Factors associated with clinical progression to severe COVID-19 in people with cystic fibrosis: A global observational study.

BACKGROUND: This international study aimed to characterise the impact of acute SARS-CoV-2 infection in people with cystic fibrosis and investigate factors associated with severe outcomes. Methods Data from 22 countries prior to 13th December 2020 and the introduction of vaccines were included. It was de-identified and included patient demographics, clinical characteristics, treatments, outcomes and sequalae following SARS-CoV-2 infection. Multivariable logistic regression was used to investigate factors associated with clinical progression to severe COVID-19, using the primary outcome of hospitalisation with supplemental oxygen. RESULTS: SARS-CoV-2 was reported in 1555 people with CF, 1452 were included in the analysis. One third were aged <18 years, and 9.4% were solid-organ transplant recipients. 74.5% were symptomatic and 22% were admitted to hospital. In the non-transplanted cohort, 39.5% of patients with ppFEV1<40% were hospitalised with oxygen verses 3.2% with ppFEV >70%: a 17-fold increase in odds. Worse outcomes were independently associated with older age, non-white race, underweight body mass index, and CF-related diabetes. Prescription of highly effective CFTR modulator therapies was associated with a significantly reduced odds of being hospitalised with oxygen (AOR 0.43 95%CI 0.31-0.60 p<0.001). Transplanted patients were hospitalised with supplemental oxygen therapy (21.9%) more often than non-transplanted (8.8%) and was independently associated with the primary outcome (Adjusted OR 2.45 95%CI 1.27-4.71 p=0.007). CONCLUSIONS: This is the first study to show that there is a protective effect from the use of CFTR modulator therapy and that people with CF from an ethnic minority are at more risk of severe infection with SARS-CoV-2.

Incidence of SARS-CoV-2 in people with cystic fibrosis in Europe between February and June 2020.

BACKGROUND: Viral infections can cause significant morbidity in cystic fibrosis (CF). The current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic could therefore have a serious impact on the health of people with CF (pwCF). METHODS: We used the 38-country European Cystic Fibrosis Society Patient Registry (ECFSPR) to collect case data about pwCF and SARS-CoV-2 infection. RESULTS: Up to 30 June 2020, 16 countries reported 130 SARS-CoV-2 cases in people with CF, yielding an incidence of 2.70/1000 pwCF. Incidence was higher in lung-transplanted patients (n=23) versus non-transplanted patients (n=107) (8.43 versus 2.36 cases/1000). Incidence was higher in pwCF versus the age-matched general population in the age groups <15, 15-24, and 25-49 years (p<0.001), with similar trends for pwCF with and without lung transplant. Compared to the general population, pwCF (regardless of transplantation status) had significantly higher rates of admission to hospital for all age groups with available data, and higher rates of intensive care, although not statistically significant. Most pwCF recovered (96.2%), however 5 died, of whom 3 were lung transplant recipients. The case fatality rate for pwCF (3.85%, 95% CI: 1.26-8.75) was non-significantly lower than that of the general population (7.46%; p=0.133). CONCLUSIONS: SARS-CoV-2 infection can result in severe illness and death for pwCF, even for younger patients and especially for lung transplant recipients. PwCF should continue to shield from infection and should be prioritized for vaccination.

