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Clinical Chemistry 43: 930-935, 1997;
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(Clinical Chemistry. 1997;43:930-935.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Use of polymerase chain reaction to identify pneumococcal infection associated with hemorrhage and shock in two previously healthy young children

Trudy O. Messmera, Cynthia G. Whitney and Barry S. Fields

National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333.
a Address correspondence to this author at: Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G05, Atlanta, GA 30333. Fax 404-639-3123; e-mail TYM2{at}CIDDBD2.EM.CDC.GOV


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A PCR assay was developed for detection of Streptococcus pneumoniae in clinical specimens including blood and paraffinized tissues. We were able to detect one organism of purified DNA or 4.5 colony-forming units in blood. The primers did not cross-react with other upper respiratory tract streptococci or with pathogens commonly found in clinical specimens. This assay was used in an investigation of an outbreak of severe illness characterized by septic shock and hemorrhage in previously healthy children. PCR detected S. pneumoniae in cerebrospinal fluid and autopsy tissues of the two infants who died. The findings from this assay indicated that PCR offers increased specificity and sensitivity over latex agglutination and counterimmunoelectrophoresis and should prove useful in the identification of additional cases of severe illness caused by S. pneumoniae.


Key Words: indexing terms: PCR • Streptococcus pneumoniae • clinical specimens • paraffinized tissues


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is the most common cause of invasive bacterial disease in the US (1). It is the leading cause of community-acquired pneumonia and a frequent cause of meningitis, sinusitis, bronchitis, and otitis media in both children and adults. Diagnosis of pneumococcal pneumonia relies, in part, on isolation and identification of the organism from a 24–48-h blood culture, but bacteremia is confirmed in <30% of cases (2)(3). In addition, culture may be negative if a patient has been treated with antimicrobial agents. Because of carriage of S. pneumoniae in the respiratory tracts of normal hosts, isolation of S. pneumoniae from this site may not reliably indicate disease. Thus, accurate diagnosis requires a spectrum of clinical and laboratory test findings, including radiologic evidence.

There is a lack of rapid, sensitive, and specific tests for diagnosing pneumococcal infections. Historically, identification of S. pneumoniae sepsis was based on obtaining a positive blood culture if other signs and symptoms consistent with the diagnosis were present. The laboratory identification of S. pneumoniae is based on the hemolysis pattern when cultured on blood agar plates and by additional biochemical tests on the cultured organisms: optochin sensitivity, the Quellung reaction, and bile solubility (4). Nonculture methods now available, such as counterimmunoelectrophoresis (CIE) and coagglutination, have failed to show uniform diagnostic sensitivity or specificity even when applied to specimens collected from patients with culture-confirmed bacteremic pneumococcal pneumonia (5)(6)1 The lack of sensitive nonculture-based methods and the limitations of culture often result in only presumptive diagnoses of pneumococcal pneumonia (7). Therefore therapy is frequently empiric and potentially suboptimal (7), and reliably identifying cases of pneumococcal infections for epidemiological studies is difficult.

We applied a new nested PCR for detection of S. pneumoniae from clinical specimens from an outbreak of invasive pneumococcal disease in a child care center (8). In December 1993, two previously healthy infants in a New Mexico child care center developed a severe illness characterized by septic shock and hemorrhage into the skin or internal organs and died. In this outbreak, cultures of blood and spinal fluid from the two patients were negative. Latex agglutination testing and CIE on cerebrospinal fluid (CSF) from the second infant affected suggested infection with S. pneumoniae as the cause of death. Clinical specimens tested included buffy coat, CSF, and autopsy tissue.

This is the first PCR application for detection of pneumococcus in autopsy tissues. Identifying the cause of death in autopsy tissues is important to treat similar cases with appropriate antibiotics, and to determine the etiology of an illness. Several reports describe PCR assays from clinical specimens of blood and (or) buffy coat (9)(10)(11)(12)(13), CSF (14)(15), sputum (16), and ear fluids (17)(18). The assay was critical in identifying the etiology of the deaths in the outbreak.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
cell cultures
All 48 type strains for the capsular types of S. pneumoniae were obtained from the Statens Serum Institute, Copenhagen, Denmark. The type strains for all the other streptococci were from the American Type Culture Collection, Rockville, MD. Streptococci were cultured overnight at 37 °C with 5% CO2 on tryptic soy agar plates with 50 mL/L defibrinated sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD), or in Pediatric TSB bottles (Fisher, Norcross, GA) with 1 mL of normal human blood added.

