Storage of Mycobacterium tuberculosis culture isolates in MicrobankTM beads at a South African laboratory.

Background: Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at -70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated. Objective: The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. Methods: In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at -70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. Results: Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. Conclusion: MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. What this study adds: This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at -70 °C and provided a suitable option for medium-term storage of MTBC.

Exhaled human breath analysis in active pulmonary tuberculosis diagnostics by comprehensive gas chromatography-mass spectrometry and chemometric techniques.

Tuberculosis (TB) is the deadliest infectious disease, and yet accurate diagnostics for the disease are unavailable for many subpopulations. In this study, we investigate the possibility of using human breath for the diagnosis of active TB among TB suspect patients, considering also several risk factors for TB for smokers and those with human immunodeficiency virus (HIV). The analysis of exhaled breath, as an alternative to sputum-dependent tests, has the potential to provide a simple, fast, non-invasive, and readily available diagnostic service that could positively change TB detection. A total of 50 individuals from a clinic in South Africa were included in this pilot study. Human breath has been investigated in the setting of active TB using the thermal desorption-comprehensive two-dimensional gas chromatography-time of flight mass spectrometry methodology and chemometric techniques. From the entire spectrum of volatile metabolites in breath, three machine learning algorithms (support vector machines, partial least squares discriminant analysis, and random forest) to select discriminatory volatile molecules that could potentially be useful for active TB diagnosis were employed. Random forest showed the best overall performance, with sensitivities of 0.82 and 1.00 and specificities of 0.92 and 0.60 in the training and test data respectively. Unsupervised analysis of the compounds implicated by these algorithms suggests that they provide important information to cluster active TB from other patients. These results suggest that developing a non-invasive diagnostic for active TB using patient breath is a potentially rich avenue of research, including among patients with HIV comorbidities.

Multidisciplinary Point-of-Care Testing in South African Primary Health Care Clinics Accelerates HIV ART Initiation but Does Not Alter Retention in Care.

BACKGROUND: Lack of accessible laboratory infrastructure limits HIV antiretroviral therapy (ART) initiation, monitoring, and retention in many resource-limited settings. Point-of-care testing (POCT) is advocated as a mechanism to overcome these limitations. We executed a pragmatic, prospective, randomized, controlled trial comparing the impact of POCT vs. standard of care (SOC) on treatment initiation and retention in care. METHODS: Selected POC technologies were embedded at 3 primary health clinics in South Africa. Confirmed HIV-positive participants were randomized to either SOC or POC: SOC participants were venesected and specimens referred to the laboratory with patient follow-up as per algorithm (∼3 visits); POC participants had phlebotomy and POCT immediately on-site using Pima CD4 to assess ART eligibility followed by hematology, chemistry, and tuberculosis screening with the goal of receiving same-day adherence counseling and treatment initiation. Participant outcomes measured at recruitment 6 and 12 months after initiation. RESULTS: Four hundred thirty-two of 717 treatment eligible participants enrolled between May 2012 and September 2013: 198 (56.7%) SOC; 234 (63.6%) POC. Mean age was 37.4 years; 60.5% were female. Significantly more participants were initiated using POC [adjusted prevalence ratio (aPR) 0.83; 95% confidence interval (CI): 0.74 to 0.93; P < 0.0001], the median time to initiation was 1 day for POC and 26.5 days for SOC. The proportion of patients in care and on ART was similar for both arms at 6 months (47 vs. 50%) (aPR 0.96; 95% CI: 0.79 to 1.16) and 12 months (32 vs. 32%) (aPR 1.05; 95% CI: 0.80 to 1.38), with similar mortality rates. Loss to follow-up at 12 months was higher for POC (36% vs. 51%) (aPR 0.82; 95% CI: 0.65 to 1.04). CONCLUSIONS: Adoption of POCT accelerated ART initiation but once on treatment, there was unexpectedly higher loss to follow-up on POC and no improvement in outcomes at 12 months over SOC.

Choosing a new CD4 technology: Can statistical method comparison tools influence the decision?

