Yes / Background: The Xpert MTB/RIF (Xpert) assay is used globally to rapidly diagnose tuberculosis and resistance to rifampicin. We investigated the frequency and predictors of false-positive findings of rifampicin resistance with Xpert. Methods: We did a prospective, observational study of individuals who were enrolled in a Rwandan nationwide diagnostic cohort study (DIAMA trial; NCT03303963). We included patients identified to have rifampicin resistance on initial Xpert testing. We did a repeat Xpert assay and used rpoB Sanger and deep sequencing alongside phenotypic drug susceptibility testing (pDST) to ascertain final rifampicin susceptibility status, with any (hetero)resistant result overriding. We used multivariable logistic regression to assess predictors of false rifampicin resistance on initial Xpert testing, adjusted for HIV status, tuberculosis treatment history, initial Xpert semi-quantitative bacillary load, and initial Xpert probe. Findings: Between May 4, 2017, and April 30, 2019, 175 people were identified with rifampicin resistance at initial Xpert testing, of whom 154 (88%) underwent repeat Xpert assay. 54 (35%) patients were confirmed as rifampicin resistant on repeat testing and 100 (65%) were not confirmed with resistance. After further testing and sequencing, 121 (79%) of 154 patients had a final confirmed status for rifampicin susceptibility. 57 (47%) of 121 patients were confirmed to have a false rifampicin resistance result and 64 (53%) had true rifampicin resistance. A high pretest probability of rifampicin resistance did not decrease the odds of false rifampicin resistance (adjusted odds ratio [aOR] 6·0, 95% CI 1·0–35·0, for new tuberculosis patients vs patients who needed retreatment). Ten (16%) of the 64 patients with true rifampicin resistance did not have confirmed rifampicin resistance on repeat Xpert testing, of whom four had heteroresistance. Of 63 patients with a very low bacillary load on Xpert testing, 54 (86%) were falsely diagnosed with rifampicin-resistant tuberculosis. Having a very low bacillary load on Xpert testing was strongly associated with false rifampicin resistance at the initial Xpert assay (aOR 63·6, 95% CI 9·9–410·4). Interpretation: The Xpert testing algorithm should include an assessment of bacillary load and retesting in case rifampicin resistance is detected on a paucibacillary sputum sample. Only when rifampicin resistance has been confirmed on repeat testing should multidrug-resistant tuberculosis treatment be started. When rifampicin resistance has not been confirmed on repeat testing, we propose that patients should be given first-line anti-tuberculosis drugs and monitored closely during treatment, including by baseline culture, pDST, and further Xpert testing. / The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.

No description available.

Influenza is a viral infection that affects global health and economy with its endemic and sometimes pandemic spread. Rapid detection of Influenza viruses enables antiviral use and can bring financial savings. It is also essential for the global surveillance of prevalent Influenza strains. RT-PCR is considered the most specific and sensitive method for detection of Influenza, but Influenza mutates at a high rate and it is therefore crucial that RT-PCR methods are updated regularly. In 2014, Cepheid released their Xpert Flu/RSV XC assay, which can detect Influenza A and B and RSV by multiplex RT-PCR in approximately one hour. The aim of this study was to evaluate this assay at Laboratoriemedicin Västernorrland by using the laboratory’s previous PCR assay for detection of Influenza viruses as reference method. Real-time RT-PCR was used to compare Xpert Flu/RSV XC to the reference method. A dilution series was performed to estimate the methods’ PCR efficiencies and precision was calculated from quadruplicates of a positive control sample. Clinical specimens (n=42) were used to evaluate the diagnostic sensitivity and specificity of Xpert Flu/RSV XC. Objective statistical analysis of PCR data was performed and discussed. The Xpert Flu/RSV XC was equivalent to the reference method and demonstrated high diagnostic sensitivity and specificity. Estimated PCR efficiencies were however low. With the introduction of Xpert Flu/RSV XC to the laboratory follows many potential benefits, primarily in form of a simplified pre analytical procedure and a shortened analysis time. The Xpert Flu/RSV XC assay enables fast diagnosis of Influenza infection.

