Prospective Evaluation of Diagnostic Accuracy of Dried Blood Spots from Finger Prick Samples for Determination of HIV-1 Load with the NucliSENS Easy-Q HIV-1 Version 2.0 Assay in Malawi

HIV-1 viral load (VL) testing is not widely available in resource-limited settings. The use of finger prick dried blood spot (FP-DBS) samples could remove barriers related to sample collection and transport. Measurement of VL using DBS from EDTA venous blood (VB-DBS) in place of plasma has previously been validated using the NucliSENS Easy-Q HIV-1 v2.0 assay, but information on the accuracy of FP-DBS samples for measuring VL is limited. This prospective study, conducted at Thyolo District Hospital in southern Malawi, compared VL levels measured on FP-DBS samples and plasma using the NucliSENS Easy-Q HIV-1 v2.0 assay. Comparability was assessed by means of agreement and correlation (131 patients with VLs of ≥100 copies/ml), sensitivity, and specificity (612 patients on antiretroviral treatment [ART]). Samples of EDTA venous blood and FP-DBS from 1,009 HIV-infected individuals were collected and prepared in the laboratory. Bland-Altman analysis found good agreement between plasma and FP-DBS VL levels, with a mean difference of −0.35 log₁₀, and 95% limits of agreement from −1.26 to 0.55 log₁₀. FP-DBS had a sensitivity of 88.7% (95% confidence interval [CI], 81.1 to 94.4%) and a specificity of 97.8% (95% CI, 96.1 to 98.9%) using a 1,000-copies/ml cut point and a sensitivity of 83.0% (95% CI, 73.4 to 90.1%) and a specificity of 100% (95% CI, 99.3 to 100%) using a 5,000-copies/ml cut point. This study shows that FP-DBS is an acceptable alternative to plasma for measuring VL using the NucliSENS Easy-Q HIV-1 v2.0. We are conducting a second study to assess the proficiency of health workers at preparing FP-DBS in primary health care clinics.     

Evaluation of Quantification of HIV-1 RNA Viral Load in Plasma and Dried Blood Spots by Use of the Semiautomated Cobas Amplicor Assay and the Fully Automated Cobas Ampliprep/TaqMan Assay,Version 2.0, in Kisumu,Kenya

In Kenya, HIV-1 viral load monitoring is commonly performed with the Cobas Amplicor using plasma specimens. Interest is growing in transitioning to real-time PCR (RT-PCR), such as the Cobas Ampliprep/Cobas TaqMan (CAP/CTM), using dried blood spots (DBS). Before implementation, direct evaluation of the two assays using DBS field specimens is required. This study compares the sensitivity, specificity, negative and positive predictive values (NPV and PPV, respectively), concordance, and agreement between HIV-1 viral load measurements using plasma and DBS specimens obtained from 512 HIV-1-infected pregnant females enrolled in the Kisumu Breastfeeding Study and tested with the Cobas Amplicor and CAP/CTM assays. The sensitivity and NPV of viral load detection in DBS specimens were higher with CAP/CTM (sensitivity, 100%; 95% confidence interval [CI], 99.1 to 100.0%; NPV, 100%; 95% CI, 59.0 to 100.0%) than the Cobas Amplicor (sensitivity, 96.6%; 95% CI, 94.3 to 98.1%; NPV, 58.8%; 95% CI, 40.7 to 75.4%). The PPVs were comparable between both assays when using DBS. The specificity of viral load detection in DBS specimens was lower with CAP/CTM (77.8%; 95% CI, 40.0 to 97.2%) than that of the Cobas Amplicor (95.2%; 95% CI, 76.2 to 99.9%). Good concordance and agreement were observed when paired plasma and DBS specimens were tested with both assays. Lower specificity with the CAP/CTM is likely due to proviral HIV-1 DNA amplification and lower detection limits with RT-PCR. However, the CAP/CTM has better sensitivity and higher throughput than the Cobas Amplicor. These findings suggest that DBS may be a suitable alternative to plasma when using RT-PCR, which could increase access to viral load monitoring in resource-limited settings.     

Qualitative Human Immunodeficiency Virus RNA Analysis of Dried Blood Spots for Diagnosis of Infections in Infants

The Gen-Probe Aptima human immunodeficiency virus type 1 (HIV-1) RNA assay was adapted for the diagnosis of HIV infection in infants by using dried blood spots. The assay was 99% sensitive (128/129) and 100% specific (162/162). This may prove useful in resource-limited settings, since it precludes the need for a phlebotomist and maintenance of a cold chain.     

Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at −20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of ≥1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 real-time PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory performance should be monitored.     

Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Infants by Use of Dried Blood Spots and an Ultrasensitive p24 Antigen Assay

We tested 617 dried blood spots (DBS) from human immunodeficiency virus-exposed infants from five countries using an ultrasensitive p24 antigen assay (Up24). The sensitivity was 94.4% (67/71) and the specificity was 100% (431/431) for infants with DBS specimens ≤20 months old; DBS older than 30 months demonstrated only 72.2% sensitivity (39/54) (P < 0.001) but displayed 100% specificity (61/61).     

Quantitation of Cytomegalovirus DNA Load in Dried Blood Spots Correlates Well with Plasma Viral Load

An assay to accurately quantitate cytomegalovirus (CMV) load in finger-stick-collected dried blood spots (DBS) could potentially be useful for field studies or for analyzing patient self-collected specimens. We therefore assessed CMV DNA load in paired venipuncture-collected plasma samples and finger-stick DBS, using a previously validated quantitative PCR assay. Assay variability, sensitivity, and changes in viral load during antiviral therapy in finger-stick DBS were compared to the reference plasma quantitative PCR assay, using 106 prospectively collected pairs of finger-stick DBS and plasma samples from 35 solid-organ transplant (SOT) patients. The DBS assay showed good agreement with the reference plasma viral load assay on the log₁₀ scale (Pearson correlation coefficient, 0.92; P < 0.001). The 95% limit of detection of the DBS assay was estimated at 2,700 plasma copies/ml (675 plasma IU/ml). In 94% (76/81) of paired DBS and plasma samples above the limit of detection, the difference in CMV load was <1 log₁₀. CMV viral load changes during antiviral treatment were comparable in plasma and DBS. We conclude that finger-stick DBS provides a convenient sample type for quantitation of CMV load that correlates well with plasma levels. Future studies to optimize and evaluate this methodology for patient self-collected samples are warranted.     

Development and Evaluation of an ompA Quantitative Real-Time PCR Assay for Chlamydia trachomatis Serovar Determination

Knowledge of circulating Chlamydia trachomatis serovars can be beneficial for sexual network surveillance, monitoring treatment success, and associating specific clinical manifestations. Typically, C. trachomatis serovars are predicted by nucleotide sequencing of four variable domains within the ompA gene. However, sequencing procedures can be labor-intensive, are not readily available, and can lack the capacity to identify multiple serovars. This study describes the development and evaluation of a quantitative real-time PCR (qPCR) test algorithm for the rapid prediction of C. trachomatis serovars, including ocular (A to C) and anogenital (D to L3) strains. This test comprises a primary qPCR to confirm C. trachomatis positivity and the phylogenetic group(s) present and a secondary set of qPCRs to determine specific serovars. Cell culture isolates from 15 prototypic C. trachomatis serovars were correctly identified using this assay, with no cross-reactivity observed among serovars or with other common pathogenic microorganisms. Five hundred clinical specimens (previously diagnosed as being C. trachomatis positive) were evaluated by qPCR, with their results compared to results obtained by conventional sequencing. The qPCR identified 88.9% (423/476) complete matches (95% confidence interval [CI], 86 to 92%) of serovars compared to the results obtained using the sequence-based approach. Among the anogenital specimens, 2.4% (12/494) (95% CI, 1.3 to 4.2%) contained multiple serovars, categorized as single-serovar infections by conventional sequencing. Overall, this test exhibited high discriminatory success for predicting C. trachomatis serovars, particularly among anogenital infections. This is the first report of a qPCR typing assay offering differentiation of C. trachomatis serovars associated with both anogenital and ocular diseases.Chlamydia trachomatis is the causative agent for one of the most common sexually transmissible infections worldwide. The majority of infections are asymptomatic and thereby left untreated, which can lead to a variety of human diseases across multiple organ systems. Urogenital infections are the most predominant, often associated with urethritis and epididymitis in men and cervicitis and pelvic inflammatory disease, with the potential outcome of ectopic pregnancy or tubal infertility, in women (3, 4, 5, 21, 30). C. trachomatis infections are also responsible for trachoma-associated blindness (16) and lymphogranuloma venereum (LGV), characterized by genital ulcers, inguinal lymphadenopathy, or proctocolitis (13).C. trachomatis strains are divided into 15 distinct serovars based on their antigenic reactivity with specific monoclonal antibodies (17, 29). These different serovars are associated with characteristic clinical manifestations; notably, serovars A to C are associated with ocular disease (trachoma and conjunctivitis), serovars D to K with typical urogenital disease (6), and serovars L1 to L3 with systemic LGV (27, 29). Currently, C. trachomatis serovar prediction relies on nucleotide sequencing of the ompA gene, which encodes the major outer membrane protein (MOMP). The ompA gene contains four symmetrically spaced variable domains (VDs; VDI to VDIV), encoding major antigenic determinants flanked and interspaced by five conserved domains (1, 24, 32). Distinct nucleotide sequence variations within the ompA VDs of different serovars have been shown to facilitate C. trachomatis serovar prediction (2, 10, 12, 27). C. trachomatis serovars can be subdivided into three distinct phylogenetic clades based on the ompA gene: the B group (comprising B/Ba, D, E, L1, and L2), the C group (comprising A, C, H, I, J, K, and L3), and the intermediate (I) group (comprising F and G). Although there is no correlation of phylogenetic clades with characteristic disease, these clades/groups have value for the development of more rapid screening assays for predicting serovars (19, 20, 33).Determination of circulating C. trachomatis serovars within a population can provide information on the epidemiology and pathogenesis of infection (7), including mapping sexual networks, can allow for monitoring treatment success, and may play a role in developing strategies for improved disease control, such as vaccine design. This study describes the development and evaluation of a quantitative real-time PCR (qPCR) approach for use in the rapid determination of infecting C. trachomatis serovars to alleviate the laborious demands associated with nucleotide sequencing and analysis.     