Factors for severe outcomes following SARS-CoV-2 infection in people with cystic fibrosis in Europe.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in people with cystic fibrosis (pwCF) can lead to severe outcomes. METHODS: In this observational study, the European Cystic Fibrosis Society Patient Registry collected data on pwCF and SARS-CoV-2 infection to estimate incidence, describe clinical presentation and investigate factors associated with severe outcomes using multivariable analysis. RESULTS: Up to December 31, 2020, 26 countries reported information on 828 pwCF and SARS-CoV-2 infection. Incidence was 17.2 per 1000 pwCF (95% CI: 16.0-18.4). Median age was 24 years, 48.4% were male and 9.4% had lung transplants. SARS-CoV-2 incidence was higher in lung-transplanted (28.6; 95% CI: 22.7-35.5) versus non-lung-transplanted pwCF (16.6; 95% CI: 15.4-17.8) (p≤0.001).SARS-CoV-2 infection caused symptomatic illness in 75.7%. Factors associated with symptomatic SARS-CoV-2 infection were age >40 years, at least one F508del mutation and pancreatic insufficiency.Overall, 23.7% of pwCF were admitted to hospital, 2.5% of those to intensive care, and regretfully 11 (1.4%) died. Hospitalisation, oxygen therapy, intensive care, respiratory support and death were 2- to 6-fold more frequent in lung-transplanted versus non-lung-transplanted pwCF.Factors associated with hospitalisation and oxygen therapy were lung transplantation, cystic fibrosis-related diabetes (CFRD), moderate or severe lung disease and azithromycin use (often considered a surrogate marker for Pseudomonas aeruginosa infection and poorer lung function). CONCLUSION: SARS-CoV-2 infection yielded high morbidity and hospitalisation in pwCF. PwCF with forced expiratory volume in 1 s <70% predicted, CFRD and those with lung transplants are at particular risk of more severe outcomes.

The global impact of SARS-CoV-2 in 181 people with cystic fibrosis.

With the growing SARS-CoV-2 pandemic, we need to better understand its impact in specific patient groups like those with Cystic Fibrosis (CF). We report on 181 people with CF (32 post-transplant) from 19 countries diagnosed with SARS-CoV-2 prior to 13 June 2020. Infection with SARS-CoV-2 appears to exhibit a similar spectrum of outcomes to that seen in the general population, with 11 people admitted to intensive care (7 post-transplant), and 7 deaths (3 post-transplant). A more severe clinical course may be associated with older age, CF-related diabetes, lower lung function in the year prior to infection, and having received an organ transplant. Whilst outcomes in this large cohort are better than initially feared overall, possibly due to a protective effect of the relatively younger age of the CF population compared to other chronic conditions, SARS-CoV-2 is not a benign disease for all people in this patient group.

Why oral antiseptic mouth rinsing before sputum collection cannot reduce contamination rate of mycobacterial culture in Burkina-Faso.

BACKGROUND: Tuberculosis (TB) diagnosis by culture in most resource-limited settings is hampered by high contamination rate varying up to 31%. Reduction of oral microorganism loads by mouth rinse with antiseptic before sputum collection showed a reduction of contamination. Moreover, knowing the characteristic of residual contaminant microorganisms would be an asset to understand contamination issues. OBJECTIVES: The aim of this study was to evaluate the effects of mouth rinsing with chlorhexidine on mycobacteria culture contaminations and to characterize morphologically the residual contaminants. METHODS: We consecutively included 158 patients in a TB center. Each of them supplied two sputa: The first before mouth rinse, and the second after 60sec of mouth rinsing with chlorhexidine (0.1%). Petroff method and Lowenstein-Jensen media were used for sputum decontamination and inoculation respectively. The contamination rates were compared, and the type of residual contaminants were characterized and compared. RESULTS: The contamination rate did not differ before and after the mouth rinse (respectively 58/150 (39 %) vs 61/150 (41 %), p=0.7). The major residual contaminants were Gram positive spore forming bacteria (94%). CONCLUSION: Chlorhexidine mouth rinsing before sputum collection did not reduce mycobacterial culture contamination rate. This is probably due to spore forming bacteria, highlighted as major residual contaminants.

Identification of spore-forming bacteria isolated from contaminated Lowenstein Jensen media and effectiveness of Vancomycin to reduce Mycobacterial culture contamination in Burkina-Faso.

