Determining the etiology of pulmonary infiltrates in patients with hematological malignancies (HM) is challenging, and early empirical treatment with broad-spectrum antimicrobial agents is therefore used when an infection is suspected, with the goal of improving patient outcomes. However, selection of the empirical antibiotics benefits considerably from the early identi-fication of organisms such as Streptococcus pneumo-niae, Haemophilus influenzae, nosocomial bacteria, Mycoplasma pneumoniae, Chlamydophila pneumo-niae, Legionella pneumophila, viruses, and fungi.
Traditionally, the identification of causative bacte-ria rests on blood cultures: sputum cultures for com-mon pathogens and M. tuberculosis; serological tests for C. pneumoniae, M. pneumoniae, and L. pneumo-phila; and cultures of specimens obtained using inva-sive techniques such as endoscopic bronchial aspiration or bronchoalveolar lavage (BAL). These methods are slow to produce results, lack sensitivity, and are influ-enced by previous antibiotic therapy. The diagnostic yield of BAL for identifying the cause of pulmonary infiltrates in patients with HM has varied between 31% and 49% [6, 28, 35, 36, 68, 85]. Thus, BAL has a lim-ited impact on early therapeutic decisions. In recent years, noninvasive techniques such as L. pneumophila or S. pneumoniae antigen detection in urine have proved capable of providing the early etiological diag-nosis of infections and, therefore, of allowing the early administration of appropriate antimicrobials. In addi-tion, recent advances in molecular diagnostic technol-ogy, such as nucleic acid amplification (PCR), have improved the ability to rapidly identify the cause of pneumonia [46]. PCR detects minute amounts of nucleic acid from potentially all respiratory pathogens and is less affected by prior antimicrobial therapy than
New Methods for Bacterial Diagnosis in Patients with Hematological Malignancies
Agnès Ferroni and Jean-Ralph Zahar
A. Ferroni (*) and J.-R. Zahar Laboratoire de Microbiologie, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades and Université René Descartes Paris 5, Faculté de Médecine, 149 Rue de Sèvres, 75015 Paris, France e-mail: [email protected]
are culture-based methods [47]. PCR is probably more sensitive than most comparator culture-based diagnos-tic tests, as it is affected neither by decreased organism viability associated with specimen transportation nor by previous antibiotic therapy. It must be kept in mind that PCR does not differentiate between viable and nonviable bacteria and that there are no clear data in the literature on DNA decay after the end of antibiotic treatment.
The use of antigen detection in clinical samples and recent advances in real-time amplification sys-tems are discussed here with reference to respiratory bacteria known to cause pneumonia in patients with HM. Unlike the identification of viruses and fungi, for which these new methods have been evaluated in immunocompromised patients [25, 35, 51, 57, 87], the identification of bacteria has been mainly evalu-ated in patients with community-acquired infec-tions. Table 18.1 lists the methods that can be used in immunocompromised patients with suspected pneumonia.
18.2 PCR
PCR techniques play a major role in the diagnosis of infections that are either very difficult or impossible to diagnose rapidly by other methods. PCR is especially useful for detecting pathogens that are not known to colonize the human respiratory tract (e.g., Legionella species), as a positive result strongly suggests infec-tion. To date, PCR methods have been developed for all the major pneumonia-causing pathogens, and com-mercial assays are available for some of them. However, few studies have compared different assays, and the techniques have not been standardized.
PCR detection involves the extraction of bacterial DNA from the sample (nasopharyngeal aspirate, BAL, sputum, or throat swabs), followed by amplification using primers. Increasingly, real-time PCR techniques, based on fluorescent probes, are superseding the tradi-tional PCR tests in which the products obtained at the end of the amplification cycles are revealed on agarose gel (end-point reaction). The main advantages of real-time PCR are rapidity; safety; ability to quantify bacte-rial DNA in the specimen; and, given that a single tube is used for all the steps, a reduced risk of cross-contamination.
Cases of culture-positive samples with negative PCR results may be due to DNA degradation, delayed assay performance, or presence of specific inhibitors. Samples, such as sputum or BAL, may include inhibi-tors, which can cause false-negative results. Therefore, an internal control coextracted with the clinical sam-ples must be used to ensure accurate control of the entire assay [67].
