RESUMEN
The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.
Asunto(s)
Bacterias , Cartilla de ADN , ARN Polimerasas Dirigidas por ADN , Análisis de Secuencia de ADN , Humanos , ARN Polimerasas Dirigidas por ADN/genética , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación , Cartilla de ADN/genética , ADN Bacteriano/genética , ARN Ribosómico 16S/genética , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Técnicas de Diagnóstico Molecular/métodos , Técnicas Bacteriológicas/métodos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Bacterianas/genéticaRESUMEN
BACKGROUND: Leishmaniasis is a rare but potentially severe tropical infectious disease, and Norwegian clinicians are generally unfamiliar with its diagnosis and treatment. This study aimed to investigate the number of cases diagnosed, performance of diagnostic methods and treatment of leishmaniasis at five university hospitals in Norway. MATERIAL AND METHOD: The number of cases, diagnosis and treatment of suspected leishmaniasis were registered prospectively in the period March 2014 - September 2017 at the university hospitals of Bergen, Oslo, Stavanger, Trondheim and Tromsø. RESULTS: A total of 13 patients with leishmaniasis were registered in the period. Visceral leishmaniasis was diagnosed in two patients infected in the Mediterranean region, after 7 and 8 weeks with symptoms. The diagnosis was made by serology as well as microscopy and/or polymerase chain reaction tests (PCR) on spleen, blood and bone marrow. Both patients were treated effectively with liposomal amphotericin B. Cutaneous leishmaniasis was diagnosed in 11 patients, and samples from 10 of these tested positive with PCR. Two patients were infected with potentially mucotropic species. Liposomal amphotericin B was the first-line choice for all those who received treatment, but one patient recovered only after local therapy with sodium stibogluconate. INTERPRETATION: Assessment of visceral leishmaniasis was undertaken according to international guidelines. The patients were diagnosed late in the disease course, presumably because the disease is rare and not well known in Norway. Cutaneous leishmaniasis was diagnosed with PCR, but none of the patients received local treatment as the first-line choice, as recommended in suitable cases, presumably because the drugs are not readily available in Norway and many clinicians are unfamiliar with the route of administration with local infiltration.
Asunto(s)
Antiprotozoarios , Leishmaniasis Visceral , Antiprotozoarios/uso terapéutico , Médula Ósea , Humanos , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/epidemiología , Región Mediterránea , Noruega/epidemiologíaRESUMEN
The role of interactions between intestinal pathogens in diarrheal disease is uncertain. From August 2010 to July 2011, we collected stool samples from 723 children admitted with diarrhea (cases) to 3 major hospitals in Dar es Salaam, Tanzania, and from 564 nondiarrheic children (controls). We analyzed the samples for 17 pathogens and assessed interactions between coinfections in additive and multiplicative models. At least one pathogen was detected in 86.9% of the cases and 62.8%, of the controls. Prevalence of coinfections was 58.1% in cases and 40.4% in controls. Rotavirus, norovirus genogroup II, Cryptosporidium, and Shigella species/enteroinvasive Escherichia coli were significantly associated with diarrhea both as monoinfections and as coinfections. In the multiplicative interaction model, we found 2 significant positive interactions: rotavirus + Giardia (odds ratio (OR) = 23.91, 95% confidence interval (CI): 1.21, 470.14) and norovirus GII + enteroaggregative E. coli (OR = 3.06, 95% CI: 1.17, 7.98). One significant negative interaction was found between norovirus GII + typical enteropathogenic E. coli (OR = 0.09, 95% CI: 0.01, 0.95). In multivariate analysis, risk factors for death were presence of blood in stool and severe dehydration. In conclusion, coinfections are frequent, and the pathogenicity of each organism appears to be enhanced by some coinfections and weakened by others. Severity of diarrhea was not affected by coinfections.
