Mbida, André Dieudonné PhD*; Sosso, Samuel PharmD†; Flori, Pierre PharmD, PhD‡; Saoudin, Henia PharmD§; Lawrence, Philip§; Monny-Lobé, Marcel MD†; Oyono, Yves*; Ndzi, Edward*; Cappelli, Giulia PhD†; Lucht, Frédéric MD, PhD‖; Pozzetto, Bruno MD, PhD§; Oukem-Boyer, Odile Ouwe Missi PhD†; Bourlet, Thomas PharmD, PhD§
The quantification of HIV-1 RNA levels in blood plasma is an important tool for determining the stage of infection and monitoring the response to antiretroviral therapy (ART) of HIV-infected patients. In resource-limited countries, where 90% of new infections occur,1 there is an increasing access to ART for HIV-infected people, especially thanks to action plans decided in 2000 during the International AIDS Conference of Durban and to the “3 by 5” initiative. However, there is a lack of access to viral load testing, in part due to the requirement of sophisticated equipments for the sampling and the amplification process, which are not readily available in many settings. Particularly, an adequate storage facility for preserving samples before testing is a crucial point. Therefore, in these resource-limited settings, the measurement of HIV-1 RNA load in dried blood spots (DBSs) or dried plasma spots (DPSs) has practical advantages over plasma, which has to be frozen at −80°C until use, and allow the transportation of samples from sites faraway from the reference laboratory. DBS and DPS have been extensively used for HIV-1 antibody testing,2,3 molecular diagnosis in pregnant women and in infants,4-6 viral load quantification of p24 antigen7,8 and RNA,8-13 and very recently for genotyping resistance testing14,15 and molecular epidemiology.16 Among the filter papers tested, Whatman specimen collection paper no. 903 seems to give the best results.10
The threshold of sensitivity of viral loads obtained from DBS or DPS is also of high importance, especially in terms of viral rebound in ART-experienced patients. Mainly 3 HIV-1 RNA quantification commercial assays have been evaluated using DBS and DPS: NASBA (Organon Technika, Durham, NC), NucliSens (BioMérieux, Lyon, France), and Amplicor HIV-1 Monitor (Roche Diagnostics Molecular Systems, Alameda, CA) tests, respectively, all exhibiting a sensitivity threshold ranging from 3 to 4 log copies per milliliter.9-11,17
However, most of the studies dedicated to the evaluation of DBS or DPS for the quantification of HIV-1 RNA have been done in reference laboratories under controlled conditions, but little data exist about their use under local conditions, especially in regions harboring a tropical climate.
Another debated point is the stability of RNA on DBS and DPS, especially after storage at room temperature; heterogeneous results are reported on this point in the literature.10,11,17-19
The aims of our study were (i) to evaluate the suitability for viral load testing of DBS and DPS specimens spotted in 2 Cameroonian rural dispensaries, by locally trained people, under tropical climate conditions; (ii) to test the stability of RNA on DBS and DPS within a 3-month period; and (iii) to assess the performance of the Abbott Real-Time HIV-1 technology in combination with these specimens.
PATIENTS AND METHODS
In November 2007, 41 individuals were sampled by venous puncture in 2 Cameroonian rural dispensaries, “La Fraternité d'Awaé” (Yaoundé) and “Obout” (M'Balmayo district), after giving their informed consent. Their mean age was 34 years (range: 18-59 years) and the male to female sex ratio was 0.38. Twenty-seven previously known HIV-positive patients, including 12 under ART (Triomune), and 14 HIV-1-negative persons were selected. Ethical clearance was given by the Ethical Committee of the “Institut de Recherches Médicales et d'Etude des Plantes Médicinales” (IMPM, Yaoundé, Cameroun).
Twenty HIV-1-infected patients monitored at the Department of Infectious Diseases of the University Hospital of Saint-Etienne (France), and exhibiting detectable HIV-1 RNA levels in plasma (>1.6 log copies/mL) were also included as a control group, after they gave their informed consent. Their mean age was 38 years (range: 24-52 years) and the male to female sex ratio was 4.