PCR techniques target a single organism (simplex PCR), multiple organisms (multiplex PCR), or all organisms (universal PCR).
18.2.1 Specific PCRs in Respiratory Samples
18.2.1.1 Intracellular Bacteria
Patients with suspected respiratory infections are gen-erally evaluated using PCR tests that target specific organisms to diagnose atypical pneumonia or pneumo-nia due to fastidious organisms. These organisms are theoretically not found as commensals in the respira-tory tract, and qualitative detection is therefore appro-priate [79].
M. pneumoniae, C. pneumoniae
M. pneumoniae can be cultured in a routine labora-tory on acellular media, but the results are obtained only after 2–3 weeks, and the test is relatively insen-sitive [19]. C. pneumoniae can be cultured only on cell lines in specialized laboratories, which requires 5–10 days. As a result, the diagnosis of these two organisms relied until recently on serological tests, whose disadvantage is that they provide only a retro-spective diagnosis. In addition, IgM antibodies to M. pneumoniae can persist for years, with levels remaining elevated in asymptomatic individuals [62], particularly healthy children. Furthermore, the vari-able specificity and sensitivity of current test kits influence the significance of the serological results [5]. Both organisms require treatment with mac-rolides, tetracyclines, or fluoroquinolones, and there-fore sensitive, specific, and fast detection methods available in routine bacteriological practice would be valuable. Real-time PCR is being increasingly used
22518 New Methods for Bacterial Diagnosis in Patients with Hematological Malignancies
Met
hod
Sam
ple
type
Tim
e-to
-res
ulta
Uri
neSe
rum
Sput
umB
ronc
hoal
veol
ar
lava
ge fl
uid
Pleu
ral fl
uid
Thr
oat s
wab
Ant
igen
det
ectio
n
(S. p
neum
onia
e
or L
. pne
umop
hila
1)
Yes
Yes
No
NE
Yes
No
10 m
in
PCR
targ
etin
g at
ypic
al
bact
eria
(M
. pne
umon
iae,
C
. pne
umon
iae)
No
No
Yes
Yes
Yes
Yes
3 h
PCR
targ
etin
g L
egio
nell
aN
oN
oY
esY
esY
esN
o3
h
PCR
AR
Nr1
6SN
oN
EN
oN
E (
only
ont
o st
erile
flu
id)
Yes
No
4–8
h
Mul
tiple
x PC
RN
oN
EN
EN
EY
esN
o3
h
PCR
targ
etin
g S.
pne
umo-
niae
or
H. i
nflue
nzae
No
NE
NE
NE
(on
ly q
uant
itativ
e PC
R)
Yes
No
3 h
PCR
targ
etin
g M
ycob
acte
ria
No
NE
Yes
NE
No
No
3 h
Tab
le 1
8.1
New
bac
teri
al d
iagn
osis
met
hods
pot
entia
lly a
pplic
able
in p
atie
nts
with
hem
atol
ogic
al m
alig
nanc
ies
NE
not
eva
luat
ed in
pat
ient
s w
ith h
emat
olog
ical
mal
igna
ncie
sa T
ime
from
DN
A e
xtra
ctio
n to
ava
ilabi
lity
of th
e PC
R r
esul
t; ho
wev
er, i
n m
ost l
abor
ator
ies,
PC
R a
nd s
eque
ncin
g ar
e no
t per
form
ed e
very
day
226 A. Ferroni and J.-R. Zahar
for the rapid diagnosis of these organisms [21, 22, 48]. Recently, PCR was found better than serology for confirming M. pneumoniae infection in the clini-cally important first week after pneumonia symptom onset [61]. Nevertheless, in case of late sampling or clearance of the organism following antibiotic treat-ment, PCR detection may be less sensitive than serol-ogy [37]. The targets most commonly used for M. pneumoniae are adhesin P1, 16S rRNA, and ATPase operon gene, and for C. pneumoniae, the outer mem-brane protein ompA and ARNr16S. These specific targets can be sought in samples contaminated with commensal flora such as sputum, potentially contam-inated BAL, or throat swab containing cells or nasopharyngeal aspirates. Sputum samples have been shown to be more useful than upper respiratory tract samples for PCR identification of M. pneumoniae and C. pneumoniae [20, 69, 77]. The time-to-result varies across methods from 50 to 90 min. Commercial real-time PCR kits showed variable sensitivities, and their cost was five times higher than that of an in-house assay [81].
