DNA isolation from bacteria by pptfiles

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									   Evaluation of 3 PCR techniques for detection of Brucella DNA in peripheral
                                     human blood
                      Manal M Baddour*, Dalal H Alkhalifa**
  *Associate Professor of Microbiology, Faculty of Medicine, Alexandria University,
               Current Affiliation: King Saud University, Saudi Arabia
       ** Lecturer of Microbiology, Girls College of Education, Saudi Arabia

                                        Abstract:
Brucellosis is a widespread zoonosis transmittable to humans. Currently the diagnosis
of this zoonosis is based on microbiological and serological laboratory tests. PCR has
been used to detect DNA from Brucella. Different target genes, primer pairs, PCR
techniques and extraction procedures have been previously published for Brucella
detection. But only a few of these primers have been used in human samples, and only
study has been carried out to compare sensitivity between them. In the present study, 3
sets of primers and 3 different PCR protocols amplifying 3 different regions of the
Brucella genome were compared for detection of Brucella DNA in a peripheral-blood
PCR assay to conclude which is most suitable for the clinical diagnostic laboratory.
These three pairs of primers amplify three different fragments included in: i) a gene
encoding a 31-kDa B. abortus antigen (B4/B5), ii) a sequence 16S rRNA of B. abortus
(F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some
modifications on the reported techniques were applied during the present work to
improve the outcome. Results showed that B4/B5 primer pair had the highest sensitivity
for detection of positive samples (98%), JPF/JPR primer pair detected 88.4% of positive
samples while F4/R2 primer pair was the least sensitive being able to detect only 53.1%
of positive samples. The specificity of the 3 techniques was 100%. B4/B5 primers were
also able to detect the smallest number of bacteria (700 cfu/ml) while JPF/JPR were able
to detect 7x105 cfu/ml and F4/R2 primers were able to detect 7x107 cfu/ml. It is thus
concluded that using the B4/B5 primer PCR with the suggested modifications is a robust
assay, which meets the sensitivity requirements to be used for testing of human blood
samples for brucellosis in the diagnostic laboratory.

                                      Introduction
Brucellosis is a widespread zoonosis which is still responsible for economic losses of
livestock in many areas of the world. It is transmittable to humans via contact with
animals or their products. Half a million new cases are reported worldwide each year,
but according to the World Health Organization, these numbers greatly underestimate
the true incidence of human disease.1 Since the disease constitutes a serious infection
necessitating treatment with a prolonged course of antibiotics, accuracy and short
turnaround time are required for the diagnostic tests.2

Global variation ranges from incidence of <1/100,000 population in UK, USA and
Australia, to 20–30/100,000 in southern European countries such as Greece and Spain,
and up to >70/100,000 in Middle Eastern countries, for example Kuwait and Saudi
Arabia.3 The countries with the highest incidence of human brucellosis are Saudi
Arabia, Iran, Palestinian Authority, Syria, Jordan and Oman. Bahrain is reported to have
zero incidence. Most human cases are caused by Brucella melitensis (B. melitensis),
particularly biovar 3. However, Brucella abortus (B. abortus) has been responsible for
an increasing number of cases in recent years, e.g. in Yemen. B. melitensis biovar 3 is
the most commonly isolated species from animals in Egypt, Jordan, Turkey, Tunisia and
Israel. B. melitensis biovar 2 was reported in Turkey and Saudi Arabia and B. melitensis


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biovar 1 in Libya, Oman and Israel. B. melitensis remains the principle cause of
brucellosis in Saudi Arabia.4

Abdou (1996)5 mentioned that brucellosis is the leading zoonotic disease in the region
and is of major concern in view of its impact on both public health and animal
production schemes. This has been stressed clearly by Memish and Mah (2001)6 in
Saudi Arabia, who mentioned that brucellosis is hyper-endemic, with more than 8000
cases reported each year to public health authorities.