Field Study of Dried Blood Spot Specimens for HIV-1 Drug Resistance Genotyping

Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at −80°C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings.     

Evaluation of the Nuclisens HIV-1 QT Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma

Nuclisens HIV-1 QT is a new version of the NASBA HIV-1 QT assay for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma. The specificity of this assay was 100% in one laboratory and 99%—with nonrepeatability of the initial false positive—in another. The test was linear between 2.0 and 6.0 log RNA copies per ml. According to the input HIV-1 RNA concentration, accuracy varied from −0.11 to +0.10 log RNA copy per ml and precision varied from 0.66 to 0.14 log RNA copy per ml. Reproducibility decreased when the HIV-1 RNA level was near the lower limit of quantitation of the test. HIV-1 RNA could be quantitated by Nuclisens HIV-1 QT in 36% (laboratory 1) and 24% (laboratory 2) of clinical samples with HIV-1 RNA levels lower than the lower limit of quantitation by NASBA HIV-1 QT. Nuclisens HIV-1 QT was not suitable for measurement of RNA from clade G and group O HIV-1 strains.     

Design and Implementation of an External Quality Assessment Program for HIV Viral Load Measurements Using Dried Blood Spots

An external quality assurance program was developed for HIV-1 RNA viral load measurements taken from dried blood spots using a reference panel and field-collected specimens. The program demonstrated that accurate and reproducible quantitation can be obtained from field-collected specimens. Residual proviral DNA may confound interpretation in virologically suppressed subjects.     

Evaluation of Bio-Rad Geenius HIV-1 and -2 Assay as a Confirmatory Assay for Detection of HIV-1 and -2 Antibodies

The performance of the new Bio-Rad Geenius HIV-1/2 confirmatory assay was compared with that of the Chiron RIBA HIV-1/HIV-2 strip immunoblot assay using 166 samples from HIV-1-, HIV-2-, or HIV-1/2-positive and HIV-negative individuals and two quality control panels. Our results indicate that the Geenius assay is a suitable alternative for confirmatory HIV testing.     

Evaluation of supplemental testing with the Multispot HIV-1/HIV-2 Rapid Test and APTIMA HIV-1 RNA Qualitative Assay to resolve specimens with indeterminate or negative HIV-1 Western blots

BackgroundThe use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis.ObjectiveTo determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm.Study designRemnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA).ResultsAccording to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive.ConclusionApplying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.     

Evaluation of Dried Blood Spots as a Feasible Alternative to Plasma for the Detection and Quantification of Hepatitis C Virus in a Tropical Setting: A Pilot Study

Introduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients’ blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903^TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log₁₀ IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.     

Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing.     

Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes

We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of bla_KPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the bla_KPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the bla_KPC PCR assay was used to confirm the presence of bla_KPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected bla_KPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of bla_KPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two bla_KPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with bla_KPC-2 and bla_KPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.