Introdução: O teste Xpert MTB / RIF está sendo cada vez mais utilizado em muitos países como diagnóstico inicial para a tuberculose (TB). Poucos estudos avaliaram o impacto do Xpert no diagnóstico em rotinas de programas de controle de TB no Brasil. O objetivo do presente estudo foi avaliar o impacto da introdução do Xpert MTB / RIF no diagnóstico de TB em uma cidade com alta incidência de TB no Brasil. Métodos: Incluímos pacientes avaliados com testes diagnósticos convencionais durante um ano antes da introdução do Xpert (grupo pré-Xpert) e pacientes avaliados usando Xpert durante um ano após a introdução do teste (grupo pós-Xpert). Resultados: 620 pacientes preencheram os critérios de inclusão (208 no grupo pré-Xpert e 412 no grupo pós-Xpert) e foram incluídos na análise. O tempo até o diagnóstico de TB foi menor no grupo pós-Xpert (0,7 dias, IQR: 0,5-1,0 dias) do que no grupo pré-Xpert (2,0 dias, IQR: 2,0-2,0 dias) (p <0,0001). Características atípicas da doença, como menor perda de peso, febre, dispneia, sudorese noturna e hemoptise; baciloscopia de escarro negativa; cultura negativa e radiografia de tórax atípica de TB foram mais comuns no grupo pós-Xpert do que no grupo pré-Xpert (p <0,0001 para todos). Conclusões: Observamos que a implementação do ensaio Xpert MTB / RIF, em rotinas de programas de controle de TB, melhora e facilita o diagnóstico de tuberculose, especialmente nos casos com manifestações da doença atípica. Esses resultados podem provavelmente ser generalizados para locais com incidência de TB similar. / Introduction: The receptor for advanced glycation end products (RAGE) is expressed in normal lungs and is upregulated during inflammation and infection. The interaction between AGEs and RAGE on the plasma membrane causes oxidative stress and apoptosis in lung cells. The objective of this study is to evaluate plasma levels of AGEs and its soluble receptor (sRAGE) in patients with active TB and healthy controls, and to investigate their relationship with food intake and nutritional status. Methods: Case-control study. AGE (carboxymethil lysine, CML) and RAGE were measured by Elisa. Nutritional assessment was performed by body mass index, triceps skin-fold thickness, mid-arm circumference, mid-arm muscle circumference, bioelectrical impedance analysis, and food frequency questionnaire. Results: 35 TB patients and 35 controls were included in the study. The mean S-RAGE levels were higher in TB patients than in controls (68.5 ± 28.1 vs 57.5 ± 24.0, p=0.046). Among cases that were current smokers, lower S-RAGE levels were associated with mortality (S-RAGE levels= 58.0 ± 36.5 [non-survivors] vs 71.3 ± 25.6 [survivors], p=0.006), and with weight loss (S-RAGE levels= 65.6 ± 27.4 [weight loss] vs 98.6 ± 16.7 [no weight loss], p=0.034). There was no statistically significant difference in CML levels and diet CML content between cases and controls. Malnutrition was more frequent in cases than in controls, but there was no correlation between nutritional parameters and CML or S-RAGE levels. Conclusions: TB patients had higher S-RAGE levels than controls. S-RAGE may play a role in disease manifestations and outcomes, being associated with weight loss and mortality.