Brucellosis is a world-wide re-emerging zoonosis and the most frequent laboratory-
acquired bacterial infection, causing severe disease in humans with unspecific clinical
signs affecting numerous organs. Patients suffering from this disease show unspecific
symptoms, e.g. fever, chills, malaise, arthralgia, headache, tiredness and weakness.
Various other febrile illnesses, e.g. malaria, tuberculosis, typhoid fever and tularemia
may present with the same symptoms. Therefore, clinical diagnosis is difficult to
establish but effective therapy requires an early diagnosis. A definite diagnosis requires
the isolation of Brucellae from blood, bone marrow or other tissues.7

Blood cultures represent the “gold standard” of laboratory diagnosis, this requires
prolonged incubation, blind subcultures, and special growth media due to their
comparatively long doubling time. Brucella species grow slowly on primary cultures
and subcultures, while their inert biochemical profiles hamper fast identification of
isolates, however, the sensitivity of this technique is low, ranging from 15 to 70%.
Consequently, detection and identification of Brucella spp. in clinical specimens by
cultures may still be a difficult task with significant delays and hazards to lab personnel8
as Brucella spp. are class III pathogens, since their handling poses considerable risk to
laboratory personnel.9

Thus diagnosis is usually based on indirect serological tests, including several
agglutination tests (Rose Bengal, Wright’s tube, Wright’s card, and Wright-Coombs)
and indirect immunofluorescence, complement fixation, and enzyme-linked
immunosorbent assays. Broad range of test sensitivity, low specificity in areas of
endemicity, lack of usefulness in diagnosing chronic disease and relapse, presence of
cross-reacting antibodies, and lack of timeliness constitute problems associated with
brucellosis serology.10,11 To overcome some of these problems, at least two serological
tests have to be combined to avoid false negative results. Usually, the serum
agglutination test is used for a first screening and complement fixation or Coombs' test
will confirm its results.

As for other fastidious pathogens, molecular methodology offers an alternative way of
diagnosing brucellosis. Nucleic acid amplification techniques, like PCR, characterized
by high sensitivity and specificity and short turnaround time can overcome the
limitations of conventional methodology. Only a few studies in the literature,12-16
however, address direct detection of Brucella spp. in clinical specimens of human origin
and in these studies the extraction procedures, the use of different primer pairs, a variety
of different target genes and different amounts of DNA were applied. Whole blood was
used as clinical specimen in all the studies except for one16 where serum was used
instead of whole-blood samples for the diagnosis of human brucellosis by PCR.
The present study addresses the issue of comparing 3 reported PCR techniques for
diagnosis of brucellosis from human blood samples and selecting the one most suitable


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for a diagnostic microbiology laboratory in terms of sensitivity, specificity, robustness
and ease of implementation.


                                  Material and Methods
Peripheral blood specimens were collected from 147 consecutive brucellosis patients
diagnosed over a period of 17 months in a semi-urban hospital setting outside Riyadh,
Saudi Arabia. All patients presented with clinical signs compatible with brucellosis, 3 of
the patients were relapses of a previous brucella infection. All patients were adults (age
range, 16 to 75 years [mean, 34.08 years]. Males constituted 66% of the cases.
Diagnosis was established by positive blood cultures and/or serology. For comparison,
50 blood samples from apparently healthy volunteers were collected and used as
negative controls. The negative control samples were obtained from individuals within
the same age, sex, and demographic categories as that of Brucella-positive samples. The
criteria used for enrollment in the negative control group included normal vital
functions, lack of prior exposure to sepsis or any chronic debilitating disease, and lack of
antibiotics intake during the study period.
Blood samples were obtained at the time of diagnosis before initiation of treatment. The
serological diagnosis was established by Wright’s tube agglutination test (Brucella
Agglutinating Sera; Remel, Dartford, UK). A titer equal to or greater than 1/160 was
considered significant. All patients tested positive by serology, while controls were
negative.
B. melitensis was kindly supplied by the Department of Microbiology (King Faisal
Specialist Hospital). It was identified by biochemical tests such as positive oxidase and
urease tests, negative H2S production, no requirement for CO2 and growth in the
presence of basic thionin and fuchsin (20µg/ml) as well as agglutination by
monospecific antisera.