Legionella
Legionella species are among the most underdiag-nosed causes of pneumonia, due to the major short-comings of existing diagnostic tests. Isolation of the organism is usually the preferred method but has sev-eral limitations, including fastidious growth require-ments, extended incubation periods (3–10 days), and overgrowth by other bacteria. Direct L. pneumophila detection in clinical specimens by immunofluores-cence methods is rapid but lacks sensitivity. Finally, soluble polysaccharide antigen detection in urine detects only L. pneumophila 1 [73]. PCR for rapid L. pneumophila diagnosis, in contrast, detects all Legionella species [16, 32, 53, 70, 89]. The most fre-quently described Legionella PCR targets are the mip gene (macrophage infectivity potentiator), a virulence gene present in the majority of Legionella species, and the ADNr 5S gene. As Legionella does not colonize the human respiratory tract, the specific target genes can be sought in samples contaminated with commen-sal flora such as sputum or potentially contaminated BAL. PCR can now be considered the test of choice for legionellosis [59], but standardized protocols need to be developed.
18.2.1.2 Common Bacteria (S. pneumoniae, H. influenzae, Staphylococcus aureus, etc.)
Pulmonary infections due to common bacteria are underdiagnosed because of the limitations of conven-tional diagnostic tests. Blood cultures lack sensitivity, particularly in patients who have received previous antibiotic treatment. Isolation of S. pneumoniae or H. influenzae from sputum or BAL may occur as a result of oropharyngeal contamination. Conversely, iso-lation may fail because of previous antibiotic therapy, poor transportation conditions, or small inoculum size.
Recently, studies have been carried out on PCRs tar-geting pyogenic bacteria. However, these tests may be difficult to interpret, as pyogenic bacteria may be pres-ent in the commensal flora, making it difficult to distin-guish between colonization and infection. These PCRs are therefore feasible in routine practice only on sam-ples that do not contain pyogenic bacteria as commen-sals, such as pleural fluid or lung biopsies. Preliminary evaluations of PCR applied to pleural fluid indicate good sensitivity for the diagnosis of pneumococcal pneumonia [23, 47]. In theory, BAL fluid and sputum, which are often colonized by S. pneumoniae or H. influenzae, particularly in children, can be used only if cultures are negative. Nevertheless, a number of investigators have assumed that quantitative PCR may be useful for assessing these contaminated respiratory samples, as the bacterial burden is higher in infection than in colonization [40, 43]. The amount of target nucleic acid in the sample is inversely related to the cycle threshold (Ct) value. This relationship can be used to establish a Ct value cutoff that provides optimal sensitivity and specificity by preventing false-positive results due to colonization with small numbers of organisms. A panel of six real-time PCR assays, includ-ing the lytA gene of S. pneumoniae and the 16S rRNA genes of H. influenzae and S. pyogenes, were used by Morozumi et al. [56] in a study of 429 clinical samples (sputum, nasopharyngeal aspirates, or throat swabs) from children and adults with pneumonia. Sensitivity and specificity, compared to clinical specimen cultures, were as follows: 96.2% and 93.2% for S. pneumoniae, 95.8% and 95.4% for H. influenzae, and 100% and 100% for S. pyogenes. The Ct values of S. pneumoniae and H. influenzae showed excellent correlations with the semi-quantitative culture results. All patients with positive PCR and negative culture results for these two pathogens had a history of previous antibiotic therapy.
