In summary, we have highlighted the large genomic diversity of the most frequent human respiratory viral infection. Our phylogenetic analysis has characterized circulating strains relative to reference strains and has identifi ed a previously unknown enterovirus genotype. We have shown that recombination also contributes to rhinovirus evolution in its natural environment. Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR BACKGROUND: Rapid and cost-effective methods for HIV-1 diagnosis and viral load monitoring would greatly enhance the clinical management of HIV-1 infected adults and children in limited-resource settings. Recent recommendations to treat perinatally infected infants within the first year of life are feasible only if early diagnosis is routinely available. Dried blood spots (DBS) on filter paper are an easy and convenient way to collect and transport blood samples. A rapid and cost effective method to diagnose and quantify HIV-1 from DBS is urgently needed to facilitate early diagnosis of HIV-1 infection and monitoring of antiretroviral therapy. METHODS AND FINDINGS: We have developed a real-time LightCycler (rtLC) PCR assay to detect and quantify HIV-1 from DBS. HIV-1 RNA extracted from DBS was amplified in a one-step, single-tube system using primers specific for long-terminal repeat sequences that are conserved across all HIV-1 clades. SYBR Green dye was used to quantify PCR amplicons and HIV-1 RNA copy numbers were determined from a standard curve generated using serially diluted known copies of HIV-1 RNA. This assay detected samples across clades, has a dynamic range of 5 log(10), and %CV <8% up to 4 log(10) dilution. Plasma HIV-1 RNA copy numbers obtained using this method correlated well with the Roche Ultrasensitive (r = 0.91) and branched DNA (r = 0.89) assays. The lower limit of detection (95%) was estimated to be 136 copies. The rtLC DBS assay was 2.5 fold rapid as well as 40-fold cheaper when compared to commercial assays. Adaptation of the assay into other real-time systems demonstrated similar performance. CONCLUSIONS: The accuracy, reliability, genotype inclusivity and affordability, along with the small volumes of blood required for the assay suggest that the rtLC DBS assay will be useful for early diagnosis and monitoring of pediatric HIV-1 infection in resource-limited settings. It is estimated that 33.2 million people were infected with HIV-1 at the end of 2007; 2.5 million were children under 15 years of age, the majority of whom acquired infection through mother-tochild transmission (MTCT; [1] ). Antiretroviral therapy (ART) is effective at blocking MTCT and can markedly improve the outcome of pediatric HIV-1 infection. However, efforts to provide widespread access to ART have been hampered by the limited availability of infant diagnostic methods. [2] [3] [4] [5] [6] Methods to diagnose and monitor HIV-1 infection in resource-poor settings are usually limited to serologic assays and CD4/CD8 counts. [2, 7, 8] However, antibody based assays can reliably guide diagnosis and management only after 18 months of age following clearance of passively transferred maternal antibodies. [9] PCR based nucleic acid amplification and quantification of HIV-1 is the gold standard for early HIV-1 diagnosis in infants and for evaluating ART efficacy. [2, 5, [10] [11] [12] [13] [14] [15] [16] Several commercial nucleic acid based molecular tests are available for HIV-1 diagnosis and viral load quantification. [15] These commercial assays require relatively large volumes of blood that need to be processed for plasma, stored, and transported under special conditions. The cost, run-time, training, maintenance and infrastructure needed to perform these assays have also limited their use in low resource settings. [6, 17, 18] Practical and reliable methods to obtain, store, and transport blood samples are necessary to develop cost effective early diagnostic assays in limited-resource settings. Spotting of whole blood onto filter paper offers technical and economic advantages over conventional venipuncture methods since it simplifies sample collection and transport to reference laboratories for diagnostic testing and viral load quantification. [17, [19] [20] [21] [22] [23] [24] In the present study, we describe a LightCycler-based real time PCR assay (rtLC) to quantify viral loads using RNA extracted from filter paper containing dried blood spots. DBS prepared either under controlled conditions in a laboratory setting by research technicians or in a clinical setting by health-professionals were successfully evaluated. This assay has comparable reproducibility, diagnostic accuracy, specificity and broader linear range at a lower cost compared to probe-based commercial assays. The rtLC HIV-1 DBS assay is also capable of detecting and quantifying different clades of HIV-1 without sacrificing sensitivity.