BACKGROUND: Method comparison tools are used to determine the accuracy, precision, agreement, and clinical relevance of a new or improved technology versus a reference technology. Guidelines for the most appropriate method comparison tools as well as their acceptable limits are lacking and not standardized for CD4 counting technologies. METHODS: Different method comparison tools were applied to a previously published CD4 dataset (n = 150 data pairs) evaluating five different CD4 counting technologies (TruCOUNT, Dual Platform, FACSCount, Easy CD4, CyFlow) on a single specimen. Bland-Altman, percentage similarity, percent difference, concordance correlation, sensitivity, specificity and misclassification method comparison tools were applied as well as visualization of agreement with Passing Bablock and Bland-Altman scatter plots. RESULTS: The FACSCount (median CD4 = 245 cells/µl) was considered the reference for method comparison. An algorithm was developed using best practices of the most applicable method comparison tools, and together with a modified heat map was found useful for method comparison of CD4 qualitative and quantitative results. The algorithm applied the concordance correlation for overall accuracy and precision, then standard deviation of the absolute bias and percentage similarity coefficient of variation to identify agreement, and lastly sensitivity and misclassification rates for clinical relevance. CONCLUSION: Combining method comparison tools is more useful in evaluating CD4 technologies compared to a reference CD4. This algorithm should be further validated using CD4 external quality assessment data and studies with larger sample sizes. © 2017 International Clinical Cytometry Society.

Implementation and Operational Research: Implementation of Multiple Point-of-Care Testing in 2 HIV Antiretroviral Treatment Clinics in South Africa.

BACKGROUND: A plethora of point-of-care (POC) tests exist in the HIV and tuberculosis diagnostic pipeline which require rigorous evaluation to ensure performance in the field. The accuracy and feasibility of nurse-operated multidisciplinary-POC testing for HIV antiretroviral therapy (ART) initiation/monitoring was evaluated. METHODS: Random HIV-positive adult patients presenting at 2 treatment clinics in South Africa for ART initiation/monitoring were consented and enrolled. POCT was performed by a dedicated nurse on a venipuncture specimen; Pima (CD4), HemoCue (hemoglobin), Reflotron (alanine aminotransferase, creatinine), Accutrend (lactate) and compared with laboratory testing. External quality assessment, training, workflow, and errors were assessed. RESULTS: n = 324 enrolled at site1; n = 469 enrolled at site 2. Clinical data on n = 305 participants: 65% (n = 198) female with a mean age of 39.8 (21-61) years; mean age of males 43.2 (26-61) years; 70% of patients required 3 or more POC tests/visit. External quality assessment material was suitable for POCT. CD4, hemoglobin and alanine aminotransferase testing showed good agreement with predicate methodology; creatinine and lactate had increased variability. Pima CD4 misclassified up to 11.6% of patients at 500 cells per microliter and reported 4.3%-6% error rate. A dedicated nurse could perform POCT on 7 patients/day; inclusion of Pima CD4 increased time for testing from 6 to 110 minutes. Transcription error rate was 1%. CONCLUSIONS: Nurses can accurately perform multidisciplinary POCT for HIV ART initiation/monitoring. This will however, require a dedicated nurse as current duties will increase if POC is added to workflow. The use of Pima CD4 will increase patients initiated on ART. Connectivity will be central to ensure quality management of results, but overall impact will need to still be addressed.

A meta-analysis of the performance of the Pima™ CD4 for point of care testing.

BACKGROUND: The Alere point-of-care (POC) Pima™ CD4 analyzer allows for decentralized testing and expansion to testing antiretroviral therapy (ART) eligibility. A consortium conducted a pooled multi-data technical performance analysis of the Pima CD4. METHODS: Primary data (11,803 paired observations) comprised 22 independent studies between 2009-2012 from the Caribbean, Asia, Sub-Saharan Africa, USA and Europe, using 6 laboratory-based reference technologies. Data were analyzed as categorical (including binary) and numerical (absolute) observations using a bivariate and/or univariate random effects model when appropriate. RESULTS: At a median reference CD4 of 383 cells/µl the mean Pima CD4 bias is -23 cells/µl (average bias across all CD4 ranges is 10 % for venous and 15% for capillary testing). Sensitivity of the Pima CD4 is 93% (95% confidence interval [CI] 91.4% - 94.9%) at 350 cells/µl and 96% (CI 95.2% - 96.9%) at 500 cells/µl, with no significant difference between venous and capillary testing. Sensitivity reduced to 86% (CI 82% - 89%) at 100 cells/µl (for Cryptococcal antigen (CrAg) screening), with a significant difference between venous (88%, CI: 85% - 91%) and capillary (79%, CI: 73% - 84%) testing. Total CD4 misclassification is 2.3% cases at 100 cells/µl, 11.0% at 350 cells/µl and 9.5 % at 500 cells/µl, due to higher false positive rates which resulted in more patients identified for treatment. This increased by 1.2%, 2.8% and 1.8%, respectively, for capillary testing. There was no difference in Pima CD4 misclassification between the meta-analysis data and a population subset of HIV+ ART naïve individuals, nor in misclassification among operator cadres. The Pima CD4 was most similar to Beckman Coulter PanLeucogated CD4, Becton Dickinson FACSCalibur and FACSCount, and less similar to Partec CyFlow reference technologies. CONCLUSIONS: The Pima CD4 may be recommended using venous-derived specimens for screening (100 cells/µl) for reflex CrAg screening and for HIV ART eligibility at 350 cells/µl and 500 cells/µl thresholds using both capillary and venous derived specimens. These meta-analysis findings add to the knowledge of acceptance criteria of the Pima CD4 and future POC tests, but implementation and impact will require full costing analysis.

