Evaluation of SAMBA II: A Qualitative and Semiquantitative HIV Point-of-Care Nucleic Acid Test.

BACKGROUND: Point-of-care (POC) nucleic acid tests (NATs) have potential to diagnose acute HIV infection and monitor persons taking pre-exposure prophylaxis or antiretroviral therapy (ART). POC NATs have not yet been evaluated in the US. METHODS: From June 2018-March 2019, we conducted a cross-sectional evaluation of the Simple Amplification-Based Assay version II (SAMBA II) POC NAT. People with HIV (PWH) and persons testing for HIV were tested with the SAMBA II qualitative (Qual) whole blood (WB) test. From April-September 2019, the Qual test was used on persons who were ART-naive, and SAMBA II Semi-quantitative (Semi-Q) WB was used with ART-experienced PWH. Both were performed on unprocessed venipuncture (VP) and, when indicated by protocol, fingerstick (FS) WB and plasma. SAMBA results were compared with Abbott RealTime HIV-1 polymerase chain reaction results on plasma. We calculated sensitivity, specificity, and concordance between tests. RESULTS: SAMBA was used in 330 visits among 280 participants: 202 (61.2%) visits from PWH, and 128 (38.8%) from HIV-negative persons. Qual test sensitivity with ART-naive participants was 91.4% [32/35, 95% confidence interval (CI): 77.6% to 97.0%] using VP WB and 100% (27/27, 95% CI: 87.5% to 100%) using FS WB. Specificity was 100% using both specimen types. Concordance between the gold standard and Semi-Q at 1000 copies/mL among PWH on ART was 97.7% (86/88, 95% CI: 92.1% to 99.4%) and 100% (30/30, 95% CI: 88.7% to 100%) using VP and FS WB, respectively. CONCLUSIONS: The SAMBA II POC NATs showed high sensitivity, specificity, and concordance with the gold standard assay, indicating its potential use in diagnostics and monitoring. Future work will evaluate POC NAT implementation in the US.

Evaluation of sample pooling using the SAMBA II SARS-CoV-2 test.

BACKGROUND: Screening of infectious asymptomatic or pre-symptomatic individuals for SARS-CoV-2 is at present a key to controling the COVID-19 pandemic. In order to expand testing capability and limit cost, pool testing of asymtomatic individuals has been proposed, provided assay performance is not significantly affected. METHODS: Combined nose and throat (N/T) swabs collected from COVID-19 infected or non-infected individuals were tested using SAMBA II individually and in pools of four (one positive and 3 negative). The evaluation was conducted by the manufacturer and an independent NHS site. Ct cycles of individual positives and pooled positives were determined by qRT-PCR. RESULTS: In 42 pools containing a single positive sample with Ct values ranging between 17 and 36, 41 pools (97.6 %) were found positive by the SARS-CoV-2 SAMBA II test. The false-negative pool by SAMBA was also negative by both reference methods used in this evaluation.The individual positive sample in this pool was positive by SAMBA (Orf only) and by one of the reference methods (S gene only, Ct 35) but negative by the second reference method indicating that the sample itself was very low viral load. All 78 pools containing 4 negative swabs were negative (100 % specificity). DISCUSSION: The preliminary data of the evaluation indicated a high level of performance in both sensitivity and specificity of the SAMBA II assay when used to test pools of 4 patient samples. The implementation of this pooled protocol can increase throughput and reduce cost/test when the prevalence of COVID is low.

Performance Evaluation of the SAMBA II SARS-CoV-2 Test for Point-of-Care Detection of SARS-CoV-2.

