Moses L. Joloba





Mycobacterium tuberculosis (MTB), is the causative agent of
tuberculosis (TB)
1.7 billion people infected with MTB worldwide
The majority have latent infection
Each year, 9 million develop disease
2 million die of TB annually
TB is the single largest cause of death from a curable
and preventable infectious disease
Millions of deaths in 2002
3.0
2.0
1.0
0.0
HIV/AIDS
Tuberculosis
Malaria
Measles

Uganda is one of the 22 countries most affected by TB in the
world.

In Uganda we detect 40,000 cases per year

This represents 50% of the cases to be detected.

About 70% of those detected are cured

WHO recommends 70% detection rate and 85% cure rates

Presented under the following areas:




A – Diagnosis
B – Identification
C – Drug susceptibility testing (DST)
D – Epidemiology
PREVIOUS (< 2006)

Light microscopy
CURRENT



Solid culture (LJ and
Middlebrook)

Semi automated Liquid
culture Bactec 460 in
JCRC

Florescent Microscope
Automated Liquid culture system
(MGIT)
Portable Liquid culture system
(Manual MGIT)

Direct phenotypic DST methods

HAIN Test for MDR detection

HAIN Test for speciation

PCR for Identification

Capillia for Identification

RFLP, Spoligotyping, RD analysis and
MIRU for epidemiology
TB diagnostic
testing in
endemic
countries
Fundamental
diagnostic: 1882
Fundamental
diagnostic: 2007
TB diagnostic
testing in
endemic
countries
Fundamental
diagnostic: 1882
Fundamental
diagnostic: 2008


Binocular microscopes – covers 80% of the country in
Uganda
Florescent microscopes (LEDs) for very busy centers
 More sensitive than light X-scopes
 Decrease workload – scan fewer fields
 LEDs do not need dark rooms and have long life bulbs


Introduction of External Quality Assurance (EQA), covers
90% of the country (Uganda) – ensures quality (2005)
Change in diagnostic policy (2008)
 2 smears instead of 3
 One positive smear instead of 2 enough to start therapy






NTRL – Wandegeya
FIND – Wandegeya
JCRC - Mengo
MRC- Entebbe
AERAS – Medical School
MSF – Epicentre - Mbarara

Solid – LJ…

Liquid Bactec 460
• Solid – LJ…
• Liquid culture
1. Automated MGIT 960
2.
Manual
MGIT
3. Bactec 9000 series for fluids
BEFORE 2006

Presumptive (accurate for
solids)
 Colony morphology
 Growth rate
 ZN

CURRENT

Presumptive for solid –
phasing out

PCR for IS6110

Capillia – key for liquid system
NAP for Bactec 460
BEFORE 2006

Indirect proportion by LJ and Bactec 460
TAT 3 weeks
TAT 3 month
1. Genotype MTBDR – Detects MDR TB in one day
PCR
2. Drect MGIT – 5d
Hybridization
Detection
3. Nitrate reductase assay – 14d
4. MODS assay – 10d
TOTAL HANDS-ON TIME = 2 MINUTES
The system is the first to fully automate and integrate all the steps required
for PCR-based DNA testing: sample preparation, DNA amplification and
detection. Designed to simplify hands-on preparation, the system provides
PCR test results from a raw sample in about an hour, enabling time-critical
DNA tests at the point of need.


Previously none
Now using PCR RD analysis we can speciate MTB
complex into








M. tuberculosis
M. africanun
M. bovis
M. bovis BCG
M. canneti
M. microti
M. pimpinedi
……
 Asiimwe BB, Koivula T, Kallenius G, Huard RC, Ghebremichael S,
Asiimwe J, Joloba ML. Mycobacterium tuberculosis Uganda Genotype
is the predominant cause of TB in Kampala, Uganda. Int J Tuberc Lung
Dis 2008; 12(4): 386 - 389.
Through use of PCR based GenoType Mycobacterium CM from HAIN we can
speciate MOTT into 14 species (2008)