The type of commensal microorganisms can influence the efficiency of sputum decontamination for TB diagnosis. A basic characterization of contaminants from LJ contaminated media showed that Gram positive Spore Forming Bacteria (SFB) were the major contaminants. This study aims to identify the species of this contaminants and to evaluate the effectiveness of VCNT at 10 µg of vancomycin to reduce mycobacterial culture contamination mainly linked to SFB. Fifty-three SFB isolated between February 2016 and May 2017 were used. The effectiveness of LJ with VCNT at 10 µg of Vancomycin were evaluated with sputum collected in the same period. SFB had been stored at -20 °C and identified after subculture onto 5% sheep blood Columbia agar and incubated at 37 °C during 24 h. Bacteria cells and isolated colonies were described. API 50CH/B was performed and MALDI-TOF MS was used for external quality control. Thirty- five (66%) isolates representing 4 genera (Bacillus, Paenibacillus, Brevisbacillus and Lysinibacillus) including 10 species were identified. The most important species were Bacillus cereus (30%) and Bacillus licheniformis (21%). Eighteen (34%) isolates were non-reactive Bacillus. The overall contamination rate on LJ with VCNT at 10 µg of vancomycin was statistically lower than which without VCNT (18.7% versus 43.8%) (p = 0.01). The most important SFB identified were B. cereus and B. licheniformis. Almost all identified strains were similar to those currently isolated in fermented traditional food suggesting in part food related contaminants. VCNT containing 10 µg of vancomycin is a good alternative method to reduce mycobacterial culture contamination.

Half of rifampicin-resistant Mycobacterium tuberculosis complex isolated from tuberculosis patients in Sub-Saharan Africa have concomitant resistance to pyrazinamide.

BACKGROUND: Besides inclusion in 1st line regimens against tuberculosis (TB), pyrazinamide (PZA) is used in 2nd line anti-TB regimens, including in the short regimen for multidrug-resistant TB (MDR-TB) patients. Guidelines and expert opinions are contradictory about inclusion of PZA in case of resistance. Moreover, drug susceptibility testing (DST) for PZA is not often applied in routine testing, and the prevalence of resistance is unknown in several regions, including in most African countries. METHODS: Six hundred and twenty-three culture isolates from rifampicin-resistant (RR) patients were collected in twelve Sub-Saharan African countries. Among those isolates, 71% were from patients included in the study on the Union short-course regimen for MDR-TB in Benin, Burkina Faso, Burundi, Cameroon, Central Africa Republic, the Democratic Republic of the Congo, Ivory Coast, Niger, and Rwanda PZA resistance, and the rest (29%) were consecutive isolates systematically stored from 2014-2015 in Mali, Rwanda, Senegal, and Togo. Besides national guidelines, the isolates were tested for PZA resistance through pncA gene sequencing. RESULTS: Over half of these RR-TB isolates (54%) showed a mutation in the pncA gene, with a significant heterogeneity between countries. Isolates with fluoroquinolone resistance (but not with injectable resistance or XDR) were more likely to have concurrent PZA resistance. The pattern of mutations in the pncA gene was quite diverse, although some isolates with an identical pattern of mutations in pncA and other drug-related genes were isolated from the same reference center, suggesting possible transmission of these strains. CONCLUSION: Similar to findings in other regions, more than half of the patients having RR-TB in West and Central Africa present concomitant resistance to PZA. Further investigations are needed to understand the relation between resistance to PZA and resistance to fluoroquinolones, and whether continued use of PZA in the face of PZA resistance provides clinical benefit to the patients.

Emergence and clonal transmission of multi-drug-resistant tuberculosis among patients in Chad.

BACKGROUND: Emergence of Multidrug-resistant (MDR) strains constitutes a significant public health problem worldwide. Prevalence of MDR tuberculosis from Chad is unavailable to date. METHODS: We collected samples from consecutive TB patients nationwide in the seven major cities of Chad between 2007 and 2012 to characterize drug resistance and the population structure of circulating Mycobacterium tuberculosis complex (MTBC) strains. We tested drug sensitivity using Line Probe Assays and phenotypic drug susceptibility testing (DST) were used for second line drugs. We genotyped the isolates using spoligotype analysis and MIRU-VNTR. RESULTS: A total of 311 cultures were isolated from 593 patients. The MDR prevalence was 0.9% among new patients and 3.5% among retreatment patients, and no second line drug resistance was identified. The distribution of genotypes suggests a dissemination of MDR strains in the Southern city of Moundou, bordering Cameroon and Central African Republic. CONCLUSION: Emerging MDR isolates pose a public health threat to Southern Chad, with risk to neighboring countries. This study informs public health practitioners, justifying the implementation of continuous surveillance with DST for all retreatment cases as well as contacts of MDR patients, in parallel with provision of adequate 2nd line regimens in the region.

