Herramientas diagnósticas - WordPress.com
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Herramientas diagnósticas
Medicina Interna 1Enfermedades Infecciosas
Dr. Chaverri [email protected]
1Monday, May 20, 13
¿Qué están buscando?
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Patógenos
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6 reinos
• Bacteria
• Protozoa
• Chromista
• Plantae
• Fungi
• Animalia
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Parásitos
• Protozooarios
• Helmintos
• Artrópodos
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Protozooarios
• Rhizopoda: Entamoeba histolytica
• Ciliophora: Paramecios
• Mastigophora: Tripanozoma cruzi
• Apicomplexa: Plasmodium spp. y Toxoplasma gondii
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Helmintos
• Nemátodos
• Intestinales: Ascaris lumbricoides, Trichuris trichiuria, Ancylostoma duodenale, Necator americanus, Anisakis, Enterobius
• Tisulares: Trichinella spiralis, Ancylostoma, Toxocara
• Trematodos: Fasciola hepatica, Paragominus westermani y mexicanus, Schistosoma mansoni y maematobium
• Céstodos: Taenia solium, Taenia saginata, Dyphilobothrium latum, Echinococcus granulosus
9Monday, May 20, 13
Artrópodos
• Sarcoptes scabiei
• Ptirius pubis
• Ptirius capitis
• Miasis
• Sarcophaga, Dermatobia, Oestrus, Gastrophilus, Cochliomyia, Lucila, Chrysomya y Musca
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Bacterias
• Filogenia
• Metabolismo
• Medio de vida
• Morfología
• Patogenicidad
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¿Cómo se hace la tinción de Gram?
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Clave dicotómica
U S I N G A T A X O N O M I C K E YBiologists often use a taxonomic key to identify or-
ganisms according to their characteristics. The most com-mon kind is a dichotomous key, which has paired state-ments describing characteristics of organisms. Pairedstatements present an “either-or” choice, so that only onestatement is true. Each statement is followed by directionsto go to another pair of statements until the name of theorganism finally appears. Figure 9.3 is a dichotomous keythat will identify each of the four most common U.S. coins:quarters, dimes, nickels, and pennies. Read statements 1aand 1b, and decide which statement applies to a givencoin. Look at the number to the right of the statement; ittells you which pair of statements to look at next. Con-tinue in this manner until you reach a group designation.If you have followed the key carefully, that designationwill name the coin.
Of course, you don’t need a taxonomic key to identifysomething as simple and as familiar as coins. But identify-ing all the many kinds of bacteria in the world is a moredifficult task. Major groups of bacteria can be identifiedwith the key in Figure 9.4. More detailed keys use stainingreactions, metabolic reactions (fermentation of particularsugars or release of different gases), growth at differenttemperatures, properties of colonies on solid media, andsimilar characteristics of cultures. By proceeding step bystep through the key, one should be able to identify an un-known organism, or even a strain, if the key is sufficientlydetailed.
Problems in TaxonomyAmong the aims of a taxonomic system are organizingknowledge about living things and establishing standardnames for organisms so that we can communicate aboutthem. Ideally, we would like to classify organisms accord-ing to their phylogenetic, or evolutionary, relationships,but this is not always easy. Evolution occurs continuouslyand at a relatively rapid rate in microorganisms, and ourknowledge of the evolutionary history of organisms is in-complete. Taxonomy must change with evolutionarychanges and new knowledge. It is far more important to
have a taxonomic system that reflects our current knowl-edge than to have a system that never changes.
Creating a taxonomic system that provides an orga-nized overview of all living things and how they are related
U S I N G A TA XO N O M I C K E Y
235
1a Smooth-edged
1b Rough-edged
Go to 2
Go to 3
2a Silver-colored
2b Copper-colored
Nickel
Penny
3a Large (about 1-in. diameter)
3b Small (about 3/4-in. diameter)
Quarter
Dime
Figure 9.3 A dichotomous key for classifying typical U.S.coins. Why would the word “flat” not be useful in this key?
