INTRODUCTION

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Community-acquired pneumonia (CAP) continues to be a significant cause of morbidity and mortality worldwide. Streptococcus pneumoniae is the most commonly defined pathogen in nearly all studies of hospitalized adults (1, 8, 12). Current criteria for a definitive diagnosis of pneumococcal pneumonia require the isolation of S. pneumoniae from blood, pleural fluid, a metastatic-site specimen, or an uncontaminated respiratory sample obtained by invasive techniques. In a substantial number of cases, despite recent improvements in diagnostic methods, the etiology of CAP cannot be established; some of these cases are probably caused by S. pneumoniae. Lately, transthoracic needle aspiration (TNA) has been used to improve the diagnostic yield of pneumonia. In several studies, TNA has proved to be safe and has provided an etiologic diagnosis in 30 to 60% of cases by standard microbiological methods (7, 17, 18). However, its diagnostic effectiveness can be reduced by a number of factors, one of the most important of which is prior antibiotic therapy. New techniques, such as antigen detection methods and PCR, which do not depend on viable organisms, are probably less affected by the administration of antimicrobial agents and might improve the overall sensitivity of TNA (3, 6).

The aim of our study was to evaluate the ability of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from patients with moderate-to-severe CAP.

	MATERIALS AND METHODS

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Setting and population studied. The study was conducted at Bellvitge Hospital, a 1,000-bed university hospital in Barcelona, Spain. From February 1995 through May 1997 all patients with moderate-to-severe CAP requiring hospitalization were prospectively monitored at our institution. They were seen by a member of the study team who filled out a previously defined computer-assisted protocol and who provided medical advice when required. TNA was regularly performed at our institution during the study period because of the good results in terms of safety and the experience of our pneumologists over the last decade. During the study period, a total of 95 TNAs were performed among the 533 patients admitted in our hospital. Therefore, use of the TNA was not the usual standard of care, and the final decision relied on the emergency team attending each patient. For the purposes of this study, we identified all patients with moderate-to-severe CAP from whom TNA samples were obtained. TNA was performed if patients gave their consent and were able to collaborate in the TNA procedure; it was not performed in the presence of any of the following contraindications: low platelet count (60,000 cells/ml), a quick ratio of >1.8 or a quick time of <60%, severe pulmonary hypertension, mechanical ventilation, AIDS, and uncontrollable cough.

TNA procedure. Premedication with 0.5 mg of atropine was administered intramuscularly 30 min before the puncture. Puncture was performed without fluoroscopic or computed-tomography control, at the patient's bedside, and before starting therapy. Intradermal and subcutaneous anesthesia with mepivacaine was administered. The procedure was carried out by using an ultrathin 25-gauge needle with its stylet. When it was believed to be on the target, a 20-ml syringe containing 5 ml of sterile saline was attached, and 4 ml was then injected. Suction was applied vigorously for at least 30 s. A second 20-ml syringe was then attached, and another 4 ml of saline was injected. One of the syringes was randomly used for conventional microbiological procedures and the latex agglutination test. The remaining sample was stored at 72°C for later PCR determination. For clinical reasons, if the amount of the TNA sample was insufficient, priority was given to standard microbiological studies.

Microbiological studies. Prior to the initiation of therapy, two sets of blood cultures were drawn at the initial evaluation. Sputum samples were processed for Gram stain and culture, when available. Paired serum samples from the acute and convalescent phases (separated by 3 to 8 weeks) were also obtained for serological studies.

Latex agglutination for pneumococcal antigen in TNA samples. After heating at 95°C for 5 min, TNA samples were centrifuged at 700 × g for 10 min. Agglutination was then performed on a 50-µl aliquot of the supernatant with the Slidex Pneumokit (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Reading of the tests was almost immediate.