22718 New Methods for Bacterial Diagnosis in Patients with Hematological Malignancies
Another controlled study evaluated the culture-PCR correlation in 235 adults with clinically and biologi-cally confirmed pneumonia. In patients not treated with antibiotics, the PCR-culture correlation was good, whereas in treated patients, PCR improved the etio-logical diagnostic yield by 10%. However, in the con-trol group, PCR performed on nasopharyngeal aspirates showed the presence of H. influenzae and S. pneumo-niae in 5% and 8% of cases, respectively [78]. Recently, Kumar et al. found S. pneumoniae and S. aureus in 14% and 8.5% of asymptomatic patients, respectively, underlining the poor specificity of PCR [46]. Specificity can be expected to increase in the near future with improvements in PCR result interpretation, including the determination of Ct cutoffs for quantitative PCR in various pulmonary samples and in well-defined patient populations. Another difficulty in interpreting PCR results stems from the specificity of the target genes used for pneumococcal PCR. The targets used in many pneumococcal PCR assays are the pneumolysin and autolysin genes, but recent evidence casts doubt on their specificity, as these genes, especially pneumo-lysin, may been found in closely related viridans group Streptococci [58, 84, 88]. Recently, the Spn9802 gene fragment compared favorably with quantitative lytA-based real-time PCR in distinguishing patients with pneumonia from control individuals [1].
18.2.1.3 Mycobacterium tuberculosis and Atypical Mycobacteria
PCR tests perform well on M. tuberculosis smear-pos-itive specimens but are less sensitive than culturing. Therefore, PCR is recommended only for smear-posi-tive samples, to determine the species without waiting for the culture result. However, in a study involving routine M. tuberculosis PCR in patients with HM, M. tuberculosis was found in 2/128 BAL specimens negative by microscopy, which considerably shortened the time to diagnosis [35].
The M. tuberculosis PCR uses two main targets: a specific portion of the 16S RNA gene and the inser-tion sequence IS6110. In recent years, real-time PCR techniques have increasingly superseded conventional PCR [11, 24, 66]. Until recently, very few commercial M. tuberculosis PCR kits were available, whereas now many real-time PCR kits are on the market. It is impor-tant to keep in mind that a negative PCR result does not
rule out tuberculosis. Commercial PCR kits are not currently available for atypical Mycobacteria.
18.2.1.4 Multiplex PCR
The multiplex format allows for multiple primer com-binations in a single reaction instead of multiple sim-plex reactions. In addition, the required patient specimen size is smaller. Several studies have evalu-ated the use of multiplex PCR for detecting atypical organisms or Mycobacteria [29, 42, 54, 63], a combi-nation of atypical and common organisms [78], or viral and bacterial pathogens in clinical specimens [46]. In recent years, these in-house assays have tended to be replaced by commercially available products, which are simpler to use in the diagnostic laboratory and allow easier quality assurance compared to in-house assays. Over the last 5 years, these commercial products have been offered mainly for detecting atypical organisms in clinical specimens: M. pneumoniae and C. pneumoniae [34]; M. pneumoniae, C. pneumoniae, L. pneumophila, Legionella spp, M. pneumoniae, and Bordetella pertus-sis [27]; C. pneumoniae, Legionella micadadei, and B. pertussis [42]; and M. pneumoniae, Coxiella burnetii, C. pneumoniae, and Legionella [12].
Multiplex PCR assays were long described as less sensitive than simplex PCR assays due to competition among primers, but two studies gave identical results with multiplex and simplex PCR tests [27, 34]. In con-trast, another study showed higher sensitivity of the simplex PCR nucleic acid sequence-based amplifica-tion (NASBA) versus multiplex PCR for detecting L. pneumophila [49]. However, the lack of an appro-priate reference standard for the quantitative analysis of intracellular pathogens is an obstacle to sensitivity comparisons across assays.
18.2.2 Specific PCR on Blood Samples
The definitive diagnosis of pneumococcal pneumonia in noninvasive respiratory samples requires isolation of the pathogen in a blood sample. However, blood cultures are rarely positive [14]. On the other hand, pneumococcal serology has low sensitivity [8]. Therefore, PCR tests on blood were mainly described
228 A. Ferroni and J.-R. Zahar
for S. pneumoniae [15, 55, 59]. A very recent study showed low sensitivity of PCR in plasma for detecting pneumococcal pneumonia (26–35%). However, this test may be useful for the rapid diagnosis in bacteremic patients, as sensitivity reaches 60–70% in this situa-tion. Specificity of ply PCR was low [2].
