RESUMO
BACKGROUND: Rapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive. METHODS: Paired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay. RESULTS: No significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood. CONCLUSIONS: No difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.
Assuntos
Capilares/parasitologia , Testes Diagnósticos de Rotina/estatística & dados numéricos , Malária/diagnóstico , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Plasmodium/isolamento & purificação , Veias/parasitologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Serra Leoa , Especificidade da Espécie , Adulto JovemRESUMO
BACKGROUND: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone. METHODS: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species. RESULTS: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens. CONCLUSIONS: Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.
Assuntos
Malária/epidemiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Plasmodium/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Testes Diagnósticos de Rotina , Feminino , Humanos , Masculino , Microscopia , Pessoa de Meia-Idade , Prevalência , Sensibilidade e Especificidade , Serra Leoa/epidemiologia , Adulto JovemRESUMO
A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.
Assuntos
Infecções Bacterianas/genética , Farmacorresistência Bacteriana/genética , Quinolonas/uso terapêutico , Bacillus/efeitos dos fármacos , Bacillus/genética , Bacillus/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Francisella/efeitos dos fármacos , Francisella/genética , Francisella/patogenicidade , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação/genética , Plasmídeos/genética , Yersinia/efeitos dos fármacos , Yersinia/genética , Yersinia/patogenicidadeRESUMO
Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.
Assuntos
Campylobacter/genética , Diarreia/microbiologia , Resistência Microbiana a Medicamentos , Escherichia coli Enteropatogênica/genética , Genes Bacterianos , Salmonella/genética , Shigella/genética , Antibacterianos/toxicidade , Campylobacter/efeitos dos fármacos , Campylobacter/isolamento & purificação , Campylobacter/patogenicidade , Diarreia/epidemiologia , Escherichia coli Enteropatogênica/efeitos dos fármacos , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/patogenicidade , Humanos , Salmonella/efeitos dos fármacos , Salmonella/isolamento & purificação , Salmonella/patogenicidade , Shigella/efeitos dos fármacos , Shigella/isolamento & purificação , Shigella/patogenicidadeRESUMO
Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase I-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91-100% sensitivity and 100% specificity in the tested strains, requiring half as many manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.
Assuntos
Bactérias/genética , DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas Biossensoriais , Desoxirribonuclease I/metabolismo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Sensibilidade e Especificidade , Coloração e RotulagemRESUMO
Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.
Assuntos
Bacillus anthracis/fisiologia , Óptica e Fotônica/métodos , Esporos Bacterianos/metabolismo , Animais , Citocalasina D/metabolismo , Dispositivos Lab-On-A-Chip , Lasers , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Microscopia Confocal , Fagocitose , Células RAW 264.7 , Esporos Bacterianos/imunologia , Proteína Vermelha FluorescenteRESUMO
A collection of 74 Enterobacteriaceae isolates found in Bo, Sierra Leone, were tested for quinolone antibiotic susceptibility and resistance mechanisms. The majority of isolates (62%) were resistant to quinolones, and 61% harbored chromosomal gyrA and/or parC mutations. Plasmid-mediated quinolone resistance genes were ubiquitous, with qnrB and aac(6')-Ib-cr being the most prevalent. Mutated LexA binding sites were found in all qnrB1 genes, and truncated qnrB pseudogenes were found in the majority of Citrobacter isolates.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Quinolonas/farmacologia , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Pseudogenes , Serra Leoa/epidemiologiaRESUMO
BACKGROUND: The rising level of antimicrobial resistance among bacterial pathogens is one of the most significant public health problems globally. While the antibiotic resistance of clinically important bacteria is closely tracked in many developed countries, the types and levels of resistance and multidrug resistance (MDR) among pathogens currently circulating in most countries of sub-Saharan Africa are virtually unknown. METHODS: From December 2013 to April 2014, we collected 93 urine specimens from all outpatients showing symptoms of urinary tract infection (UTI) and 189 fomite swabs from a small hospital in Bo, Sierra Leone. Culture on chromogenic agar combined with biochemical and DNA sequence-based assays was used to detect and identify the bacterial isolates. Their antimicrobial susceptibilities were determined using a panel of 11 antibiotics or antibiotic combinations. RESULTS: The 70 Enterobacteriaceae urine isolates were identified as Citrobacter freundii (n = 22), Klebsiella pneumoniae (n = 15), Enterobacter cloacae (n = 15), Escherichia coli (n = 13), Enterobacter sp./Leclercia sp. (n = 4) and Escherichia hermannii (n = 1). Antimicrobial susceptibility testing