Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada).

Chlamydia pneumoniae has been associated with atherosclerosis and several other chronic diseases, but reports from different laboratories are highly variable and "gold standards" are lacking, which has led to calls for more standardized approaches to diagnostic testing. Using leading researchers in the field, we reviewed the available approaches to serological testing, culture, DNA amplification, and tissue diagnostics to make specific recommendations. With regard to serological testing, only use of microimmunofluorescence is recommended, standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin (Ig) G titers for determining acute infection and IgA for determining chronic infection are discouraged. Confirmation of a positive culture result requires propagation of the isolate or confirmation by use of polymerase chain reaction (PCR). Four of 18 PCR assays described in published reports met the proposed validation criteria. More consistent use of control antibodies and tissues and improvement in skill at identifying staining artifacts are necessary to avoid false-positive results of immunohistochemical staining. These standards should be applied in future investigations and periodically modified as indicated.

Comparison of two commercial microimmunofluorescence kits and an enzyme immunoassay kit for detection of serum immunoglobulin G antibodies to Chlamydia pneumoniae.

We compared the MRL and the Labsystems Chlamydia pneumoniae microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to C. pneumoniae. The correlation between the results obtained with these two MIF kits was excellent (r = 0.95; P = 0.001). The cross-reactivity of the C. pneumoniae-positive sera with C. trachomatis- and C. psittaci-positive sera was assessed for each MIF kit. For C. pneumoniae-positive sera with titers of > or =32, the Labsystems MIF kit exhibited more cross-reactivity to C. psittaci than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to C. pneumoniae (for the MRL MIF kit, r = 0.79 [P = 0.001]; for the Labsystems MIF kit, r = 0.78 [P = 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of C. pneumoniae antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation.

Detection of chlamydiosis in a shipment of pet birds, leading to recognition of an outbreak of clinically mild psittacosis in humans.

Avian chlamydiosis was detected in a shipment of > 700 pet birds from a Florida bird distributor that were sold to nine Atlanta-area pet stores in August 1995. Respiratory illness among persons who had recently acquired birds from this shipment was reported to local public health officials. The attack rate of acute respiratory illness was 10.7% among persons in households exposed to birds from the implicated flock vs. 1.8% among control households (odds ratio, 6.60; 95% confidence interval, 1.39-31.2). Illness and serological evidence of infection in the absence of symptoms were more common among persons in households with recently purchased birds that were sick or that had died and among persons who had had direct contact with the birds. Clinical psittacosis or serological evidence of Chlamydia psittaci infection was found in 30.7% of households with birds from the infected flock. Mild illnesses and asymptomatic infections in exposed persons were unusual features of this outbreak.

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

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

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.

ELISPOT assay for Chlamydia-specific, antibody-producing cells correlated with conventional complement fixation and microimmunofluorescence.

Chlamydia antigens cross-reactive with pneumoniae (TWAR), psittaci, and trachomatis strains were used to evaluate the ELISPOT assay for detecting antigen-specific, antibody-secreting cells (ASC). Human blood specimens from healthy and hospitalized persons were randomly collected and tested by coating the nitrocellulose membrane at the base of microtiter wells. Ficoll-separated mononuclear cells from blood specimens collected in EDTA were incubated in the wells with Iscove's growth medium in CO2 atmosphere at 37 degrees C. An IgG-specific conjugate labeled with biotin was used in an avidin-peroxidase chromogen system for indicating the areas (spots) of immunologically committed lymphocytes. Positive specimens had median levels of ASC above 8 per 10(6) cells (range 15-23 ASC/10(6) cells). Evidence that the ELISPOT is reliable, sensitive, and specific includes the following:(1) immunized animal and clinical human specimens in control experiments were selectively reactive in the presence of antigen, but negative without antigen, (2) serologically characterized reference sera demonstrated homologous rather than heterologous reactions with the antigens, (3) conventional complement fixation and microimmunofluorescence on serum fractions of clinical specimens correlated well (P < 0.02) with ELISPOT results that were both TWAR- and psittaci-positive, and (4) the array of specimens (from healthy donors, community hospitalized, and pulmonary service patients) selected for their increasing likelihood in that order for being positive due to illness was then confirmed and supported by their respectively increasing positivity rates (6, 15, and 25%) for TWAR/psittaci combined. The incidence of positive specimens for either TWAR or psittaci was greatest (23/54, 43%) in specimens from the hospitalized patients and least (8/33, 24%) in specimens from healthy individuals. These findings suggest that ELISPOT detects chalmydial antibody production at the cellular level. ELISPOT positivity thus indicates previous exposure and would favor earlier detection.