detection of s. pneumoniae in blood
A fresh culture of type 19F S. pneumoniae was removed from a sheep blood agar (SBA) plate (Becton Dickinson Microbiology Systems) by flooding the plate with 3 mL of sterile isotonic saline and scraping the colonies off with a sterile inoculating loop. The solution was vortex-mixed and diluted with sterile saline to a MacFarland reading of 0.5. This reading is ~1.5 x 108 cells per mL. Further dilutions in sterile saline were made to adjust the cell number to 15 cells per µL. Aliquots equivalent to ~1–150 organisms were added to 150 µL of fresh blood collected in EDTA tubes from healthy volunteers. Identical amounts of the cell suspensions that were added to blood were plated onto fresh SBA plates, incubated overnight at 37 °C in a CO2 incubator, and the number of colonies counted the next day for an accurate determination of the cell number added to blood. Similarly, S. pneumoniae could be harvested by centrifugation from overnight growth in Pediatric TSB bottles supplemented with 1 mL of normal human blood and processed in a like manner as pneumococci grown on SBA plates. There was no difference in sensitivity of detection of plate-grown vs broth-grown organisms.

The heat detergent method (HD) of Dawson et al. (19) was used to prepare blood samples for PCR. Briefly, the red cells were lysed by hypotonic treatment with ammonium chloride followed by addition of sterile water. The remaining cells were collected by centrifugation, resuspended in 100 µL of detergent buffer (Nonidet P40 and Tween 20), and boiled in a boiling water bath for 30 min. Five to 10 µL were used in a final PCR reaction volume of 50 µL.

dna preparation of streptococci and other bacteria for pcr
After overnight growth on blood agar plates, the cultures of streptococci or other bacteria were harvested by flooding each plate with 3 mL of isotonic saline and dislodging the colonies with a sterile inoculation loop. The suspension was removed to a sterile 1.5-mL Eppendorf tube and centrifuged at 3000g for 10 min. The supernatant was removed and the pellet was treated with 50 µL of 0.5 mol/L potassium hydroxide for 30 min at 37 °C. Fifty microliters of 0.5 mol/L Tris, pH 8, was added, and the tubes were boiled in a boiling water bath for 20 min. The tubes were centrifuged at 16 000g in an IEC MicroMax microcentrifuge (International Equipment Co., Needham Heights, MA). The supernatants were diluted at least 1:10 for PCR.

Quantified DNA was prepared by reading the absorbance at 260 nm after extraction on an ABI model 340-A DNA extractor (Perkin-Elmer, Applied Biosystems Division, Foster City, CA). This DNA was used to measure sensitivity of the PCR.

pcr amplification
Samples for PCR were prepared in a class II laminar flow hood, and amplification and analysis of PCR products were each performed in separate locations. Reaction volumes of 50 µL containing 10 mmol/L Tris-HCl, pH 8.8, 75 mmol/L KCl, 2.8 mmol/L MgCl2, 200 µmol/L of each deoxynucleoside triphosphate, 0.1 g/L bovine serum albumin (Sigma Chemical Co., St. Louis, MO), 1.25 U of Taq polymerase (Boehringer Mannheim, Indianapolis, IN), 0.2 µmol/L of each outer primer, and 5 µL of DNA sample were overlaid with one drop of mineral oil and placed in a Perkin-Elmer Thermal Cycler Model 480 (Perkin-Elmer, Norwalk, CT) for 1 cycle of 94 °C for 4 min, followed by 30 cycles of 94 °C for 1 min, 62 °C for 30 s, and 72 °C for 1 min. A final 5-min extension at 72 °C concluded the amplification. The nested or inner PCR reaction mixture was similar to the first except that it contained 0.1 µL of the product of the outer PCR and 0.2 µmol/L of each inner primer. The cycling conditions were identical.

The primers target the pneumococcal autolysin gene sequenced by Garcia et al. (20), which is unique and characteristic of S. pneumoniae (21). A computation at the National Center for Biotechnology Information with the BLAST network service confirmed the uniqueness of the target sequence: The only matches were with the phage HB-3 of S. pneumoniae. There was no human sequence homology.

A750 Outer sense 5'-GGCTACTGGTACGTACATTC-3'

A1145 Outer antisense 5'-AATCAAGCCATCTGGCTCTA-3'

A781 Inner sense 5'-ATCCAAAAGACAAGTTTGAGA-3'

A1055 Inner antisense 5'-CTGGATAAAGGCATTTGATAC-3'

The outer PCR product is 395 bp and the inner product is 274 bp.

Amplification products were separated by electrophoresis through 2.5% agarose gels [1.5% Nusieve GTG agarose (FMC Bioproducts, Rockland, ME) and 1.0% agarose (Bio-Rad, Richmond, CA)] in Tris–borate–EDTA and were visualized by ethidium bromide fluorescence.