Asunto(s)
Coinfección/epidemiología , Diarrea/epidemiología , Heces , Microbioma Gastrointestinal , Parásitos/patogenicidad , Virus/patogenicidad , Animales , Estudios de Casos y Controles , Coinfección/complicaciones , Coinfección/microbiología , Coinfección/parasitología , Diarrea/microbiología , Diarrea/parasitología , Ensayo de Inmunoadsorción Enzimática , Heces/química , Heces/microbiología , Heces/parasitología , Femenino , Humanos , Lactante , Modelos Logísticos , Masculino , Parásitos/clasificación , Parásitos/aislamiento & purificación , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Tanzanía/epidemiología , Virulencia , Virus/clasificación , Virus/aislamiento & purificaciónRESUMEN
BACKGROUND: The spread of Extended Spectrum ß-lactamases (ESBLs) among Enterobacteriaceae and other Gram-Negative pathogens in the community and hospitals represents a major challenge to combat infections. We conducted a study to assess the prevalence and genetic makeup of ESBL-type resistance in bacterial isolates causing community- and hospital-acquired urinary tract infections. METHODS: A total of 172 isolates of Enterobacteriaceae were collected in Dar es Salaam, Tanzania, from patients who met criteria of community and hospital-acquired urinary tract infections. We used E-test ESBL strips to test for ESBL-phenotype and PCR and sequencing for detection of ESBL genes. RESULTS: Overall 23.8% (41/172) of all isolates were ESBL-producers. ESBL-producers were more frequently isolated from hospital-acquired infections (32%, 27/84 than from community-acquired infections (16%, 14/88, p < 0.05). ESBL-producers showed high rate of resistance to ciprofloxacin (85.5%), doxycycline (90.2%), gentamicin (80.5%), nalidixic acid (84.5%), and trimethoprim-sulfamethoxazole (85.4%). Furthermore, 95% of ESBL-producers were multi-drug resistant compared to 69% of non-ESBL-producers (p < 0.05). The distribution of ESBL genes were as follows: 29/32 (90.6%) bla CTX-M-15, two bla SHV-12, and one had both bla CTX-M-15 and bla SHV-12. Of 29 isolates carrying bla CTX-M-15, 69% (20/29) and 31% (9/29) were hospital and community, respectively. Bla SHV-12 genotypes were only detected in hospital-acquired infections. CONCLUSION: bla CTX-M-15 is a predominant gene conferring ESBL-production in Enterobacteriaceae causing both hospital- and community-acquired infections in Tanzania.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/genética , Infecciones Urinarias/epidemiología , Infecciones Urinarias/microbiología , beta-Lactamasas/genética , Infecciones Comunitarias Adquiridas/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Farmacorresistencia Bacteriana/genética , Resistencia a Múltiples Medicamentos/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Femenino , Hospitales , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Tanzanía/epidemiología , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Infecciones Urinarias/tratamiento farmacológico , beta-Lactamasas/análisisRESUMEN
BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea in resource-limited regions. It is also an important cause of diarrhea in travellers to these areas.To evaluate the protective efficacy of new ETEC vaccines that are under development, there is a need to increase the capacity to undertake Phase IIB (human challenge) clinical trials and to develop suitable challenge models. METHODS: An in-hospital study was performed where fasting adult volunteers were experimentally infected with 1 × 106 to 1 × 109 colony forming units (CFUs) of the wild-type ETEC strain TW10598, which had been isolated from a child with diarrhea in West Africa in 1997. We recorded symptoms and physical signs and measured serum immune response to the TW10598 bacterium. RESULTS: We included 30 volunteers with mean age 22.8 (range 19.8, 27.4) years. The most common symptoms were diarrhea (77%), abdominal pain (67%), nausea (63%), and abdominal cramping (53%). Seven subjects (23%) experienced fever, none were hypotensive. Most of the volunteers responded with a substantial rise in the level of serum IgA antibodies against the challenge strain. CONCLUSIONS: We established the capacity and methods for safely undertaking challenge studies to measure the efficacy of ETEC vaccine candidates in a hospital ward. Strain TW10598 elicited both clinical symptoms and an immune response across the doses given.
Asunto(s)
Escherichia coli Enterotoxigénica/fisiología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Dolor Abdominal/microbiología , Adulto , Anticuerpos Antibacterianos/inmunología , Diarrea/inmunología , Diarrea/microbiología , Diarrea/prevención & control , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Vacunas contra Escherichia coli/inmunología , Femenino , Voluntarios Sanos , Experimentación Humana , Humanos , Masculino , Adulto JovenRESUMEN
We hereby present the first descriptions of human-invasive infections caused by Escherichia marmotae, a recently described species that encompasses the former "Escherichia cryptic clade V." We describe four cases, one acute sepsis of unknown origin, one postoperative sepsis after cholecystectomy, one spondylodiscitis, and one upper urinary tract infection. Cases were identified through unsystematic queries in a single clinical lab over 6 months. Through genome sequencing of the causative strains combined with available genomes from elsewhere, we demonstrate Es. marmotae to be a likely ubiquitous species containing genotypic virulence traits associated with Escherichia pathogenicity. The invasive isolates were scattered among isolates from a range of nonhuman sources in the phylogenetic analyses, thus indicating inherent virulence in multiple lineages. Pan genome analyses indicate that Es. marmotae has a large accessory genome and is likely to obtain ecologically advantageous traits, such as genes encoding antimicrobial resistance. Reliable identification might be possible by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), but relevant spectra are missing in commercial databases. It can be identified through 16S rRNA gene sequencing. Escherichia marmotae could represent a relatively common human pathogen, and improved diagnostics will provide a better understanding of its clinical importance. IMPORTANCE Escherichia coli is the most common pathogen found in blood cultures and urine and among the most important pathogenic species in the realm of human health. The notion that some of these isolates are not Es. coli but rather another species within the same genus may have implications for what Es. coli constitutes. We only recently have obtained methods to separate the two species, which means that possible differences in important clinical aspects, such as antimicrobial resistance rates, virulence, and phylogenetic structure, may exist. We believe that Es. marmotae as a common pathogen is new merely because we have not looked or bothered to distinguish between the thousands of invasive Escherichia passing through microbiological laboratories each day.