Nucleic acid amplification for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory samples is the standard method for diagnosis. The majority of this testing is centralized and therefore has turnaround times of several days. Point-of-care (POC) testing with rapid turnaround times would allow more effective triage in settings where patient management and infection control decisions need to be made rapidly. The inclusivity and specificity of the Simple AMplification-Based Assay (SAMBA) II SARS-CoV-2 test were determined by both in silico analyses of the primers and probes and wet testing. The SAMBA II SARS-CoV-2 test was evaluated for performance characteristics. Clinical performance was evaluated in residual combined throat/nose swabs and compared to that of the Public Health England real-time PCR assay targeting the RdRp gene. The SAMBA II SARS-CoV-2 test has an analytical sensitivity of 250 copies/ml for detecting two regions of the genome (open reading frame 1ab [ORF1ab] and nucleocapsid protein [N]). The clinical performance was evaluated in 172 residual combined nose/throat swabs provided by the Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge (CMPHL), which showed an estimated positive percent agreement of 98.9% (95% confidence interval [CI], 93.83 to 99.97) and negative percent agreement of 96.4% (95% CI, 89.92 to 99.26) compared to testing by the CMPHL. The data show that the SAMBA II SARS-CoV-2 test performs equivalently to the centralized testing methods, but with a shorter turnaround time of 86 to 101 min. Point-of-care tests such as SAMBA should enable rapid patient management and effective implementation of infection control measures.

Combined Point-of-Care Nucleic Acid and Antibody Testing for SARS-CoV-2 following Emergence of D614G Spike Variant.

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity.

Point of Care Nucleic Acid Testing for SARS-CoV-2 in Hospitalized Patients: A Clinical Validation Trial and Implementation Study.

There is an urgent need for rapid SARS-CoV-2 testing in hospitals to limit nosocomial spread. We report an evaluation of point of care (POC) nucleic acid amplification testing (NAAT) in 149 participants with parallel combined nasal and throat swabbing for POC versus standard lab RT-PCR testing. Median time to result is 2.6 (IQR 2.3-4.8) versus 26.4 h (IQR 21.4-31.4, p < 0.001), with 32 (21.5%) positive and 117 (78.5%) negative. Cohen's κ correlation between tests is 0.96 (95% CI 0.91-1.00). When comparing nearly 1,000 tests pre- and post-implementation, the median time to definitive bed placement from admission is 23.4 (8.6-41.9) versus 17.1 h (9.0-28.8), p = 0.02. Mean length of stay on COVID-19 "holding" wards is 58.5 versus 29.9 h (p < 0.001). POC testing increases isolation room availability, avoids bed closures, allows discharge to care homes, and expedites access to hospital procedures. POC testing could mitigate the impact of COVID-19 on hospital systems.

Multicountry Validation of SAMBA - A Novel Molecular Point-of-Care Test for HIV-1 Detection in Resource-Limited Setting.

INTRODUCTION: Early diagnosis of HIV-1 infection and the prompt initiation of antiretroviral therapy are critical to achieving a reduction in the morbidity and mortality of infected infants. The Simple AMplification-Based Assay (SAMBA) HIV-1 Qual Whole Blood Test was developed specifically for early infant diagnosis and prevention of mother-to-child transmission programs implemented at the point-of-care in resource-limited settings. METHODS: We have evaluated the performance of this test run on the SAMBA I semiautomated platform with fresh whole blood specimens collected from 202 adults and 745 infants in Kenya, Uganda, and Zimbabwe. Results were compared with those obtained with the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 assay as performed with fresh whole blood or dried blood spots of the same subjects, and discrepancies were resolved with alternative assays. RESULTS: The performance of the SAMBA and CAP/CTM assays evaluated at 5 laboratories in the 3 countries was similar for both adult and infant samples. The clinical sensitivity, specificity, positive predictive value, and negative predictive value for the SAMBA test were 100%, 99.2%, 98.7%, and 100%, respectively, with adult samples, and 98.5%, 99.8%, 99.7%, and 98.8%, respectively, with infant samples. DISCUSSION: Our data suggest that the SAMBA HIV-1 Qual Whole Blood Test would be effective for early diagnosis of HIV-1 infection in infants at point-of-care settings in sub-Saharan Africa.

Performance of the SAMBA I and II HIV-1 Semi-Q Tests for viral load monitoring at the point-of-care.

