Download Detection limit of mycobacteria in blood following the

Systemic Infections by Mycobacterium tuberculosis in HIV-Positive Patients as
Determined by Direct Molecular Testing: Influence of Blood Volume
Bwanga F1, Worodria W1, Luyombya A2, Najjingo I2, Asege L2, Nalwoga T 1, Disqué C3, Lorenz MG3, Allerheiligen V4, Weizenegger M5
University College of Health Sciences, Kampala, Uganda; 2MBN Clinical Laboratories Kampala, Uganda
3Molzym, Bremen, Germany; 4Hain Lifescience, Nehren, Germany;
5Labor Dr. Limbach und Kollegen, Heidelberg, Germany
Objectives
Results and Conclusions
MTB-DNA Blood
FluoroType® MTB
DNA Isolation
Blood samples
2x 1 ml (study 1)
9 ml (study 2)
Removal of
human and
free MTB DNA
Analysis
Lysis of
MTB
Isolation of
MTB DNA
Detection of the
MTB complex
Spike
(cfu/volume)
850
Positive/total
1 ml
10 ml
3/3
3/3
150
75
30
15
3
1.5
3/3
3/3
3/3
2/3
1/3
0/3
3/3
3/3
3/3
2/3
0/3
0/3
LOD (cfu/ml)
≤30
≤3
 The detection limit of M. bovis BCG in blood was 1.5 cfu/FT
MTB assay (5 µl of the 100 µl eluate) in both the small
(1 ml) and large (10 ml) volume extraction protocol.
 This translates to 30 cfu/ml and 3 cfu/ml when 1 ml and 10
ml blood was extracted, respectively.
 Conclusion: The use of a large volume (10 ml) for DNA
extraction increases the detection sensitivity of M. bovis
BCG in blood. This probably is a combined effect of the
removal of human DNA and the use of a large volume.
Detection of mycobacteria in the blood of HIV patients under suspect of MTB
infection
Study 2
(9 ml blood)
Sensitivity
71%
Specificity
96%
PPV
83%
NPV
92%
Concordance
90%
BC+ BC- Sum
BC+ BC- Sum
PCR+
3
1
4 PCR+
5
1
6
PCR6 32 38 PCR2 23 25
7 24 31
Sum
9 33 42 Sum
Fig. 3: Time to positivity of culture (BC;
median of both studies) and the molecular
detection assay (PCR).
days
days
25
20
Study 1
(2x 1 ml blood)
33%
97%
75%
84%
83%
25
Table 2: Influence of blood volume on the diagnostic performance
of FT MTB assay with DNA extracted by MTB-DNA Blood kit.
20
15
Criteria for participation of patients included HIV
infection with clinical suspicion of mycobacteremia or
disseminated tuberculosis (TB) with or without
pulmonary TB, and absence of TB- or HIV-drug
treatment. MTB-DNA Blood (Molzym, Germany) was
used for DNA extraction from 1 ml (study 1) and 9 ml
EDTA blood (study 2). DNA eluates were analysed
with the Fluorotype® MTB assay (Hain Lifescience,
Germany) (FT MTB). Cultures (5 ml blood, BD
Bactec® 9000 Myco/F Lytic-Medium) were analysed
for MTB, and confirmed with the Capilia TB test
(Tauns, Switzerland). MTB DNA was extracted from
sputum using the FluoroLyse kit and analysed using
the FT MTB assay.
Table 1: Detection of M. bovis BCG spiked into EDTA blood. DNA was extracted using MTB-DNA Blood and
detected in the FT MTB assay.
15
days
10
Methods
Detection limit of mycobacteria in blood following the molecular approach
(see Fig. 1 and 2)
10
5
Mycobacteraemia is common among HIV-infected
individuals turning rapidly fatal because culturebased diagnosis takes weeks. Molecular tests have
yet not been shown to have acceptable diagnostic
values justifying their use in the direct diagnosis of
mycobacteraemia. The aim of the present study was
to evaluate the influence of the blood volume
extracted on the sensitivity of detection of
mycobacteraemia by a Real-Time PCR test. For this,
two study populations of patients under suspect of
Mycobacterium tuberculosis (MTB) infection were
employed.
5
0
1Makerere
0
BC
BC
PCR
PCR
Fig. 1: Culture-independent molecular approach for the
detection of mycobacteraemia.
 A considerably higher diagnostic sensitivity was observed with the 9 ml (71 %) compared with the 2x 1 ml
(33 %) extraction protocol of MTB-DNA Blood kit (Table 2). The specificities were equally high with both
extraction protocols (97 and 96 %).
 The predictive values were higher with the large (PPV: 83 %; NPV: 92 %) than the small (PPV: 75 %; NPV:
84 %) volume extraction protocol (Table 2).
 Cultures took 3 weeks (median) until positive in both studies (Fig. 3). In contrast, results by the molecular
test were obtained after only 4-4.5 hours (2 h extraction; 2-2.5 h assaying).
 Conclusions: The general diagnostic performance was better analysing a high blood volume (9 ml) than a
small blood volume (2x 1 ml). The molecular approach diagnosed one culture-negative mycobacteraemia
each in both studies (Table 2). The molecular assay is rapid and can aid culture diagnosis by a weeks
earlier initiation of antimycobacterial therapy in positive cases.
Fig. 2: Detection of the MTB complex with FluoroType® MTB.
Positive control
Negative control
U97
positive for M.
tuberculosis
complex
U104
Amplification Control Amplification of
M. tuberculosis complex
The studies were approved by the ethics committee of Makerere University College of Health Sciences
contact: [email protected]