Asunto(s)
Antiinfecciosos , Infecciones por Escherichia coli , Sepsis , Escherichia , Escherichia coli/genética , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/microbiología , Humanos , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Colonization in HIV-infected populations with extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) is particularly worrisome in low-income settings. This study describes the prevalence of ESBL-PE carriage and associated risk factors among newly HIV-diagnosed adults in a community setting in Tanzania. A total of 595 newly diagnosed HIV adults with a median age of 35 years with interquartile range (IQR) 29-42 years and a median CD4 count of 492 cells/µL (IQR 390-666 cells/µL) were recruited. Among these, 194/595 (32.6%, 95% confidence interval [CI] 28.9-36.6) were ESBL-PE carriers. Participants with low CD4 count (<350 cells/µL) had significantly higher prevalence of ESBL-PE carriage compared with those with CD4 count ≥350 cells/µL (26/58, 44.8%, vs. 168/537, 31.3%, p = 0.04). Antibiotic use in last 4 weeks (odds ratio [OR] 1.55, 95% CI 1.08-2.22, p = 0.02) and CD4 count ≥350 cells/µL (OR 1.78, 95% CI 1.03-3.09, p = 0.04) were independent risk factors for fecal carriage of ESBL-PE. In total, 244 isolates of ESBL-PE were isolated from 194 participants. Of these, 238/244 (97.5%) harbored blaCTX-M genes, with blaCTX-M-15 being predominant (219/238 (92%), followed by blaCTX-M-27 (9/238 (3.8%), blaCTX-M-14 (8/238 (3.4%), blaCTX-M-55 (1/238), and blaCTX-M 211/3 (1/238). blaSHV-2a genes were detected in four isolates, whereas the blaSHV-12 gene was detected in one isolate. Phenotypic carbapenemase-producing Enterobacteriaceae was detected in one HIV-positive person with CD4 count 132 cells/µL. In conclusion prevalence of ESBL-PE carriage is high among newly diagnosed HIV adults in Dar es Salaam, and is significantly associated antibiotic use and low CD4 count.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/genética , Infecciones por VIH/epidemiología , Resistencia betalactámica/genética , Adulto , Factores de Edad , Recuento de Linfocito CD4 , Portador Sano , Método Doble Ciego , Enterobacteriaceae/efectos de los fármacos , Heces/microbiología , Femenino , Genes Bacterianos , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Prevalencia , Índice de Severidad de la Enfermedad , Factores Sexuales , Factores Socioeconómicos , Tanzanía/epidemiología , Resistencia betalactámica/efectos de los fármacosRESUMEN
Increased knowledge about the role of horizontal gene transfer is key to improve our understanding of the spread of antimicrobial resistance (AMR) in human populations. We therefore studied the dissemination of the blaCTX-M-15 extended-spectrum-ß-lactamase (ESBL) gene in Klebsiella pneumoniae isolates obtained from stool samples from hospitalized children and healthy controls below 2 years of age in Dar es Salaam, Tanzania, from August 2010 to July 2011. We performed Illumina whole-genome sequencing (WGS) to characterize resistance genes, multilocus sequence type (MLST), plasmid incompatibility group (Inc), and plasmid MLST of 128 isolates of K. pneumoniae with blaCTX-M-15 recovered from both healthy and hospitalized children. We assessed the phylogenetic relationship using single nucleotide polymorphism (SNP)-based analysis and resolved the sequences of five reference plasmids by Oxford Nanopore technology to investigate plasmid dissemination. The WGS analyses revealed the presence of a blaCTX-M-15-positive IncFIIK5/IncR plasmid with a highly conserved backbone in 70% (90/128) of the isolates. This plasmid, harboring genes encoding resistance to most ß-lactams, aminoglycosides, trimethoprim-sulfamethoxazole, and chloramphenicol, was present in phylogenetically very diverse K. pneumoniae strains (48 different MLSTs) carried by both hospitalized and healthy children. Our data strongly suggest widespread horizontal transfer of this ESBL-carrying plasmid both in hospitals and in the general population.IMPORTANCE Horizontal spread of plasmids carrying multiple resistance genes is considered an important mechanism behind the global health problem caused by multidrug-resistant bacteria. Nevertheless, knowledge about spread of plasmids in a community is limited. Our detailed molecular analyses of K. pneumoniae isolated from hospitalized and healthy children in Tanzania disclosed an epidemic spread of a resistance plasmid. In this study population, we revealed horizontal plasmid transfer among K. pneumoniae as the key factor for dissemination of ESBLs. Traditional outbreak investigation and surveillance focus on the spread of bacterial clones, and short-read sequencing can result in erroneous plasmid composition. Our approach using long-read sequencing reveals horizontal gene transfer of antimicrobial resistance, and therefore has a potential impact on outbreak investigations and approaches to limit spread of AMR.
