herramientas diagnósticas - wordpress.com

Herramientas diagnósticas

Medicina Interna 1Enfermedades Infecciosas

Dr. Chaverri Murillojchaverri@unibe.ac.cr

1Monday, May 20, 13

¿Qué están buscando?

2Monday, May 20, 13

3Monday, May 20, 13

4Monday, May 20, 13

Patógenos

5Monday, May 20, 13

6 reinos

• Bacteria

• Protozoa

• Chromista

• Plantae

• Fungi

• Animalia

6Monday, May 20, 13

Parásitos

• Protozooarios

• Helmintos

• Artrópodos

7Monday, May 20, 13

Protozooarios

• Rhizopoda: Entamoeba histolytica

• Ciliophora: Paramecios

• Mastigophora: Tripanozoma cruzi

• Apicomplexa: Plasmodium spp. y Toxoplasma gondii

8Monday, May 20, 13

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

10Monday, May 20, 13

Bacterias

• Filogenia

• Metabolismo

• Medio de vida

• Morfología

• Patogenicidad

11Monday, May 20, 13

12Monday, May 20, 13

13Monday, May 20, 13

14Monday, May 20, 13

¿Cómo se hace la tinción de Gram?

15Monday, May 20, 13

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

16Monday, May 20, 13

¿De qué color se tiñe una bacteria Gram

positiva?

17Monday, May 20, 13

Virus

• ADN

• ARN

• Retrovirus

18Monday, May 20, 13

Familias virales• Adenovirus

• Herpesviridae

• Papillomaviridae

• Polyomaviridae

• Poxviridae

• Hepadnaviridae

• Parvoviridae

• Atroviridae

• Caliciviridae

• Picornaviridae

• Coronaviridae

• Flaviviridae

• Togaviridae

• Togaviridae

• Retroviridae

• Orthomyxoviridae

• Arenaviridae

• Bunyaviridae

• Filoviridae

• Parmyxoviridae

• Rhabdoviridae

• Reoviridae

19Monday, May 20, 13

¿Cómo hacer el diagnóstico?

20Monday, May 20, 13

¿Cómo hacer el diagnóstico?

• Demostrar el patógeno

• Demostrar el patógeno

• Demostrar el patógeno

• Demostrar el patógeno

• Demostrar el patógeno

• Demostrar el patógeno

20Monday, May 20, 13

Pruebas no específicas

• Conteo leucocitario y diferencial

• Leucocitosis

• Leucopenia

• Velocidad de eritrosedimentación (VES)

• Proteina C reactiva (PCR)

• Procalcitonina

• Interleucinas

21Monday, May 20, 13

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

22Monday, May 20, 13

Examinación directa

• Tinción Gram

• Cocos, Bacilos, Cocobacilos

• Cocos en racimo, en parejas, en cadenas

• Bacilos pequeños, grandes, filamentosos

23Monday, May 20, 13

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

24Monday, May 20, 13

Reacciones bioquímicas

• Fermentadores de lactosa

• Enterobacteriaceae

• Oxidasa positivo: Aeromonas, Pasteurella, Vibrio

• Oxidasa negativo: E. coli, Klebsiella spp., Enterobacter spp., Citrobacter spp.

25Monday, May 20, 13

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.

26Monday, May 20, 13

Cultivos

• Estándar de oro

27Monday, May 20, 13

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)

28Monday, May 20, 13

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

29Monday, May 20, 13

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

30Monday, May 20, 13

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

31Monday, May 20, 13

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

32Monday, May 20, 13

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

33Monday, May 20, 13

¿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

34Monday, May 20, 13

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

35Monday, May 20, 13

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

36Monday, May 20, 13

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

37Monday, May 20, 13

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

38Monday, May 20, 13

Hibridización con probetas de ADN

• Extracción de ADN o ARN

• Detectar la información genética para gérmenes específicos

39Monday, May 20, 13

Mycobacterium tuberculosis, Neisseria gonorrhoeae, hongosGenes de resistenciaVHB, VHC, VIH

Amplificación de ácidos nucleicos

• Reacción en cadena de polimerasa (PCR)

• ADN polimerasa, primers de oligonucleótidos

40Monday, May 20, 13

VIH, M. tuberculosis, B. burgdorferi, H. pylori, genes mecA, resistencia a isonizida o rifampicina