Discrimination of Streptococcus pneumoniae from other upper respiratory tract streptococci by arbitrarily primed PCR.

OBJECTIVES: In the clinical laboratory, identification of Streptococcus pneumoniae can be confused with other streptococci. Conventional biochemical tests such as optochin sensitivity and bile solubility can give inconsistent results. This report presents a method to distinguish true S. pneumoniae from other upper respiratory tract streptococci when conventional tests fail. DESIGN AND METHODS: We used arbitrarily primed polymerase chain reaction with the single primer M13 universal as a method to distinguish S. pneumoniae from other upper respiratory tract streptococci. RESULTS: The fingerprint pattern of S. pneumoniae was established by amplifying DNA of S. pneumoniae type strains 1-48 and of other common upper respiratory tract streptococci at three different DNA concentrations with the single primer M13 universal. From these type strains, a common arbitrarily primed-polymerase chain reaction pattern was identified characterized by two predominant bands of equal intensity at 800 base pairs and at 1100 base pairs. Fingerprint patterns of viridans streptococci were easily distinguishable from those of S. pneumoniae. Many of the clinical isolates used in this study were equivocal by conventional tests but were distinguishable by their fingerprint patterns. CONCLUSIONS: Our results indicated that the fingerprint pattern of S. pneumoniae is species specific and distinguishes true S. pneumoniae of clinical isolates from other streptococci when conventional biochemical tests are unclear.

Mycoplasma contamination of chlamydiae isolated from clinical specimens.

Ten Chlamydia pneumoniae strains were screened for Mycoplasma contamination using two differently designed Mycoplasma-specific polymerase chain reactions (PCR). The primers of the Mycoplasma-specific PCR designed by Spaepen et al. (9) cross-reacted with all of the C. pneumoniae strains giving false-positive results. When the 10 strains of C. pneumoniae were tested for mycoplasmas with the PCR designed by Harasawa et al. (5), only 3 were positive. Mycoplasmas were cultured from these three C. pneumoniae strains confirming the latter PCR results. The PCR of Harasawa et al. (5) was highly specific for mycoplasmas and did not cross-react with C. pneumoniae. These findings suggest that chlamydiae should be periodically screened for Mycoplasma contamination. Careful attention to primer design is important if PCR is chosen as the screening method.

Detection of Chlamydia pneumoniae in clinical specimens by polymerase chain reaction using nested primers.

A nested primers strategy was used to develop a two-step PCR test for the direct species-specific detection of the 16s rRNA gene of Chlamydia pneumoniae. This test was applied to 58 nasopharyngeal or oropharyngeal swab specimens collected from patients in studies of community-acquired pneumonia and in a local outbreak of respiratory disease. Twelve patients (21%) showed evidence of Chlamydia pneumoniae infection in serological tests (7/56; 13%), culture (8/58; 14%) or PCR (10/58; 17%). Nested PCR but not single-step PCR was found to be as sensitive as culture or serology for detection of infection with this organism. In summary, nested PCR can be useful in direct testing of clinical specimens for Chlamydia pneumoniae, making additional DNA purification steps unnecessary.

Detection of streptococcal pyrogenic exotoxin genes by a nested polymerase chain reaction.

Severe invasive disease associated with group A Streptococcus (GAS) has recently increased in frequency. Isolates of GAS from normally sterile sites were examined for the streptococcal pyrogenic exotoxin genes spe A, spe B and spe C to determine if they play a role in this disease. Four primers for each gene were used in a nested polymerase chain reaction (PCR) configuration. The first PCR generated fragments of 818, 1106, and 801 bp, respectively, for the extotoxin genes. The second PCR generated fragments of 500, 912 and 654 bp for the spe A, spe B and spe C genes using the fragments from the first PCR as template. Of 62 strains tested, 35 (56%) contained the spe A gene, and 17 (27%) contained the spe C gene. All GAS strains studied, regardless of disease association, contained the spe B gene. These data corroborate accumulating evidence that the genes encoding pyrogenic exotoxin types B and C are not associated with severe invasive streptococcal illness including streptococcal toxic shock-like syndrome. This PCR-based gene detection system has clinical and epidemiologic applications because of its ease of performance, non-isotope labelling, high specificity and sensitivity, and lack of requirement for purified DNA.