Performance of FACSPresto Point-of-Care Instrument for CD4-T Cell Enumeration in Human Immunodeficiency Virus (HIV)-Infected Patients Attending Care and Treatment Clinics in Belgium and Tanzania.

BACKGROUND: CD4 T-cell counts are widely used to assess treatment eligibility and to follow-up HIV-infected patients. The World Health Organization prequalification of in vitro diagnostics program conducted a performance evaluation of the FACSPresto (BD Biosciences), a new point-of-care instrument to measure absolute CD4-T cell (CD4) counts and percentages in venous and capillary blood samples from HIV-infected patients. METHODS: Patients were recruited in Belgium (200 patients) and in Tanzania (247 patients). Venous blood samples were analyzed in two nearby reference laboratories. In addition, nurses/technicians collected a capillary blood sample by finger prick directly into a FACSPresto CD4 cartridge. Assay precision was assessed on fresh blood and on external quality control samples. Trueness (bias) was assessed by comparing results from FACSPresto with the reference (single-platform FACSCalibur). Clinical misclassification was measured at 200, 350 and 500 cells/µL thresholds. RESULTS: Intra-assay precision was < 6%, and inter-assay < 8%. CD4 results from FACSPresto and reference method resulted in regression slopes of 0.99-1.11 using either venous or capillary blood. Correlation was better for venous than for capillary blood (minimum 0.97 vs 0.93 respectively). Capillary blood resulted in a larger bias than venous blood, with 24 and 83 cells/µL for absolute CD4 counts on capillary blood in Antwerp and Dar es Salaam respectively, vs 12 and 41 cells/µL on venous blood. Bias on CD4% was < 1% on both venous and capillary blood, and was proportionally better than for absolute CD4 counts. Clinical misclassification was in line with the average overestimation, showing a very good specificity, but sensitivity around 70-90%. The rejection rate was 11% on first reading, leading to 6% of all samples without final result after a second reading. CONCLUSIONS: The FACSPresto performed very well on venous blood samples, and well on capillary blood samples.

CD4 results with a bias larger than hundred cells per microliter can have a significant impact on the clinical decision during treatment initiation of HIV patients.

BACKGROUND: CD4 counts are currently used to assess HIV patients for treatment eligibility and to monitor antiretroviral response to treatment. The emerging point-of-care devices could fill an important gap in resource-limited settings. However, the accuracy of CD4-counting instruments is diverse and data on how CD4 measurement errors have an impact on clinical decisions are lacking. METHODS: Clinicians were queried on the use of CD4 results in their clinical setting. Subsequently, the effect of CD4 measurement errors on treatment initiation was put in a statistical model. Based on clinical CD4 databases from Belgium, Cambodia, and Senegal, the percentage of unchanged clinical decisions was calculated (treatment initiation should start within a 3-month delay [one visit]) for escalating CD4 measurement errors, taking into account the strict or preventive application of CD4 thresholds at 350 or 500 cells/µl used by clinicians. RESULTS: To ensure that the treatment was initiated appropriately for at least 95% of patients, an error of 5 - 10 cells/µl was allowed. This is significantly smaller than the bias of ±50 cells/µl most clinicians considered acceptable. For limits of agreement (LOA, 1.96 x error) of 100 cells/µl, corresponding to most CD4 instrument evaluations, the misclassification rate of patients was found to be 3 - 28% at the threshold of 350 cells/µl (strict or flexible), and 13 - 20% at 500 cells/µl. CONCLUSIONS: The maximum allowed CD4 bias on results from new CD4 technologies should not exceed 50 cells/µl (LOA 100 cells/µl) when applied for treatment initiation, to ensure at least 72% of correct clinical decisions. © 2016 International Clinical Cytometry Society.

Human Immunodeficiency Virus (HIV)-Infected Patients Accept Finger Stick Blood Collection for Point-Of-Care CD4 Testing.