After a training period, the spotting of the cards was done by local employees of the dispensaries (a technician and a nurse). For each sample of blood, 4 Whatman 903 cards were used, 2 for DBS and 2 for DPS, respectively. For DBS, 5 spots of 50 μL of whole blood were spotted onto each card. For DPS, after 20 minutes of centrifugation at 3000g, 5 spots of 50 μL of plasma were deposited onto each Whatman 903 card. The remaining liquid plasma (LP) was kept in a refrigerated bag and transported by car to the “Chantal Biya” International Reference Centre (CIRCB), within 12 hours after sampling and kept frozen at −80°C until use. Cards were dried overnight at ambient temperature in the dispensary (Cameroonian samples) or under biosafety hood at the Laboratory of Virology of Saint-Etienne (control group samples). Once dried, each card was packaged in a ziplock bag and stored at ambient temperature before testing. An additional blood sample was taken from HIV-positive subjects from the dispensaries for CD4 cell count.
Routing of the Samples
One series of DBS and DPS cards collected in Cameroon was routed back with the investigators and tested at the Laboratory of Virology of Saint-Etienne Hospital, within 2-6 weeks after sampling (series 1). The second series was sent by mail from Yaoundé to Saint-Etienne and tested after a delay of 3-4 months of storage at ambient temperature (series 2) at the Saint-Etienne Laboratory of Virology. This period was selected to be longer than that within which samples are usually tested. LP samples were tested at the CIRCB in Yaoundé.
Viral Load Quantification
All the LP samples were prepared manually using the Abbott mSample Preparation System, followed by manual reaction assembly. DBS and DPS samples were tested using the Abbott Real-Time HIV-1 Assay combining an automated extraction by the m2000sp apparatus and a real-time amplification by the m2000rt instrument. For the DBS and DPS samples, the RNA extraction was done according to the following procedure: briefly, two half-in spots were cut from each card using a hole puncher. Between each card, the puncher was cleaned by punching through a clean card according to the procedure described by Driver et al.20 Spots were transferred to tubes containing 1.7 mL of Abbott Lysis Buffer and incubated 2 hours under intermittent shaking. The eluate was then transferred to a 12 × 75 mm polypropylene reaction vessel. Specimens were processed along with controls and calibrators using the 1 mL DBS/DPS protocol on m2000sp, according to the manufacturer's instructions. Real-time detection was performed on m2000rt using the respective 1 mL DBS/DPS protocol, according to the manufacturer's instructions. LPs were tested using the 0.6 mL m2000rt plasma protocol. Because of the low quantity of remaining LP samples and according to the manufacturer's recommendations, we decided to use the 0.6 mL protocol instead of the 1 mL protocol, considering that the thresholds of detection between the 2 protocols are comparable from a clinical point of view. When a discrepancy of more than 0.5 log copies per milliliter was observed between results obtained from DBS, DPS, or LP sample from the same patient, the outlier was tested again (when the number of spots was sufficient).
CD4 Cell Count
This test was done at the IMPM using the CyFlow Counter (Partec Afrique Centrale, Douala, Cameroun), less than 12 hours after sampling.
Student t test was used to compare the mean values of viral loads. Spearman rank correlation test was used to measure the correlation between the RNA viral loads obtained on DBS, DPS, and LP. HIV-1 RNA values below the detection limit were assigned 1.6 log copies per milliliter. The statistical significance was set at P < 0.05.
The demographical, clinical, and biological characteristics of the 27 patients sampled in the 2 rural dispensaries and tested positive for HIV-1 are reported in Table 1. All patients under ART exhibited a viral load <1.6 log copies per milliliter, except 1 (1.83 log copies/mL).
Impact of the Sampling Conditions on HIV-1 RNA Quantification
The means of difference between viral loads obtained with LP and DBS were not statistically different for cards sampled in the 2 rural dispensaries in Cameroon (series 1) and those sampled from the control group, under a biohood in the laboratory of Virology of Saint-Etienne (−0.01 ± 0.5 vs −0.09 ± 0.29 log copies/mL); the same result was also obtained when comparing viral loads from LP and DPS (−0.32 ± 0.44 vs −0.06 ± 0.51 log copies/mL).
