This document discusses techniques for diagnosing tuberculous meningitis. It describes direct diagnostic techniques that detect the causal agent and indirect techniques that detect the host's immune response. Adenosine deaminase (ADA) is an indirect diagnostic technique discussed in detail. ADA levels are often elevated in tuberculosis and other diseases. The document outlines the principle, definition, and determination of the ADA technique. Studies comparing ADA to polymerase chain reaction and examining ADA cutoff levels for diagnosing tuberculous meningitis in cerebrospinal fluid are summarized.

1. Técnicas en Diagnóstico
de Meningitis
Tuberculosa
Alejo Albornóz

2. Diagnóstico Directo
Investigan la presencia del agente etiológico o, uno de sus componentes.
Diagnóstico Indirecto
No investigan presencia del agente en sí, sino la huella que dejó éste al
pasar por su huésped en términos de una respuesta inmune contra el
agente y que indirectamente permite suponer que el agente en cuestión
estuvo presente en el huésped en algún momento.

3. Micobacterias
Pertenecen a la familia de Mycobacteriaceae , son bacilos grampositivos aeróbicos obligados, ácidoalcohol resistentes. La fuerza del ácido es una característica de las micobacterias y se detecta en la
tinción de Ziehl-Neelsen. Las micobacterias son gram-positivos con un alto contenido de G + C
genómico (59-66%). La presencia de ácidos micólicos de cadena larga en la pared celular favorece la
fuerza del ácido y justificó la resistencia de las micobacterias.
El género Mycobacterium comprende más de 120 especies diferentes del complejo M. tuberculosis. Las
bacterias pueden causar enfermedades graves en humanos y animales, tales como la tuberculosis, la
lepra o Micobacteriosis. Debido a su patogenicidad se distinguen los siguientes tres grupos:
M. tuberculosis complex (cepas estrechamente relacionadas):
M. tuberculosis
M. bovis ssp. bovis y ssp. caprae
M. bovis BCG (Bacilo de Calmette-Guérin)
M. africanum
M. canettii
M. microti
M. pinnipedii
NTM (”micobacterias no tuberculosas o atípicas”)
M. leprae

4. FIGURE 2.1
Coverage of country consultations on estimates of TB disease burden, 2008–2013

6. Resistencia a Drogas
Ethiopia
Georgia
300
India
Indonesia
800
500
15000
400
200
600
300
10000
200
100
400
5000
100
0
8000
Number of cases
MDR-TB cases (orange) and
additional rifampicin-resistant
TB cases (blue) detected compared with TB cases enrolled
on MDR-TB treatment (green)
2009–2012, globally and in 27
high MDR-TB burden
countries, 2009–2012
200
Kazakhstan
0
1000
Kyrgyzstan
150
0
Latvia
350
330
750
6000
Lithuania
100
310
500
4000
290
50
2000
250
0
0
270
0
Nigeria
Myanmar
250
Pakistan
Philippines
2000
1500
2000
1000
800
2500
1500
500
1000
0
500
300
600
200
400
100
200
0
0
Russian Federation
Republic of Moldova
South Africa
Tajikistan
20000
1100
800
15000
900
600
15000
10000
700
10000
5000
5000
Uzbekistan
Ukraine
200
0
500
300
0
2000
800
1500
200
0
0
Global
400
500
Viet Nam
600
1000
8000
400
100 000
80 000
6000
60 000
40 000
4000
2000
2009
2010
2011
2012
2009
2010
2011
2012
2009
20 000
2010
2011
2012
0
2009
2010
2011
2012

