Proper use of the clinical microbiology laboratory.

The interaction between clinicians and microbiology laboratory staff has to be one of mutual benefit. The more the laboratory personnel know about your patients, the more meaningful and thorough will be the results. Communication is the key to success. Visit the microbiology laboratory and get to know the staff. The clinician also needs to be familiar with and use the most commonly used diagnostic tests for individual bacterial pathogens appropriately.

Identification of influenza A virus by shell vial culture and two commercially available antigen detection methods.

BACKGROUND: Effective use of amantidine and rimantidine for treating patients and for reducing transmission requires rapid diagnosis of influenza A. Rapid culture methods require 1-2 days to detect influenza A virus. Direct fluorescent antibody (DFA) staining and enzyme immunoassay (EIA) can detect influenza A antigen within 1-4 h. OBJECTIVES: We compared DFA staining using the Bartels viral respiratory panel and the Directigen FLU-A EIA with shell vial centrifugation culture. STUDY DESIGN: Ninety-seven fresh specimens from a variety of respiratory sources and transported from hospitals throughout the USA to our national referral laboratory were tested. A true positive was defined as culture positive or both antigen tests positive. RESULTS: Fifteen specimens were true positive. Sensitivity with culture was 93%, EIA 67%, and DFA 47%. Specificity was excellent with all three methods: 100%, 98%, 99%. Culture detected additional viruses that can cause respiratory tract disease: herpes simplex, cytomegalovirus, respiratory syncytial, influenza B, and adenovirus. Fourteen (70%) of 20 frozen specimens previously positive for influenza A were positive on retest by EIA. Overall sensitivity of EIA compared with culture using 35 positive specimens was 69%. CONCLUSIONS: These results suggest that the rapid EIA is useful to screen for influenza A, but that critical antigen-negative specimens should be submitted to a virology laboratory for culture for optimal sensitivity and for recovery of other viruses.

Identification of influenza A virus by shell vial culture and two commercially available antigen detection methods.

BACKGROUND: Influenza continues to be a major cause of morbidity and mortality especially in the elderly and persons with underlying disease. Shell vial cell culture and antigen detection techniques may speed up diagnosis and enable better patient treatment and management. OBJECTIVES: To compare shell vial centrifugation culture with commercially available direct fluorescence and enzyme immunoassay kits using a variety of respiratory specimens. STUDY DESIGN: To detect influenza A virus, we compared direct fluorescent antibody (DFA) staining using the Bartels Viral Respiratory Panel and the Directigen FLU-A enzyme immunoassay (EIA) with shell vial centrifugation culture. Ninety-seven fresh specimens from a variety of respiratory sources, and transported from hospitals throughout the U.S. to our national referral laboratory, were tested. RESULTS: Fifteen specimens were true positives: culture positive or both antigen tests positive. Sensitivity with culture was 93%, EIA 67%, and DFA 47%. Specificity was excellent with all three methods: 100%, 98%, 99%. Culture detected additional viruses that can cause respiratory tract disease: herpes simplex, cytomegalovirus, respiratory syncytial, influenza B, and adenovirus. Fourteen (70%) of 20 frozen specimens previously positive for influenza A were positive on retest by EIA. Overall sensitivity of EIA compared with culture using 35 positive specimens was 69%. CONCLUSIONS: The rapid EIA is useful to screen for influenza A, but critical antigen-negative specimens should be submitted to a virology laboratory for culture. Shell vial cultures can provide a sensitive and universal diagnostic system for influenza A and a variety of other viruses.

Herpes simplex virus infection of the fetus and newborn.

Although infrequent, untreated neonatal herpes results in death in half the cases and neurologic sequelae in three quarters of the survivors. Neonatal infection is usually acquired from maternal genital herpes, which is asymptomatic or unrecognized in 60% to 80% of women. The greatest risk of neonatal infection occurs when the mother has primary genital herpes involving the cervix at delivery, and the infant is premature and delivered with instrumentation (eg, scalp electrodes). More than 80% of neonates with herpes will have typical herpetic lesions of the skin, eye, or mouth, and most of the remainder will have either encephalitis or a sepsis syndrome with pneumonitis and hepatitis and negative bacterial cultures. Because herpes can mimic other neonatal infections, laboratory diagnosis is important, using cultures of the virus from lesions, peripheral blood white cells, or CSF. Treatment with intravenous acyclovir does reduce mortality and neurologic sequelae, but outcome is still guarded in babies with disseminated disease or encephalitis. Prevention focuses on caesarean section in women with active lesions at the time of impending delivery and avoidance of postnatal exposure. Further studies are needed to determine whether maternal screening (eg, HSV-2 type specific antibodies and vaginal cultures in selected women at delivery) will be cost effective in preventing neonatal herpes.

Is it bacterial or viral? Laboratory differentiation.

