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

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 log10, and %CV \u3c8% up to 4 log10 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

Enhanced survival following oral and systemic <i>Salmonella enterica</i> serovar Typhimurium infection in polymeric immunoglobulin receptor knockout mice

<div><p>Background</p><p>Polymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. <i>Typhimurium</i>), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. <i>Typhimurium</i> virulence in pIgR knockout (KO) and wildtype (WT) mice.</p><p>Methods</p><p>In uninfected animals, we harvested jejunal segments for Ussing chamber analyses of transepithelial resistance (TER); mesenteric lymph nodes (mLN) for bacterial culture; and serum and stool for IgA. Separately, we infected mice either orally or intravenously (IV) with <i>S</i>. <i>Typhimurium</i> to compare colonization, tissue dynamics, and inflammation between KOs and WTs.</p><p>Results</p><p>Uninfected KOs displayed decreased TER and dramatically increased serum IgA and decreased fecal IgA vs. WT; however, KO mLNs yielded fewer bacterial counts. Remarkably, WTs challenged orally with <i>S</i>. <i>Typhimurium</i> exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection.</p><p>Conclusions</p><p>Absence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor <i>S</i>. <i>Typhimurium</i> infection. These findings raise the possibility that immune adaptation to increased gut permeability and elevated serum IgA in the setting of SIgA deficiency provides compensatory protection against invasive gut pathogens.</p></div

Increased survival of pIgR KO following high-dose oral <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were gavaged with 10⁹ CFU of <i>S</i>. <i>Typhimurium</i> following a 4-hour fast and followed for 2 weeks. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.05, log-rank Mantel-Cox test). Data are representative of 3 experiments, with equivalent infectious doses between KO and WT in each experiment.</p

Increased survival of pIgR KO following intravenous <i>S</i>. <i>Typhimurium</i> challenge.

<p>Mice were injected IV via the tail vein with 3.2x10² CFU of <i>S</i>. <i>Typhimurium</i> and followed for 26 days. Mean survival for C57BL/6 mice was statistically significantly shorter than the pIgR KO mice (<i>P</i><0.0001, log-rank Mantel-Cox test). Data are representative of 2 experiments.</p

Elevated serum IgA, decreased stool IgA, and decreased IgA coating of fecal bacteria in pIgR KO mice.

<p>(A) Serum IgA is significantly increased in pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.0001, Mann-Whitney). (B) No significant difference in the serum IgG between pIgR KO and C57BL/6 mice. (C) Stool IgA is significantly decreased in pIgR KO mice compared to C57BL/6 mice (<i>P</i><0.0001, Mann-Whitney). (D) pIgR KO mice showed a 3-fold reduction in the percentage of stool bacteria coated with IgA (<i>P</i><0.0001, Mann-Whitney).</p

Serum pro-inflammatory cytokines TFNα, IL-1β, and IL-6 are significantly lower in pIgR KO mice vs wildtype mice 7 days post oral <i>S</i>. <i>Typhimurium</i> infection.

<p>C57BL/6 mice showed significantly increased (A) TNFα (<i>P</i><0.0001, Mann-Whitney), (B) IL-1β (<i>P</i><0.005, Mann-Whitney test), and (C) IL-6 (<i>P</i><0.0001, Mann-Whitney) compared to pIgR KO. (D) No difference in IFNγ levels between groups.</p

Intestinal and systemic indicators of <i>S</i>. <i>Typhimurium</i> infection intensity are reduced in pIgR KO mice following oral challenge.

<p>(A) Spleen weight was significantly elevated in C57BL/6 mice 7 days post-oral infection compare to both uninfected C57BL/6 (<i>P</i><0.001) and infected pIgR KO (<i>P</i><0.01, Kruskal-Wallis with Dunn multiple comparison test). (B) Cecum weight was significantly diminished in C57BL/6 mice compared to pIgR KO mice at 7 days post-oral infection (<i>P</i><0.05, Kruskal-Wallis with Dunn multiple comparison test). Data are representative of 3 experiments. Baseline weights of spleen and cecum did not differ in uninfected C57BL/6 and pIgR KO mice.</p