Introdução: O teste Xpert MTB / RIF está sendo cada vez mais utilizado em muitos países como diagnóstico inicial para a tuberculose (TB). Poucos estudos avaliaram o impacto do Xpert no diagnóstico em rotinas de programas de controle de TB no Brasil. O objetivo do presente estudo foi avaliar o impacto da introdução do Xpert MTB / RIF no diagnóstico de TB em uma cidade com alta incidência de TB no Brasil. Métodos: Incluímos pacientes avaliados com testes diagnósticos convencionais durante um ano antes da introdução do Xpert (grupo pré-Xpert) e pacientes avaliados usando Xpert durante um ano após a introdução do teste (grupo pós-Xpert). Resultados: 620 pacientes preencheram os critérios de inclusão (208 no grupo pré-Xpert e 412 no grupo pós-Xpert) e foram incluídos na análise. O tempo até o diagnóstico de TB foi menor no grupo pós-Xpert (0,7 dias, IQR: 0,5-1,0 dias) do que no grupo pré-Xpert (2,0 dias, IQR: 2,0-2,0 dias) (p <0,0001). Características atípicas da doença, como menor perda de peso, febre, dispneia, sudorese noturna e hemoptise; baciloscopia de escarro negativa; cultura negativa e radiografia de tórax atípica de TB foram mais comuns no grupo pós-Xpert do que no grupo pré-Xpert (p <0,0001 para todos). Conclusões: Observamos que a implementação do ensaio Xpert MTB / RIF, em rotinas de programas de controle de TB, melhora e facilita o diagnóstico de tuberculose, especialmente nos casos com manifestações da doença atípica. Esses resultados podem provavelmente ser generalizados para locais com incidência de TB similar. / Introduction: The receptor for advanced glycation end products (RAGE) is expressed in normal lungs and is upregulated during inflammation and infection. The interaction between AGEs and RAGE on the plasma membrane causes oxidative stress and apoptosis in lung cells. The objective of this study is to evaluate plasma levels of AGEs and its soluble receptor (sRAGE) in patients with active TB and healthy controls, and to investigate their relationship with food intake and nutritional status. Methods: Case-control study. AGE (carboxymethil lysine, CML) and RAGE were measured by Elisa. Nutritional assessment was performed by body mass index, triceps skin-fold thickness, mid-arm circumference, mid-arm muscle circumference, bioelectrical impedance analysis, and food frequency questionnaire. Results: 35 TB patients and 35 controls were included in the study. The mean S-RAGE levels were higher in TB patients than in controls (68.5 ± 28.1 vs 57.5 ± 24.0, p=0.046). Among cases that were current smokers, lower S-RAGE levels were associated with mortality (S-RAGE levels= 58.0 ± 36.5 [non-survivors] vs 71.3 ± 25.6 [survivors], p=0.006), and with weight loss (S-RAGE levels= 65.6 ± 27.4 [weight loss] vs 98.6 ± 16.7 [no weight loss], p=0.034). There was no statistically significant difference in CML levels and diet CML content between cases and controls. Malnutrition was more frequent in cases than in controls, but there was no correlation between nutritional parameters and CML or S-RAGE levels. Conclusions: TB patients had higher S-RAGE levels than controls. S-RAGE may play a role in disease manifestations and outcomes, being associated with weight loss and mortality.

Introdução: O teste Xpert MTB / RIF está sendo cada vez mais utilizado em muitos países como diagnóstico inicial para a tuberculose (TB). Poucos estudos avaliaram o impacto do Xpert no diagnóstico em rotinas de programas de controle de TB no Brasil. O objetivo do presente estudo foi avaliar o impacto da introdução do Xpert MTB / RIF no diagnóstico de TB em uma cidade com alta incidência de TB no Brasil. Métodos: Incluímos pacientes avaliados com testes diagnósticos convencionais durante um ano antes da introdução do Xpert (grupo pré-Xpert) e pacientes avaliados usando Xpert durante um ano após a introdução do teste (grupo pós-Xpert). Resultados: 620 pacientes preencheram os critérios de inclusão (208 no grupo pré-Xpert e 412 no grupo pós-Xpert) e foram incluídos na análise. O tempo até o diagnóstico de TB foi menor no grupo pós-Xpert (0,7 dias, IQR: 0,5-1,0 dias) do que no grupo pré-Xpert (2,0 dias, IQR: 2,0-2,0 dias) (p <0,0001). Características atípicas da doença, como menor perda de peso, febre, dispneia, sudorese noturna e hemoptise; baciloscopia de escarro negativa; cultura negativa e radiografia de tórax atípica de TB foram mais comuns no grupo pós-Xpert do que no grupo pré-Xpert (p <0,0001 para todos). Conclusões: Observamos que a implementação do ensaio Xpert MTB / RIF, em rotinas de programas de controle de TB, melhora e facilita o diagnóstico de tuberculose, especialmente nos casos com manifestações da doença atípica. Esses resultados podem provavelmente ser generalizados para locais com incidência de TB similar. / Introduction: The receptor for advanced glycation end products (RAGE) is expressed in normal lungs and is upregulated during inflammation and infection. The interaction between AGEs and RAGE on the plasma membrane causes oxidative stress and apoptosis in lung cells. The objective of this study is to evaluate plasma levels of AGEs and its soluble receptor (sRAGE) in patients with active TB and healthy controls, and to investigate their relationship with food intake and nutritional status. Methods: Case-control study. AGE (carboxymethil lysine, CML) and RAGE were measured by Elisa. Nutritional assessment was performed by body mass index, triceps skin-fold thickness, mid-arm circumference, mid-arm muscle circumference, bioelectrical impedance analysis, and food frequency questionnaire. Results: 35 TB patients and 35 controls were included in the study. The mean S-RAGE levels were higher in TB patients than in controls (68.5 ± 28.1 vs 57.5 ± 24.0, p=0.046). Among cases that were current smokers, lower S-RAGE levels were associated with mortality (S-RAGE levels= 58.0 ± 36.5 [non-survivors] vs 71.3 ± 25.6 [survivors], p=0.006), and with weight loss (S-RAGE levels= 65.6 ± 27.4 [weight loss] vs 98.6 ± 16.7 [no weight loss], p=0.034). There was no statistically significant difference in CML levels and diet CML content between cases and controls. Malnutrition was more frequent in cases than in controls, but there was no correlation between nutritional parameters and CML or S-RAGE levels. Conclusions: TB patients had higher S-RAGE levels than controls. S-RAGE may play a role in disease manifestations and outcomes, being associated with weight loss and mortality.