Although access to antiretroviral therapy for HIV infection is increasing in resource-poor countries, viral load testing for monitoring of treatment efficacy remains limited, expensive, and confined to centralized laboratories. The SAMBA HIV-1 Semi-Q Test is a nucleic acid-based amplification assay developed for viral load monitoring performed on either the semi-automated SAMBA I system for laboratory use or the fully automated SAMBA II system for point-of care use. We have assessed the performance characteristics of the SAMBA HIV-1 Semi-Q Test on SAMBA I and SAMBA II systems according to the Common Technical Specifications of the European Community's 98/79 In Vitro Diagnostic Medical Devices Directive. The sensitivity, specificity, reproducibility, and viral subtype coverage of the test were similar on the SAMBA I and SAMBA II platforms. The clinical performance on the SAMBA I system was compared with the Roche CAP/CTM assay and evaluated in-house with 130 patient specimens from London as well as in the field with 390 specimens in Kenya and Zimbabwe. The overall concordance between the SAMBA and CAP/CTM assays was 98.1%. The clinical performance of the test on the SAMBA II platform in comparison with the Abbott HIV-1 RealTime Assay was evaluated in-house with 150 specimens from Ukraine, yielding a concordance of 98.0%. The results thus show that the SAMBA HIV-1 Semi-Q Test performs equivalently on SAMBA I and SAMBA II, and they suggest that the test is suitable for implementation at the point-of-care in resource-poor regions where viral load testing is desperately needed but often unavailable.

Performance evaluation of the point-of-care SAMBA I and II HIV-1 Qual whole blood tests.

The SAMBA HIV-1 Qual Whole Blood Test is a nucleic acid-based amplification assay for the qualitative detection of HIV-1 in whole blood of adults or infants. The test can be run on either the semi-automated SAMBA I system for clinical use or the fully automated, including readout, SAMBA II system for point-of-care use in resource-limited settings. We have assessed the performance characteristics of the SAMBA HIV-1 Qual Whole Blood Test on SAMBA I and SAMBA II. The limit of detection obtained for the two tests were 518IU/ml and 399copies/ml on SAMBA I and 457IU/ml and 433copies/ml on SAMBA II. Test specificity on both systems was 100% with a panel of 503 HIV-1 negative samples. Evaluation of test reproducibility showed 100% concordance with expected gold standard results as well as 100% agreement between operators, days, and runs as well as within runs on both SAMBA I and SAMBA II. Our results thus show that the SAMBA HIV-1 Qual Whole Blood Test performs equivalently on SAMBA I and SAMBA II, and also suggest that the test is suitable for implementation in medium-throughput clinical facilities (SAMBA I) or low-throughput point-of-care (POC) settings (SAMBA II) in resource-poor regions.

SAMBA HIV semiquantitative test, a new point-of-care viral-load-monitoring assay for resource-limited settings.

Routine viral-load (VL) testing of HIV-infected individuals on antiretroviral therapy (ART) is used to monitor treatment efficacy. However, due to logistical challenges, implementation of VL has been difficult in resource-limited settings. The aim of this study was to evaluate the performance of the SAMBA semi-Q (simple amplification-based assay semiquantitative test for HIV-1) in London, Malawi, and Uganda. The SAMBA semi-Q can distinguish between patients with VLs above and below 1,000 copies/ml. The SAMBA semi-Q was validated with diluted clinical samples and blinded plasma samples collected from HIV-1-positive individuals. SAMBA semi-Q results were compared with results from the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test, v2.0. Testing of 96 2- to 10-fold dilutions of four samples containing HIV-1 subtype C as well as 488 samples from patients in the United Kingdom, Malawi, and Uganda yielded an overall accuracy for the SAMBA semi-Q of 99% (95% confidence interval [CI], 93.8 to 99.9%) and 96.9% (95% CI 94.9 to 98.3%), respectively, compared to to the Roche test. Analysis of VL data from patients in Malawi and Uganda showed that the SAMBA cutoff of 1,000 copies/ml appropriately distinguished treated from untreated individuals. Furthermore, analysis of the viral loads of 232 patients on ART in Malawi and Uganda revealed similar patterns for virological control, defined as either <1,000 copies/ml (SAMBA cutoff) or <5,000 copies/ml (WHO 2010 criterion; WHO, Antiretroviral Therapy for HIV Infection in Adults and Adolescents: Recommendations for a Public Health Approach, 2010). This study suggests that the SAMBA semi-Q has adequate concurrency with the gold standard measurements for viral load. This test can allow VL monitoring of patients on ART at the point of care in resource-limited settings.

Nucleic acid dipstick test for molecular diagnosis of pandemic H1N1.