INTRODUCTION: HIV-infected patients require antiretroviral treatment for life. To improve access to care, CD4 enumeration and viral load tests have been redesigned to be used as point-of-care techniques using finger-stick blood. Accurate CD4 counting in capillary blood requires a free flowing blood drop that is achieved by blade incision. The aim of this study was to assess the attitude of the patients toward blade-based finger-stick blood donation. METHODS: Four hundred and ninety-nine patients were included (299 patients from South Africa and 200 from Belgium). They completed a questionnaire to express their preference for finger stick or venipuncture, after undergoing both. The South African patient cohort was divided in two groups, receiving either single or multiple finger stick for CD4 and other HIV-related tests. The Belgian patients received a single finger stick for CD4 testing, and were asked to respond directly and again after two days. RESULTS: The majority of the patients preferred the finger stick to the venipuncture. The perceived pain using the blade was superior to a small needle, but similar to a large needle. They preferred up to three finger sticks over one venipuncture. Up to 30% of the patients changed their mind over two days. The main reason for choosing a finger stick was continued bleeding after venipuncture. The most cited objection to finger stick was pain/soreness. CONCLUSION: Patient perceptions support the implementation of donating capillary blood with blade-based finger stick during CD4 point-of-care testing.

Use of RODAC plates to measure containment of Mycobacterium tuberculosis in a Class IIB biosafety cabinet during routine operations.

OBJECTIVE/BACKGROUND: Guidelines for the manipulation of Mycobacterium tuberculosis (MTB) cultures require a Biosafety Level 3 (BSL-3) infrastructure and accompanying code of conduct. In this study, we aimed to validate and apply detection methods for viable mycobacteria from surfaces in a BSL-3 MTB laboratory. METHODS: We evaluated phenotypic (Replicate Organism Detection and Counting [RODAC] plates) and molecular (propidium monoazide [PMA]-based polymerase chain reaction [PCR]) approaches for the detection of viable mycobacteria, as well as the effect of 70% ethanol applied for 5min for disinfection against mycobacteria. For validation of the method, recovery of serial dilutions of Mycobacterium bovis bacillus Calmette-Guérin from glass slides was measured. Subsequently, we stamped surfaces in and around the biosafety cabinet (BSC) after different technicians had manipulated high bacterial load suspensions for routine drug-susceptibility testing in a Class II BSC. RESULTS: RODAC stamping could detect as few as three bacteria on slides stamped either 5min or 60min after inoculation. PMA-based PCR, tested in parallel, did not pass validation. Mycobacteria were still detected after 5-min disinfection with ethanol 70%. In the BSL-3, from 201 RODAC-stamped surfaces, MTB was detected in four: three inside a BSC-on a tube cap and on an operator's gloves-and one outside, on an operator's gown. CONCLUSION: RODAC plates detect mycobacteria at low numbers of microorganisms. In addition, this method allowed us to show that 70% ethanol does not reliably kill mycobacteria when applied for 5min to a dried surface, and that MTB bacilli may arrive outside a Class II BSC during routine practice, although the route could not be documented.

The majority of patients with multidrug-resistant tuberculosis in Sub-Saharan Africa present a concomitant resistance to pyrazinamide.