1a Gram-positive
1b Not Gram-positive
Go to 2
Go to 3
2a Cells spherical in shape
2b Cells not spherical in shape
Gram-positive cocci
Go to 4
3a Gram-negative
3b Not Gram-negative (lack cell wall)
Go to 5
Mycoplasma
4a Cells rod-shaped
4b Cells not rod-shaped
Gram-positive bacilli
Go to 6
5a Cells spherical in shape
5b Cells not spherical in shape
Gram-negative cocci
Go to 7
6a Cells club-shaped
6b Cells variable in shape
Corynebacteria
Propionibacteria
7a Cells rod-shaped
7b Cells not rod-shaped
Gram-negative bacilli
Go to 8
8a Cells helical with several turns
8b Cells comma-shaped
Spirochetes
Vibrioids
Figure 9.4 A dichotomous key for classifying majorgroups of bacteria.
Do you wonder when you sip wine or eatcheese, where the microorganisms that madethose products come from? They might be fromthe American Type Culture Collection (ATCC) in
Manassas, Virginia, which keeps some preserved cultures in asecured vault to protect them against theft. Yes, theft—some strains of organisms are valuable enough to be kepthere because their characteristics are important in researchor in industrial applications such as winemaking. Many or-ganisms are preserved in a dormant state to prevent themfrom undergoing genetic changes that might alter their char-acteristics. Different researchers can order particular strainsfrom the ATCC, ensuring that all tests done anywhere aroundthe world with these mail-ordered organisms are using ge-netically identical microbes. Manufacturers of wine orcheese can be sure that they will always be able to obtain theorganisms that make products with particular distinctive fla-vors or other characteristics.
C L O S E - U P
Mail-Order Microbes
U S I N G A T A X O N O M I C K E YBiologists often use a taxonomic key to identify or-
ganisms according to their characteristics. The most com-mon kind is a dichotomous key, which has paired state-ments describing characteristics of organisms. Pairedstatements present an “either-or” choice, so that only onestatement is true. Each statement is followed by directionsto go to another pair of statements until the name of theorganism finally appears. Figure 9.3 is a dichotomous keythat will identify each of the four most common U.S. coins:quarters, dimes, nickels, and pennies. Read statements 1aand 1b, and decide which statement applies to a givencoin. Look at the number to the right of the statement; ittells you which pair of statements to look at next. Con-tinue in this manner until you reach a group designation.If you have followed the key carefully, that designationwill name the coin.
Of course, you don’t need a taxonomic key to identifysomething as simple and as familiar as coins. But identify-ing all the many kinds of bacteria in the world is a moredifficult task. Major groups of bacteria can be identifiedwith the key in Figure 9.4. More detailed keys use stainingreactions, metabolic reactions (fermentation of particularsugars or release of different gases), growth at differenttemperatures, properties of colonies on solid media, andsimilar characteristics of cultures. By proceeding step bystep through the key, one should be able to identify an un-known organism, or even a strain, if the key is sufficientlydetailed.
Problems in TaxonomyAmong the aims of a taxonomic system are organizingknowledge about living things and establishing standardnames for organisms so that we can communicate aboutthem. Ideally, we would like to classify organisms accord-ing to their phylogenetic, or evolutionary, relationships,but this is not always easy. Evolution occurs continuouslyand at a relatively rapid rate in microorganisms, and ourknowledge of the evolutionary history of organisms is in-complete. Taxonomy must change with evolutionarychanges and new knowledge. It is far more important to
have a taxonomic system that reflects our current knowl-edge than to have a system that never changes.
Creating a taxonomic system that provides an orga-nized overview of all living things and how they are related
U S I N G A TA XO N O M I C K E Y
235
1a Smooth-edged
1b Rough-edged
Go to 2
Go to 3
2a Silver-colored
2b Copper-colored
Nickel
Penny
3a Large (about 1-in. diameter)
3b Small (about 3/4-in. diameter)
Quarter
Dime
Figure 9.3 A dichotomous key for classifying typical U.S.coins. Why would the word “flat” not be useful in this key?