Recently, a commercial test using multiplex PCR in serum for numerous bacterial pathogens was evaluated in neutropenic patients [82]. However, this test has not been specifically assessed in patients with lung infiltrates.
18.2.3 Universal PCR
Universal PCR detects a gene found in all bacteria, usually the gene coding for 16S ribosomal RNA. The sequence of this gene allows the identification of most bacteria, without a presumptive diagnosis. This sequence includes conserved and variable regions. Universal primers are selected for conserved regions flanking a variable region, which allows differentia-tion of bacterial species. The amplified product is then sequenced, and the sequence is compared to all bacte-rial sequences contained in a database (e.g., GenBank). The sensitivity of this PCR varies across bacterial spe-cies (10–100 organisms/mL). Universal PCR has been developed chiefly for diseases such as endocarditis, meningitis, and bacteremia. It is useful in case of prior antibiotic treatment, fastidious organisms, or small inoculums. It should be used only for specimens from normally sterile sites, where it can detect only mono-microbial infections, as the presence of two or more organisms produces a mixture of non-interpretable sequences. This technique proved useful for diagnos-ing pleural empyema in children (40% increase in diagnostic yield compared to culture) and adults (31% increase) [47, 52]. The main organisms detected by universal PCR were S. pneumoniae, anaerobic bacteria (Fusobacterium, Prevotella), S. aureus, H. influenzae, and Streptococcus milleri group. The role for universal PCR in pulmonary samples, which are theoretically contaminated by saprophytic flora, remains to be eval-uated. If cultures are negative, then universal PCR is theoretically appropriate. The likelihood of finding a mixture of bacteria by sequencing is not known.
A microarray method has been found promising for the detection and identification of nine bacterial
pathogens. For this method, universal primers ampli-fying a conserved region of the bacterial gyrB/parE gene are used, and the single-stranded PCR products are then characterized by hybridization on an oligo-nucleotide array. This technique can be used for mul-tibacterial infections, for which PCR products cannot be directly analyzed by DNA sequencing [72].
18.3 Antigen Detection
The diagnosis of pneumonia using antigen detec-tion has made progress in recent years. Commercial antigen detection assays are now widely available for two bacterial pathogens, L. pneumophila and S. pneumoniae.
18.3.1 Legionella
Urine antigen testing allows the early diagnosis and appropriate antibiotic therapy of legionellosis [41]. The capture antibody technique used in the majority of these assays is considered specific for L. pneumo-phila serogroup 1, which causes most legionellosis cases in humans. However, as many as 40% of cases are related to other serogroups and are missed by the assay. In a recent study on the effectiveness of urine antigen detection, pooled sensitivity was 0.74 (95% CI, 0.68–0.81) and specificity was 0.991 (95% CI, 0.984–0.997) [74].
The antigen detected is a heat-stable component of the lipopolysaccharide portion of the Legionella cell wall [44, 86]. It is generally detectable in urine as soon as 3 days after symptom onset and can persist for more than 300 days. In one study, antigen was detected in 88% of patients tested on days 1–3 after symptom onset, 80% on days 4–7, 89% on days 8–14, and 100% after day 14 [45]. The most recent method for detect-ing antigenuria is the immunochromatographic test (ICT) membrane assay. The ICT assay is similar to a home pregnancy test and is commercially available. The test is simple to perform and does not require spe-cial laboratory equipment, and the results are obtained within 15 min. A study found that the ICT assay for L. pneumophila serogroup 1 was 80% sensitive and 97% specific [33]. More recently, it was suggested that
22918 New Methods for Bacterial Diagnosis in Patients with Hematological Malignancies
specificity and sensitivity were higher after 30 min of incubation [17]. The use of concentrated urine samples has been suggested to increase sensitivity [7].
There is still a need for developing antigen capture assays that can diagnose infections with all Legionella species and serogroups. Twenty years ago, Tang and Toma developed a broad-spectrum ELISA that detected soluble antigens from numerous L. pneumophila sero-groups and species by using rabbit antisera from a combination of intradermal, intramuscular, and intra-venous inoculations over a period of 8 months [80]. Unfortunately, this test was not commercialized.
