31 research outputs found

    The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

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    INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

    Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum -lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan

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    Background: This study aimed to characterize blaCTX-M, blaTEM, and blaSHV genes among extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae species in different regions of Sudan. Methods: In this cross-sectional study, different clinical samples (n = 985) were collected randomly from symptomatic patients from four geographical regions of Sudan and cultured on chromogenic media. Following bacterial identification, phenotypic screening of ESBLs was done according to CLSI guidelines using cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. The DNA was extracted by guanidine hydrochloride protocol, and then conventional PCR was used to detect blaCTX-M, blaTEM, and blaSHV genes. The presence of genes’ subtypes was characterized by DNA Sanger sequencing for selected samples. Results: Enterobacteriaceae represented 31% (305/985) of all isolates, 42 (128/305) of which were ESBLs producer, confirmed by  phenotypic confirmatory test (75% [96/128] of them were positive for blaCTX-M genes, 61% [78/128] for blaTEM genes, and 38% [48/128] for blaSHV genes). Fourteen isolates (11%) were negative for all genes. Forty-eight percent (63/75) of Escherichia coli isolates were positive for blaCTX-M, while in Klebsiella pneumoniae, the dominant gene was blaTEM (82%) and had a low amount of blaSHV (59%). There was a significant association (P-value = 0.001 for all except for chloramphenicol, P = 0.014, and amikacin, P = 0.017) between resistance to third-generation cephalosporins and ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin. Forty-two percent (40/96) of CTX-M-positive isolates were in Gizera State, 33% (32.96) in Sinnar, 24% (23/96) in Khartoum, and 1% (1/96) in White Nile. Conclusion: We conclude that blaCTX-M genes are the most dominant genes in ESBLsproducing isolates and are more prevalent in big cities than in rural areas. Keywords: phenotypic, blaCTX-M, blaTEM, and blaSHV ESBLs genes, Enterobacteriaceae, Suda

    Prevalence of blaCTX-M, blaTEM, and blaSHV Genes among Extended-spectrum β-lactamases-producing Clinical Isolates of Enterobacteriaceae in Different Regions of Sudan

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    Background: This study aimed to characterize blaCTX-M, blaTEM, and blaSHV genes among extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae species in different regions of Sudan. Methods: In this cross-sectional study, different clinical samples (n = 985) were collected randomly from symptomatic patients from four geographical regions of Sudan and cultured on chromogenic media. Following bacterial identification, phenotypic screening of ESBLs was done according to CLSI guidelines using cefotaxime (30 μg), ceftazidime (30 μg), and cefepime (30 μg) discs with and without clavulanic acid. The DNA was extracted by guanidine hydrochloride protocol, and then conventional PCR was used to detect blaCTX-M, blaTEM, and blaSHV genes. The presence of genes’ subtypes was characterized by DNA Sanger sequencing for selected samples.  Results: Enterobacteriaceae represented 31% (305/985) of all isolates, 42 (128/305) of which were ESBLs producer, confirmed by phenotypic confirmatory test (75% [96/128] of them were positive for blaCTX-M genes, 61% [78/128] for blaTEM genes, and 38% [48/128] for blaSHV genes). Fourteen isolates (11%) were negative for all genes. Forty-eight percent (63/75) of Escherichia coli isolates were positive for blaCTX-M, while in Klebsiella pneumoniae, the dominant gene was blaTEM (82%) and had a low amount of blaSHV (59%). There was a significant association (P-value = 0.001 for all except for chloramphenicol, P = 0.014, and amikacin, P = 0.017) between resistance to third-generation cephalosporins and ciprofloxacin, nalidixic acid, meropenem, chloramphenicol, and amikacin.  Forty-two percent (40/96) of CTX-M-positive isolates were in Gizera State, 33% (32.96) in Sinnar, 24% (23/96) in Khartoum, and 1% (1/96) in White Nile.  Conclusion: We conclude that blaCTX-M genes are the most dominant genes in ESBLs-producing isolates and are more prevalent in big cities than in rural areas. Keywords: phenotypic, blaCTX-M, blaTEM, and blaSHV ESBLs genes, Enterobacteriaceae, Suda