Asunto(s)
Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Brotes de Enfermedades , Farmacorresistencia Bacteriana Múltiple , Femenino , Transferencia de Gen Horizontal , Hospitalización , Humanos , Lactante , Infecciones por Klebsiella/microbiología , Masculino , Tipificación de Secuencias Multilocus , Filogenia , Plásmidos/genética , Tanzanía/epidemiología , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Febrile illness is the commonest cause of hospitalization in children <5 years in sub-Saharan Africa, and bacterial bloodstream infections and malaria are major causes of death. METHODS: From March 2017 to July 2018, we enrolled 2,226 children aged 0-5 years hospitalized due to fever in four major public hospitals of Dar es Salaam, namely, Amana, Temeke, and Mwananyamala Regional Hospitals and Muhimbili National Hospital. We recorded social demographic and clinical data, and we performed blood-culture and HIV-antibody testing. We used qPCR to quantify Plasmodium falciparum parasitaemia and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) to identify bacterial isolates. Disk diffusion method was used for antimicrobial susceptibility testing. RESULTS: Nineteen percent of the children (426/2,226) had pathogens detected from blood. Eleven percent (236/2,226) of the children had bacteraemia/fungaemia and 10% (204/2,063) had P. falciparum malaria. Ten children had concomitant malaria and bacteraemia. Gram-negative bacteria (64%) were more frequent than Gram-positive (32%) and fungi (4%). Over 50% of Gram-negative bacteria were extended-spectrum beta-lactamase (ESBL) producers and multidrug resistant. Methicillin resistant Staphylococcus aureus (MRSA) was found in 11/42 (26.2%). The most severe form of clinical malaria was associated with high parasitaemia (>four million genomes/µL) of P. falciparum in plasma. Overall, in-hospital death was 4% (89/2,146), and it was higher in children with bacteraemia (8%, 18/227) than malaria (2%, 4/194, p = 0.007). Risk factors for death were bacteraemia (p = 0.03), unconsciousness at admission (p < 0.001), and admission at a tertiary hospital (p = 0.003). CONCLUSION: Compared to previous studies in this region, our study showed a reduction in malaria prevalence, a decrease in in-hospital mortality, and an increase in antimicrobial resistance (AMR) including ESBLs and multidrug resistance. An increase of AMR highlights the importance of continued strengthening of diagnostic capability and antimicrobial stewardship programs. We also found malaria and bacteraemia contributed equally in causing febrile illness, but bacteraemia caused higher in-hospital death. The most severe form of clinical malaria was associated with P. falciparum parasitaemia.
RESUMEN
TRIAL REGISTRATION: ClinicalTrials.gov ClinicalTrials.gov, Project ID: NCT02870751.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Escherichia coli Enterotoxigénica/inmunología , Enterotoxinas/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Recuento de Colonia Microbiana , Diarrea/microbiología , Diarrea/prevención & control , Proteínas de Escherichia coli/inmunología , Femenino , Calor , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Masculino , Metaloproteasas , Voluntarios , Adulto JovenRESUMEN
Septicemia caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae was associated with high mortality in Tanzanian children. Conjugation experiments on the SHV-12-producing Enterobacteriaceae isolates showed that ESBL-encoding genes were transferred on large plasmids together with genes encoding resistance to aminoglycosides, resistance to ceftazidime, gentamicin, doxycycline, trimethoprim-sulfamethoxazole, and chloramphenicol.