demonstrated that 85.7 % of these isolates were MDR while 64.3 % produced an extended-spectrum ß-lactamase (ESBL). The most notable observations included widespread resistance to sulphonamides (91.4 %), chloramphenicol (72.9 %), gentamycin (72.9 %), ampicillin with sulbactam (51.4 %) and ciprofloxacin (47.1 %) with C. freundii exhibiting the highest and E. coli the lowest prevalence of multidrug resistance. The environmental cultures resulted in only five Enterobacteriaceae isolates out of 189 collected with lower overall antibiotic resistance. CONCLUSIONS: The surprisingly high proportion of C. freundii found in urine of patients with suspected UTI supports earlier findings of the growing role of this pathogen in UTIs in low-resource countries. The isolates of all analyzed species showed worryingly high levels of resistance to both first- and second-line antibiotics as well as a high frequency of MDR and ESBL phenotypes, which likely resulted from the lack of consistent antibiotic stewardship policies in Sierra Leone. Analysis of hospital environmental isolates however suggested that fomites in this naturally ventilated hospital were not a major reservoir for Enterobacteriaceae or antibiotic resistance determinants.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Adulto , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Análise de Sequência de DNA , Serra Leoa , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , beta-Lactamases/genéticaRESUMO
Lassa virus (LASV) is endemic to parts of West Africa and causes highly fatal hemorrhagic fever. The multimammate rat (Mastomys natalensis) is the only known reservoir of LASV. Most human infections result from zoonotic transmission. The very diverse LASV genome has 4 major lineages associated with different geographic locations. We used reverse transcription PCR and resequencing microarrays to detect LASV in 41 of 214 samples from rodents captured at 8 locations in Sierra Leone. Phylogenetic analysis of partial sequences of nucleoprotein (NP), glycoprotein precursor (GPC), and polymerase (L) genes showed 5 separate clades within lineage IV of LASV in this country. The sequence diversity was higher than previously observed; mean diversity was 7.01% for nucleoprotein gene at the nucleotide level. These results may have major implications for designing diagnostic tests and therapeutic agents for LASV infections in Sierra Leone.
Assuntos
Variação Genética , Febre Lassa/epidemiologia , Febre Lassa/virologia , Vírus Lassa/classificação , Vírus Lassa/genética , Filogeografia , Animais , Genes Virais , Genoma Viral , Genótipo , Geografia , Febre Lassa/transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Ratos , Serra Leoa/epidemiologiaRESUMO
Laboratories associated with small hospitals often have limited expertise, personnel, and equipment to rapidly identify rare and emerging infectious diseases. We describe the successful use of the FilmArray system for rapid detection of Ebola virus directly from clinical samples in 6 out of 83 tested subjects in a small health care center in Sierra Leone.
Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Virologia/métodos , Sangue/virologia , Ebolavirus/genética , Hospitais , Humanos , Faringe/virologia , Serra LeoaRESUMO
Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.
Assuntos
Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Genes Bacterianos , Hospitais Militares , beta-Lactamases/genética , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Militares , Anotação de Sequência Molecular , Família Multigênica , Plasmídeos , Estados Unidos , beta-Lactamases/metabolismoRESUMO
OBJECTIVE: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM). RESULTS: Whole genome amplicons from 22 B. mallei (BM) and 37 B. pseudomallei (BP) isolates were tested for > 500 ARDs using ARDM v.3.1. ARDM detected the following Burkholderia spp.-derived genes, aac(6), blaBP/MBL-3, blaABPS, penA-BP, and qacE, in both BM and BP while blaBP/MBL-1, macB, blaOXA-42/43 and penA-BC were observed in BP only. The method of denaturing template for whole genome amplification greatly affected the numbers and types of genes detected by the ARDM. BlaTEM was detected in nearly a third of BM and BP amplicons derived from thermally, but not chemically denatured templates. BlaTEM results were confirmed by PCR, with 81% concordance between methods. Sequences from 414-nt PCR amplicons (13 preparations) were 100% identical to the Klebsiella pneumoniae reference gene. Although blaTEM sequences have been observed in B. glumae, B. cepacia, and other undefined Burkholderia strains, this is the first report of such sequences in BM/BP/B. thailandensis (BT) clade. These results highlight the importance of sample preparation in achieving adequate genome coverage in methods requiring untargeted amplification before analysis.
Assuntos
Anti-Infecciosos , Burkholderia mallei , Burkholderia pseudomallei , Burkholderia , Síndrome do Desconforto Respiratório , Humanos , Burkholderia mallei/genética , Burkholderia/genéticaRESUMO
We diagnosed 400 possible IgM-positive cases of chikungunya virus in Bo, Sierra Leone, during July 2012-January 2013 by using lateral flow immunoassays. Cases detected likely represent only a small fraction of total cases. Further laboratory testing is required to confirm this outbreak and characterize the virus.