1a Gram-positive
1b Not Gram-positive
Go to 2
Go to 3
2a Cells spherical in shape
2b Cells not spherical in shape
Gram-positive cocci
Go to 4
3a Gram-negative
3b Not Gram-negative (lack cell wall)
Go to 5
Mycoplasma
4a Cells rod-shaped
4b Cells not rod-shaped
Gram-positive bacilli
Go to 6
5a Cells spherical in shape
5b Cells not spherical in shape
Gram-negative cocci
Go to 7
6a Cells club-shaped
6b Cells variable in shape
Corynebacteria
Propionibacteria
7a Cells rod-shaped
7b Cells not rod-shaped
Gram-negative bacilli
Go to 8
8a Cells helical with several turns
8b Cells comma-shaped
Spirochetes
Vibrioids
Figure 9.4 A dichotomous key for classifying majorgroups of bacteria.
Do you wonder when you sip wine or eatcheese, where the microorganisms that madethose products come from? They might be fromthe American Type Culture Collection (ATCC) in
Manassas, Virginia, which keeps some preserved cultures in asecured vault to protect them against theft. Yes, theft—some strains of organisms are valuable enough to be kepthere because their characteristics are important in researchor in industrial applications such as winemaking. Many or-ganisms are preserved in a dormant state to prevent themfrom undergoing genetic changes that might alter their char-acteristics. Different researchers can order particular strainsfrom the ATCC, ensuring that all tests done anywhere aroundthe world with these mail-ordered organisms are using ge-netically identical microbes. Manufacturers of wine orcheese can be sure that they will always be able to obtain theorganisms that make products with particular distinctive fla-vors or other characteristics.
C L O S E - U P
Mail-Order Microbes
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¿De qué color se tiñe una bacteria Gram
positiva?
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Virus
• ADN
• ARN
• Retrovirus
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Familias virales• Adenovirus
• Herpesviridae
• Papillomaviridae
• Polyomaviridae
• Poxviridae
• Hepadnaviridae
• Parvoviridae
• Atroviridae
• Caliciviridae
• Picornaviridae
• Coronaviridae
• Flaviviridae
• Togaviridae
• Togaviridae
• Retroviridae
• Orthomyxoviridae
• Arenaviridae
• Bunyaviridae
• Filoviridae
• Parmyxoviridae
• Rhabdoviridae
• Reoviridae
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¿Cómo hacer el diagnóstico?
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¿Cómo hacer el diagnóstico?
• Demostrar el patógeno
• Demostrar el patógeno
• Demostrar el patógeno
• Demostrar el patógeno
• Demostrar el patógeno
• Demostrar el patógeno
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Pruebas no específicas
• Conteo leucocitario y diferencial
• Leucocitosis
• Leucopenia
• Velocidad de eritrosedimentación (VES)
• Proteina C reactiva (PCR)
• Procalcitonina
• Interleucinas
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Conteo normal de leucocitos: 4500 a 11000 células/mLGranulocitos (neutrófilos, eosinófilos, basófilos) y agranulocitos (linfocitos, monocitos)VES > 100 mm/H: se relaciona a endocarditis, osteomielitis, infecciones inraabdominalesInterleucinas: IL-1, IL-6, IL-8, FNT,
Leucograma
• Neutrofilia
• Linfocitosis
• Monocitosis
• Desviación izquierda
• Formas inmaduras
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Examinación directa
• Tinción Gram
• Cocos, Bacilos, Cocobacilos
• Cocos en racimo, en parejas, en cadenas
• Bacilos pequeños, grandes, filamentosos
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Reacciones bioquímicas
• Coagulasa
• Positivo: Staphylococcus aureus
• Negativo: Staphylococcus epidermidis, saprophyticus, hominis, haemolyticus, warneri
• Diplococos: Streptococcus penumoniae
• Cadenas: reacciones de hemólisis
• Alfa: Streptococcus viridians
• Beta: Streptococcus pyogenes, agalactiae
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Reacciones bioquímicas
• Fermentadores de lactosa
• Enterobacteriaceae
• Oxidasa positivo: Aeromonas, Pasteurella, Vibrio
• Oxidasa negativo: E. coli, Klebsiella spp., Enterobacter spp., Citrobacter spp.