7. Técnica
Cinético-colorimétrica
Diagnóstico Indirecto

8. Adenosin Deaminasa (ADA)
Es una enzima esencial para el metabolismo de las purinas y principalmente de ciertos tipos de células del
organismo, en especial, de las células qque se ocupan del desarrollo del sistema inmune, como por ejemplo de los
linfocitos T.
La adenosina deaminasa(ADA)se encuentra en los eritrocitos, leucocitos, pulmón, hígado, estómago, tracto
genitourinario y suero. La enzima contiene dos grupos tiol reactivos. Se afirma qque las actividades de la enzima son
mucho más altas en la tuberculosis qque en otras enfermedades.
La adenosina deaminasa es una de las pocas enzimas del suero qque se encuentra consistentemente baja en
enfermedades del tracto biliar y frecuentemente altas en enfermedad hepática crónica.
La adenosina deaminasa(ADA)se encuentra elevada en el suero en enfermedades como la hepatitis, cirrosis,
hemocromatosis, ictericia obstructiva asociada con enfermedad neoplásica, cancer de próstata y vejiga, anemia
hemolítica, fiebre reumática, fiebre tifoidea, gota, talasemia mayor, leucemia mieloide, tuberculosis, enfermedades
autoinmunes, mononucleósis infecciosa, falla cardiaca.
La ADA tiene varias isoenzimas: ADA 1 es fundamentalmente intracelular y ADA 2 predomina en plasma y suero, el
sistema monocito-macrófago puede ser la principal fuente de ADA2
La isoenzima 2 del enzima ADA, se considera un indicador del recambio de la línea monocito/macrófago y por lo
tanto, la determinación de la actividad del ADA en fluídos biológicos se emplea en el diagnóstico de la tuberculosis
pleural y peritoneal, así como en la meningitis tuberculosa. La medida de la actividad de ADA se realiza habitualmente
mediante métodos espectrofotométricos qque pueden ser facilmente utilizados en los analizadores de bioquímica
clínica.
Las muestras hemolizadas presentan valores falsamente elevados, probablemente debidos a un aumento del isoenzima 1 del
ADA o también llamado eritrocitario, por ser la única forma que posee el hematíe.
ADA en sangre, tiene utilidad como marcador para enfermedades infecciosas tales como: Mononucleosis, Fiebre tifoidea y
Hepatitis. Valores de ADA muy bajos, reflejan una inmunodeficiencia.

9. Determinación cuantitativa de Adenosina Deaminasa (ADA)
PRINCIPIO DEL MÉTODO
La prueba de ADA se basa en la desaminación enzimática de adenosina a inosina que se convierte en hipoxantina por purina
nucleósido fosforilasa (PNP). A continuación la hipoxantina se convierte en ácido úrico e hidrógeno peróxido (H2O2) por xantina
oxidasa (XOD). H2O2 se reactiva con N-Etil-N-(2-hidroxi-3-sulfopropil)-3-metilanilina (EHSPT) y 4-aminoantipirina (4-AA) en
presencia de peroxidasa (POD) para generar tinte quinona que se monitoriza de forma cinética.
ADA
Adenosina + H2O
Inosina + Pi
Inosina + NH3
PNP
Hipoxantina + Ribosa 1-fosfato
XOD
Hipoxantina + 2H2O + 2O2
2H2O2 + Ácido úrico
POD
2H2O2 + 4-AA + EHSPT
4H2O + tinte quinona (máx. 556nm)
Una unidad de ADA se define como la cantidad de ADA que genera un
adenosina por min. a 37ºC.
º
mol de inosina a partir de

10. Lakkana Boonyagars, M.D., Sasisopin Kiertiburanakul, M.D., MHS
J Infect Dis Antimicrob Agents 2010;27:111-8.
Cerebrospinal fluid
A study comparing the ADA activity in cerebrospinal fluid (CSF) between patients with tuberculous and non-tuberculous meningitis was conducted.
The ROC curve identified a CSF ADA level of 15.5 U/l as the best cut-off value to differentiate between the 2 groups, with a sensitivity of 75 percent, specificity of 93
percent and area under the curve of
0.92.24 In bacterial meningitis, mean ADA is quite high when compared with non-tuberculous and non-bacterial meningitis group. The yield of ADA may be low in
setting to differentiate bacterial from tuberculosis meningitis. The possible explanation may be from ADA value in most assays detected total ADA which includes ADA-1
and ADA-2. Thus, fluid with high cell counts (e.g. bacterial meningitis) can have high total ADA and may be undifferentiated from tuberculous meningitis.
ADA activity in the CSF of HIV-infected patients had limited value for diagnosis of tuberculous meningitis. A retrospective study was conducted to determined ADA
levels in 417 CSF samples from HIV-infected patients with neurological symptoms. HIV-associated neurological disorders and progressive multifocal leukoencephalopathy
were not associated with elevated ADA in CSF. When using a cut-off point of 8.5 IU/l for the diagnosis of tuberculous meningitis, sensitivity was only 57 percent and
specificity was 87 percent. A cut-off value of 10 IU/l gave a specificity of 90 percent but very low sensitivity (36%). False-positive results were found in patients with
neurological cytomegaloviral disease, cryptococcal disease, lymphomatous and probable candidal meningitis. The results of this study indicated that ADA determination in
CSF has limited utility for the diagnosis of tuberculous meningitis in HIV-infected patients.
Recommendation from British Infection Society for the diagnosis and treatment of TB of the central nervous system in adults and children26 suggests that the
activity of ADA is raised in the CSF of patients with tuberculous meningitis and has been evaluated as a diagnostic assay. The major problem was lacking of specificity.
High CSF ADA activity has been reported from patients with lymphomas, malaria, brucellosis and pyogenic meningitis. Thus, CSF ADA activity is not recommended as a
routine diagnostic test for TB of the central nervous system. However, prevalence of tuberculous meningitis in Thailand is high and positive predictive value for ADA in
diagnosis of tuberculous meningitis is much higher than that of European countries. The value of CSF ADA may have usefulness in Thailand.
Adenosine deaminase versus polymerase chain reaction
Nucleic acid identification by PCR is a rapid, sensitive and specific tool for the detection of Mycobacterium tuberculosis. It permits direct identification of the M. tuberculosis complex and results are available in a day or two. However, sensitivity depends on a target site. PCR targets such as IS6110 and hsp65 kDa yield a sensitivity of
42-100 percent and a specificity of 85-100 percent.29 Sensitivity of PCR was achieved when devR and IS6110 test results were combined; the sensitivity and specificity
values were 83 percent and 94 percent respectively in pleural fluid.30
A cross-sectional study was performed in a total of 179 body fluid samples. All specimens were analyzed for AFB smear, ADA activity (by a method based on the Berthlot
reaction) and multiplex PCR using amplicons such as IS6110, dnaJ gene and hsp65 genes. On comparing AFB and ADA results with PCR, the PCR is clearly more
effective than AFB smear (p < 0.001) and ADA estimation (p < 0.02) in all types of body fluids.
Disadvantages for PCR are; it needs more resources and sophisticated equipments than ADA, price is higher, needs longer time for test results and not every hospitals can
set PCR lab (especially small to medium sizes hospitals).