Assuntos
Infecções por Alphavirus/epidemiologia , Vírus Chikungunya/imunologia , Surtos de Doenças , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções por Alphavirus/sangue , Infecções por Alphavirus/diagnóstico , Anticorpos Antivirais/sangue , Criança , Doenças Transmissíveis Emergentes/sangue , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/epidemiologia , Feminino , Humanos , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Monitorização Imunológica , Serra Leoa/epidemiologia , Adulto JovemRESUMO
We describe the results of a molecular epidemiological survey of 15 carbapenemase-encoding genes from a recent collection of clinical isolates from Mercy Hospital in Bo, Sierra Leone. The most salient findings revealed that (i) 60% of the isolates harbored multiple carbapenemase genes; (ii) the blaDIM-1 gene, which has previously only been reported in The Netherlands, is also circulating in this environment; and (iii) blaOXA-51-like and blaOXA-58 genes, which were thought to reside exclusively in Acinetobacter species, can also be found in members of the Enterobacteriaceae.
Assuntos
Proteínas de Bactérias/genética , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , beta-Lactamases/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Genótipo , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Análise de Sequência de DNA , Serra Leoa/epidemiologiaRESUMO
Human health depends on reliable access to safe drinking water, but in many developing countries only a limited number of wells and boreholes are available. Many of these water resources are contaminated with biological or chemical pollutants. The goal of this study was to examine water access and quality in urban Bo, Sierra Leone. A health census and community mapping project in one neighborhood in Bo identified the 36 water sources used by the community. A water sample was taken from each water source and tested for a variety of microbiological and physicochemical substances. Only 38.9% of the water sources met World Health Organization (WHO) microbial safety requirements based on fecal coliform levels. Physiochemical analysis indicated that the majority (91.7%) of the water sources met the requirements set by the WHO. In combination, 25% of these water resources met safe drinking water criteria. No variables associated with wells were statistically significant predictors of contamination. This study indicated that fecal contamination is the greatest health risk associated with drinking water. There is a need to raise hygiene awareness and implement inexpensive methods to reduce fecal contamination and improve drinking water safety in Bo, Sierra Leone.
Assuntos
Nível de Saúde , Poluição da Água/análise , Abastecimento de Água/estatística & dados numéricos , Água Potável/química , Monitoramento Ambiental , Humanos , Medição de Risco , Serra Leoa , Poluentes da Água/análise , Poluição da Água/estatística & dados numéricosRESUMO
BACKGROUND: Resource-limited tropical countries are home to numerous infectious pathogens of both human and zoonotic origin. A capability for early detection to allow rapid outbreak containment and prevent spread to non-endemic regions is severely impaired by inadequate diagnostic laboratory capacity, the absence of a "cold chain" and the lack of highly trained personnel. Building up detection capacity in these countries by direct replication of the systems existing in developed countries is not a feasible approach and instead requires "leapfrogging" to the deployment of the newest diagnostic systems that do not have the infrastructure requirements of systems used in developed countries. METHODS: A laboratory for molecular diagnostics of infectious agents was established in Bo, Sierra Leone with a hybrid solar/diesel/battery system to ensure stable power supply and a satellite modem to enable efficient communication. An array of room temperature stabilization and refrigeration technologies for reliable transport and storage of reagents and biological samples were also tested to ensure sustainable laboratory supplies for diagnostic assays. RESULTS: The laboratory demonstrated its operational proficiency by conducting an investigation of a suspected avian influenza outbreak at a commercial poultry farm at Bo using broad range resequencing microarrays and real time RT-PCR. The results of the investigation excluded influenza viruses as a possible cause of the outbreak and indicated a link between the outbreak and the presence of Klebsiella pneumoniae. CONCLUSIONS: This study demonstrated that by application of a carefully selected set of technologies and sufficient personnel training, it is feasible to deploy and effectively use a broad-range infectious pathogen detection technology in a severely resource-limited setting.
