Novel Automation of an Enzyme-Linked Immunosorbent Spot Assay Testing Method: Comparable Diagnostic Performance of the T-SPOT.TB Test Using Manual Density Gradient Cell Isolation versus Automated Positive Selection with the T-Cell Select Kit

The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval [CI], 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape

Background and objectives. Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acidfast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. Design. A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. Setting. Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. Outcome measures. Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. Results. Previously treated patients were at a high risk of MDRTB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0 - 99.8%), 97.2% (95% CI: 94.5 - 99.9%) and 96.5% (95% CI: 94.1 - 98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28 - 85 days with conventional DST. However, only 70.6% of FASTPlaque- Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicin resistant strains were MDR-TB. Conclusions. Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission

Bacteriophage- based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis

BACKGROUND: Sputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated. METHODS: We did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses. RESULTS: The data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons. CONCLUSION: Phage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity

Diagnostic accuracy of a three-gene Mycobacterium tuberculosis host response cartridge using fingerstick blood for childhood tuberculosis: a multicentre prospective study in low-income and middle-income countries

BACKGROUND: Childhood tuberculosis remains a major cause of morbidity and mortality in part due to missed diagnosis. Diagnostic methods with enhanced sensitivity using easy-to-obtain specimens are needed. We aimed to assess the diagnostic accuracy of the Cepheid Mycobacterium tuberculosis Host Response prototype cartridge (MTB-HR), a candidate test measuring a three-gene transcriptomic signature from fingerstick blood, in children with presumptive tuberculosis disease. METHODS: RaPaed-TB was a prospective diagnostic accuracy study conducted at four sites in African countries (Malawi, Mozambique, South Africa, and Tanzania) and one site in India. Children younger than 15 years with presumptive pulmonary or extrapulmonary tuberculosis were enrolled between Jan 21, 2019, and June 30, 2021. MTB-HR was performed at baseline and at 1 month in all children and was repeated at 3 months and 6 months in children on tuberculosis treatment. Accuracy was compared with tuberculosis status based on standardised microbiological, radiological, and clinical data. FINDINGS: 5313 potentially eligible children were screened, of whom 975 were eligible. 784 children had MTB-HR test results, of whom 639 had a diagnostic classification and were included in the analysis. MTB-HR differentiated children with culture-confirmed tuberculosis from those with unlikely tuberculosis with a sensitivity of 59·8% (95% CI 50·8–68·4). Using any microbiological confirmation (culture, Xpert MTB/RIF Ultra, or both), sensitivity was 41·6% (34·7–48·7), and using a composite clinical reference standard, sensitivity was 29·6% (25·4–34·2). Specificity for all three reference standards was 90·3% (95% CI 85·5–94·0). Performance was similar in different age groups and by malnutrition status. Among children living with HIV, accuracy against the strict reference standard tended to be lower (sensitivity 50·0%, 15·7–84·3) compared with those without HIV (61·0%, 51·6–69·9), although the difference did not reach statistical significance. Combining baseline MTB-HR result with one Ultra result identified 71·2% of children with microbiologically confirmed tuberculosis. INTERPRETATION: MTB-HR showed promising diagnostic accuracy for culture-confirmed tuberculosis in this large, geographically diverse, paediatric cohort and hard-to-diagnose subgroups. FUNDING: European and Developing Countries Clinical Trials Partnership, UK Medical Research Council, Swedish International Development Cooperation Agency, Bundesministerium für Bildung und Forschung; German Center for Infection Research (DZIF)

European recommendations for short-term surveillance of health problems in childhood, adolescent and young adult cancer survivors from the end of treatment to 5 years after diagnosis: a PanCare guideline

\ua9 The Author(s) 2023. Purpose: Childhood, adolescent and young adult (CAYA) cancer survivors require ongoing surveillance for health problems from the end of cancer treatment throughout their lives. There is a lack of evidence-based guidelines on optimal surveillance strategies for the period from the end of treatment to 5 years after diagnosis. We aimed to address this gap by developing recommendations for short-term surveillance of health problems based on existing long-term follow-up (LTFU) care guidelines. Methods: The guideline working group, consisting of healthcare professionals, parents and survivor representatives from 10 countries, worked together to identify relevant health problems that may occur in survivors between the end of treatment and 5 years after diagnosis and to develop recommendations for short-term surveillance of health problems. The recommendations were drawn from existing LTFU guidelines and adapted where necessary based on clinical expertise. Results: The working group developed 44 recommendations for short-term surveillance of health problems, which were divided into four categories based on the level of surveillance required: awareness only (n = 11), awareness, history and/or physical examination without surveillance test (n = 15), awareness, history and/or physical examination with potential surveillance test (n = 1) and awareness, history and/or physical examination with surveillance test (n = 17). Conclusion: The development of a guideline for short-term surveillance of health problems fills a critical gap in survivorship care for CAYA cancer survivors, providing much-needed support immediately after treatment up to 5 years after diagnosis. Implications for Cancer Survivors. This guideline will support healthcare professionals to provide appropriate follow-up care and improve the quality of life of CAYA cancer survivors