OBJECTIVE/BACKGROUND: Pyrazinamide (PZA) is an antibacterial used in the first-line regimen against tuberculosis (TB) for its action against dormant bacilli. PZA is also included in the new short regimen to treat multidrug-resistant TB (MDR-TB). However, the prevalence and significance of PZA resistance is not known in Central and West Africa. METHODS: Between 2013 and 2016, we collected samples from MDR-TB patients recruited in an observational study implementing the new short MDR-TB regimen in six countries: Burundi (n=35), Cameroon (n=135), Niger (n=57), Central African Republic (n=35), Democratic Republic of the Congo (n=99), and Rwanda (n=16). Resistance to rifamipicine, isoniazide, injectables, and fluoroquinolones was tested by phenotypic (live strains) or genotypic methods (inactivated strains). Resistance to PZA was analyzed through sequencing of the pncA gene. Relevance of mutations was established based on recent literature. RESULTS: From 377 patients with MDR-TB, 354 (94%) samples were successfully sequenced. Among those, 53% (189) presented a mutation in pncA that confers a reported (121), potential (56), or unclear (12) resistance. Furthermore, six isolates presented a mutation associated with PZA sensitivity. The frequency of resistance per country was 26% in Central African Republic, 39% in Niger, 49% in Cameroon, 66% in Burundi, 68% in Democratic Republic of the Congo, and 87% in Rwanda. Isolates presented 109 different profiles of mutations, including 73 occurring only once. Codon 12 was most frequently affected (15 isolates), including 10 isolates with Asp12Ala. These 10 isolates came from three different countries, and presented different profiles of resistance to other drugs. The two next most frequent mutations, Met175Ile and Gln10Pro (8 isolates and 7 isolates, respectively), each suggest clusters of transmission, with similar geographical and resistance characteristics. Moreover, four isolates presented two simultaneous genetic variations, and 11 patients had a mix of sensitive and resistant bacilli. Preliminary data tend to indicate that patients carrying a PZA-resistant isolate had a higher failure rate on the new short MDR-TB treatment regimen (7% vs. 3%). All isolates resistant to injectables (4) and most (19/21) of those resistant to fluoroquinolones, including two extremely-resistant TB isolates, were also resistant to PZA. CONCLUSION: Similar to other regions in the world, the majority of MDR-TB strains from Sub-Saharan Africa countries are resistant to PZA, albeit with diverse rates between countries. We identified a diverse range of mutations in pncA, with 30% of them not previously reported. The impact of such resistance on the success of the short MDR-TB regimen will require more investigation.

Laboratory and field evaluation of the Partec CyFlow miniPOC for absolute and relative CD4 T-cell enumeration.

BACKGROUND: A new CD4 point-of-care instrument, the CyFlow miniPOC, which provides absolute and percentage CD4 T-cells, used for screening and monitoring of HIV-infected patients in resource-limited settings, was introduced recently. We assessed the performance of this novel instrument in a reference laboratory and in a field setting in Senegal. METHODOLOGY: A total of 321 blood samples were obtained from 297 adults and 24 children, all HIV-patients attending university hospitals in Dakar, or health centers in Ziguinchor. Samples were analyzed in parallel on CyFlow miniPOC, FACSCount CD4 and FACSCalibur to assess CyFlow miniPOC precision and accuracy. RESULTS: At the reference lab, CyFlow miniPOC, compared to FACSCalibur, showed an absolute mean bias of -12.6 cells/mm3 and a corresponding relative mean bias of -2.3% for absolute CD4 counts. For CD4 percentages, the absolute mean bias was -0.1%. Compared to FACSCount CD4, the absolute and relative mean biases were -31.2 cells/mm3 and -4.7%, respectively, for CD4 counts, whereas the absolute mean bias for CD4 percentages was 1.3%. The CyFlow miniPOC was able to classify HIV-patients eligible for ART with a sensitivity of ≥ 95% at the different ART-initiation thresholds (200, 350 and 500 CD4 cells/mm3). In the field lab, the room temperature ranged from 30 to 35°C during the working hours. At those temperatures, the CyFlow miniPOC, compared to FACSCount CD4, had an absolute and relative mean bias of 7.6 cells/mm3 and 2.8%, respectively, for absolute CD4 counts, and an absolute mean bias of 0.4% for CD4 percentages. The CyFlow miniPOC showed sensitivity equal or greater than 94%. CONCLUSION: The CyFlow miniPOC showed high agreement with FACSCalibur and FACSCount CD4. The CyFlow miniPOC provides both reliable absolute CD4 counts and CD4 percentages even under the field conditions, and is suitable for monitoring HIV-infected patients in resource-limited settings.

WHO multicenter evaluation of FACSCount CD4 and Pima CD4 T-cell count systems: instrument performance and misclassification of HIV-infected patients.