Sphingolipid biosynthesis by rat liver cells: effects of serine, fatty acids and lipoproteins.

The effects of circulating factors that might influence de novo sphingolipid biosynthesis were examined with rat liver cells by following the incorporation of [14C]serine into sphingosine and sphinganine, the predominant long-chain base backbones of hepatic sphingolipids. The rate of long-chain base formation depended on the concentration of [14C]serine in the medium and exhibited saturation kinetics. Long-chain base formation was stimulated by another precursor, palmitic acid, but stearic, oleic, linoleic and linolenic acids were inhibitory. This kinetic behavior indicates that long-chain base formation in liver is affected by the availability of the substrates of the initial enzyme of this pathway, serine palmitoyltransferase. Since liver is also exposed to sphingolipids associated with circulating lipoproteins, the effects of various lipoprotein fractions were determined and each appeared to decrease long-chain base formation. These results suggest that hepatic long-chain base biosynthesis can be stimulated by increases in the circulating levels of the precursors serine and palmitic acid whereas some other fatty acids and lipoproteins decrease the flux through this pathway.

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes.

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.

Stimulation of DNA synthesis any myo-inositol incorporation in mammalian cells.

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [3H]-myo-inositol into trichloroacetic acid precipitable material during 0--60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G1 by amino acid starvation is not accompanied by increased incorporation of [3H[-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G1 than cells arrested by serum depletion. Inhibition of PI synthesis by variation of-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0--4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G1 and subsequent entry into S phase.

Regulation of growth of fibroblasts.

Present knowledge on regulation of fibroblast growth is based on in vitro culture of fibroblasts from different sources. The research has focused on 2 problems: identification of the signal that reaches the fibroblast from outside and tells it to grow and identification of metabolic reactions inside the cell that commit it to initiate DNA synthesis after the signal arrives. Although the signal and the metabolic reactions have not yet been clearly identified, and the relationship between in vivo conditions and the result of these in vitro studies still has to be determined, the large body of data collected so far and the steadily growing information concerning these problems suggest a complex interrelation between cellular environment and metabolic processes involved in growth regulation.

The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum.

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5- to 10-fold over the control and is extremely potent, exerting a saturating response at a a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids in 5-fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.

Control of growth of benzo(a)pyrene-transformed 3T3 cells.

The growth controls observed in benzo[a]pyrene-transformed 3T3 cells (BP3T3) are compared with those of virus-transformed and normal 3T3 cells. Superficially, the chemically transformed BP3T3 cells have the same behavior as virus-transformed SV3T3 cells. Both grow to high cell density in culture medium with 10% serum, both form colonies in Methocel, and both are tumorigenic. Closer examination, however, has disclosed that BP3T3 cells exhibit "normal" growth controls at low serum concentrations. In contrast to the behavior of SV3T3 cells, the initiation of DNA synthesis in BP3T3 cells is still dependent on a serum factor. If BP3T3 cells are grown in medium with 0.2% serum, the cells become quiescent, with growth arrested in the Gu or G0 phase of the cell cycle. The addition of serum or the fibroblast growth factor (FGF) to such quiescent cells leads to the initiation of DNA synthesis and the resumption of growth. As with normal 3T3 cells, if the growth rate of BP3T3 cells is limited by a suboptimal concentration of serum, the growth rate of the cells is increased by the addition of FGF. Also, BP3T3 cells show density-dependent regulation of growth, if the medium contains a low concentration of serum. BP3T3 cells, therefore, have the behavior of "transformed" cells when cultured in medium with 10% serum, but behave as "normal" cells in medium with low serum. In comparison with normal 3T3 cells, the difference in growth behavior of BP3T3 cells appears to be due to a substantial decrease in the cells' requirement for a serum growth factor of the FGF type. Exploration of possible causes of this substantial decrease indicates that the primary cause is a lower rate of depletion of the serum growth factor from the culture medium by BP3T3 cells. The decrease in rate of depletion is sufficient to account for the uncontrolled growth of BP3T3 cells in medium with 10% serum. It is suggested that a decreased rate of depletion of a growth factor may contribute to tumorigenicity of cells in vivo.