DNA extraction from blood samples:
Peripheral blood samples from patients and controls were collected in citrated
vacutainers. All samples were aliquoted and stored at -20°C until tested. A 0.5-ml
portion of anticoagulated whole blood was mixed with 1 ml of erythrocyte lysis solution
(320 mM saccharose, 5 mM MgCl2, 1% Triton X-100, 10 mM Tris-HCl [pH 7.5]) and
centrifuged at 15,000 x g for 2 min. The cell pellet was washed with 1 ml of water four
times (Miller et al, 1988)[24]. DNA was isolated from whole-blood pellets with a PURE
GENE Nucleic Acid Extraction Kit (Gentra Systems) as per manufacturer's instructions,
briefly: 50 µL of pelleted cells were mixed thoroughly with 250 µL of cell lysis solution
in a 1.5-mL microfuge tube. The cell lysate was incubated at 65°C for 15 min before
adding 1.5µl Proteinase K Solution (20 mg/ml), mixing thoroughly and incubating at
55°C for 1 hour. Then 100 µL of protein precipitation solution were added to precipitate
proteins. After brief vortexing, the suspension was chilled on ice for 5 min and then
centrifuged at 14000 x g for 3 min to precipitate residual proteins as a tight pellet.
Subsequently, the supernatant containing DNA was poured into a clean 1.5-mL
microfuge tube, mixed with 300 µL of isopropanol (2-propanol) by gently inverting the
tube for at least 5 min, and finally centrifuged at 14000 x g for 5 min to precipitate
purified DNA. The precipitated DNA was reconstituted in 10 µL DNA hydration
solution (Gentra Systems), quantified spectrophotometrically, and stored at -80°C until
use.




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Isolation of genomic DNA from bacteria:
The positive control was genomic DNA isolated from a B. melitensis strain by the
following protocol: the strain was grown in a 5-ml culture until saturated, then 1.5 ml of
the culture was microcentrifuged until a compact pellet was formed. The pellet was
resuspended in 567 µl TE buffer (10 mM Tris-Cl, 1mM EDTA [pH 8]). Then 30 µl of
10% SDS and 3µl of 20 mg/ml proteinase K (Promega,) were added, mixed thoroughly,
and incubated 1 hr at 37°C. To this, 100µl of 5M NaCL were added and mixed
thoroughly. Then 80 µl CTAB/NaCL (4.1 g NaCl, 10 g CTAB in 100 ml H2O) solution
were added, mixed thoroughly, and incubated 10 min at 65°C. One volume (0.7 to 0.8
ml) of 24:1 chloroform/isoamyl alcohol was added, mixed thoroughly, and
microcentrifuged 4 to 5 min. The supernatant was transferred to a fresh tube and 1
volume of 25:24:1 phenol/chloroform/isoamyl alcohol was added with thorough
extraction and microcentrifuged for 5 minutes. The supernatant was transferred to a
fresh tube and 0.6 volume of isopropanol was added and mixed gently. After brief
centrifugation, supernatant was discarded and 70% ethanol was added to the pellet.
After further centrifugation, pellet was dried and resuspended in 100µl TE buffer.17

DNA amplification:
The three pairs of primers chosen amplified regions of three different Brucella genes:

(i) primers B4 (5`-TGG CTC GGT TGC CAA TAT CAA-3`) and B5 (5`-CGC GCT
TGC CTT TCA GGT CTG-3`) amplified a 223-bp fragment present on a gene encoding
a 31-kDa B. abortus antigen;18 (ii) oligonucleotides JPF (5`-GCG CTC AGG CTG CCG
ACG CAA-3`) and JPR (5`-ACC AGC CAT TGC GGT CGG TA-3`) amplified a 193-
bp fragment from a gene encoding an outer membrane protein (omp-2);12 and (iii) a 905-
bp fragment was amplified with primers F4/R2, derived from the 16S rRNA sequence
on B. abortus.19
PCR reactions for each pair of primers were carried out as follows with slight
modifications from the original published protocols:

(i) the B4/B5 pair: in a 50µL reaction mixture which contained 10mM Tris-HCl, 2.25
mM MgCl2, 50mM KCl, 200µM each deoxynucleotide triphosphate (dNTP) (Promega),
1µL each of primers B4 and B5 (5pmol/µL) (TIB MOLBIOL, Berlin, Germany), 1.25U
Taq polymerase (Promega) and 5µL extracted DNA. The PCR profile was set at: initial
denaturation 93°C for 5 min, then 40 cycles of template denaturation at 90°C for 60s,
60s of primer annealing at 60°C and 60s of primer extension at 72°C with a final
extension at 72°C for 7 min.18