11. (16)
Chotmongkol et al in their study reported 75% sensitivity and 93%
specificity for CSF-ADA level in diagnosis of TBM with a 15.5IU/L
cutoff
Kashyap et al(17) in their study reported that with a 11.39 IU/L cut-off ,
the sensitivity and specificity of ADA measurement in diagnosis of
TBM in CSF samples of their patients were 82% and 83% respectively.
Corral et al.(18) reported a 57% sensitivity and 87% specificity
with a 8.5IU/L cut-off for CSF-ADA level in the diagnosis of TBM in
HIV infected patients and
Gautam etal.(19) in their study reported the sensitivity and specificity of
85% and 88.0% respectively for CSFADA levels in diagnosis of
TBM with a 6.97IU/L cutoff value.

12. COMPARACIÓN ENTRE DIFERENTES ESTUDIOS DE LA SENSIBILIDAD, ESPECIFICIDAD, VPP Y VPN PARA LA
MEDICIÓN DE ADA EN LCR EN EL DIAGNÓSTICO DE MT.
AUTOR
NUMERO DE
POBLACIÓN
PACIENTES
CON
MT
ADA EN
SENSIBILIDAD
LCR (UI/L)
ESPECIFICIDAD
VPP
VPN
MISHRA ET AL (1996) (10)
27
NIÑOS
>5
89%
92%
SD
SD
BARO ET AL (1996) (12)
12
ADULTOS
>6.5
83%
85%
SD
SD
MANN ET AL (1982) (22)
33
ADULTOS
>5
85%
84%
SD
SD
ZUÑIGA-RAMIREZ ET AL (2005) (23)
23
ADULTOS
>7
39%
96%
42
96
GAMBHIR ET AL (1999) (8)
36
ADULTOS
>=8
44%
75%
SD
SD
LÓPEZ L.F-CORTÉS ET AL (1995) (24)
20
ADULTOS
>10
48%
96%
1
0,91
COOVADIA ET AL (1986) (25)
38
NIÑOS
>=10
73%
71%
SD
SD
KASHYAP RS ET AL (2006) (20)
27
ADULTOS
>11.39
82%
83%
SD
SD
MT MENNGITIS TUBERCULOSA; VPP VALOR PREDICTIVO POSITIVO; VPN VALOR PREDICTIVO NEGATIVO