Both the outer and inner PCRs were optimized with the Opti-Prime PCR optimization kit (Stratagene, La Jolla, CA). The PCR product was verified by sequence analysis (National Biosciences, Plymouth, MN).

preparation of clinical specimens
We received CSF, buffy coat, and four paraffinized autopsy blocks (liver\/spleen\/thymus, diaphragm, and two lung) from one patient. From the second patient we received three paraffinized blocks: lung, pancreas\/ heart\/liver, and spleen\/lymph node. Buffy coat and CSF were prepared for PCR with standard chloroform\/phenol extraction methods. Buffy coat, CSF, and autopsy tissues from the two patients in New Mexico were provided by Kurt Nolte of the New Mexico Office of the Medical Investigator and by the New Mexico Department of Health laboratory personnel. The procedures followed for human subjects were in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 1983.

Autopsy tissues were prepared for PCR in a nonrespiratory disease laboratory by using the method of Heller et al. (22). Briefly, 8-µm tissue sections were deparaffinized, and the dried tissue was digested and then extracted with chloroform\/phenol, followed by ethanol precipitation. The pellet was dissolved in sterile, endotoxin-free water, and 5 µL were used in the PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The nested PCR was both sensitive and specific. Neither the outer nor inner primer sets cross-reacted with other streptococci or with other respiratory pathogens. Table 1 lists the microorganisms tested for specificity. Fifty femtograms of S. pneumoniae DNA was included in all the tests on other bacteria to confirm that the PCR was able to amplify target tissues had they been present.


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  		Table 1. Microorganisms tested for cross-reactivity in the PCR assay with both the first- and second-step primers.

The detection limit of the nested PCR was 1.25 fg of purified S. pneumoniae DNA (less than one genome equivalent). An example of the sensitivity and specificity of the nested reaction is shown in Fig. 1 . The figure illustrates the dilution effect of detecting very low concentrations of DNA in a sample. Five femtograms and 1.25 fg amplify, whereas 2.5 fg do not. This is a concentration effect at limiting dilution of target in the sample. Five femtograms represents ~2 genome equivalents. At a concentration of 1 fg/µL, a random 5-µL aliquot may not contain any genome equivalent, whereas another 5-µL aliquot may contain more than two genome equivalents.



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  		Figure 1. Sensitivity and specificity of the nested PCR.

The detection limit of the PCR is shown to be 1.25–5 fg of S. pneumoniae DNA. The specificity of the PCR is demonstrated by the failure of the primers to amplify even microgram quantities of other streptococcal DNA, examples of which are listed on the figure. Samples were tested at least three different times and none of the other upper respiratory tract streptococci amplified. At least one of the positive controls always amplified, although the concentration (5, 2.5, or 1.25 fg) at which amplification was observed varied because of the effect of sampling only 5 µL of very dilute DNA.

Figure 2 shows the detection of S. pneumoniae by nested PCR in human blood. Four and one-half colony-forming units (CFU) of S. pneumoniae were detectable. Counted S. pneumoniae were added to 200 µL of human blood, and the blood was prepared for PCR with the HD method described in Materials and Methods. The figure shows the results of varying the volume of the HD buffer used to boil the final cell pellet on detection by PCR. A high (too numerous to count) and a low number (1 CFU per µL) of pneumococci were added to the blood before isolation. There was no effect on detection by PCR if the final HD buffer volume decreased.



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  		Figure 2. Detection of pneumococci from whole blood by nested PCR.

Lanes 1 and 2 are positive controls and show detection of 50 fg of purified pneumococcal DNA from a capsular type 3 strain and a capsular type 19F strain, respectively. The remaining lanes contained 200 µL of human blood supplemented with counted pneumococci and processed with various amounts of HD buffer. Lane 3 had 5 µL (4.5 CFU) from 100 µL of HD buffer, lane 4 had 10 µL (9 CFU) from 100 µL of HD buffer, lane 5 had 5 µL (4.5 CFU) from 50 µL of HD buffer, lane 6 had 10 µL (9 CFU) from 50 µL of HD buffer, lane 7 had 5 µL (4.5 CFU) from 25 µL of HD buffer, lane 8 had 10 µL (9 CFU) from 25 µL of HD buffer, lane 9 had 5 µL of too numerous to count (TNTC) CFU from 100 µL of HD buffer, lane 10 had 10 µL (TNTC CFU) from 100 µL of HD buffer, lane 11 had 5 µL (TNTC CFU) from 50 µL of HD buffer, lane 12 had 10 µL (TNTC CFU) from 50 µL of HD buffer, lane 13 had 5 µL (TNTC CFU) from 25 µL of HD buffer, and lane 14 had 10 µL (TNTC CFU) from 25 µL HD buffer. DNA was prepared for PCR from whole blood as described in Materials and Methods.