Assuntos
Surtos de Doenças/prevenção & controle , Influenza Aviária/diagnóstico , Laboratórios/organização & administração , Análise em Microsséries/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Comunicação , Países em Desenvolvimento , Surtos de Doenças/veterinária , Estabilidade de Medicamentos , Diagnóstico Precoce , Fontes de Energia Elétrica , Indicadores e Reagentes , Influenza Aviária/epidemiologia , Infecções por Klebsiella/diagnóstico , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/veterinária , Pessoal de Laboratório/educação , Análise em Microsséries/veterinária , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/veterinária , Aves Domésticas , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Serra Leoa/epidemiologia , Manejo de EspécimesRESUMO
Introduction: In spite of promising medical, sociological, and engineering strategies and interventions to reduce the burden of disease, malaria remains a source of significant morbidity and mortality, especially among children in sub-Saharan Africa. In particular, progress in the development and administration of chemotherapeutic agents is threatened by evolved resistance to most of the antimalarials currently in use, including artemisinins. Methods: This study analyzed the prevalence of mutations associated with antimalarial resistance in Plasmodium falciparum from 95 clinical samples collected from individuals with clinically confirmed malaria at a hospital in Bo, Sierra Leone between May 2017 and December 2018. The combination of polymerase chain reaction amplification and subsequent high throughput DNA sequencing was used to determine the presence of resistance-associated mutations in five P. falciparum genes - pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13. The geographic origin of parasites was assigned using mitochondrial sequences. Results: Relevant mutations were detected in the pfcrt (22%), pfmdr1 (>58%), pfdhfr (100%) and pfdhps (>80%) genes while no resistance-associated mutations were found in the pfkelch13 gene. The mitochondrial barcodes were consistent with a West African parasite origin with one exception indicating an isolate imported from East Africa. Discussion: Detection of the pfmdr1 NFSND haplotype in 50% of the samples indicated the increasing prevalence of strains with elevated tolerance to artemeter + lumefantrine (AL) threatening the combination currently used to treat uncomplicated malaria in Sierra Leone. The frequency of mutations linked to resistance to antifolates suggests widespread resistance to the drug combination used for intermittent preventive treatment during pregnancy.
RESUMO
A significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, including Mycobacterium, Brucella, Coxiella burnetii, Clostridium perfringens, and Bacillus. The presence of Coxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments.
Assuntos
Microbiologia do Ar , Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Poeira , Microbiologia do Solo , Bactérias/genética , DNA Bacteriano/genética , Humanos , Iraque , Kuweit , Análise em Microsséries/métodosRESUMO
Military recruits experience a high incidence of febrile respiratory illness (FRI), leading to significant morbidity and lost training time. Adenoviruses, group A Streptococcus pyogenes, and influenza virus are implicated in over half of the FRI cases reported at recruit training center clinics, while the etiology of the remaining cases is unclear. In this study, we explore the carriage rates and disease associations of adenovirus, enterovirus, rhinovirus, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis in military recruits using high-density resequencing microarrays. The results showed that rhinoviruses, adenoviruses, S. pneumoniae, H. influenzae, and N. meningitidis were widely distributed in recruits. Of these five agents, only adenovirus showed significant correlation with illness. Among the samples tested, only pathogens associated with FRI, such as adenovirus 4 and enterovirus 68, revealed strong temporal and spatial clustering of specific strains, indicating that they are transmitted primarily within sites. The results showed a strong negative association between adenoviral FRI and the presence of rhinoviruses in recruits, suggesting some form of viral interference.
Assuntos
Adenoviridae/isolamento & purificação , Infecções por Adenovirus Humanos/epidemiologia , Militares , Infecções por Picornaviridae/epidemiologia , Rhinovirus/isolamento & purificação , Adolescente , Bactérias/isolamento & purificação , Sequência de Bases , DNA Viral/análise , Feminino , Febre/etiologia , Febre/virologia , Humanos , Masculino , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Faringe/virologia , Doenças Respiratórias/etiologia , Doenças Respiratórias/virologia , Adulto JovemRESUMO
The Bacillus cereus group includes three closely related species, B. anthracis, B. cereus, and B. thuringiensis, which form a highly homogeneous subdivision of the genus Bacillus. One of these species, B. anthracis, has been identified as one of the most probable bacterial biowarfare agents. Here, we evaluate the sequence and length polymorphisms of the Bacillus collagen-like protein bcl genes as a basis for B. anthracis detection and fingerprinting. Five genes, designated bclA to bclE, are present in B. anthracis strains. Examination of bclABCDE sequences identified polymorphisms in bclB alleles of the B. cereus group organisms. These sequence polymorphisms allowed specific detection of B. anthracis strains by PCR using both genomic DNA and purified Bacillus spores in reactions. By exploiting the length variation of the bcl alleles it was demonstrated that the combined bclABCDE PCR products generate markedly different fingerprints for the B. anthracis Ames and Sterne strains. Moreover, we predict that bclABCDE length polymorphism creates unique signatures for B. anthracis strains, which facilitates identification of strains with specificity and confidence. Thus, we present a new diagnostic concept for B. anthracis detection and fingerprinting, which can be used alone or in combination with previously established typing platforms.