Comparison of RNA Viral Loads From DBS, DPS, and LP Sampled Under Tropical Conditions (Series 1)
The overall sensitivity values of DBS and DPS were 88.2% and 91.1%, respectively. DBS and DPS percentage of detected samples were 20 and 0 with LP results in the 1.6-2 log copies per milliliter range; 33.3 and 66.6% in the 2-3 log copies per milliliter range; and 100% with LP tested >3 log copies per milliliter (Fig. 1).
The overall specificity was 100% for DBS and DPS assays: All patients tested seronegative for HIV-1 were tested undetectable for HIV-1 RNA from LP, DBS, and DPS. All samples tested undetectable in LP were also undetectable in DBS and DPS.
The coefficients of correlation between the HIV-1 RNA viral loads were calculated by comparing results of LP to DBS and DPS of the first series of cards. The coefficients of correlation of viral loads from LP compared with DBS and from LP compared with DPS were 0.90 (Fig. 2A) and 0.92 (Fig. 2B), respectively (Spearman rank correlation test). In addition, no statistical differences were observed between the mean viral loads obtained from DBS (4.79 ± 1.11 log copies/mL), DPS (4.30 ± 1.09 log copies/mL), and LP (4.88 ± log copies/mL), respectively (Fig. 2C).
Interassay Variability According to the Sampling Method
The difference between viral loads obtained from LP and DBS, and from LP and DPS, was calculated for each patient (series 1 and control group). The differences between DBS and LP viral loads and between DPS and LP with regard to LP values are shown, respectively, in Figures 3A, B; 86.1% of results were within 0.5 log copies per milliliter and 94.4% within 1 log copies per milliliter, when comparing LP to DBS results; 77.7% of results were within 0.5 log copies per milliliter and 97.2% within 1 log copies per milliliter, when comparing LP to DPS results.
The verification of results exhibiting a discrepancy of at least 0.5 log copies per milliliter obtained from DBS, DPS, and LP of the same patient was systematically done. Because similar results were always obtained, with minimal variations (0-0.11 log copies/mL) between the 2 measurements, the first series of values were taken into account for the statistical analysis.
Impact of the Storage Conditions on HIV-1 RNA Quantification
As illustrated in Figure 4A, the coefficient of correlation between the viral loads obtained from series 1 (tested within 2-6 weeks of storage at room temperature after sampling) and series 2 (tested after at least 3 months of storage at room temperature) of DBS was 0.99. For these specimens, 100% of results were within 0.5 log copies per milliliter, when comparing DBS from series 1 and series 2.
With regard to DBS, for DPS, the coefficient of correlation between the values obtained from series 1 and 2 was found to be lower (0.66); the mean decrease of viral load was 1.26 ± 0.69 log copies per milliliter (Fig. 4B).
In the present study, we demonstrate the ability of the Real-Time HIV-1 assay (Abbott Molecular Diagnostics), combining an automated RNA extraction by the m2000sp apparatus and viral load measurement by the m2000rt protocol, to quantify HIV RNA from DBS and DPS. We found excellent correlation rates between viral loads tested from LP compared with those tested from DBS or DPS. Indeed, numerous commercial reverse transcriptase-polymerase chain reaction (RT-PCR) assays have been previously tested with DBS and/or DPS supports including NASBA HIV-1 RNA QT System (Organon Teknika);9-10,18,21 Amplicor HIV Monitor kit (Roche Diagnostics Molecular Systems),9,17,21 Primagen Retina Rainbow Assay,13 and recently NucliSens EasyQ HIV-1 assay (BioMérieux).19 The majority of these studies evidenced a good correlation rate between RNA viral loads obtained from DBS and LP.9-13,18,19 One study reported a lack of reproducibility using NASBA test from DBS spotted on 903 filter papers, probably due to the presence of RNAses on the cards;10 Brambilla et al9 also described less variation with the NucliSens assay compared with the Monitor test, in a multicenter study. A good correlation was also found between viral loads obtained from DBS spotted on Guthrie's paper and whole blood samples, using the NASBA system.11 Concerning DPS samples, comparable data to those obtained using LP samples are reported in several studies,9,13 but 1 recent paper described a significant mean decrease of 0.64 log copies per milliliter between LP and DPS results obtained with the Amplicor monitor 1.5 assay.17 In our experience, we also observed a mean decrease of 0.58 log copies per milliliter between LP and DPS results; however, this difference was not statistically significant.