    Neutrophil elastase promotes Leishmania donovani infection via interferon-β

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    Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major named inhibitor of serine peptidases (ISP2) inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF and IFN production and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knock-out mice for NE (ela-/-), TLR4 or TLR2. NE and TLR4 co-localized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased nitric oxide and decreased TGF production. Expression of ISP2 was not detected in L. donovani and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN mRNA than background mice macrophages and the intracellular growth of was fully restored by exogenous IFN. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN necessary for parasite survival/growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway

    Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP)

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    <div><p>Background</p><p>Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp <i>Leishmania</i> Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.</p><p>Methodology/Principal findings</p><p>The Loopamp <i>Leishmania</i> Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite <i>Leishmania</i> rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.</p><p>Conclusions/Significance</p><p>Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.</p></div

    A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis

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    Background. Poor access to diagnosis stymies control of visceral leishmaniasis (VL). Antibody-detecting rapid diagnostic tests (RDTs) can be performed in peripheral health settings. However, there are many brands available and published reports of variable accuracy. Methods. Commercial VL RDTs containing bound rK39 or rKE16 antigen were evaluated using archived human sera from confirmed VL cases (n = 750) and endemic non-VL controls (n = 754) in the Indian subcontinent (ISC), Brazil, and East Africa to assess sensitivity and specificity with 95 confidence intervals. A subset of RDTs were also evaluated after 60 days' heat incubation (37°C, 45°C). Interlot and interobserver variability was assessed. Results. All test brands performed well against ISC panels (sensitivity range, 92.8-100; specificity range, 96-100); however, sensitivity was lower against Brazil and East African panels (61.5-91 and 36.8-87.2, respectively). Specificity was consistently > 95 in Brazil and ranged between 90.8 and 98 in East Africa. Performance of some products was adversely affected by high temperatures. Agreement between lots and readers was good to excellent ( > 0.73-0.99). Conclusions. Diagnostic accuracy of VL RDTs varies between the major endemic regions. Many tests performed well and showed good heat stability in the ISC; however, reduced sensitivity against Brazilian and East African panels suggests that in these regions, used alone, several RDTs are inadequate for excluding a VL diagnosis. More research is needed to assess ease of use and to compare performance using whole blood instead of serum and in patients coinfected with human immunodeficiency virus

    A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis

    Get PDF
    Background: Poor access to diagnosis stymies control of visceral leishmaniasis (VL). Antibody-detecting rapid diagnostic tests (RDTs) can be performed in peripheral health settings. However, there are many brands available and published reports of variable accuracy. Methods: Commercial VL RDTs containing bound rK39 or rKE16 antigen were evaluated using archived human sera from confirmed VL cases (n = 750) and endemic non-VL controls (n = 754) in the Indian subcontinent (ISC), Brazil, and East Africa to assess sensitivity and specificity with 95% confidence intervals. A subset of RDTs were also evaluated after 60 days' heat incubation (37°C, 45°C). Interlot and interobserver variability was assessed. Results: All test brands performed well against ISC panels (sensitivity range, 92.8%-100%; specificity range, 96%-100%); however, sensitivity was lower against Brazil and East African panels (61.5%-91% and 36.8%-87.2%, respectively). Specificity was consistently > 95% in Brazil and ranged between 90.8% and 98% in East Africa. Performance of some products was adversely affected by high temperatures. Agreement between lots and readers was good to excellent (κ > 0.73-0.99). Conclusions: Diagnostic accuracy of VL RDTs varies between the major endemic regions. Many tests performed well and showed good heat stability in the ISC; however, reduced sensitivity against Brazilian and East African panels suggests that in these regions, used alone, several RDTs are inadequate for excluding a VL diagnosis. More research is needed to assess ease of use and to compare performance using whole blood instead of serum and in patients coinfected with human immunodeficiency virus
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