PCR assay for S. pneumoniae. We used the method described by Zhang et al. (19), with minor modifications adapted to the specific conditions of our specimen. An 86 bp fragment of the PBP2b pneumococcal gene was amplified by this protocol. DNA extraction from TNA samples was accomplished with a commercial kit (QIAamp Blood Kit; Qiagen, Hilden, Germany) in order to avoid PCR inhibition by the hemoglobin frequently present in the specimen. The extraction procedure was completed after ca. 2 h of processing time.

Limit of detection of the PCR procedure. An overnight culture of a clinical strain of S. pneumoniae was suspended in a Mueller-Hinton broth to achieve a turbidity similar to that of the 0.5 McFarland standard. Then, serial 10-fold dilutions were made in the same broth supplemented with 5% human blood in order to mimic blood contamination in TNA samples. A 100-µl aliquot from each tube was plate subcultured before the remaining suspension was extracted by the same procedure as for TNA specimens and then amplified by PCR. Plates were incubated at 37°C for 24 h; the colonies were counted and referred to the exact number of CFU present in each tube suspension.

Definitions. CAP was defined as a febrile, acute respiratory illness with the presence of a new infiltrate evident on a chest radiograph. Patients with a prior hospitalization within 2 weeks of a current diagnosis of pneumonia were excluded. Hospitalization was considered if one or more of the following conditions was present: age, 70 years; respiratory insufficiency (a PaO₂ level of 60 mm Hg or a PaO₂/FiO₂ ratio of 300); multilobar radiological involvement; shock; underlying diseases; and/or unresponsiveness to previous antibiotic therapy. To calculate the severity of the pneumonia we used the Simplified Acute Physiology Score described elsewhere (9).

Statistical methods. The final diagnosis of cases according to both the strict and the expanded criteria, as described above, was used as the standard for determining the diagnostic usefulness of the PCR and latex agglutination methods in terms of sensitivity, specificity, and positive and negative predictive values. For the purposes of the calculations, patients were classified into two groups: (i) those with pneumococcal pneumonia and (ii) those with nonpneumococcal pneumonia, including cases with other known causes and pneumonia of unknown etiology.

	RESULTS

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A total of 95 patients with moderate-to-severe CAP in whom TNA was performed were analyzed. There were 69 men and 26 women, with a mean age of 65 years (range, 19 to 87 years). The mean Simplified Acute Physiology Score was 8.7 (range, 1 to 27). In 34 patients, pneumonia involved more than one lobe on the initial chest radiograph evaluation. In 52 patients (54.7%) there were underlying diseases, mostly diabetes mellitus (n = 18), chronic heart diseases (n = 15), and chronic obstructive pulmonary disease (n = 13). In 18 patients there was more than one underlying disease. A total of 39 patients had received influenza vaccine, and only 2 patients had been immunized with pneumococcal vaccine in the previous 5 years. A total of 34 patients (37.8%) had received antibiotic treatment prior to hospital admission. Sputum samples were attempted from all study patients, but they were obtained only in 58 cases. In 28 of these the sample was considered an acceptable specimen for cultivation, as previously defined. A predominant microorganism was isolated in 14 cases, yielding S. pneumoniae in 9 cases and Haemophilus influenzae in 5 cases. In 3 additional cases in which the sputum sample was considered not acceptable for culture of conventional pathogens, a culture in selective media yielded L. pneumophila strains.

In 82 TNA samples, both the latex agglutination test and PCR assay were performed. In 3 of the total 95 specimens, there was not enough sample to perform both the latex agglutination test and the PCR (1 in the pneumococcal-pneumonia culture-positive group, 1 in the group with an etiology other than S. pneumoniae, and 1 in the unknown-etiology group). In three cases the sample amount was insufficient to perform PCR (one in the pneumococcal-pneumonia culture-positive group, two in the unknown-etiology group). Finally, in seven cases latex agglutination for pneumococcal antigen was not performed (one in the pneumococcal-pneumonia culture-positive group, one in the unknown-etiology group, and five in the other-etiologies group).