Yes / We compared cetylpyridinium chloride (CPC), ethanol (ETOH), and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics. Three sputum samples were collected from each of 133 smear-positive tuberculosis (TB) patients (399 sputum samples). Each patient's sputum was stored with either CPC, ETOH, or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification targeting a short fragment (81 bp, GeneXpert MTB/RIF [Xpert]) or a long fragment (1,764 bp, in-house nested PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputum samples. After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested PCR was obtained with OMNI (n = 125, 94%) than with ETOH (n = 114, 85.7%; P = 0.001). Smears with scanty acid-fast bacilli (AFB) had lower rpoB PCR positivity with ETOH storage (n = 10, 41.7%) than with CPC (n = 16, 66.7%; difference, 25%; 95% confidence interval [CI], 3.5 to 46.5; P = 0.031) or OMNI (n = 16, 69.6%; difference, 26.1%; 95% CI, 3.8 to 48.4; P = 0.031), with no difference between CPC and OMNI. Poststorage, the threshold cycle (CT ) values significantly decreased compared to those prestorage with ETOH (difference, -1.1; 95% CI, -1.6 to -0.6; P = 0.0001) but not with CPC (P = 0.915) or OMNI (P = 0.33). For one patient's ETOH- and CPC-stored specimens with a CT of <10, Xpert gave results of rifampin false resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen. In conclusion, 28-day storage of sputum in OMNI, CPC, or ETOH at ambient temperature does not impact short-fragment PCR (Xpert), including for low smear grades. However, for long-fragment PCR, ETOH yielded a lower PCR positivity for low smear grades, while the performance of OMNI and CPC was excellent for all smear grades. (The study has been registered at ClinicalTrials.gov under registration number NCT02744469.). / Directorate General for Development (DGD), Belgium (FA4 to C.N.S., B.C.D.J., D.A., and L.R.), and the European Research Council-INTERRUPTB starting grant (number 311725 to B.C.D.J., C.J.M., and L.R.)

Anaerobe opportunist Clostridium difficile causes the majority of hospital-acquired antibiotic-associated diarrhea. Infections can be severe because of its ability to withstand many antibiotics, to sporulate and to produce toxins (A, B and binary).     In Sundsvall Hospital C. difficile is detected with real-time PCR, which targets the sequences of toxin B, binary toxin and a regulatory gene deletion seen in the virulent ribotype 027. All positive samples are stored frozen for one month, available for further analysis or outbreak investigation. The aim of this study was to investigate if temperature and transportation time may affect the viability of C. difficile, and the PCR-result.     Frozen feces samples were cultivated, identified with MALDI-TOF and analyzed with real-time PCR after at least one month of storage. To simulate the effect of transportation time, samples were stored at 4-8°C for three and seven days before cultivation and identification. Controls were cultivated after freezing for comparison.     Ninety percent of the frozen samples contained viable C. difficile. Discrepancies between PCR-results were found for two of the oldest samples collected (six months), which turned negative. Fresh samples showed lower amount of viable C. difficile after three days (50 %) than after seven days (60 %) of storage, perhaps because of competition with other bacteria and sporulation. The frozen control group contained a higher viable amount, 75 %. The results indicate that C. difficile tolerates to be stored at low temperatures as practiced today at the laboratory. Transportation time seem to affect the outcome of cultivation, but not the PCR-result.