A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements. The amplification step is monitored with an internal control to ensure correct interpretation of test results. The clinical performance of this assay was evaluated using blinded RNA samples extracted from nasal/throat swab specimens from 262 patients exhibiting influenza-like illness. Compared with the United Kingdom National Standard Method, based on quantitative reverse transcription-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the new assay were 95.3% (95% confidence interval, 88.5 to 98.7%), 99.4% (95% confidence interval, 96.9 to 99.9%), 98.8% (95% confidence interval, 93.5 to 99.9%), and 97.8% (95% confidence interval, 94.4 to 99.4%), respectively. The SAMBA for pandemic H1N1 provides a new technology that could potentially facilitate timely diagnosis and management of infected individuals, thereby informing decision making with regard to patient isolation during a pandemic outbreak.

Simple amplification-based assay: a nucleic acid-based point-of-care platform for HIV-1 testing.

BACKGROUND: A new nucleic acid-based assay (simple amplification-based assay [SAMBA]) for rapid visual detection of human immunodeficiency virus-type 1 (HIV-1) by dipstick is described. The assay was designed to be simple, stable, robust, self-contained, and capable of detecting a broad spectrum of HIV-1 subtypes and recombinant forms. METHODS: The performance of the SAMBA HIV-1 test (amplification and detection chemistry) was evaluated using the World Health Organization HIV-1 RNA Genotype Reference Panel, with clinical samples representing various viral subtypes and recombinant forms common in sub-Saharan Africa. Sixty-nine randomly selected and blinded clinical samples that had undergone HIV-1 genotypic resistance analyses in a large London teaching hospital were also tested. These samples included 14 different viral subtypes or recombinant forms with viral loads of 78-9.5 x 10(6) copies/mL. RESULTS: The sensitivity and viral subtype coverage of the SAMBA HIV-1 test were either comparable to or better than those of the commercially available nucleic acid-based HIV-1 diagnostic tests. CONCLUSIONS: The unique characteristics and competitive performance of the SAMBA HIV-1 test render it suitable for point-of-care and near-patient testing in both developed and developing countries.

From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas.

Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.

Evolutionary origin of cAMP-based chemoattraction in the social amoebae.

Phenotypic novelties can arise if integrated developmental pathways are expressed at new developmental stages and then recruited to serve new functions. We analyze the origin of a novel developmental trait of Dictyostelid amoebae: the evolution of cAMP as a developmental chemoattractant. We show that cAMP's role of attracting starving amoebae arose through recruitment of a pathway that originally evolved to coordinate fruiting body morphogenesis. Orthologues of the high-affinity cAMP receptor (cAR), cAR1, were identified in a selection of species that span the Dictyostelid phylogeny. The cAR1 orthologue from the basal species Dictyostelium minutum restored aggregation and development when expressed in an aggregation-defective mutant of the derived species Dictyostelium discoideum that lacks high-affinity cARs, thus demonstrating that the D. minutum cAR is a fully functional cAR. cAR1 orthologues from basal species are expressed during fruiting body formation, and only this process, and not aggregation, was disrupted by abrogation of cAR1 function. This is in contrast to derived species, where cAR1 is also expressed during aggregation and critically regulates this process. Our data show that coordination of fruiting body formation is the ancestral function of extracellular cAMP signaling, whereas its derived role in aggregation evolved by recruitment of a preexisting pathway to an earlier stage of development. This most likely occurred by addition of distal cis-regulatory regions to existing cAMP signaling genes.

Heterogeneity of ITS1 sequences in the biting midge Culicoides impunctatus (Goetghebuer) suggests a population in Argyll, Scotland, may be genetically distinct.

Ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, variation in ITS1 from eight Culicoides species was analysed by PCR, DNA restriction analysis, cloning, and sequencing. ITS1 variants were essentially homogenized within a species, as sequences were identical or closely related. However, Culicoides impunctatus ITS1 sequences derived from one (Argyll) of five populations contained considerable genomic diversity. The secondary structure of each ITS1 was computed. The structure aided the production of an accurate alignment and the identification of a large indel. A phylogenetic analysis was performed. Some of the sequences from the diverse Argyll C. impunctatus population were more related to Culicoides imicola, a vector of animal pathogens in the Old World, than they were to the other C. impunctatus sequences. Thus, the rDNA ITS1 regions of individuals in the Argyll C. impunctatus population were not conforming to the general theory of rDNA homogenization through molecular drive.