Cultures of CSF, blood, and tissues from patients in this study were negative for S. pneumoniae. Gram-positive cocci were detected in blood buffy coat of one patient, and a latex agglutination test of CSF indicated infection with S. pneumoniae. CIE of the CSF specimen was positive for S. pneumoniae (personal communication of D.M. Musher, VA Medical Center, Houston, TX).

Figure 3 shows the results of the second-step PCR amplification of clinical specimens from the two children in this study. Even though we optimized the reaction conditions for all primers, the first-step PCR on the clinical specimens used in this report failed to show bands in ethidium bromide-stained agarose gels. All of the specimens from both infants were positive in our second-step PCR; however the CSF-positive specimen is not shown in this figure. There were four paraffinized blocks from patient 1, including two from lung, and three blocks from patient 2. The tissue blocks, sectioned by the Bacterial Diseases Pathology Laboratory, were deparaffinized and extracted along with the buffy coat and CSF in a nonrespiratory disease laboratory at CDC. To confirm our PCR results, the tissue blocks were given to the Viral Diseases Pathology Laboratory where new sections were made, deparaffinized, and digested. We found identical PCR positive results on all of the tissue specimens sectioned and prepared from two different laboratories (data not shown).



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  		Figure 3. Second-step PCR products amplified from clinical specimens of the two children in this study.

The positive control is 25 fg of S. pneumoniae DNA. The negative control was a mock CSF control, prepared in parallel with the CSF clinical specimen of one patient but containing no CSF material. The CSF clinical specimen was PCR positive but is not shown in this figure. The remaining lanes show amplification of autopsy specimens prepared from paraffinized tissue blocks of the two infants. The buffy coat and the last four lanes on the right of the gel are from patient one (*); the remaining three specimens directly to the right of the buffy coat are from the second patient (**). All of the clinical specimens were positive. The PCR was repeated on the same specimen extracts at least three different times with identical results. All of the specimens shown here were prepared in another CDC laboratory that did not work with respiratory pathogens. The 100-bp ladder is from Gibco BRL (Gaithersburg, MD).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lack of sensitive and specific tests for pneumococcal pneumonia make it difficult to identify etiology of disease when results with traditional methods are negative or samples and specimens are scarce. Because CSF, blood, and tissue cultures of patients were negative in this outbreak, determining the etiology of the cases in New Mexico required use of alternative diagnostic methods.

We designed a nested PCR to detect pneumococci in respiratory infection. The target sequence was from the autolysin gene common to all S. pneumoniae. A nested strategy was used to increase specificity and sensitivity of detection of target in clinical specimens. The nested reaction is a two-step PCR. One percent or less of the first-step product is amplified in the second-step PCR, and the second-step primers target a sequence internal to the first-step sequence. Clinical specimens often contain inhibitors to PCR even after purification steps such as chloroform\/phenol extraction. The two-step PCR often circumvents the effects of inhibitors in clinical specimens because while the first-step product may be too small for detection by ethidium bromide staining, enough product is synthesized for amplification and detection in the second step or nested reaction.

The detection limit of the nested PCR was equivalent to the amount of target contained in one bacterium as determined by using purified DNA or 4.5 CFU in blood. All of the type strains of S. pneumoniae were amplifiable with the expected size products. The most common forms of streptococci found in respiratory airways that could cross-react are S. viridans and group C; however, PCR products were not detected with any of these organisms tested, nor with other respiratory pathogens that might be present in clinical specimens of interest.

The PCR we developed was used to identify the etiology (S. pneumoniae) of an unknown illness causing the deaths of two children in an Albuquerque child care center. One of the two infant deaths occurred 6 weeks before the investigation, and autopsy tissues were the only specimens remaining for one of the infants. This method was crucial to linking the two deaths to one etiology.

Because this study involved only two cases, the general use of PCR to retrospectively diagnose pneumococcal disease from paraffinized tissues must be interpreted with caution. Asymptomatic carriage of S. pneumoniae in up to 20% of adults requires the exclusion of detection of pneumococcus in control tissues. A prospective study of detection in control tissues removed and handled sterilely at autopsy is required to establish the efficacy of this test as a general method.


   Acknowledgments
 
We thank the members of the New Mexico State Health Department of Pathology for their collection and analysis of the specimens used in this study. We also thank both the Bacterial Diseases Pathology Laboratory of Leo Gorelkin and Jeanine Bartlett and the Viral Diseases Pathology Laboratory of Sherif Zaki for shaving the tissue blocks and digesting the tissues for PCR.


   Footnotes
 
1 Nonstandard abbreviations: CIE, counterimmunoelectrophoresis; CSF, cerebrospinal fluid; SBA, sheep blood agar; HD, heat detergent; and CFU, colony forming unit.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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