13. . Resultados de la ADA con punto de corte de 9U/L en algunas enfermedades
neurológicas en pacientes VIH positivos (IC 95%)
Etiología de la
infeccion del
Velasquez G, Betancur J, Estrada B, Ospina S y
colaboradores. Infecciones em 193 pacientes com SIDA.
SNC en
Sensibilidad
Especificidad
VP +
VP –
RV +
92,5
2,5
RV -
pacientes VIH
positivos
Acta 18: 56-65.
Tuberculosis
Lizarazo J, Castro F, De Arco M, Chaves
O, Peña M.
73,9
70,5
meníngea
(53,8-94,0)
(61,3-79,7)
(20,8-50,0)
(86,1-98,9)
(1,7-3,7)
(0,2-0,7)
58,8
70,2
41,7
82,5
1,97
0,59
(40,8-76,8)
(60,4-80,0)
(26,7-56,7)
(73,6-91,5)
(1,3-3,0)
(0,38-0,9)
40
62,8
12,5
88,7
1,08
0,95
(11,9-68,1)
(53,5-72,3)
(2,1-22,9)
(81,2-96,3)
(0,5-2,1)
(0,6-1,5)
Neurolúes
10
60,2
2,1
88,7
0,25
1,5
n=10
(0-33,6)
(50,9-69,4)
(0-7,2)
(81,2-96,3)
(0-1,6)
(1,2-1,9)
Otros Dx
8,7
46,3
8,3
47,5
0,16
1,97
(0-17,9)
(34,9-57,7)
(0-17,2)
(35,9-59,1)
(0-0,4)
(1,5-3,5)
35,4
0,37
n= 23
Infecciones oportunistas del sistema nervioso central en
pacientes com VIH atendidos en el hospital Erasmo Meoz,
Cucuta, 1995 – 2005. Infectio. 2006; 10(4): 226 – 31.
Criptococosis
meníngea
n= 34
Castañeda E, Torrado E, Arango M, De
Bedout C,
Tobón AM, Restrepo A. Grupo Colombiano de estúdio de
la criptococosis. Criptococosis en Colombia: estudio
interinstitucional. Inf Quinc Epidemiol Nac. 2000; 5: 115-9
Plan nacional de respuesta ante el VIH-SID A
Colombia 2008-2011. Ministerio de Protección Social.
Dirección general de salud pública. ONUSIDA.
Toxoplasmosis
cerebral
n= 15
neurológicos
n= 46
VP: Valor predictivo
RV: Razón de verosimilitud

14. Valores de ADA, glucosa y proteínas en LCR por enfermedad neurológica.
GLUCOSA
Velasquez G, Betancur J, Estrada B, Ospina S y
PROTEINAS
(mg/dl)
(mg/dl)
ENFERMEDAD
ADA (U/L)
Tuberculosis
16,0 ± 9,8
30,8 ± 12,2
134,0 ± 93,0
10,4 ± 6,0
33,8 ± 15,7
95,2 ± 63,1
9,4 ± 7,2
41,8 ± 5,7
88,4 ± 75,1
Neurolúes
5,3 ± 3,5
48,6 ± 18,8
42,8 ± 16,2
Otros diagnósticos *
5,7 ± 8,4
46,2 ± 13,6
50,1 ± 49,4
Total
9,3 ± 8,5
39,8 ± 15,2
81,1 ± 71,2
colaboradores. Infecciones em 193 pacientes con SIDA.
Acta 18: 56-65.
Lizarazo J, Castro F, De Arco M, Chaves
meníngea
Infecciones oportunistas del sistema nervioso
pacientes com VIH atendidos en el hospital
Cucuta, 1995 – 2005. Infectio. 2006; 10(4): 226 – 31.
Castañeda E, Torrado E, Arango M, De
Criptococosis
meníngea
Toxoplasmosis
Tobón AM, Restrepo A. Grupo Colombiano de
la criptococosis. Criptococosis en Colombia:
cerebral
interinstitucional. Inf Quinc Epidemiol Nac. 2000; 5: 115-9
Plan nacional de respuesta ante el VIH-SID
Colombia 2008-2011. Ministerio de Protección S
Dirección general de salud pública. ONUSIDA.
* Encefalopatía asociada al VIH, encefalitis por histoplasma, meningitis aséptica y sin diagnóstico definido

