Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost.

Control of Bordetella pertussis in the community is hampered by slow and insensitive diagnostic tests. We therefore examined the accuracy and cost of culture, direct fluorescent antibody (DFA) staining, and PCR in a routine clinical laboratory. Six hundred thirty seven nasopharyngeal swabs and aspirates in casamino acids transport medium were cultured, stained with polyclonal (Difco), and monoclonal (BL-5 and Accu-Mab) anti-B. pertussis reagents, and amplified by an IS481-specific PCR. PCR products were detected by a hybridization-enzyme immunoassay kit (Gen-eti-k DEIA, DiaSorin), with confirmation by a second PCR in a separate laboratory. Sensitivities and specificities of culture, polyclonal DFA, monoclonal DFA, and PCR were 36 and 100%, 11.4 and 94.6%, 8.3 and 98. 4%, and 95.0 and 99.3%, respectively, with a prevalence of 15.7%. The DFA tests were the most economical, and the PCR cost was 31% higher than culture. This study suggests that with minor improvements in economy, pertussis PCR can be implemented in a clinical laboratory with marked improvement in diagnostic accuracy.

Detection of Mycobacterium-specific interferon-gamma-producing human T lymphocytes by flow cytometry.

Flow cytometry has proven to be a useful tool for the investigation of cytokine synthesis by selected cell subpopulations. While most reports have used mitogen stimulation or long-term cultures with antigen, we describe here a novel method to allow the detection of rare mycobacterial antigen-specific cytokine synthesizing cells within one day. The most important feature of this method is the use of an FITC-conjugated isotype-matched control antibody to identify and exclude cells which fluoresce non-specifically. With this technique, we demonstrate interferon-gamma (IFN-gamma) staining in 785 cells per 1 x 10(5) T cells counted, in mycobacterial antigen-stimulated peripheral blood mononuclear cells from a BCG-vaccinated subject. In comparison, only 14 IFN-gamma-staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1 x 10(5) T cells are stained by an irrelevant control antibody. Specific responses are detectable after 12 h of in vitro culture, and peak at 24 h. In volunteer health care workers, IFN-gamma staining correlated with IFN-gamma production using a published ELISPOT assay (r=0.927). IFN-gamma staining was also higher in PBMC from mantoux skin test-positive volunteers, compared to cells from skin test-negative subjects (p=0.0045). Flow cytometry following short-term culture can thus be used for enumeration of antigen-specific IFN-gamma synthesizing cells.

Bacteremia with Acinetobacter species: risk factors and prognosis in different clinical settings.

Acinetobacter species are widespread environmental gram-negative coccobacilli that are associated with nosocomial infections; these bacteria are considered to be of relatively low virulence and rarely cause invasive disease. Fifty-two cases of bacteremic episodes due to Acinetobacter species were reviewed, and risk factors and outcomes of these cases were examined. It was noted that these cases belonged to a few clinical groups with markedly different outcomes. Eighteen patients had malignancies (predominantly hematologic), and bacteremia often developed after respiratory infection. Nine patients suffered traumatic injuries and developed bacteremia with Acinetobacter species after endotracheal intubation in the intensive care unit and respiratory colonization. Four burn patients, three of whom had burns covering > or = 50% of their body surface areas, had burn infections prior to bacteremia. While many patients had central venous catheters, in only four cases were the catheters clearly infected prior to the positive blood culture. Prior use of antibiotics was widespread in all groups of patients, and isolates showed high levels of antimicrobial resistance, particularly to beta-lactam agents. The outcome of infection correlated more closely with the type of underlying illness than with other factors such as biovar, polymicrobial bacteremia, or appropriate therapy. Patients with malignancies and burn patients fared poorly (10 of 18 and 2 of 4 patients, respectively, died), while trauma patients and patients with other illnesses did well.

In vitro effects of acephate on carbonic anhydrase activity in the blood and gills of rainbow trout, Salmo gairdneri.

Acephate, a water-soluble organophosphate pesticide used to control terrestrial insect pests, may enter aquatic ecosystems in the course of its use and adversely affect fish populations. The in vitro effects of this insecticide on gill and red blood cell (RBC) carbonic anyhdrase (CA) activity in rainbow trout were investigated over a range of 100 mg/1 (0.55 mM) to 50,000 mg/l (273 mM) to assess the manner in which acephate might affect respiratory capacity in exposed fish. Concentrations required to produce 50% inhibition of CA activity in the gill and RBC preparations were 38,000 mg/l (207 mM) and 8,900 mg/l (48 mM) respectively. The toxic action of acephate may be related to inhibition of CA activity in the blood and gills with resultant disturbances of respiratory capacity and salt balance.