(ii) the JPF/JPR pair: in a 50 µL reaction mixture which contained 10 mM Tris-HCl 2
mM MgCl2, 50mM KCl, 200µM each deoxynucleotide triphosphate (dNTP) (Promega),
0.2 µL each of primers JPF and JPR (50pmol/µL) (TIB MOLBIOL, Berlin, Germany),
1.25U Taq polymerase (Promega) and 5µL extracted DNA.
The PCR profile was set at: initial denaturation 94°C for 4 min, then 40 cycles of
template denaturation at 94°C for 60s, 60s of primer annealing at 60°C and 60s of
primer extension at 72°C with a final extension at 72°C for 3 min.12

(iii) the F4/R2 pair: in a 50 µL reaction mixture which contained 10 mM Tris-HCl, 1.5
mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200µM each deoxynucleotide
triphosphate (dNTP) (Promega), 0.2µL each of primers F4 and R2 (50pmol/µL) (TIB



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MOLBIOL, Berlin, Germany), 0.5U Taq polymerase (Promega) and 5µL extracted
DNA.
The PCR profile was set at: initial denaturation 95°C for 5 min, then 35 cycles of
template denaturation at 95°C for 30s, 90s of primer annealing at 54°C and 90s of
primer extension at 72°C with a final extension at 72°C for 6 min.19

MgCl2 concentration was optimized for each technique separately. All PCR reactions
were performed in a Mycycler (Bio-Rad Laboratories, Hercules, Calif, USA). In each
PCR run, a positive control (obtained by DNA extraction from B. melitensis culture) and
a negative control (PCR grade water instead of the DNA) were included to monitor
adequate performance of the run and absence of cross contamination. Further specificity
testing (e.g., involving other significant bacteria, patients with fever of unknown origin,
or hybridization after PCR) was not performed, since these studies have already been
conducted12-14,19,20. Amplicons were detected and photographed by fluorescence after
electrophoresis in a 1% agarose gel (Sigma) in the presence of ethidium bromide (1
µg/ml) in a gel documentation system (Gel Doc XR; Bio-Rad Laboratories, Hercules,
Calif, USA). All standard precautions recommended for prevention of contamination
with DNA and amplicons were undertaken (Kwok & Higushi,1989).21 To ensure the
reliability of the results, all samples were processed in duplicates and the test was
considered positive if the signal from the amplified product was clearly visible in both
samples. A range of positive control (B. melitensis culture) concentrations was tested
with each technique to eliminate the possibility of bacterial DNA concentration (amount
of target DNA in a sample) causing a false negative effect and positive results were
obtained with all concentrations tested.


PCR limit of detection in inoculated blood:
Detection limit of PCR assay was evaluated for blood by the 3 studied protocols. To
determine the cfu, a concentrated suspension of the 48 h culture of B. melitensis was
prepared in sterile saline and 10 fold serial dilutions (10-1 to 10-10) were made. From the
last 5 dilutions, 0.1 ml suspensions were inoculated onto 2 tryptic soy agar plates and
incubated at 37°C for 5 days. Colonies on plates were counted [22]. The concentration
of the undiluted brucella culture was estimated to be 14 x 109. The number of cells in
the bacterial suspension was also estimated spectrophotometrically at OD 600 nm. OD
of 1.4 x109 cfu/ml bacterial cells at 600 nm was determined as 0.15. To determine the
sensitivity of PCR from blood, known numbers of bacterial cells were added to PCR
negative blood samples. Aliquots of 0.5 ml were extracted and processed by PCR by the
different protocols as described above. These experiments were run in triplicate.

Results:

The PCR limit of detection in inoculated blood was found to vary for the 3 tested
techniques, the B4/B5 primers were remarkably able to detect 7 x 102 cfu/ml while the
JPF/JPR primers were able to detect 7 x105 cfu/ml, and F4/R2 primers were the least
sensitive being able to detect 7 x 107 cfu/ml (Fig. 1). As 20 bacterial cells are equivalent
to 60 fg of bacterial DNA [18], it is thus estimated that B4/B5 primers are able to detect
2.1 ng of DNA while primers JPF/JPR can detect 2.1 pg DNA and F4/R2 can detect 210
pg DNA.