15. Técnicas de
Biología Molecular
Dianóstico Directo

16. Xpert MTB/RIF
En el “Xpert MTB/RIF”, el único paso que se realiza de forma manual, es la adición del reactivo bactericida
a la muestra (esputo) antes de introducirlo en la máquina, elimina así el inconveniente de necesitar
laboratorios de alta bioseguridad ya que todas las reacciones se realizan después de inactivar los
gérmenes presentes en el esputo.
El test automático MTB/RIF integra una modificación de la reacción convencional de polimerasa en cadena,
PCR, con la que se reduce la contaminación del producto amplificado, en este caso el gen llamado rpoB ;
mutaciones en este gen, que codifica para la subunidad beta de la ARN polimerasa, están implicadas en la
resistencia a rifampicina, además también amplifica las secuencias que rodean el gen que son específicas
del complejo M. tuberculosis. Contiene además todos los reactivos necesarios para la lisis bacteriana,
extracción del ADN y detección del producto amplificado.
En el “Xpert MTB/RIF” los pasos finales del ensayo, los que dependen de la reacción en cadena de la
polimerasa, un procedimiento muy sensible pero que es muy propenso a las contaminaciones de la
muestra, tienen lugar en un recipiente herméticamente sellado, lo que elimina la posibilidad de
contaminación, y por ello de falsos positivos.
Otra ventaja del “Xpert MTB/RIF” es que para su manipulación no se necesita personal especializado, lo
que supone una gran ventaja para realizar el diagnóstico en las áreas rurales mal comunicadas y con
pocos recursos de los países con mayor índice de esta enfermedad, puesto que el entrenamiento que se
necesita para llevar a cabo el test es mínimo.
Por otra parte, el tiempo que tarda la máquina en dar el resultado del análisis es de menos de 2 horas, esto
es otra ventaja en comparación con la técnica utilizada hoy en día, el crecimiento de la muestra en medio
selectivo y la tinción de Ziehl-Neelsen que, además del peligro que acarrea su manipulación, tarda como
mínimo unas 2 semanas en obtener resultados.

20. GRADE summary of findings – Diagnostic accuracy of the Xpert MTB/RIF assay in multi-centre clinical validation studies
Review question: What is the diagnostic accuracy of Xpert MTB/RIF for i) detection of pulmonary tuberculosis; and ii) detection of resistance to rifampicin?
Patients/population: Adult pulmonary TB suspects (for TB detection); Confirmed TB cases (for rifampicin resistance detection)
Setting: Multi-centre clinical validation studies
Index test: Conventional culture and DST
Importance: A rapid, accurate, simple test could replace conventional culture and DST and expand testing to lower levels of the health service
Reference standard: Conventional microscopy, culture, drug susceptibility testing, clinical diagnosis of pulmonary TB
Studies: Cross-sectional or cohort
Outcomes: TP, TN, FP, FN
Effect %
No. of
What do these results mean given
What do these results mean given
(95% CI)
participants
10% prevalence among suspects
30% prevalence among suspects being
(studies)
being screened for TB?
screened for TB?
Quality of evidence
Diagnostic accuracy for M.
tuberculosis
All specimens
Sensitivity 92% (90, 94)
Specificity 99% (98, 100)
AFB smear positive/culture
positive
Sensitivity 98% (97, 99)
AFB smear negative /culture
positive
With a prevalence of 10%, 100/1000
will have TB. Of these, 92 (TP) will
be identified; 8 (FN) will be missed
by Xpert MTB/RIF. Of the 900
patients without TB, 891 (TN) will
not be treated; 9 (FP) may be
unnecessarily treated.
With a prevalence of 30%, 300/1000
will have TB. Of these, 276 (TP) will be
identified; 24 (FN) will be missed by
the Xpert MTB/RIF. Of the 700 patients
without TB, 693 (TN) will not be
treated; 7 (FP) may be unnecessarily
treated.
Moderate

No. of
participants
(studies)
720 (5)
What do these results mean given
10% prevalence of rifampicin
resistance among persons with TB?
With a prevalence of 10%, 100/1000
will have rifampicin resistance. Of
these, 98 (TP) will be identified; 2
(FN) will be missed by Xpert
MTB/RIF. Of the 900 patients with
TB susceptible to rifampicin, 891
(TN) will not be treated for MDR; 9
(FP) may be unnecessarily treated.
What do these results mean given
30% prevalence of rifampicin
resistance among persons with TB?
With a prevalence of 30%, 300/1000
will have rifampicin resistance. Of
these, 294 (TP) will be identified; 6 (FN)
will be missed by Xpert MTB/RIF. Of
the 700 patients with TB susceptible to
rifampicin, 693 (TN) will not be treated
for MDR; 7 (FP) may be unnecessarily
treated for MDR.
Quality of Evidence
Sensitivity 72% (65, 79)
Diagnostic accuracy for
rifampicin resistance
Effect %
(95% CI)
All specimens
Sensitivity 98% (94, 99)
Specificity 98% (96, 99)
WHO/HTM/TB/2011.4
1,341 (5)
Moderate