Diagnosing childhood pulmonary tuberculosis using a single sputum specimen on Xpert MTB/RIF at point of care.

BACKGROUND: The GeneXpertMTB/RIF (Cepheid, USA) (Xpert) has proved successful for pulmonary tuberculosis (TB) diagnosis on decontaminated/concentrated induced sputum specimens from children. Capacity to perform induction in many settings is limited. OBJECTIVE: To assess: (i) volumes of 'routinely obtained' sputum in a district-level academic hospital; (ii) whether sputum specimens not meeting Xpert-required testing volumes could still be tested; and (iii) performance of Xpert on a single paediatric sputum specimen at point of care (POC). METHODS: Two sputa were collected from paediatric TB suspects (£14 years) at Rahima Moosa Mother and Child Hospital, Johannesburg, South Africa. One specimen was weighed at POC; if the volume was ≥0.1 mL but <0.5 mL, it was increased to 0.5 mL using saline. On-site Xpert testing (G3 cartridge) was performed by a dedicated laboratory technician. The second specimen was referred for TB smear microscopy and culture as per standard of care (SOC). RESULTS: A total of 484 patients presumed to have TB (median age 24 months) were eligible for this study, performed between June 2011 and May 2012. Xpert could not be used on 4.1% of specimens because of volumes<0.1 mL, and 62.8% required addition of saline prior to Xpert testing. Xpert generated a 2.2% error and 3.7% invalid rate, compared with the SOC that rejected 2.3% because of insufficient volume and 2.3% that were contaminated. The diagnostic performance compared with culture was 62.5% (95% confidence interval (CI) 24.7-91) and 99.1% (95% CI 97.4-99.8) sensitivity and specificity, respectively, for Xpert (n=345) and 33.3% (7.9-69.9) and 99.5% (98.1-99.9) sensitivity and specificity, respectively, for smear microscopy (n=374). CONCLUSIONS: Up to 67% of 'routinely obtained' sputum specimens from children (£14 years) are below the required volume for Xpert testing but can be 'topped up' with saline. XpertMTB/RIF performed better than microscopy and generated clinically relevant, timeous results, but sensitivity did not reach the same levels as culture in children.

Feasibility of HIV point-of-care tests for resource-limited settings: challenges and solutions.

Improved access to anti-retroviral therapy increases the need for affordable monitoring using assays such as CD4 and/or viral load in resource-limited settings. Barriers to accessing treatment, high rates of loss to initiation and poor retention in care are prompting the need to find alternatives to conventional centralized laboratory testing in certain countries. Strong advocacy has led to a rapidly expanding repertoire of point-of-care tests for HIV. point-of-care testing is not without its challenges: poor regulatory control, lack of guidelines, absence of quality monitoring and lack of industry standards for connectivity, to name a few. The management of HIV increasingly requires a multidisciplinary testing approach involving hematology, chemistry, and tests associated with the management of non-communicable diseases, thus added expertise is needed. This is further complicated by additional human resource requirements and the need for continuous training, a sustainable supply chain, and reimbursement strategies. It is clear that to ensure appropriate national implementation either in a tiered laboratory model or a total decentralized model, clear country-specific assessments need to be conducted.

Evaluation of the use of plasma preparation tubes for HIV viral load testing on the COBAS AmpliPrep/COBAS TaqMan HIV-1 version 2.0.