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              A                                    B                                          C


                                          M 1 2 3 4 5 6   7    8   9 10 11 12   M 1 2    3   4 5 6 7 8 9 10 11 12
  M 1 2 3 4 5 6 7 8 9 10 11 12 13 14




 223 bp                                   193 bp                                905 bp



Fig 1: Sensitivity of three different PCR assays for detection of B. melitensis in the
spiked blood samples. The indicated amounts of B. melitensis cells were amplified in the
blood of a healthy subject (free of Brucella infection according to clinical, serological
and microbiological tests) with different primer sets, as follows:
A: primers B4/B5; B: primers JPF/JPR; C: primers F4/R2. The 223-bp,193-bp, and 905-
bp PCR products are indicated. Lanes: M, 100 bp molecular weight marker; (1-8: spiked
blood samples with different amount of B. melitensis cells) 1: 7x108, 2: 7x107, 3: 7x106,
4: 7x105, 5: 7x104, 6: 7x103, 7: 7x102, 8: 7x101, 9: negative blood sample, 10 positive
control in B, 11: positive control in B and C, 13: positive control in A, 14: negative
control in A, 12: negative control in B and C.

This finding was depicted while testing the samples as the B4/B5 primers gave the best
results with only 3 false negative results for positive samples. Among the 147 patient
samples tested, 144 were positive by B4/B5 primers (98 % sensitivity) while 130 were
positive by JPF/JPR primers (88.4 % sensitivity) and only 78 were positive by F4/R2
primers (53.1 % sensitivity). Only 55 (37.4 %) samples gave positive reactions in the 3
techniques (Table 1). Out of eleven samples that gave weak bands by the B4/B5
primers, 4 gave strong bands by the JPF/JPR primers, 6 were negative and one gave a
weak band. Interestingly though, using the F4/R2 primers for those samples yielded 5
positives and 6 negatives. Of the 9 samples giving weak bands by the JPF/JPR primers,
8 gave strong bands by B4/B5 and one gave a weak band. Eight samples gave weak
bands by F4/R2 primers while all of them gave strong bands by the JPF/JPR primers and
all but one gave strong bands by the B4/B5 primers.


Table 1: Comparative values of test results between different primer pairs

              Test results of positive samples     Test results of control samples

Primer pair       No. positive    No. negative     No. positive          No. negative        Limit of detection
                   out of 147      out of 147       out of 50             out of 50              (cfu/ml)
                      (%)             (%)              (%)                   (%)

  B4/B5        144 (98.0*)             3 (2.0)         0 (0)              50 (100§)               7x102

 JPF/JPR       130 (88.4*)         17(11.6)            0 (0)              50 (100§)               7x105

  F4/R2         78 (53.1*)         69 (46.9)           0 (0)              50 (100§)               7x107

   * = sensitivity, § = specificity


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The three relapse patients were detected by B4/B5 primers while only 2 were detected
by JPF/JPR primers and none were detected by the F4/R2 primers. All samples obtained
from the control group (healthy subjects) tested negative with the three primer pairs,
conferring an assay specificity of 100%.



Discussion:
Brucellosis is not an emerging disease but rather one that is overlooked by the majority
of the scientific community. Brucellosis is the most common reported infectious disease
among National Guard soldiers and their families.6 Brucella melitensis is the most
pathogenic for humans among the six brucella species.22

Currently, the diagnosis of this zoonosis is based on microbiological and serological
laboratory tests. The diagnostic value of antibody assays is unsatisfactory in the early
stages of the disease due to low sensitivity, serological cross-reactions, and the inability
to distinguish between active and inactive infection due to antibody persistence after
therapy. Some may show false positive serological reactions because of infection by
other gram-negative organisms, Yersinia enterocolitica in particular. The pathogen can
also be detected by blood cultures (which represent the ‘gold standard’ of laboratory
diagnosis). However, false negative cultures could be attributed to antibiotic intake.

When performing the techniques exactly according to the published protocols, no bands
were obtained and after several trials at modifications of the techniques, positive results
were finally acquired. These modifications were then used throughout the study and
namely were: increasing the number of cycles in each technique by 5 extra cycles and
increasing the annealing time for the B4/B5 primers to 60 seconds rather than 30
seconds, the Taq polymerase concentration used with the JPF/JPR primers was reduced
to 1.25U instead of 2.5U, as well as changing the MgCl2 concentration to the optimum
value for each technique as obtained by calibration. The amount of primers used in the
JPF/JPR reaction were also reduced to decrease the intensity of formation of primer
dimmers which were consistently observed when using the original concentrations. The
F4/R2 primers yielded the least sensitivity and several attempts were made to increase
that value including increasing the Taq polymerase concentration from 0.5 U per
reaction to 1.25 U and using a range of primer concentrations from 0.5 µM up to 1 µM
as well as eliminating Triton X-100 from the reaction mixture without success in
improving the sensitivity of the technique. It is speculated that increasing the number of
cycles has improved the diagnostic sensitivities of all primers used as some weak bands
were obtained after increasing the number of cycles that were not detectable using the
original cycle numbers published.

