An emerging clade of Chikungunya West African genotype discovered in real-time during 2023 outbreak in Senegal.

Chikungunya (CHIKV) is a re-emerging endemic arbovirus in West Africa. Since July 2023, Senegal and Burkina Faso have been experiencing an ongoing outbreak, with over 300 confirmed cases detected so far in the regions of Kédougou and Tambacounda in Senegal, the largest recorded outbreak yet. CHIKV is typically maintained in a sylvatic cycle in Senegal but its evolution and factors contributing to re-emergence are so far unknown in West Africa, leaving a gap in understanding and responding to recurrent epidemics. We produced, in real-time, the first locally-generated and publicly available CHIKV whole genomes in West Africa, to characterize the genetic diversity of circulating strains, along with phylodynamic analysis to estimate time of emergence and population growth dynamics. A novel strain of the West African genotype, phylogenetically distinct from strains circulating in previous outbreaks, was identified. This suggests a likely new spillover from sylvatic cycles in rural Senegal and potential of seeding larger epidemics in urban settings in Senegal and elsewhere.

Heterogeneity of the Omicron variant in Senegal.

•Omicron variant continues to progress in Senegal with the appearance of new contaminations.•IRESSEF detected the first positive case of the Omicron variant on Friday, December 3, 2021.•Since this date, the number of Omicron variant infections has increased over the weeks.•Molecular surveillance of the Omicron variant allowed us to identify a strong variation of this variant in our country.

Emergence of novel combinations of SARS-CoV-2 spike receptor binding domain variants in Senegal.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that carry mutations in the spike gene are of concern for potential impact to treatment and prevention efforts. To monitor for new SARS-CoV-2 mutations, a panel of specimens were sequenced from both wave one (N = 96), and wave two (N = 117) of the pandemic in Senegal by whole genome next generation sequencing. Amongst these genomes, new combinations of SARS-CoV-2 spike mutations were identified, with E484K + N501T, L452R + N501Y, and L452M + S477N exclusively found in second wave specimens. These sequences are evidence of local diversification over the course of the pandemic and parallel evolution of escape mutations in different lineages.

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.

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.

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.

Multisite evaluation of a point-of-care instrument for CD4(+) T-cell enumeration using venous and finger-prick blood: the PIMA CD4.

BACKGROUND: CD4(+) T-cell enumeration (CD4 count) is used as a criterion to initiate antiretroviral therapy (ART) in HIV patients and to monitor treatment efficacy. However, simple, affordable, and reliable point-of-care (POC) instruments adapted to resource-limited settings are still lacking. The PIMA CD4 analyzer is a new POC instrument for CD4 counting that uses disposable cartridges and a battery-powered analyzer. METHODS: Whole blood samples were taken by venipuncture or by finger prick from 300 subjects, including HIV-infected patients and HIV (-) controls. CD4 counts were measured by PIMA (using venous or capillary blood) and by FACSCount (using venous blood) considered as the reference. RESULTS: Similar CD4 counts were obtained by PIMA and FACSCount using either HIV+ venous blood or HIV+ finger-prick blood samples. However, with a concordance coefficient of 0.88 and a Pearson correlation of 0.89, finger-prick blood performed not as good as venous blood (0.97 and 0.98, respectively). For a clinical decision to start ART at 200 CD4 cells per microliter, sensitivity of PIMA was 90%/91% and specificity 98%/96% for venous/finger-prick blood, respectively, and for a treatment threshold of 350 CD4 cells per microliter, the sensitivity was 98%/91% and the specificity was 79%/80% for venous/finger-prick blood, respectively. Repeatability (precision) on venous blood resulted in a coefficient of variation of 4%. Using finger-prick blood, the average instrument error frequency resulting in aborted analyses was 14%. CONCLUSIONS: PIMA is a good POC instrument for screening adult HIV-infected patients in resource-limited settings for treatment eligibility. Its performance on finger-prick blood is not as good as on venous blood. Adequate training for correct use of finger-prick blood samples is mandatory.

Evaluation of a flow cytometry method for CD4 T cell enumeration based on volumetric primary CD4 gating using thermoresistant reagents.