18.3.2 Streptococcus pneumoniae
The recent development of a rapid ICT assay that detects the C polysaccharide cell wall antigen common to all S. pneumoniae strains has renewed interest in antigen detection. The Binax Now test uses a rabbit anti-S. pneumoniae antibody that binds to soluble pneumococcal C antigen present in the sample (NOW Streptococcus pneumoniae test; Binax, Portland, ME). The resulting complex is immobilized by a band of rabbit anti-S. pneumoniae antibodies adsorbed onto a nitrocellulose membrane sample line. A second band of goat antirabbit IgG (control line) captures excess visualizing complex. Usually, a swab is dipped into 200 mL of sample fluid and inserted into the test device. A buffer solution is added, and the device is closed. The result can be read visually after 15 min. A pink to purple color on both the sample and control lines indi-cates a positive result. Color on the control line only indicates a negative result and absence of color on the control line an invalid test. Pneumococcal pneumonia can be diagnosed using antigen detection in urine, BAL fluid, or pleural fluid.
18.3.2.1 Pneumococcal Antigen Detection in Urine
The reported sensitivity of the ICT assay applied on urine samples ranges from 44.2% to 88.8% [18, 30, 50, 60, 71, 76]. Sensitivity is higher in bacteremic than in nonbacteremic patients and increases when urine sam-ples are concentrated, which is usually achieved by ultracentrifugation and may last for 1–4 h [50, 60, 71].
Several studies of the Binax Now S. pneumoniae urine antigen showed good performance for diagnosing bac-teremic pneumococcal infections in adults. Sensitivity is good, ranging from 77% to 87% [10, 75, 76]. Specificities of 97–100% were found in studies that used controls with nonpneumococcal bacteremia or noninfectious disorders [75, 76].
Given its excellent specificity, this test can be con-sidered an important tool for detecting S. pneumoniae in patients with community-acquired pneumonia of unknown etiology. It provided the diagnosis of pneu-mococcal pneumonia in one-fourth of cases [26]. It is important to note that the test may remain positive for several weeks after pneumococcal pneumonia [3, 50] and that patients with detectable antigen may have been colonized with S. pneumoniae. Indeed, specific-ity of the ICT urine test has been reported to be low in children due to nasopharyngeal carriage [31]. In adult patients, specificity was 89.7–100%, depending on the reference standard [18, 50, 71]. Antigen-positive results should be interpreted carefully in pneumonia patients with chronic bronchitis, because detectable antigen may be caused by pneumococcal carriage in the lower respiratory tract [9, 10].
In a recent study, pneumococcal urinary antigen detection (Binax Now S. pneumoniae Antigen Test) was assessed for diagnosing pneumococcal exacerba-tions of chronic obstructive pulmonary disease (COPD). Forty-six patients with S. pneumoniae in spu-tum cultures were studied during a stable period and during an exacerbation [4]. During the stable period, the antigen was detected in 10.3% of patients in non-concentrated urine and in 41.4% of patients in concen-trated urine. Corresponding proportions during exacerbations were 17.6% in nonconcentrated urine and 76.5% in concentrated urine. Specificity was eval-uated by testing 72 patients whose sputum samples were negative for S. pneumoniae. ICT was positive in nonconcentrated urine from one patient and in concen-trated urine from nine patients. Factors significantly associated with a positive test on concentrated urine were a history of at least one exacerbation (P = 0.024), admission for a previous exacerbation (P = 0.027), and pneumonia within the past year (P = 0.010). The only factor significantly associated with a positive test on nonconcentrated urine was pneumonia within the past year (P = 0.006). In conclusion, in COPD patients, a positive pneumococcal urinary antigen test during bronchial exacerbation or pneumonia should be
230 A. Ferroni and J.-R. Zahar
interpreted with caution, as it may reflect a prior S. pneumoniae infection [3, 4].