BACKGROUND: CD4⁺ T-cell counts are used to screen and follow-up HIV-infected patients during treatment. As part of the World Health Organization prequalification program of diagnostics, we conducted an independent multicenter evaluation of the FACSCount CD4 and the Pima CD4, using the FACSCalibur as reference method. METHODS: A total of 440 paired capillary and venous blood samples were collected from HIV-infected patients attending the HIV outpatient clinic in Antwerp, Belgium, and the HIV care and treatment center in Dar es Salam, Tanzania. Capillary blood was run on Pima analyzer, whereas venous blood was analyzed on FACSCount, Pima, and FACSCalibur instruments. Precision and agreement between methods were assessed. RESULTS: The FACSCount CD4 results were in agreement with the FACSCalibur results with relative bias of 0.4% and 3.1% on absolute CD4 counts and an absolute bias of -0.6% and -1.1% on CD4% in Antwerp and Dar es Salam, respectively. The Pima CD4 results were in agreement with the FACSCalibur results with relative bias of -4.1% and -9.4% using venous blood and of -9.5% and -0.9% using capillary blood in Antwerp and Dar es Salam, respectively. At the threshold of 350 cells per microliter, the FACSCount CD4 and Pima CD4 using venous and capillary blood misclassified 7%, 9%, and 13% of patients, respectively. CONCLUSIONS: The FACSCount CD4 provides reliable CD4 counts and CD4% and is suitable for monitoring adult and pediatric HIV patients in moderate-volume settings. The Pima CD4 is more suitable for screening eligible adult HIV patients for antiretroviral treatment initiation in low-volume laboratories.

Impact of pre-analytical factors on mycobacterium cultures contaminations rates in Burkina Faso, West Africa.

INTRODUCTION: For a high quality level diagnosis, mycobacterium culture must comply with the pre-analytical and analytical conditions recommended by the WHO and the country National Tuberculosis Program (NTP). In this study, we determined whether temperature and duration of sputum storage were associated with culture contamination in Burkina Faso. METHODS: Sputa were collected in 5 districts labs in Burkina Faso. Temperature and duration of sputum storage were recorded. After the collection, sputa were decontaminated using Petroff modified method, and the pellet was inoculated on LJ media and LJ media supply with 2% sodium pyruvate. Risk of culture contamination associated with temperature and duration of sputum storage was measured by Chi2 test and logistic regression. RESULTS: Out of 404 specimens, 61% (246/404) were stored between 2 and 8°C, and 15% (61/404) were processed within three days. The global contamination rate was 24%, with only 8% for samples respecting WHO recommendations, up to 35% for others. Storage at room temperature was associated with a significantly higher risk of contamination compared to storage at 2-8°C (OR 2.24, p = 0.001, IC 95%). CONCLUSION: The recommendations about the temperature and the duration of sputum storage before cultures are not completely respected. This leads to high contamination rate of mycobacterium culture. It will be necessary to take logistics measures in peripherals health services or to develop more selective medium for mycobacterium culture in low income countries.

CD4 T-cell enumeration in a field setting: evaluation of CyFlow counter using the CD4 easy count kit-dry and Pima CD4 systems.

BACKGROUND: Flow Cytometry (FCM) is still considered to be the method of choice for accurate CD4 enumeration. However, the use of FCM in developing countries is problematic due to their cost and complexity. Lower-cost technologies have been introduced. We evaluated CyFlow Counter together with its lyophilized reagents, and Pima CD4 in high-temperature area, using FACSCount as reference. MATERIALS AND METHODS: Whole blood samples were consecutively collected by venipuncture from 111 HIV+ patients and 17 HIV-negative donors. CD4 T-cell enumeration was performed on CyFlow Counter, Pima CD4 and FACSCount. RESULTS: CyFlow Counter and Pima CD4 systems showed good correlation with FACSCount (slope of 0.82 and 0.90, and concordance ρc of 0.94 and 0.98, respectively). CyFlow Counter showed absolute or relative biases (LOA) of -63 cells/mm(3) (-245 to 120) or -9.8% (-38.1 to 18.4) respectively, and Pima CD4 showed biases (LOA) of -30 cells/mm(3) (-160 to 101) or -3.5% (-41.0 to 33.9%). CyFlow Counter and Pima CD4 showed respectively 106.7% and 105.9% of similarity with FACSCount. According to WHO-2010 ART initiation threshold of 350 cells/mm(3), CyFlow Counter and Pima CD4 showed respectively sensibility of 100% and 97%, and specificity of 91% and 93%. CyFlow Counter and Pima CD4 were strongly correlated (slope of 1.09 and ρc of 0.95). These alternative systems showed good agreement with bias of 33 cells/mm(3) (-132 to 203) or 6.3% (-31.2 to 43.8), and similarity of 104.3%. CONCLUSION: CyFlow Counter using CD4 easy count kit-dry and Pima CD4 systems can accurately provide CD4 T-cell counts with acceptable agreement to those of FACSCount.