HIV viral load monitoring forms an essential part of the management of patients receiving antiretroviral therapy, but transport of samples without loss of RNA integrity may be problematic in resource limited settings. The use of plasma preparation tubes (PPT) which can be centrifuged to separate cellular components before transport may provide a simple and cost-effective alternative to standard EDTA samples. We investigated whether PPT generated reliable results using the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 test version 2.0 (CAP/CTM HIV-1 v2.0). The mean difference between EDTA and PPT prepared samples (n=261) was acceptable (log 0.04 copies/ml, percentage similarity CV 3.53%). PPT can be used for viral load testing on the CAP/CTM HIV-1 v2.0.

Use of a prequalification panel for rapid scale-up of high-throughput HIV viral load testing.

Increased access to antiretroviral drugs expands needs for viral load (VL) testing. South Africa's National Health Laboratory Service responded to demands by implementing two testing platforms in 17 laboratories within 8 months. An industry partner's collaboration, training programs, and method verification with a VL prequalification panel ensured testing quality and rapid implementation.

Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study.

BACKGROUND: The Xpert MTB/RIF (Cepheid) non-laboratory-based molecular assay has potential to improve the diagnosis of tuberculosis (TB), especially in HIV-infected populations, through increased sensitivity, reduced turnaround time (2 h), and immediate identification of rifampicin (RIF) resistance. In a prospective clinical validation study we compared the performance of Xpert MTB/RIF, MTBDRplus (Hain Lifescience), LightCycler Mycobacterium Detection (LCTB) (Roche), with acid fast bacilli (AFB) smear microscopy and liquid culture on a single sputum specimen. METHODS AND FINDINGS: Consecutive adults with suspected TB attending a primary health care clinic in Johannesburg, South Africa, were prospectively enrolled and evaluated for TB according to the guidelines of the National TB Control Programme, including assessment for smear-negative TB by chest X-ray, clinical evaluation, and HIV testing. A single sputum sample underwent routine decontamination, AFB smear microscopy, liquid culture, and phenotypic drug susceptibility testing. Residual sample was batched for molecular testing. For the 311 participants, the HIV prevalence was 70% (n = 215), with 120 (38.5%) culture-positive TB cases. Compared to liquid culture, the sensitivities of all the test methodologies, determined with a limited and potentially underpowered sample size (n = 177), were 59% (47%-71%) for smear microscopy, 76% (64%-85%) for MTBDRplus, 76% (64%-85%) for LCTB, and 86% (76%-93%) for Xpert MTB/RIF, with specificities all >97%. Among HIV+ individuals, the sensitivity of the Xpert MTB/RIF test was 84% (69%-93%), while the other molecular tests had sensitivities reduced by 6%. TB detection among smear-negative, culture-positive samples was 28% (5/18) for MTBDRplus, 22% (4/18) for LCTB, and 61% (11/18) for Xpert MTB/RIF. A few (n = 5) RIF-resistant cases were detected using the phenotypic drug susceptibility testing methodology. Xpert MTB/RIF detected four of these five cases (fifth case not tested) and two additional phenotypically sensitive cases. CONCLUSIONS: The Xpert MTB/RIF test has superior performance for rapid diagnosis of Mycobacterium tuberculosis over existing AFB smear microscopy and other molecular methodologies in an HIV- and TB-endemic region. Its place in the clinical diagnostic algorithm in national health programs needs exploration. Please see later in the article for the Editors' Summary.

Abbott RealTime HIV-1 m2000rt viral load testing: manual extraction versus the automated m2000sp extraction.

The Abbott RealTime HIV-1 assay is a real-time nucleic acid amplification assay available for HIV-1 viral load quantitation. The assay has a platform for automated extraction of viral RNA from plasma or dried blood spot samples, and an amplification platform with real time fluorescent detection. Overall, this study found no clinically relevant differences in viral load, if samples were extracted manually.

Quantifying HIV for monitoring antiretroviral therapy in resource-poor settings.

There is increasing evidence to support the inability of CD4 cell count monitoring to predict virological failure in human immunodeficiency virus-infected individuals receiving antiretroviral therapy. There is renewed interest in improving access to viral load monitoring in resource-constrained regions to monitor adherence to treatment and to switch therapy. The field is rapidly changing as new technology platforms are made available for evaluation. This article presents an up to date summary of the assays available for viral load monitoring and suggests approaches for their implementation.

Evaluation of the Abbott m2000rt RealTime HIV-1 assay with manual sample preparation compared with the ROCHE COBAS AmpliPrep/AMPLICOR HIV-1 MONITOR v1.5 using specimens from East Africa.