Asunto(s)
Infecciones por Enterobacteriaceae , Enterobacteriaceae/genética , Plásmidos/genética , Sepsis/microbiología , Resistencia betalactámica/genética , beta-Lactamasas/genética , Niño , Farmacorresistencia Bacteriana Múltiple/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/enzimología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/genética , Humanos , Pruebas de Sensibilidad Microbiana , Plásmidos/clasificación , Plásmidos/efectos de los fármacos , Estudios Prospectivos , Sepsis/tratamiento farmacológico , Sepsis/epidemiología , Tanzanía/epidemiologíaRESUMEN
Cotrimoxazole is widely used, particularly as a prophylactic drug in HIV patients. We assessed resistance mechanisms among cotrimoxazole resistant-Gram negative bacterial isolates (n = 123) obtained from blood (n = 69) and urine (n = 54) from Tanzanian patients. sul genes were detected in 98% (121/123) of the isolates. Coexistence of sul1 and sul2 was common (49/123). The dfr genes were found in 63% (77/123) of all isolates. sul1, dfrA15, and dfrA5 genes predominated among Klebsiella pneumoniae, while sul2 and dfrA1 genes were frequent in Escherichia coli isolates. Two isolates, both K. pneumoniae, carried sul3. Integrons were detected in 81.3% (100/123) of all isolates. Class 1 integrons were found in 95% (42/44), 53% (23/43), and 80.6% (25/31) of K. pneumoniae, E. coli, and other Enterobacteriaceae isolates, respectively. Class 2 integrons were found in 14% of E. coli, but not in K. pneumoniae. All sul1 genes in K. pneumoniae were carried in class 1 integrons. Gene cassette arrays dfrA5 and dfrA15-aadA1 were most frequently associated with class 1 integrons, while class 2 integrons contained only dfrA1-sat2-aadA1 gene cassettes. This is the first report of sul3 gene in K. pneumoniae from human sources. The finding that mechanisms differ between E. coli and K. pneumoniae may broaden our understanding of cotrimoxazole resistance.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/genética , Genes Bacterianos , Integrones , Klebsiella pneumoniae/genética , Antibacterianos/farmacología , Estudios Transversales , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Humanos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Estudios Prospectivos , Tanzanía , Combinación Trimetoprim y Sulfametoxazol/farmacología , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiologíaRESUMEN
The aim of the present study was to compare serotyping, PFGE and AFLP for typing of Streptococcus pneumoniae with regard to discriminatory power, typeability and typing system concordance. Thirty-four isolates from cerobrospinal fluid and 34 time-matched blood culture isolates collected from in-patients at two hospitals in western Norway during the period from January 1994 to May 2002 were included in the study. The discriminatory powers of serotyping, PFGE and AFLP were 0.93, 0.99 and 0.95, respectively. The typeabilities for serotyping, PFGE and AFLP were 1, 1 and 0.99, respectively. A good concordance was shown between all the typing methods. Serotyping would most probably have a higher discriminatory power if further subtyping had been performed. PFGE was more discriminatory than AFLP, and AFLP grouped more-distantly related isolates together. The two typing methods thus provided different information, and therefore both could be useful adjuncts to serotyping for the characterization of S. pneumoniae.
Asunto(s)
Serotipificación , Streptococcus pneumoniae/clasificación , Electroforesis en Gel de Campo Pulsado , Humanos , Infecciones Neumocócicas/microbiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Especificidad de la Especie , Streptococcus pneumoniae/genéticaRESUMEN
Leishmaniasis is one of the world's most neglected infectious diseases, affecting around 12 million people and more than 350 million at risk of infection. The clinical picture varies from self-healing cutaneous lesions to severe visceral infections, but still no commercial vaccines for humans are available and the currently used drugs have unpleasant side effects. Here we report a real-time PCR assay targeting the arginine permease gene AAP3 that can be applied for all the nine different species of the Leishmania genus tested; 4 Old World species and 5 New World species, from both L. (Leishmania) and L. (Viannia) subgenera. No cross-reaction was seen with Trypanosoma cruzi, Trypanosoma brucei, human or mouse genomic DNA. The assay has a high sensitivity, with a limit of detection of 10fg DNA for L. (L.) major and L. (L.) donovani, and 100fg DNA for L. (V.) braziliensis, and can be used for both qualitative and quantitative purposes. This AAP3-Assay, run in duplex with a host specific gene-assay, was also successfully used for quantification of parasite load of footpads from L. (L.) major-infected mice. It can therefore be a valuable tool in applications like monitoring effects of drugs, the selection of vaccine candidates and in screening patients, including asymptomatic carriers.