	45,700	74,600	60,150	4.77
HI-31	2	29,300	94,700	62,000	4.79
HI-31	4	29,300	39,700	34,500	4.53
HI-31	6	26,700	191,000	108,850	5.03

HI: HIV

Although the sample size is small, the results of this study indicate that storage and transportation of samples at ≤37°C for a limited duration may not have much effect on test results and their clinical correlation. Therefore, in Indian setup in winters, the samples can be transported without cool/gel pack or any cooling device. Furthermore, there are many areas in India where the temperature remains below 37°C throughout the year. However, in areas where the temperature exceeds 37°C in summers, the temperature of samples during the transport should be maintained at least at 37°C. Further, the study could not be performed with large sample size due to their unavailability; therefore, the outcome of this pilot study needs to be tested with large sample size for better statistical interpretation to draw a final decision.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.