The performance of the Abbott m2000rt RealTime HIV-1 assay (RealTime HIV-1) with manual sample preparation was compared against the ROCHE COBAS AmpliPrep/AMPLICOR HIV-1 MONITOR Test v1.5 (CAP/CA HIV-1) using samples collected from 100 donors infected with HIV and 20 donors not infected with HIV in northern Tanzania where HIV-1 subtypes A, C, D, and their recombinant forms predominate. The RealTime HIV-1 appeared to have more within-run variability at high HIV-1 RNA concentrations, but total assay variability over the dynamic range tested was within the manufacturer's claim of <0.3 SD copies/mL. Accuracy studies showed 100% concordance for positive and negative values. When continuous values were examined, CAP/CA HIV-1 yielded higher values than the RealTime HIV-1 at higher nominal HIV-1 RNA concentrations. The RealTime HIV-1 assay showed excellent linearity between 2.5 and 7.0 log copies/mL. Of negative samples, 100% showed negative results, and >95% of samples with nominal concentrations of 40 copies/mL were detected at > or = 40 copies/mL by RealTime HIV-1. Manual sample preparation may contribute to higher total assay variability. This study suggests that the Abbott m2000rt RealTime HIV-1 assay with manual sample preparation is an acceptable and feasible alternative to the conventional ROCHE COBAS AmpliPrep/AMLICOR HIV-1 Monitor v1.5 assay and that the RealTime HIV-1 assay performs well on samples from East Africa.

Evaluation of the Abbott m2000 RealTime human immunodeficiency virus type 1 (HIV-1) assay for HIV load monitoring in South Africa compared to the Roche Cobas AmpliPrep-Cobas Amplicor, Roche Cobas AmpliPrep-Cobas TaqMan HIV-1, and BioMerieux NucliSENS EasyQ HIV-1 assays.

The implementation of antiretroviral therapy demands the need for increased access to viral load (VL) monitoring. Newer real-time VL testing technologies are faster and have larger dynamic ranges and fully automated extraction to benefit higher throughputs in resource-poor environments. The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay was evaluated as a new option for testing for HIV-1 subtype C in South Africa, and its performance was compared to the performance of existing assays (the Cobas AmpliPrep-Cobas TaqMan HIV-1, version 1, assay; the AmpliPrep-Cobas Monitor standard HIV-1 assay; and the NucliSENS EasyQ-EasyMag HIV-1 assay) in a high-throughput laboratory. The total precision of the RealTime HIV-1 assay was acceptable over all viral load ranges. This assay compared most favorably with the Cobas AmpliPrep-Cobas TaqMan HIV-1 assay (R(2) = 0.904), with a low standard deviation of difference being detected (0.323 copies/ml). The bias against comparator assays ranged from -0.001 copies/ml to -0.228 copies/ml. Variability in the reporting of VLs for a 20-member subtype panel compared to the variability of other assays was noted with subtypes G and CRF02-AG. The RealTime HIV-1 assay can test 93 samples per day with minimal manual preparation, less staff, and the minimization of contamination through automation. This assay is suitable for HIV-1 subtype C VL quantification in South Africa.

Ultra-high-throughput, automated nucleic acid detection of human immunodeficiency virus (HIV) for infant infection diagnosis using the Gen-Probe Aptima HIV-1 screening assay.

The early diagnosis of human immunodeficiency virus (HIV) infection in infants is critical to ensure the initiation of treatment before significant immunological compromise. Each year an estimated 300,000 HIV-exposed infants in South Africa require access to tests for the diagnosis of HIV infection. Currently, testing is performed at several facilities by using PCR amplification of HIV DNA at 6 weeks of age by the use of dried blood spots (DBSs) and whole blood (WB). The Gen-Probe Aptima HIV type 1 (HIV-1) screening assay (the Aptima assay) is a qualitative nucleic acid test based on transcription-mediated amplification (TMA), a technology routinely used in blood banks in South Africa. The performance characteristics of Gen-Probe's TMA technology compared well to those of the Roche Amplicor HIV-1 DNA (version 1.5) assay. The sensitivity of the assay with WB and DBS samples was 100%, and the specificities were 99.4% and 99.5% for DBSs and WB, respectively. The detection of HIV by the Aptima assay at greater levels of dilution in samples negative by the comparator assay indicates an improvement in sensitivity by the use of the TMA technology. The ability to process 1,900 samples in a 24-h period on the Tigris instrument makes the Aptima assay an attractive option for high-volume, centralized laboratories.