The TNA culture was positive in 29 cases (30.5%). S. pneumoniae was the most frequently isolated pathogen (16 cases), followed by L. pneumophila (9 cases), Haemophilus influenzae (2 cases), Escherichia coli (1 case), and mixed aerobic-anaerobic respiratory flora (1 case). In two cases, in addition to S. pneumoniae, a second pathogen (H. influenzae and Moraxella catarrhalis) was isolated in the TNA samples.

Overall, considering all standard techniques, 57 patients (60.0%) had an etiological diagnosis. TNA alone provided an etiological diagnosis in 10 cases (9 S. pneumoniae, 1 E. coli) and confirmed a presumptive diagnosis in 5 others (4 S. pneumoniae, 1 H. influenzae). Seven patients fulfilled strict diagnostic criteria for two different infectious agents.

Distributions of the final diagnosis and the PCR and latex agglutination test results are shown in Tables 1 and 2. In one case of M. pneumoniae pneumonia, the PCR determination was inconclusive (borderline readings after repeated testing), and it was considered true negative for the calculations.

The calculated limit of detection for our PCR assay was between 2 and 27 CFU/ml. As Table 3 shows, PCR was more sensitive than latex agglutination. Focusing on the 82 samples in whom both diagnostic tests were performed, the additional diagnosis provided by PCR assay was 32.7% (95% confidence interval of the difference from 9.5 to 36.4%) in the subgroup of culture-proven pneumococcal-pneumonia group and 12.2% (95% confidence interval of the difference from 0.4 to 20.1%) when calculated for all pneumonias. Table 4 shows clinical and laboratory data of false-positive results for either the PCR assay or the latex agglutination test when the strict diagnostic criteria are used.

PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy. Of the 34 patients who had received antibiotic treatment prior to hospital admission, 6 fulfilled the criteria for pneumococcal pneumonia. As shown in Table 5, TNA culture yielded S. pneumoniae in two cases (33.3%), while PCR was positive in five cases (83.3%). For its part, latex agglutination was positive in one of the four cases in which it was performed (25.0%).

The latex agglutination test result was available for physicians in the emergency room in the majority of cases and provided information before the initial antibiotic therapy was selected. In fact, only two patients with a positive latex agglutination test were admitted on combined -lactam and macrolide therapy. In none of the patients admitted on a single -lactam therapy was a macrolide added to therapy after a negative latex agglutination result.

	DISCUSSION

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Although previous studies (7, 11, 17, 18) have shown TNA to be a highly specific technique for the diagnosis of pneumonia, current indications of TNA in the CAP setting have not been well defined. Our study did not address this issue. We studied a large number of hospitalized patients with moderate-to-severe CAP in whom TNA was performed and evaluated the effectiveness of the latex agglutination test and PCR in these specimens. As expected, TNA improved the diagnostic yield in our study; in fact, more than a quarter of the definitive etiologic diagnosis (15 of 57) were made by culture of TNA samples. As for our two false-negative PCR results, they were from non-bloody samples, and so a possible negative effect by hemoglobin was reasonably ruled out. However, the possibility of a negative test resulting from the use of separate syringes for doing the different microbiological studies should be born in mind.

It is assumed that culture-positive pneumococcal pneumonia represents only a portion of the cases of pneumonia caused by this pathogen, so there is considerable room for alternative, more-sensitive diagnostic tests. The new laboratory techniques used in the diagnosis of pneumococcal pneumonia mainly confront two challenges related to their sensitivity and specificity. The first is the lack of a satisfactory standard for comparison. This is especially true of methods based on gene amplification, which is assumed to have a very low limit of detection. The second is the increasing number of coinfections reported in several recent studies (2, 4, 10). This phenomenon may lead us to consider cases of undiagnosed coinfections as false-positive results of these techniques.