21. GRADE summary of findings – Diagnostic accuracy of the Xpert MTB/RIF assay in multi-centre demonstration studies
Review question: What is the diagnostic accuracy of Xpert MTB/RIF for i) detection of pulmonary tuberculosis; and ii) detection of resistance to rifampicin?
Patients/population: Adult pulmonary TB suspects (for TB detection); Confirmed TB cases (for rifampicin resistance detection)
Setting: Multi-centre clinical validation studies
Index test: Conventional culture and DST
Importance: A rapid, accurate, simple test could replace conventional culture and DST and expand testing to lower levels of the health service
Reference standard: Conventional microscopy, culture, drug susceptibility testing, clinical diagnosis of pulmonary TB
Studies: Cross-sectional or cohort
Outcomes: TP, TN, FP, FN
Effect %
No. of
What do these results mean given
What do these results mean given
(95% CI)
participants
10% prevalence among suspects
30% prevalence among suspects being
(studies)
being screened for TB?
screened for TB?
Quality of Evidence
Diagnostic accuracy for MTB
All specimens
Sensitivity 91% (88, 93) 2,530 (6)
Specificity 99% (98, 99)
AFB smear positive/culture
positive
Sensitivity 99% (97-100)
AFB smear negative/culture
positive
Sensitivity 80% (75, 84)
Diagnostic accuracy for
Rifampicin resistance
Effect %
(95% CI)
All specimens
Sensitivity 95% (91, 97)
Specificity 98% (97, 99)
WHO/HTM/TB/2011.4
No. of
participants
(studies)
2,530(6)
With a prevalence of 10%, 100/1000
will have TB. Of these, 91 (TP) will
be identified; 9 (FN) will be missed
by Xpert MTB/RIF. Of the 900
patients without TB, 891 (TN) will
not be treated; 9 (FP) may be
unnecessarily treated.
With a prevalence of 30%, 300/1000
will have TB. Of these, 273 (TP) will be
identified; 27 (FN) will be missed by
the Xpert MTB/RIF. Of the 700 patients
without TB, 693 (TN) will not be
treated; 7 (FP) may be unnecessarily
treated.
What do these results mean given
10% prevalence of rifampicin
resistance among persons with TB?
With a prevalence of 10%, 100/1000
will have rifampicin resistance. Of
these, 95 (TP) will be identified; 5
(FN) will be missed by Xpert
MTB/RIF. Of the 900 patients with
TB susceptible to rifampicin, 882
(TN) will not be treated for MDR; 12
(FP) may be unnecessarily treated
for MDR.
What do these results mean given
30% prevalence of rifampicin
resistance among persons with TB?
With a prevalence of 30%, 300/1000
will have Rifampicin resistance. Of
these, 285 (TP) will be identified;
15(FN) will be missed by Xpert MTBRIF. Of the 700 patients with TB
susceptible to rifampicin, 686 (TN) will
not be treated for MDR; 14 (FP) may
be unnecessarily treated for MDR.
Moderate

Quality of Evidence
Moderate


22. GRADE summary of findings – Diagnostic accuracy of the Xpert MTB/RIF assay in single-centre unpublished studies1
Review question: What is the diagnostic accuracy of Xpert MTB/RIF for i) detection of pulmonary tuberculosis; and ii) detection of resistance to rifampicin?
Patients/population: Adult pulmonary TB suspects (for TB detection); confirmed TB cases (for rifampicin resistance detection)
Setting: Multi-centre clinical validation studies
Index test: Conventional culture and DST
Importance: A rapid, accurate, simple test could replace conventional culture and DST and expand testing to lower levels of the health service
Reference standard: Composite reference standards (LJ culture, histology/cytology, ADA for CSF and fluids, CT for CSF, follow-up at 3 months)
Studies: Cross-sectional or cohort
Outcomes: TP, TN, FP, FN
Crude pooled effect %1
No. of
What do these results mean given
What do these results mean given
participants
10% prevalence among suspects
30% prevalence among suspects being
(studies)
being screened for TB?
screened for TB?
Quality of Evidence
Diagnostic accuracy for MTB
All specimens pulmonary and
extrapulmonary
Sensitivity 92.5%
4,373 (10)
Specificity 98.0%
Diagnostic accuracy for
rifampicin resistance
No. of
participants
(studies)
917(3)
With a prevalence of 30%, 300/1000
will have TB. Of these, 278 (TP) will be
identified; 12(FN) will be missed by the
Xpert MTB/RIF. Of the 700 patients
without TB, 686(TN) will not be
treated; 14 (FP) may be unnecessarily
treated.
Moderate