Retesting the 22 JPF/JPR negative samples using 2.5U of Taq polymerase lead to
development of 5 very weak bands which were then considered positive, these bands
would not have been detected had the number of cycles not been increased. It is thus
concluded that using a 1.25U Taq polymerase (together with decreasing the amount of
primer from 50 pmol to 10 pmol) would be more cost effective in this technique
provided that the cycle numbers are increased.

In the present study, to eliminate the possibility of PCR inhibitors such as heparin
(which binds Taq polymerase) and EDTA (which chelates Mg+2 ions from the PCR


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mixture), sodium citrate was used as the anticoagulant. Additionally, the extraction
technique was unified so that the results obtained would depend solely on the primer
sensitivity. Moreover, during the primary phase of the study, different concentrations of
target DNA were tested (ranging from 1µl to 5µl of control DNA) to eliminate the
possibility of reaction inhibition due to excess target DNA.

The possible cause for false negatives in JPF/JPR protocol could therefore, be the
presence of porphyrins or the heme compounds, which remain in the DNA sample due
to the lack of sufficient washings. The F4/R2 technique was the least sensitive and
needed the highest number of cells to give a positive band, all attempts at improving its
results were unsuccessful. Different specimens, sample pretreatment, and DNA
extraction methods could account for discrepant results in comparison to the original
reports12,19 but not for the differences obtained with analytical sensitivity.

In the present study, the sensitivity of these primer pairs was different from that
described in the original reports. However, considering the complexity of PCR methods
and differences between procedures, these results are not surprising. Despite use of the
same primer pair, parameters like sample selection, anticoagulants, storage conditions,
sample pretreatment methods, extraction methods, and finally the actual PCR assay all
were variable. Similar to the findings of the present study, in a previous report, the
analytical sensitivity of the JPF/JPR assay using isolated genomic B. melitensis control
strain was lower than the sensitivity of the B4/B5 assay. All attempts to improve the
analytical sensitivity by changing assay parameters were unsuccessful.16 Only in 4 out
of 10 whole-blood specimens was the 193-bp band amplified. The presence of inhibitors
in PCR-negative specimens was ruled out by examining samples diluted in water. In
order to exclude the possibility of inefficient DNA extraction, aliquots of the original
samples were thawed and DNA was re-extracted and used as a template for both PCR
assays (B4/B5 and JPF/JPR). The same results were obtained. The diagnostic
sensitivities of the JPF/JPR assay for whole-blood specimens thus corresponded to 40%.
No examination of further patient samples was undertaken due to apparent insufficient
test performance.16 In the present study, use of diluted samples (1 to 10 in water) did
not improve the overall sensitivity of the B4/B5 or JPF/JPR primer pairs so it was not
adopted as a routine (data not shown). Although detection limit of the assay using the
JPF/JPR primers was (7 x 105 cfu/ml), 88.4 % of blood samples were found positive by
PCR. It seems that this detection limit is not a problem in testing blood samples for
brucellosis because enough bacterial DNA is present in blood.

Only few reports in the literature have evaluated the application of PCR for the
diagnosis of human brucellosis, and most of them used the primers B4/B5. The first
study examined samples from 20 brucellosis patients diagnosed by serology.
Mononuclear cells were isolated from EDTA-whole blood; DNA was extracted with a
lysis buffer containing proteinase K and used directly for PCR without purification. All
patients tested positive; however, two successive rounds of PCR were required in order
to enhance band intensity, an approach prone to lead to contamination with amplicons.
All negative controls were negative.13 Another study examined peripheral blood samples
from 47 brucellosis patients retrospectively. Specimens were collected in sodium citrate,
depleted of red blood cells, and digested with a proteinase K-containing lysis buffer, and
DNA was extracted by a salting-out procedure. Excellent sensitivity (100%) was
reported in comparison to blood culture and serology (70 and 84%, respectively).14
Extensive washing of cell pellets, determination and adjustment of the isolated DNA


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concentration, and incubation of DNA with H2O2 were recommended for avoiding false
negatives; however, this method of optimization resulted in a lengthy, complicated
procedure. The specificity was 98%.14 However, since the aim of the present study is to
recommend a procedure that would be practical, cost effective, with short turn around
time and limited hazards to lab personnel as well as being simple enough to be routinely
done in a diagnostic lab, these modifications do not seem suitable.