In addition to better sensitivity and good specificity, other important advantages of the new laboratory tests when applied on a routine basis are rapidity, simplicity, and cost-effectiveness. This is particularly the case when latex agglutination and PCR are compared for the diagnosis of pneumococcal pneumonia. Data from the literature have shown variable results from PCR assays with regard to sensitivity. In cases of bacteremic pneumococcal pneumonia, sensitivity with blood specimens has ranged from 37.5 to 100.0%, depending on the technical procedure used (5, 14, 16, 19). Overall, sensitivity is lower when PCR was applied to whole blood than when applied to buffy coat preparations or sera, probably as a consequence of the inhibitory effect of hemoglobin on the amplification. This could also partially explain why other authors have reported PCR assays to be less sensitive than pneumococcal antigen detection in TNA samples (13, 15).

In an attempt to avoid this negative effect and to increase sensitivitiy, we used a column DNA extraction method and amplicon detection by hybridization with a biotinylated probe. In our study, detection of pneumococcal DNA by PCR proved to be more sensitive than latex agglutination, although, when considering all patients, the difference was limited. In contrast, latex agglutination compares favorably with the PCR assay in terms of cost, simplicity, and rapidity. For instance, latex agglutination can be performed in less than half an hour of processing time. PCR assay, on the other hand, is labor-intensive; the complete procedure takes about 10 h, which precludes its use as a realistic alternative method to conventional diagnostic techniques.

There were three false-positive results in the latex agglutination test among patients with other known etiologies when the strict criteria were applied. In all three cases the patients had a final diagnosis of C. pneumoniae pneumonia. In one case, the clinical and laboratory data and the presence of gram-positive diplococci in a sputum Gram stain with growth of normal flora supported the assumption of a coinfection. This patient was considered as true positive in the analysis with expanded criteria. False-positive results of latex agglutination in patients with C. pneumoniae pneumonia have been reported elsewhere (15). It should be noted that we used serology as the standard diagnosis of C. pneumoniae infection; this is considered a sensitive technique, although its specificity in diagnosing acute infection is currently under debate.

As for PCR, there were also three false-positive results in patients with other known etiologies. One patient previously treated with amoxicillin had a serologically diagnosed C. pneumoniae pneumonia, so a possible coinfection cannot be excluded. The second patient presented with aspiration pneumonia in which mixed aerobic and anaerobic bacteria of the upper respiratory tract were isolated by TNA. There are two possible explanations for this result. First, the presence of multiple bacteria might have inhibited the growth of a small inoculum of S. pneumoniae which could be detected by gene amplification. Second, this might be a real false-positive result, given the relationship in the PBP-2b genes of viridans streptococci and S. pneumoniae, although Zhang et al. (19) found no such cross-reactivity with the same primers and probe as the ones we used in our PCR assay. E. coli pneumonia was diagnosed in the third patient because of TNA-positive culture for this microorganism and negative blood cultures (a sputum sample was not obtained). PCR test results were repeatedly positive for S. pneumoniae. Because the E. coli strain was not available for further studies, we considered this PCR result a false positive.

Clinical presentation of all patients with pneumonia of unknown etiology and positive results in PCR and/or latex agglutination tests was compatible with bacterial pneumonia, and it is our understanding that most of these cases are, in fact, caused by S. pneumoniae. In many cases etiologic diagnosis was not performed because of the impossibility of obtaining a sputum sample of good quality or because of other circumstances, such as previous antibiotic treatment. In fact, PCR was more sensitive than TNA culture and latex agglutination in this subgroup of patients. For these reasons, we considered the expanded criteria to be more suitable for evaluating the real performance of both PCR and latex agglutination assays.

In summary, our study shows that latex agglutination and PCR improve the diagnostic yield of TNA. Latex agglutination is a simple, inexpensive technique whose results can be made readily available in the emergency room setting. Although less sensitive than PCR, it appears to offer practical advantages for the choice of initial antibiotic therapy. PCR is a very sensitive and specific technique which, due to its complexity, should perhaps be reserved for research purposes. A sensitive and specific PCR assay such as the one we used here could be a good standard for future evaluations of other diagnostic techniques.