Background: Tuberculosis (TB) is a major global health threat that results not only in health consequences but also economic consequences. Since 2015 the World Health Organization (WHO) has developed a strategy with the aim of ending the global burden of TB by reducing TB-related deaths, reducing TB incidence, and eliminating the burden of TB-related catastrophic costs for patients and their families. In order to reach these targets, we need to implement effective TB diagnostic and care strategies that are feasible for both patients as well as the health care system. -- Methods: This study consists of two manuscripts which assess the economic burden of TB - one from the patient perspective and the other from the health system perspective. The first manuscript is a systematic review aimed to determine the costs incurred by patients and their households while receiving TB care with direct (medical and non-medical) as well as indirect costs being examined for the pre-diagnostic, post-diagnostic and total phase of care. It analyzed studies with varying patient populations from low-, middle-, and high-income settings to help estimate key factors that drive patient costs. Furthermore, it assessed the proportion of patients that incurred catastrophic costs and the coping strategies that they resorted to in order to offset the costs of TB care. The second manuscript is a modelling study which aimed to develop, parameterize and analyze a decision analytic model to determine the cost, health outcomes as measured by disability-adjusted life years (DALYs) averted and the cost-effectiveness of second-generation lateral flow lipoarabinomannan assay (SG LF-LAM) diagnostic algorithms in people living with HIV (PLHIV) per DALY averted. This model examined four different strategies - 1) the standard of care (SOC) Gene Xpert MTB/RIF only, 2) Gene Xpert MTB/RIF plus LF-LAM for all patients, 3) Gene Xpert MTB/RIF plus LF-LAM for patients with a negative Xpert result, and 4) Gene Xpert MTB/RIF plus LF-LAM for patients who are symptom negative. -- Results: A systematic review showed that total patient costs related to TB care ranged from a mean of $2.80 to $19,153.80 (2019 USD) with costs largely dependent on geographic location as well as patient population, Direct medical and indirect cost components were the largest source of costs for patients and their families while receiving TB care. Direct medical costs included the cost of medication, consultations, diagnostics, follow-up testing, and hospitalization while indirect costs mainly consisted of loss of income. The costs of TB care were considered catastrophic for the majority of patients resulting in them using coping strategies to offset the burden of costs. In the second manuscript, the cost-effectiveness analysis Xpert only was found to be dominated by Xpert + FujiLAM conditional on a negative Xpert with an ICER of 1,000 USD/per DALY averted compared to the standard of care (SOC) Xpert only. Sensitivity analysis found that variations in the key model parameters had an impact on the cost and effectiveness calculations obtained through the model. -- Conclusions: The burden of TB-related costs impact both patients and the health care system at all stages of TB care. Novel diagnostic strategies like the inclusion of FujiLAM for TB diagnosis in PLHIV are cost-effective tools that can aid in case detection and reduce severe outcomes of TB. In order to reduce the TB burden and achieve the "End TB" strategy goals, studies need to work to understand the key components involved in these costs as well as work to develop and implement effective, feasible interventions for TB diagnostics and care.

Yes / Background: Mycobacterium tuberculosis rapid diagnostic tests (RDTs) are widely employed in routine laboratories and national surveys for detection of rifampicinresistant (RR)-TB. However, as next-generation sequencing technologies have become more commonplace in research and surveillance programs, RDTs are being increasingly complemented by whole genome sequencing (WGS). While comparison between RDTs is difficult, all RDT results can be derived from WGS data. This can facilitate continuous analysis of RR-TB burden regardless of the data generation technology employed. By converting WGS to RDT results, we enable comparison of data with different formats and sources particularly for low- and middle-income high TB-burden countries that employ different diagnostic algorithms for drug resistance surveys. This allows national TB control programs (NTPs) and epidemiologists to utilize all available data in the setting for improved RR-TB surveillance. Methods: We developed the Python-based MycTB Genome to Test (MTBGT) tool that transforms WGS-derived data into laboratory-validated results of the primary RDTs—Xpert MTB/RIF, XpertMTB/RIF Ultra, GenoType MDRTBplus v2.0, and GenoscholarNTM+MDRTB II. The tool was validated through RDT results of RR-TB strains with diverse resistance patterns and geographic origins and applied on routine-derived WGS data. Results: The MTBGT tool correctly transformed the single nucleotide polymorphism (SNP) data into the RDT results and generated tabulated frequencies of the RDT probes as well as rifampicin-susceptible cases. The tool supplemented the RDT probe reactions output with the RR-conferring mutation based on identified SNPs. The MTBGT tool facilitated continuous analysis of RR-TB and Xpert probe reactions from different platforms and collection periods in Rwanda. Conclusion: Overall, the MTBGT tool allows low- and middle-income countries to make sense of the increasingly generated WGS in light of the readily available RDT. / Erasmus Mundus Joint Doctorate Fellowship grant 2016- 1346.