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Reacciones bioquímicas
• No fermentadores de lactosa
• Oxidasa positivo: Pseudomonas spp., Flavobacterium spp., Alcaligenes spp., Achromobacter spp.
• Oxidasa negativo: Proteus spp., Proficendia spp., Serratia spp., Morganella spp., Salmonella spp., Shigella spp., Stenotrophomonas, Acinetobacter spp.
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Cultivos
• Estándar de oro
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Recolección de secreciones
• Limpieza del área
• Aspirar con jeringa la mayor cantidad posible de material
• Pasar dos aplicadores (colocar los aplicadores en tubos estériles)
• Frotis Y Cultivo (por separado)
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Hemocultivos
• Buscar el equipo y frascos de hemocultivo
• Encontrar las áreas de venopunción
• Cubrebocas
• Lavado de manos
• Delantal estéril, campos abiertos y gorro
• Guantes estériles
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Hemocultivos
• Con técnica limpiar el sitio de punción vascular
• La limpieza debe ser con clorhexidina
• Cambio de guantes al cambiar el sitio de punción vascular
• No se recomienda el cambio de aguja para inocular el frasco
• Transporte rápido al laboratorio
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Los frascos
• A nivel de hospitales nacionales
• Equipo BAC-Alert y VITEK-2
• Tapa azul: antes del inicio de antibióticos
• Tapa verde: tras inicio de antibióticos (contiene carbón activado)
• Tapa anaranjada: anaerobios
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Medio de frijol de soya-caseina digerida
El volumen
• Niños: 1 mL/botella
• Adultos: 10 mL/botella
• Cada mL menos de muestra reduce la posibilidad de aislamiento un 10%
• DOS frascos
• Antes del inicio de la terapia antibiótica
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VolumenPeso del paciente mL de sangre por botella
< 8 Kg 0,5
8-14 Kg 1,5
15-27 Kg 2,5
28-40 Kg 5
41-55 Kg 7,5
> 55 Kg 10
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¿Cuándo hemocultivar?• Bacteremia
• SRIS
• Sepsis severa
• Choque séptico
• Endocarditis bacteriana
• Sepsis neutropénica
• Meningitis
• Bronconeumonía
• Infección del tracto urinario complicada
• Artritis séptica
• Fiebre de causa no clara
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SRIS: síndrome de respuesta inflamatoria sistémicaEndocarditis: se requieren al menos 3 pares de hemocultivos en sitios y momentos distintos
Catéteres para acceso venoso
• Cuando el paciente tiene dispositivo de acceso venoso central o de gran calibre
• Tomar cultivo periférico primero
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Una vez tomada la muestra
• No tapar lo códigos de barras
• No tapar el fondo del frasco
• Rotular los frascos con el nombre del paciente, número de id., hora y fecha de toma
• Indicar en los frascos y en las solicitudes si la sangre es periférica o de catéter y si tiene varios lúmenes de cual se ha extraído
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Antígenos y anticuerpos
• Detección y cuantificación de anticuerpos
• Los antígenos pueden seguir circulando aunque el patógeno ya no esté vivo
• Los anticuerpos se mantendrán positivos
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Inmunofluorescencia: CMV, VRS, VVZ, Treponema pallidum, Borrelia burgdorferi, Chlamydia trachomatisAglutinación por látex: antígeno capsular meningocóccico, Legionella pneumophilaELISA: VIH, VRS, anticuerpo neumocóccico en sangre, Neisseria gonorrhoeae, H. pylori
Métodos moleculares
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Hibridización con probetas de ADN
• Extracción de ADN o ARN
• Detectar la información genética para gérmenes específicos
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Mycobacterium tuberculosis, Neisseria gonorrhoeae, hongosGenes de resistenciaVHB, VHC, VIH
Amplificación de ácidos nucleicos
• Reacción en cadena de polimerasa (PCR)
• ADN polimerasa, primers de oligonucleótidos
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VIH, M. tuberculosis, B. burgdorferi, H. pylori, genes mecA, resistencia a isonizida o rifampicina