To differentiate viral, chlamydial, and mycoplasmal infections from bacterial disease in office and in emergency room practice, a combination of epidemiologic and clinical features usually will suggest one or a few microorganisms. Following that, laboratory diagnosis can be more targeted. Definition of the specific etiology will enable the proper management choice of antibiotics, antivirals, or symptomatic therapy.

Tsukamurella paurometabolum: a novel pathogen causing catheter-related bacteremia in patients with cancer.

Tsukamurella paurometabolum is a weakly acid-fast, pleomorphic gram-positive bacterium found in soil. Human infection due to this organism has rarely been described, and there are no published accounts of bacteremia. Three cases of bacteremia due to T. paurometabolum and related to long-term use of a central venous catheter in patients with cancer who were receiving chemotherapy are described.

Is vaginal douching related to cervical carcinoma?

The association between cervical carcinoma and vaginal douching was examined in a population-based case-control study conducted in the low-risk population of Utah between 1984 and 1987. The authors compared 266 cases of in situ and invasive cervical carcinoma with 408 group-matched controls by vaginal douching behavior, controlled for age, lifetime number of sex partners, cigarette smoking history, religious activity, and educational level. Essentially no association was found in women who douched once per week or less, but in those who douched more than once per week, a consistent relation was demonstrated (adjusted odds ratio = 4.7, 95% confidence interval 1.9-11). Few differences were found with type of douching preparation used. The authors hypothesize that frequent douching alters the vaginal chemical environment, making the cervix more susceptible to pathologic change.

Cross contamination of viral specimens related to shell vial caps.

A cluster of poliovirus type 3-positive specimens cultured in shell vials led to the discovery of significant cross contamination between vials related to the type of vial cap. Simulated testing with colored media in the shell vials indicated that the most contamination occurred with the plastic snap cap and that the least occurred with the screw cap. Measures to prevent cross contamination in clinical virology laboratories are discussed.

Dietary vitamins A, C, and E and selenium as risk factors for cervical cancer.

The relation between cervical cancer and dietary intake of vitamins A, C, and E, beta-carotene, and selenium was examined in a population-based case-control study in Utah. Cervical cancer cases (n = 266) and population-based controls (n = 408) were interviewed between 1984 and 1987. Protective effects were observed for vitamins A, C, and E and beta-carotene but were attenuated by age, level of education, and lifetime cigarette use. Associated risk (comparing highest with lowest quartiles of intake) went from 0.53 (crude) to 0.71 (adjusted) for vitamin A; from 0.55 (crude) to 0.82 (adjusted) for beta-carotene; from 0.45 (crude) to 0.55 (adjusted) for vitamin C; from 0.58 (crude) to 0.60 (adjusted) for vitamin E; and from 0.95 (crude) to 0.70 (adjusted) for selenium. Adjustment for number of sex partners and church attendance, factors significantly related to cervical cancer risk, only slightly attenuated these adjusted risk estimates.

Sexual activity, contraception, genital infections, and cervical cancer: support for a sexually transmitted disease hypothesis.

A case-control study was conducted in Utah between 1984 and 1987 to examine risk factors for cervical cancer. Interviews were completed with 266 histologically confirmed carcinoma in situ and invasive squamous cell cervical cancer cases who were categorically matched by age to 408 controls. Among the factors identified as altering risk for cervical cancer, after adjustment for age, education, church attendance, and cigarette smoking, were: having numerous sex partners (odds ratio (OR) = 8.99 for 10 or more partners); the current mate having several sex partners (adjusted OR for 10 or more partners = 8.62); using foam or jelly as a contraceptive method (OR, adjusted for number of sex partners, = 0.44); reported Trichomonas infection (OR, adjusted for number of sex partners, = 2.10); and herpes simplex virus type 2 infection as determined by 2:1 neutralization index values above 100 (OR = 2.70). A protective effect was noted from the use of diaphragms (OR = 0.67) or condoms (OR = 0.53) in women who reported more than one sex partner. These data support the hypothesis that cervical cancer is a sexually transmitted disease.

Cigarette smoking and exposure to passive smoke are risk factors for cervical cancer.

Personal cigarette smoking and exposure to passive smoke as risk factors for cervical cancer were examined in a population-based, case-control study conducted in Utah. Personal cigarette smoking was found to increase the risk of cervical cancer, after adjusting for age, educational level, church attendance, and sexual activity. The adjusted risk estimate associated with being a current smoker was 3.42 (95% confidence interval [Cl], 2.10 to 5.57); for having smoked for 5 or more pack-years, it was 2.81 (95% Cl, 1.73 to 4.55); and for having smoked at least 100 lifetime cigarettes, it was 2.21 (95% Cl, 1.44 to 3.39). The adjusted risk estimate (also adjusted for actual cigarettes smoked) associated with passive smoke exposure for 3 or more hours per day was 2.96 (95% Cl, 1.25 to 7.03). Risk from passive smoking was greater in women who were not smokers (odds ratio, 3.43; 95% Cl, 1.23 to 9.54) than in women who smoked (odds ratio, 2.59; 95% Cl, 0.23 to 29.24).