An important goal to achieve when using DBS or DPS is a sensitive threshold of RNA viral load detection, at least comparable to that established from LP. Although our study was not designed to determine the detection threshold of the protocol, it can be estimated to be around 3 log copies per milliliter. Other comparable thresholds of detection have been reported varying from 3 to 4 log copies per milliliter, using NASBA, NucliSens, or Monitor Amplicor assays.9-11,17 Although this sensitivity level allows a proper decision to be made concerning the initiation of therapy, according to the actual guidelines (CD < 350 cells/mm3 or viral load >5 log copies/mL), DBS or DPS could fail to detect early virological rebound due to ART resistant strains. However, in poor resource setting countries, the World Health Organization recommend to use a higher cutoff (ie, 4 log copies/mL) before changing regimen from the first-line to the second-line therapy.22 Even though, further studies are needed to establish the appropriate cutoff for switching therapy in resource-limited settings, an increase of sensitivity of commercial RT-PCR viral loads tests coupled to DBS or DPS samples should be pursued.
The ability of these RT-PCR assays to encompass the genetic diversity is also a crucial point. Even if 2 recent studies suggest the good capacity of the Abbott Real-Time PCR assay and of the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time RT-PCR test to amplify non-B strains,23,24 a large comparative study and the definition of an international standard is urgently needed to improve the accuracy of all HIV-1 RNA assays.
The contribution of the cell package to the viral load in DBS seemed to be negligible in our experience. By contrast with a previous report,21 and in agreement with numerous others, we found no evidence of amplification of intracellular RNA in DBS cards because no statistical differences were found when comparing the mean viral loads obtained from DBS, DPS, and LP.13,18,19,25 Therefore, a hematocrit adjustment is not required for the calculation of viral loads obtained from DBS with the Real-Time Abbott assay. In the same way, no proviral DNA was amplified because all LP values found below the limit of detection of the assay (1.6 log copies/mL) were also found undetectable on DBS. These findings are in contrast with a recent report showing that more than 20% of patients under highly active antiretroviral therapy and with undetectable viremia on LP were positive when tested on DBS but negative on DPS with the Roche COBAS TaqMan real-time RT-PCR assay.26 Previous studies have also shown that polymerase chain reaction (PCR) inhibitors such as proteins, the porphyrin moiety of hemoglobin from erythrocytes, and iron seem to be increasingly resistant to elution during storage and do not impair the quantification of HIV RNA from DBS.21,25,27,28 Our results confirm these observations because a significant correlation was found between viral loads obtained from DBS and DPS.