What do these results mean given
What do these results mean given
Quality of Evidence
10% prevalence of rifampicin
30% prevalence of rifampicin
resistance among persons with TB? resistance among persons with TB?
All specimens (pulmonary and
With a prevalence of 10%, 100/1000 With a prevalence of 30%, 300/1000
Moderate
Sensitivity 98.6%
extrapulmonary)
will have rifampicin resistance. Of
will have rifampicin resistance. Of

these, 98 (TP) will be identified; 2
these, 296 (TP) will be identified; 4(FN)
Specificity 98.8%
(FN) will be missed by Xpert
will be missed by Xpert MTB/RIF. Of
MTB/RIF. Of the 900 patients with
the 700 patients with TB susceptible to
TB susceptible to rifampicin, 889
rifampicin, 692(TN) will not be treated;
(TN) will not be treated for MDR; 11 8 (FP) may be unnecessarily treated for
(FP) may be unnecessarily treated
MDR.
for MDR.
1
These studies were evaluated individually and crude pooled sensitivity and specificity estimates calculated since meta-analyses was not possible given variability in study design, use of
various reference standards and availability of preliminary data only. The quality of evidence was consequently downgraded (on directness) by 1 point.
WHO/HTM/TB/2011.4
Pooled effect %
With a prevalence of 10%, 100/1000
will have TB. Of these, 92 (TP) will
be identified; 8 (FN) will be missed
by Xpert MTB/RIF. Of the 900
patients without TB, 882 (TN) will
not be treated; 18 (FP) may be
unnecessarily treated.

23. TUBERCULOSIS
DIAGNOSTICS
Xpert MTB/RIF Test
WHO RECOMMENDATIONS
The rapid TB test – known as Xpert MTB/RIF- is a fullyautomated diagnostic molecular test. It has the potential to
revolutionize and transform TB care and control. The test:
• simultaneously detects TB and rifampicin drug resistance
• provides accurate results in less than two hours so that
patients can be offered proper treatment on the same day
• has minimal bio-safety requirements and training needs, and
can be housed in non-conventional laboratories
UPDATED WHO RECOMMENDATIONS
AS OF OCTOBER 2013
For diagnosis of extrapulmonary TB and
For diagnosis of pulmonary TB and rifampicin
rifampicin resistance:
resistance:
Strong recommendation:
• Xpert MTB/RIF should be used as the
• Xpert MTB/RIF should be used as the initial
diagnostic test in adults and children presumed to initial diagnostic test in testing
cerebrospinal fluid specimens from
have MDR-TB or HIV-associated TB
patients presumed to have TB
meningitis
Conditional recommendations (recognising major
Strong recommendation:
resource implications):
• Xpert MTB/RIF may be used as the initial
diagnostic test in adults and children presumed to
have TB
Conditional recommendation:
• Xpert MTB/RIF may be used as a replacement test
for usual practice (including conventional microscopy,
culture, and/or histopathology) for testing of specific
• Xpert MTB/RIF may be used as a follow-on test to
non-respiratory specimens (lymph nodes and other tismicroscopy in adults presumed to have TB but not
sues) from patients presumed to have extrapulmonary
at risk of MDR-TB or HIV-associated TB, especially
TB
in further testing of smear-negative specimens
For all WHO TB diagnostics policy documents: http://

24. Time to detection
Multidrug-resistant tuberculosis
MDR-TB diagnosis with solid culture and DST
Microscopy
Solid culture
1st line DST
2nd line DST
24 hrs
6-8 weeks
3-4 weeks
3-4w
MDR-TB diagnosis
after 9 to 12 weeks
MDR-TB diagnosis with liquid culture and DST
Microscopy
Liquid culture
1st line DST
2nd line DST
24hrs
2-3 weeks
1-3 weeks
1-2w
MDR-TB diagnosis
after 3 to 5 weeks
MDR-TB diagnosis with line probe assay, liquid culture and DST
Line probe assay
Microscopy
2-4 hrs
24 hrs
Line probe assay
2-4 hrs
MDR-TB line DST
diagnosis
2nd
after 2 to 4 hours
+
-
1-2w
Liquid DST
1st line culture
1st line DST
2nd line DST
2-3 weeks
1-2w
1-3 weeks
1-2w
MDR-TB diagnosis
after 3 to 5 weeks