Acyclovir treatment of disseminated herpes simplex virus type 2 infection in weanling mice: alteration of mortality and pathogenesis.

Intranasal inoculation of weanling mice with herpes simplex virus type 2 (HSV-2) provides an experimental infection that closely resembles disseminated and central nervous system HSV infections of human neonates. Intraperitoneal treatment with acyclovir (ACV) successfully reduced mortality even when therapy was begun as late as 2 days and oral therapy as late as 4 days after viral challenge. Treatment with ACV beginning on day 1 completely inhibited HSV-2 replication in lung, spleen, kidney, olfactory lobe, and cerebrum and decreased viral titers in the pons by 2-3 logs. Comparison of these data with our previous experiments using adenine arabinoside and adenine arabinoside 5' monophosphate indicates that ACV is more effective in the murine model of neonatal disease and suggests that ACV may also be more effective in treating the disease in humans.

Rapid detection of herpes simplex virus in clinical specimens by centrifugation and immunoperoxidase staining.

The effect of immunoperoxidase staining and centrifugation on the sensitivity and rapidity of herpes simplex virus detection in mink lung cell cultures was determined with 730 clinical specimens. In standard tube cultures, the use of immunoperoxidase staining resulted in detection of 31 (91%) of 34 positive cultures after overnight incubation, compared with 25 (74%) detected without the stain (P less than 0.05). The effect of centrifugation of specimens onto the monolayer followed by overnight incubation and immunoperoxidase staining was studied with 431 specimens. Of 107 positive specimens, 103 (96%) were detected by this method, compared with 91 (85%) detected in standard cell cultures observed for 5 days (P less than 0.02). Standard cell cultures that were examined after overnight incubation detected only 62 (58%) of the 107 positive specimens (P less than 0.001). Centrifugation of clinical specimens onto cell monolayers followed by overnight incubation and immunoperoxidase staining is more rapid and sensitive than are standard cell culture techniques for the laboratory diagnosis of herpes simplex virus infection.

Synergism between recombinant human interferon and nucleoside antiviral agents against herpes simplex virus: examination with an automated microtiter plate assay.

An automated, quantitative, cytopathic effect (CPE) inhibition assay with human fibroblasts in 96-well microtiter plates was used to examine the combination of recombinant human interferon-alpha (rIFN-alpha A) and acyclovir, vidarabine, or dihydroxypropoxymethyl guanine against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) in vitro. Fifty percent CPE (CPE50) end points, calculated from optical density readings of crystal violet-stained monolayers in an automated spectrophotometer, represented 1.7 log reduction in viral yield (50-fold or 98% decrease). Using CPE50 end points of drugs alone and in combination, we defined synergism, additivism, or antagonism with an isobologram plot and a combination index equation. The combinations of rIFN-alpha A plus acyclovir and rIFN-alpha A plus dihydroxypropoxymethyl guanine were highly synergistic against both HSV-1 and HSV-2, whereas the combination of rIFN-alpha A plus vidarabine was additive to mildly synergistic. Combinations of antiviral agents synergistic in cell cultures should be pursued with further studies in animal models of human viral disease and potentially in clinical trials.

Recurrent genital herpes simplex virus infection in guinea pigs.

After recovery from initial genital herpes simplex virus (HSV) infections, female guinea pigs developed spontaneous recurrent infections characterized by discrete erythematous or vesicular herpetic lesions on the external genital skin. HSV type 2 (HSV2) caused significantly more recurrent infections in guinea pigs than did HSV type 1 (HSV1). HSV2-infected animals demonstrated a significant decline in frequency of recurrences over time. The viral nature of the recurrent lesions was confirmed by recovery of infectious HSV, detection of HSV antigen, and histologic examination. Latent HSV2 could be demonstrated in dorsal root ganglia and external genital skin after recovery from the primary infection. Recurrent genital HSV infection in the guinea pig shares many features with recurrent genital herpes in humans and provides a model for studying the relationship between latency and recurrences and for exploring methods for control of recurrent disease.

More rapid isolation of herpes simplex virus in a continuous line of mink lung cells than in Vero or human fibroblast cells.

Herpes simplex virus (HSV) was isolated from clinical specimens more rapidly in mink lung (ML) cells, a continuous cell line available from a commercial supplier, than in Vero cells or human fibroblast (HF) cells. Stock strains of HSV type 1 (HSV-1) and HSV-2 titered higher in ML cells than in Vero or HF cells. ML cells were equivalent to rabbit kidney (RK) cells in the isolation of HSV in clinical specimens, but titers of stock HSV strains were lower. ML cells could be employed to type strains of HSV-1 and HSV-2, using the technique of differential susceptibility to bromovinyldeoxyuridine (BVDU). ML cells, therefore, are a convenient and useful cell line for the isolation and typing of HSV in diagnostic virology laboratories.