The quantification of viral load in blood of HIV-infected patients remains the best marker for the follow-up of ART efficacy. Recent initiatives like the US President's Emergency Plan for AIDS Relief, the Global Fund against AIDS, and also the Clinton Foundation for children have dramatically increased the availability of antiretroviral drugs for HIV-infected populations in developing countries. In Cameroun since 2007, ART therapy is free for all eligible HIV-infected patients. Therefore, the necessity of the access to both CD4+ cells count and viral load assays for the initiating and the monitoring of ART in infected patients of resource-poor areas is no longer disputed. With regard to commercial viral load assays, several alternative options are available. Some authors have recently suggested the use of an ultrasensitive p24 assay as an alternative to replace HIV RNA quantification.7,8,29,30 If this approach seems promising for the diagnosis, especially in the pediatric population, the lack of specificity on DBS samples, and sensitivity for the amplification of some subtypes, and the poor correlation between kinetics of p24 and HIV RNA in infected patients dramatically reduce its usefulness for the therapeutic management of patients. To improve the sensitivity level of p24, a low-cost dynamic model technology so-called SMARThivVLmos including a “modified PCR-based” protocol for viral load values below 5000 copies per milliliter (3.70 log copies/mL) has been recently described, but not yet extensively evaluated.31 Another approach is to measure the reverse transcriptase (RT) activity of HIV-1 in plasma using the Cavidi Exavir load test. This assay exhibits a sensitivity threshold of around 1000 copies per milliliter and seems especially adapted to low-volume samples. But, unfortunately, the RT activity of strains resistant to RT inhibitory drugs is decreased.32
For these reasons and to allow comparable management in terms of the decision to start therapy and the surveying of viral escape, it seems reasonable to consider that quantification of RNA by standardized real-time PCR assays is the most appropriate approach to manage HIV-infected people in resource-limited areas, as is the case in northern countries. Indeed, 1 critical point of these assays is their cost. Recently, a low-cost real-time RT-PCR assay developed by the French Agence Nationale de Recherches sur le SIDA,33 packaged and distributed by Biocentric (Bandol, France), has been compared with the Amplicor Monitor assay (version 1.5; Roche Diagnostics Molecular Systems).34 The authors reported a good correlation between the 2 assays, but false-positive results were generated; they also underlined the need for an expensive PCR instrument for this generic HIV viral load assay. Until now, no data have been published on the use of this test on DBS or DPS samples.
DBS and DPS potentially represent suitable methods of collection, storage, and routing of blood samples, which can be used for RNA HIV quantification, especially in areas lacking adequate infrastructures for the management of frozen plasma. Using spots has technical and economic advantages over collecting plasma because no plasma separation (for DBS) and no cold chain are required and it also represents a noninfectious mean of routing. However, the realization of spots on filter paper requires several precautions during pipetting, hand manipulation of cards, and drying steps to avoid false-negative results related to the presence of RNAses or to the degradation of RNA during the drying step and/or false-positive results due to cross-contaminations. Our study demonstrates that DBS and DPS can be done by trained local nonspecialists, dried, and transported under tropical climate conditions because no differences were observed with results obtained with cards spotted under an air-controlled cabinet at the Laboratory of Virology of Saint-Etienne (France) by trained personnel. Little data exist about their use under local conditions, especially in regions harboring a tropical climate. Solomon et al have previously described DBS as a valuable tool for widespread HIV serosurveillance in remote areas within tropical countries. So far, only 2 studies reported the realization of DBS for HIV-1 viral load testing and their shipment under field conditions in Mexico and in Senegal.18,19 In our study, venous puncture was done to obtain a large volume of blood, allowing us to undertake this comparative study. Provided that the correct sample input volume is used, peripheral blood obtained by finger puncture could easily replace venous puncture to obtain DBS because it requires minimal training and decreases the risks associated with disposal of needles and syringes. Because DBS does not need venous puncture sampling, special handling for collection, and transportation through a cold chain, this seems a cheap and adequate method for resource-limited areas.
Another important finding of our study was that whole blood but not plasma could be stored on Whatman 903 filter paper cards for up to 3 months at room temperature, before testing HIV-1 RNA viral load. Very heterogeneous data are present in the literature on this topic. It has been shown that DBS could be stored at 4, 22, and 37°C for 7 days,18 8 and 15 days at 37°C,19 or 1 year at −70°C.11 Concerning DPS, a good conservation was obtained after 3 days at 37°C and 2 weeks at 20°C in 1 study,10 but a degradation of RNA was observed after 7 days of storage at 22°C in another.17
As a conclusion, our study clearly demonstrates that DBS represent an inexpensive means of collection and storage of blood, which can be performed under local conditions in resource-limited settings and are suitable for the differed quantification of HIV-1 RNA.
We are grateful to the study participants for their dedication to the study and to Mother Anne Morin from the monastery “Les Trappistines d'Obout” for her excellent welcome and accommodation during the study. We also thank Abbott Molecular Diagnostics for technical support and for providing the kits for the assays and Joseph Aubin Nanfack at CIRCB for his excellent technical assistance.
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