18.3.2.2 Pneumococcal Antigen Detection in Bronchoalveolar Lavage Fluid
Little is known about the use of the ICT assay on BAL fluid samples. However, this test may be useful, as at the time of bronchoscopy most patients are receiving antibiotics, which are known to damage bacterial mor-phology and prevent growth in cultures. ICT could be used as an adjunct to standard cytological and micro-biological studies. However, when the result is posi-tive, the possibility of a mixed flora or superinfecting pathogen should always be considered. In a retrospec-tive study of the Binax Now Streptococcus pneumo-niae test on BAL fluid samples from patients with suspected pneumonia, 96 BAL fluid samples were tested [38]. Sensitivity was assessed using 20 samples from patients with documented pneumococcal pneu-monia. Specificity was tested using BAL fluid samples from patients with nonpneumococcal disease (n = 41) and in samples containing no respiratory pathogens and a total bacterial count <104 CFU/mL (n = 35). Pneumococcal antigen was detected in 29 (30.2%) samples, with 95% sensitivity and 86.8% specificity [38]. An older study of the ICT assay found 50% sen-sitivity and 100% specificity [39].
18.3.2.3 Pneumococcal Antigen Detection in Pleural Fluid
Pleural empyema rarely complicates community-acquired pneumonia. The rapid identification of the causal agent is required for effective treatment and a good outcome [83]. The classical microbiological tech-nique for the diagnosis of pleural empyema is standard culture and microscopic examination. However, false-negative results related to a small sample volume or previous antibiotic therapy are common. Testing for soluble pneumococcal antigens is a complementary approach, as the results are immediately available, and S. pneumoniae can be detected even after antibiotic initiation.
In a recent retrospective study, the ICT Binax Now Streptococcus pneumoniae assay was evaluated in adults to determine whether the detection of
pneumococcal antigen in pleural fluid was better than conventional microbiological methods for the etiologic diagnosis of pneumonia [65]. In this study, the ICT assay was performed on pleural fluid sam-ples from 34 patients with pneumonia due to S. pneu-moniae, 89 patients with effusions of nonpneumococcal origin, and 17 patients with pneumonia of unknown etiology. The ICT result was positive in 24 (70.6%) of 34 patients with pneumococcal pneumonia and negative in 83 (93.3%) of 89 patients without pneu-mococcal pneumonia. The pleural ICT assay was more sensitive than blood cultures (37.5%) and pleu-ral fluid cultures (32.3%), but less sensitive than urine pneumococcal antigen detection (82.1%). However, three patients with pneumococcal pneu-monia and negative ICT urine test results had a posi-tive pleural fluid antigen test. Moreover, previous antibiotic exposure did not influence pneumococcal antigen detection in either pleural fluid or urine spec-imens [65]. In a retrospective study in children, S. pneumoniae was identified in 22% of samples by culture and in 69% by the Binax Now assay [64]. This test was positive in all 15 pleural fluid samples that yielded S. pneumoniae in culture, two samples that yielded Streptococcus oralis and Streptococcus salivarius in culture, and 34 culture-negative sam-ples. Fifteen of these 34 culture-negative samples were retrospectively tested by PCR methods, and 14 were shown to contain S. pneumoniae DNA [64].
Similar high sensitivity was found in another study in children. Among 60 children with pneumococcal empyema, 17 were diagnosed only by PCR (43%) and 23 by both PCR and culture. Compared to culture and/or PCR, the sensitivity of antigen detection was 90% [47]. More recently, the Binax Now test was evaluated on pleural fluid from 73 children admitted with pleural effusion over a period of 4 years. Sensitivity was 88% and specificity 71%, with a positive predictive value of 96% [13].
18.4 Conclusion
Although these new diagnostic methods have not been specifically evaluated in patients with HM, they seem to hold considerable promise for increasing the perfor-mance of current diagnostic strategies. They should be used simultaneously on blood, BAL fluid, sputum, and
23118 New Methods for Bacterial Diagnosis in Patients with Hematological Malignancies
throat swabs, and compared with conventional cul-tures. In the near future, molecular methods will com-plete, rather than replace, culture-based methods for detecting pathogens for which antibiotic resistance is a concern. Antigen detection methods may also have a role. However, a positive L. pneumophila test indicates a pulmonary infection due to serogroup 1, but a nega-tive result does not rule out active infection due to other Legionella species. For S. pneumonia, the inter-pretation of the urinary antigen test results appears more difficult. A positive test may indicate simple col-onization or previous pneumococcal infection, whereas the meaning of a negative test has not been evaluated in patients with HM.
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