Finally, a short report described a study involving a small number of brucellosis patients
that tried to reproduce results obtained with the methodology described above. The use
of identical procedures, however, did not reproduce the previous results; the sensitivity
and specificity were 50 and 60%, respectively. Different inoculum sizes and degradation
of target DNA in clinical samples due to different storage conditions were assumed to
account for discrepant results, as did the well-known fact that in-house PCR results are
difficult to reproduce in different laboratories.15

The excellent sensitivity reported by Matar and Queipo-Ortuno,13,14 using the primers
B4/B5, in the diagnosis of human brucellosis has not been reproduced by other
groups.15,16 In the present study, the diagnostic sensitivity of B4/B5 primers was 98%
and not 100% but it was able to detect the 3 relapse cases (which was also the case in
Queipo-Ortuno et al, 1997).14 The reasons why these PCR methods, using the same
primers, showed different sensitivities are unknown, but these results are not surprising,
considering the complexity of PCR methods and the differences between procedures. In
the present study, increasing the number of PCR cycles to 40 rather than 35 as well as
increasing the annealing time to 60 seconds instead of 30 seconds had its toll on
increasing the detection limit as some weak bands were observed that were not seen
when the 35 cycle protocol was followed.

Navarro et al in 200225 showed F4/R2 to be the most sensitive primers, which was not
the case in the present study. They found that this pair of primers was affected by the
presence of human DNA. One study used F4/R2 primers for diagnosis of brucellosis
with a sensitivity of 72.1% which was higher than that found in the present work (Nimri,
2003).23 In accordance with previous results,13,14,18 the B4/B5 PCR assay specificity as
well as the other primer pair assays was excellent.

The short turnaround time of PCR (less than 4 h) compares favorably with that of blood
cultures and Wright’s tube and Wright-Coombs tests (3 to 7 days, 24 h, and 48 h,
respectively). Finally, costs of in-house PCR methods are low for laboratories already
equipped with the necessary infrastructure. The B4/B5 primers amplify a 223bp
fragment from the gene which encodes the synthesis of an immunogenic protein on the
external membrane of B. abortus (BCSP31). This protein, with a molecular mass of 31
kDa, is specific to the genus Brucella and is present in all its biovars. Requirements for
performing B4/B5 primer PCR should be available for any diagnostic lab with the
facilities to run in house PCR. The apparent high sensitivity of the assay (detection of
700 cfu/ml) without being affected by the different concentrations of DNA, the small
concentration of primer required (1 µl of a 5 pmol/µl stock) in addition to the ability to
use ready made 10X PCR buffers as well as detection, with high diagnostic sensitivity
(98%) of DNA extracted by a commercially available, easy to use DNA extraction kit
(Pure Gene), from both acute samples as well as samples from relapsed cases, all these
combined qualify this primer pair to be used for routine diagnosis of brucellosis from
human blood by PCR.


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Conclusions:
Brucellosis remains a serious public health issue, and much remains to be done to reach
the goal of controlling human and animal brucellosis. In the present work, we have
compared three different PCR methods for the detection of Brucella spp. in human blood
samples. We conclude that primers B4/B5 are the most effective of the three PCR
methods evaluated in this study for the detection of Brucella DNA, and they could
provide a useful tool for diagnosis of human brucellosis in a clinical diagnostic
laboratory setting.

Acknowledgements:
This work was partially supported by the Research Centre, King Saud University.

References:
1- World Health Organization. Fact sheet N173, July 1997. World Health Organization,
Geneva, Switzerland.

2- Solera J, Martinez-Alfaro E, Espinosa A. Recognition and optimum treatment of
brucellosis. Drugs 1997; 53: 245–256.

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