27. THE USE OF MOLECULAR LINE PROBE ASSAY FOR THE DETECTION RESISTANCE TO SECOND-LINE ANTI-TUBERCULOSIS DRUGS
LPA technology involves the following steps: First, DNA is extracted from M. tuberculosis isolates (indirect testing) or directly from clinical specimens (direct testing). Next, polymerase
chain reaction (PCR) amplification of the resistance-determining region of the gene under question is performed using biotinylated primers. Following amplification, labeled PCR products
are hybridized with specific oligonucleotide probes immobilized on a strip. Captured labeled hybrids are detected by colorimetric development, enabling detection of the presence of
M. tuberculosis complex, as well as the presence of wild-type and mutation probes for resistance. If a mutation is present in one of the target regions, the amplicon will not hybridize
with the relevant probe. Mutations are therefore detected by lack of binding to wild-type probes, as well as by binding to specific probes for the most commonly occurring mutations.
The post hybridization reaction leads to the development of coloured bands on the strip at the site of probe binding.
Hain Lifescience new LPA, the Genotype MTBDRsl® test, for the rapid determination of genetic mutations associated with resistance to fluoroquinolones, aminoglycosides
(kanamycin, amikacin), cyclic peptides (capreomycin), ethambutol, and streptomycin. The identification of resistance to fluoroquinolones is enabled by the detection of the most
significant mutations of the gyrA gene (coding for DNA gyrase). For the detection of resistance to aminoglycosides/cyclic peptides, the 16S rRNA gene (rrs) and for detection of
resistance to ethambutol the embB gene (which, together with the genes embA and embC, codes for arabinosyl transferase) are examined. The assay format is similar to the Genotype MTBDRplus assay for the detection of mutations conferring rifampicin and isoniazid resistance, endorsed by WHO in 2008, and allows for testing and reporting results within 24
hours.
Resistencia
a fármacos
Fluoroquinolonas
Aminoglucósidos
Péptidos Cíclicos
Etambutol

28. Farmacogenética:
El Futuro de la Medicina
con Técnicas de
Biología Molecular

29. FARMACOGENÉTICA
Estudia las variaciones genéticas que determinan el efecto farmacológico
de una droga: eficacia, rango terapéutico, efectos adversos, toxicidad.
•FARMACOS MAS PODEROSOS
•DROGAS MAS SEGURAS Y MEJORES, EN LA PRIMERA VEZ
•METODOS MAS PRECISOS PARA DETERMINAR LAS DOSIS
APROPIADAS
•MEJORAS EN EL DESCUBRIMIENTO DE DROGAS Y PROCESOS
DE APROBACION
•DISMINUCION DEL COSTO GENERAL DEL CUIDADO DE LA
SALUD

30. CITOCROMO P-450
HAY POR LO MENOS 12 FAMILIAS DE GENES P-450
HAY ALELOS POLIMORFICOS EN CASI TODAS LAS FAMILIAS
ENZIMA
FENOTIPO
FARMACOS
EFECTO
CYP2C9
METABOLIZADOR
lento
AINES – WARFARINA
– FENITOINA –
losartan - torasemida
RIESGO DE
TOXICIDAD
CYP2C19
CYP2D6
METABOLIZADORES
lentos Y RAPIDOS
METABOLIZADORES
lentos Y RAPIDOS
antidepresivos –
DIAZEPAM OMEPRAZOL
Antidepresivos
-Antipsicoticos –
antiarritmicos
Antianginosos
lentos: RIESGO
DE TOXICIDAD
RAPIDOS:
DISMINUCION
EFICACIA
lentos: RIESGO
DE TOXICIDAD
RAPIDOS:
DISMINUCION
EFICACIA

31. GEN
FARMACO
EFECTO ASOCIADO CON
POLIMORFISMOS
ECA
INHIBIDORES DE LA
ECA
DISMINUCION DE LA PRESION
ARTERIAL – TOS SECA
RECEPTOR
B2
INHIBIDORES DE LA
BK
ECA
RECEPTOR
2
AGONISTAS 2
TOS SECA
BRONCODILATACION – AUMENTO DE LA
FRECUENCIA CARDIACA –
SUCEPTIBILIDAD A LA
DESENSIBILIZACION INDUCIDA POR
AGONISTAS

32. GenBank
www.ncbi.nlm.nih.gov

33. Muchas gracias por su atención!
Tengamos ideales elevados y pensemos en alcanzar grandes cosas,
porque como la vida rebaja siempre y
no se logra sino una parte de lo que se ansía,
por eso soñando alto alcanzaremos mucho más.
Para una voluntad firme, nada es imposible, no hay fácil ni difícil;
fácil es lo que ya sabemos hacer,
difícil, lo que aún no hemos aprendido a hacer bien”.
Bernardo Houssay (1887 – 1971)
Premio Nobel de Medicina y Fisiología -1947