HIV and Tuberculosis co-infection impacts T-cell activation markers but not the numbers subset of regulatory T-cells in HIV-1 infected patients.

BACKGROUND: Tuberculosis (TB) has been shown to accelerate the clinical course of HIV infection, but the mechanisms by which this occurs are not well understood. Regulatory T-cells (Tregs) are known to dampen hyperactivation of the immune cells, but it remains unclear whether hyperactivation of T-cells in HIV infection is associated with a decrease of Tregs and what the effect Mycobacterium tuberculosis (MTB) co-infection has on T-cell activation and Tregs. OBJECTIVES: In this study, we aim to evaluate whether active TB is associated with the increased expression of T-cell activation markers and reduced number of Treg cells in HIV-1-infected patients. METHODS: This study was conducted on 69 subjects consisting of 20 HIV-infected patients, 20 HIV and MTB co-infected patients, 19 MTB-infected patients and 10 uninfected control subjects negative for both MTB and HIV. The frequencies of T-cell activation markers (CD38 and HLA-DR) and Treg cells (CD4+CD25+CD127-) were measured by flow cytometry. RESULTS: Significantly higher expression of CD38 and HLA-DR on CD4+ and CD8+ T-cells was found in MTB and HIV co-infected patients compared with HIV-infected patients. However, no significant difference in the percentage of Treg cells was reported between HIV patients with TB and those without. The study also showed a negative correlation between regulatory T-cells frequency and CD4+ T-cell counts. CONCLUSION: These results suggest that TB enhances the expression of peripheral T-cell activation markers during HIV infection, whilst having no impact on the percentages of Treg cells.

Performance of the PointCare NOW system for CD4 counting in HIV patients based on five independent evaluations.

INTRODUCTION: Point-of-care (POC) CD4 testing can improve access to treatment by enabling decentralization and reducing patient loss-to-follow-up. As new POC CD4 technologies become available, their performance should be assessed before widespread deployment. This study reports the findings of five independent evaluations of the PointCare NOW CD4 system. MATERIALS/METHODS: Evaluations were conducted in Southern Africa (Mozambique, South Africa) and North America (Canada, USA). 492 blood samples (55 from HIV-negative blood donors and 437 from HIV-infected patients, including 20 children aged between 12 and 59 months) were tested with both the PointCare NOW and reference flow cytometry instruments. Assessment of bias, precision and levels of clinical misclassification for absolute and percent CD4 count was conducted. RESULTS: PointCare NOW significantly overestimated CD4 absolute counts with a mean relative bias of +35.0%. Bias was greater in samples with CD4 counts below ≤ 350 cells/µl (+51.3%) than in the CD4 >350 cells/µl stratum (15.1%). Bias in CD4% had a similar trend with an overall relative mean bias of +25.6% and a larger bias for low CD4 stratum (+40.2%) than the higher CD4 stratum (+5.8%). Relative bias for CD4% in children was -6.8%. In terms of repeatability, PointCare NOW had a coefficient of variation of 11%. Using a threshold of 350 cells/µl, only 47% of patients who qualified for antiretroviral therapy with reference CD4 testing, would have been eligible for treatment with PointCare NOW test results. This was 39% using a 200 cells/µl threshold. Agreement with infant samples was higher, with 90% qualifying at a 25% eligibility threshold. CONCLUSION: The performance of the PointCare NOW instrument for absolute and percent CD4 enumeration was inadequate for HIV clinical management in adults. In children, the small sample size was not large enough to draw a conclusion. This study also highlights the importance of independent evaluation of new diagnostic technology platforms before deployment.