Laboratory follow-up of HIV patients in resource-limited settings requires appropriate instruments for CD4 T cell enumeration. In this study, we evaluated the application of a simplified, mobile and robust flow cytometry system, the Apogee Auto 40 analyzer (Auto40) using thermoresistant reagents, for CD4 T cell enumeration. We measured the absolute CD4 counts in fresh whole blood samples from 170 Senegalese subjects, including 129 HIV-positive (HIV+) patients and 41 HIV-negative (HIV-) controls. Based on volumetric primary CD4 gating, cells were stained with commercially available reagents (Easy MoAb CD4;Bio-D, Valenzano, Italy) and analyzed on the Auto40. The results were compared with those from the FACSCount system (Becton Dickinson, San Jose, USA). Repeatability analysis was performed on duplicate testing of 49 samples on both FACSCount and Auto40. The intra-run precision was measured by 10 replicates using 3 clinical blood samples with low, intermediate and high CD4 concentrations. The results from the two instruments were in good agreement. The percent similarity between the results of both instruments was 99%±relative standard deviation of 12.7%. The concordance correlation coefficient was 0.99. The absolute bias and limits of agreement (LOA) between the two instruments, calculated by Bland-Altman analysis, were clinically acceptable (bias: +4 cells/µl; LOA: -111 to +120 cells/µl). The clinical agreement between the two instruments at a cutoff of 200 CD4 cells/µl was 94%. The repeatability of measurements on the Auto40 was also similar to that observed with FACSCount system (bias +0.1 cells/µl, coefficient of variation 2.5% vs bias -1.1cells/µl, coefficient of variation 2.9% respectively). In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings.

Low-level CD4+ T cell activation in HIV-exposed seronegative subjects: influence of gender and condom use.

Immune activation has been suggested to increase susceptibility to human immunodeficiency virus type 1 (HIV-1) transmission, while at the same time it could be deemed essential for mounting an effective antiviral immune response. In this study, we compared levels of T cell activation between exposed seronegative (ESN) partners in HIV-1 discordant couples and HIV-unexposed control subjects in Dakar, Senegal. ESN subjects showed lower levels of CD38 expression on CD4(+) T cells than did control subjects. However, this was found to be associated with concurrent differences in the use of condoms: ESN subjects reported a higher degree of condom use than did control subjects, which correlated inversely with CD38 expression. In addition, we observed markedly higher levels of T cell activation in women compared with men, irrespective of sexual behavior. These findings question the relevance of low-level CD4(+) T cell activation in resistance to HIV-1 infection and underscore the need to take gender and sexual behavior characteristics of high-risk populations into account when analyzing correlates of protective immunity.

Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal.

Measurement of viral load in plasma remains the best marker for the follow-up of antiretroviral therapy. However, its use is limited in developing countries due to the lack of adequate facilities and equipment, and cryopreservation of plasma during storage and transportation. Practical and reliable methods adapted to field conditions for the collection, transportation and accurate measurement of HIV-1 viral load are needed for the optimum use of antiretroviral therapy in resource-limited countries. This study evaluated the use of dried blood spots (DBS) for the real-time quantitation of HIV-1 RNA levels with the NucliSENS EasyQ((R)) HIV-1 assay (bioMérieux, Lyon, France) under field conditions in Senegal (Africa). Dried blood spots and plasma from 41 patients living in suburban Dakar were used for determination of HIV-1 RNA concentrations and stability at 37 degrees C. Analysis was performed at the Dakar University Hospital laboratory. Extraction was done with the bioMérieux NucliSENS((R)) miniMAGtrade mark, and real-time detection was done with the bioMérieux NucliSENS((R)) EasyQ system. HIV-1 RNA concentrations in plasma were compared with concentrations in dried blood spots after 8 and 15 days at 37 degrees C. The study showed a strong concordance in RNA levels between plasma and dried blood spots, which appear to be very stable over time with no apparent degradation observed after 2 weeks at 37 degrees C (mean difference 0.065logIU/ml). These results suggest that the use of dried blood spots in combination with the NucliSENS EasyQ HIV-1 assay is well adapted for HIV-1 RNA level monitoring in centralized laboratories in developing countries.

Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments.

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.