Vasectomy under local anaesthesia performed free of charge as a family planning service : complications and results

Includes bibliographyThe original publication is available at http://www.samj.org.zaObjective. To evaluate the safety and efficacy of vasectomy performed under local anaesthesia by junior doctors at a secondary level hospital as part of a free family planning service. Method. Men requesting vasectomy were counselled and given written instructions to use alternative contraception until two semen analyses 3 and 4 months after vasectomy had confirmed azoospermia. Bilateral vasectomy was performed as an outpatient procedure under local anaesthesia by junior urology registrars. Statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, Fisher's exact or Spearman's rank correlation tests as appropriate. Results. Between January 2004 and December 2005, 479 men underwent vasectomy at Karl Bremer Hospital, Western Cape, South Africa; their average age was 36.1 (range 21 - 66) years, they had a median of 2 (range 0 - 10) children, and only 19% had 4 or more children. The average operation time was 15.5 (range 5 - 53) minutes. Complications occurred in 12.9%; these were pain (7.3%), swelling (5.4%), haematoma (1.3%), sepsis (1%), difficulty locating the vas (1%), vasovagal episode (0.6%), bleeding (0.6%), wound rupture (0.4%) and dysuria (0.2%) (some men had more than one complication). Of the men 63.3% returned for one semen analysis and 17.5% for a second. The vasectomy failure rate ranged from 0.4% (sperm persisting >365 days after vasectomy) to 2.3% (sperm seen >180 days after vasectomy and/or in the second semen specimen). No pregnancies were reported. The complication (5.6%) and failure rates (0%) were lowest for the registrar who had performed the smallest number of vasectomies and whose average operation time was longest. Comparing the first one-third of procedures performed by each of the doctors with the last one-third, there was a significant decrease in average operating times but not in complication rates. Conclusions. Vasectomy can be performed safely and effectively by junior doctors as an outpatient procedure under local anaesthesia, and should be actively promoted in South Africa as a safe and effective form of male contraception

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape

ArticleThe original publication is available at http://www.samj.org.zaBibliographyBackground and objectives. Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acid-fast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. Design. A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. Setting. Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. Outcome measures. Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. Results. Previously treated patients were at a high risk of MDR-TB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0-99.8%), 97.2% (95% CI: 94.5-99.9%) and 96.5% (95% CI: 94.1-98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28-85 days with conventional DST. However, only 70.6% of FASTPlaque-Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicin-resistant strains were MDR-TB. Conclusions. Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission.Publishers' Versio

Mutations in the rrs A1401G gene and phenotypic resistance to amikacin and capreomycin in mycobacterium tuberculosis

The aminoglycosides amikacin (AMK)/kanamycin (KAN) and the cyclic polypeptide capreomycin (CAP) are important injectable drugs in the treatment of multidrug-resistant tuberculosis. Cross-resistance among these drug classes occurs and information on the minimum inhibitory concentrations (MICs), above the normal wild-type distribution, may be useful in identifying isolates that are still accessible to drug treatment. Isolates from the Eastern Cape Province of South Africa were subjected to DNA sequencing of the rrs (1400-1500 region) and tlyA genes. Sequencing data were compared with (i) conventional susceptibility testing at standard critical concentrations (CCs) on Middlebrook 7H11 agar and (ii) MGIT 960-based MIC determinations to assess the presence of AMK- and CAP-resistant mutants. Isolates with an rrs A1401G mutation showed high-level resistance to AMK (>20 mg/L) and decreased phenotypic susceptibility to CAP (MICs 10-15 mg/L). The MICs of CAP were below the bioavailability of the drug, which suggests that it may still be effective against multi- or extensively drug resistant tuberculosis [M(X)DR-TB]. Agar-based CC testing was found to be unreliable for resistance recognition of CAP in particular

A comparative sudy of two pressure relieving techniques on three different wheelchair cushions

Introduction: Pressure relief done by a spinal cord injured patient is of utmost importance in order to prevent pressure sore formation. Some pressure-relieving techniques are described in previous literature, but their effectivity has not yet been deter- mined on different wheelchair cushions. Null Hypothesis (H0): The null hypothesis (H0) stated for thi study is that there is no difference in the effectivity of forward leaning and forward leaning towards the left as pressure relieving techniques for quadriplegic wheelchair users over the differen wheelchair cushions. Design: An analytical experimental study design using a convenient sample group of ten complete lesion quadriplegics (C6 to T1) was performed at the Physiotherapy Department, University of Pretoria. Method: Interface pressure (in mmHg) over the ischial tuberosities and upper thigh areas was measured using the Talley Oxford Pressure Monitor MKII with a 12-way matrix cell system. Results and Conclusion: The Friedman test for associated observations indicated statistically that the leaning  diagonally forward pressure relieving technique is more effective for all three wheelchair cushions used in this study