M. avium spp.
•

M. chelonae
• M. Kansasii

M. abscessus
• M. malmoense

M. fortiutum
• M. peregrinum

M. gordonae
• M. marinum/M. ulcerans

M. intracellulare
• M. tuberculosis
complex

M. scrofulaceum
M. interjectum
• M. xenopi.

RFLP

Spoligotyping – PCR Based

MIRU – PCR based
No. of
IS6110
Isolates copies
2
8
5
2
6
3
2
3
3
3
2
2
2
3
2
2
3
3
2
2
3
2
3
3
2
2
2
2
2
3
3
4
6
2
11
14
14
01
17
12
10
12
01
11
15
18
17
13
09
14
15
15
17
16
17
13
16
17
16
15
08
15
15
16
10
14
11
04
Sex
F (M)
HIV
+ (-)
1 (1)
3 (5)
4 (1)
1 (1)
3 (3)
2 (1)
1 (1)
1 (2)
2 (1)
2 (1)
0 (2)
0 (2)
0 (2)
2 (1)
0 (2)
1 (1)
2 (1)
1 (2)
1 (1)
1 (1)
1 (2)
0 (2)
2 (1)
0 (3)
0 (2)
1 (1)
1 (1)
0 (2)
2 (0)
1 (2)
1 (2)
4 (0)
2 (4)
1 (1)
0 (2)
4 (4)
2 (3)
1 (1)
4 (2)
2 (1)
0 (2)
1 (2)
1 (2)
3 (0)
0 (2)
0 (2)
0 (2)
0 (3)
2 (0)
1 (1)
0 (3)
2 (1)
0 (2)
1 (1)
3 (0)
0 (2)
2 (1)
0 (3)
2 (0)
1 (1)
1 (1)
1 (1)
1 (1)
0 (3)
2 (1)
2 (2)
2 (4)
1 (1)
Age, years
<40 (>40)
2 (0)
7 (1)
4 (1)
2 (0)
3 (3)
3 (0)
2 (0)
3 (0)
3 (0)
2 (1)
2 (0)
2 (0)
2 (0)
3 (0)
2 (0)
2 (0)
2 (1)
3 (0)
0 (2)
1 (1)
3 (0)
0 (2)
3 (0)
3 (0)
2 (0)
2 (0)
1 (1)
2 (0)
2 (0)
3 (0)
3 (0)
4 (0)
5 (1)
2 (0)
Benon B.Asiimwe,1,2 Moses L. Joloba2, Solomon Ghebremichael1,3, Tuija Koivula4, David P. Kateete2,
Fred K. Ashaba2, Alexander Pennhag3, Ramona Petersson3, and Gunilla Kallenius*,1,3. DNA Restriction
Fragment Length Polymorphism Analysis of Mycobacterium
tuberculosis Isolates from HIV-Seropositive and HIV-seronegative Patients in Kampala,
Uganda. (Submitted to Clinical Microbiology and Infection)
IS6110-RFLP clustering
Fig. 1. Description of 33 clustered spoligotypes (2 to 49 cases) in the study
Order STa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
1
128
135
52
UGA1
590
21
125
UGA2
42
26
UGA3
78
UGA4
UGA5
UGA6
UGA7
UGA8
UGA9
4
142
UGA10
59
UGA11
UGA12
UGA13
UGA14
UGA15
UGA16
UGA17
64
288
UGA18
Nb (%)
04 (1.2)
49 (14.2)
38 (11.0)
26 (7.6)
17 (4.5)
15 (4.4)
12 (3.5)
11 (3.2)
10 (2.9)
09 (2.6)
09 (2.6)
09 (2.6)
09 (2.6)
09 (2.6)
09 (2.6)
09 (2.6)
08 (2.3)
07 (2.0)
07 (2.0)
06 (1.7)
06 (1.7)
06 (1.7)
05 (1.5)
05 (1.5)
04 (1.2)
04 (1.2)
03 (0.9)
03 (0.9)
03 (0.9)
02 (0.6)
02 (0.6)
02 (0.6)
02 (0.6)
Spoligotype Patternc
Labeld
Beijing
T2
T2-Uganda
T2
ND
T2
CAS1_KILI
T2
T2
LAM9
CAS1_DELHI
T2
T1-T2
T2
T2
T2
T2
T2
T2
LAM3/S
CAS1
T2
LAM11-ZWE
T2
ND
ND
ND
ND
ND
ND
LAM6
CAS2
ND
aAs
identified in SpolDB4.0; ST, Shared Type; Nb, Number of isolates (as a percentage of total M. tuberculosis strains in the
study); cFilled boxes represent positive hybridization while empty boxes represent absence of spacers; dLabel defining the
lineage/sub lineage; ND, Not yet determined in SpolDB4.0.
Asiimwe BB, Ghebremichael S, Kallenius G, Koivula T, Joloba ML. Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis
patients in peri-urban Kampala, Uganda. BMC Infect Dis. 2008 Jul 28;8(1):101. [Epub ahead of print] PMID: 18662405 [PubMed - as supplied by publisher]
 Need to strengthen systems to utilize existing and new
technology
Develop good tools to monitor lab performance


Train TB teams at point of care
Searching for Point
of care test