Rapid Laboratory Diagnosis Research Articles

Hemorrhagic viral keratoconjunctivitis in Taiwan caused by adenovirus types 19 and 37: applicability of polymerase chain reaction-restriction fragment length polymorphism in detecting adenovirus genotypes.

Acute keratoconjunctivitis with prominent subconjunctival hemorrhage (SCH) is usually perceived by a clinician as acute hemorrhagic conjunctivitis (AHC) associated with enteroviruses; however, SCH can also be an adenoviruses infection. A rapid and sensitive laboratory diagnosis is helpful for differential diagnosis. Therefore, the sensitivity and applicability of polymerase chain reaction (PCR) and reverse transcription (RT)-PCR diagnoses were evaluated for keratoconjunctivitis associated with viral infection. Conjunctival swabs from patients with acute conjunctivitis were tested using a PCR-restriction fragment length polymorphism (PCR-RFLP) for adenovirus detection and RT-PCR for enterovirus detection. The results were compared with those using the culture isolation and neutralization test; also, the clinical findings of the patients were analyzed with special attention to SCH patterns. Neither coxsackievirus A type 24 variant (CA24v) nor enterovirus type 70 (EV70) was detected in 113 patients with acute conjunctivitis. The positive results of adenovirus (Ad) were 39.9% by the PCR method and 37.1% by culture isolation. For the patients with adenoviral conjunctivitis, 68.1% was owing to Ad37 and 19.2% was owing to Ad19. SCH was present in 51.5% of the positive cases, and 44.7% of the Ad-positive patients had secondary illnesses. SCH can be a predominant presentation of Ad19 and Ad37 keratoconjunctivitis and may herald a new stage in the evolution of adenoviruses. PCR and PCR-RFLP are rapid and reliable methods for Ad detection and typing; however, if the amplified genes and restriction enzymes are not properly selected, they may not be able to detect new genotypes of adenoviruses or the evolution of these viruses.

Screening tests of disseminated intravascular coagulation: guidelines for rapid and specific laboratory diagnosis.

To study the clinician's ordering pattern in the diagnosis of disseminated intravascular coagulation (DIC) and to analyze the utility of selected tests by assessing their sensitivity, specificity, and overall efficiency. Retrospective, nonrandomized, clinical study. University hospital intensive care units. A total of 82 inpatients treated in our intensive care units were identified from the hospital computer system as having been tested for DIC in a 3-month period. Screening tests for DIC were ordered for the suspected patients. Prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen/fibrin degradation products (FDP), and fibrinogen were used most frequently as DIC diagnostic tests. The FDP and D-dimer combination (n = 39) had the highest diagnostic efficiency of 95%, with sensitivity being 91% and specificity 94%. This is followed by FDP (n = 71), efficiency 87%, sensitivity 100%, and specificity 67%; PT/PTT and FDP combination (n = 71), efficiency 86%, sensitivity 91%, and specificity 71%; and D-dimer (n = 44), efficiency 80%, sensitivity 91%, and specificity 68%. The rest of the commonly used tests, such as PT, PTT, thrombin time, platelet count, fibrinogen, and the presence of schistocytes (n = 80), had individually either low specificity or low sensitivity and, therefore, low efficiency scores (57%, 57%, 70%, 67%, 65%, and 51%, respectively). The D-dimer and FDP tests offered the best test panel in the diagnosis of DIC. We propose the use of D-dimer, FDP, and antithrombin as the DIC diagnostic test panel, with D-dimer and FDP providing a rapid and specific diagnosis, antithrombin providing insight to the severity and prognosis, and FDP (rapid and less expensive than D-dimer) to follow-up the progress of the condition once the diagnosis is established.

Dengue situation in Brazil by year 2000.

Dengue virus types 1 and 2 have been isolated in Brazil by the Department of Virology, Instituto Oswaldo Cruz, in 1986 and 1990 respectively, after many decades of absence. A successful continental Aedes aegypti control program in the Americas, has been able to eradicate the vector in most countries in the 60's, but the program could not be sustained along the years. Dengue viruses were reintroduced in the American region and the infection became endemic in Brazil, like in most Central and South American countries and in the Caribbean region, due to the weaning of the vector control programs in these countries. High demographic densities and poor housing conditions in large urban communities, made the ideal conditions for vector spreading. All four dengue types are circulating in the continent and there is a high risk of the introduction in the country of the other two dengue types in Brazil, with the development of large epidemics. After the Cuban episode in 1981, when by the first time a large epidemic of dengue hemorrhagic fever and dengue shock syndrome have been described in the Americas, both clinical presentations are observed, specially in the countries like Brazil, with circulation of more than one dengue virus type. A tetravalent potent vaccine seems to be the only possible way to control the disease in the future, besides rapid clinical and laboratory diagnosis, in order to offer supportive treatment to the more severe clinical infections.

Rapid diagnosis of tuberculous meningitis by a dot-immunobinding assay.

In this study, a dot-immunobinding assay (Dot-Iba) was standardized to measure circulating antimycobacterial antibodies in the cerebrospinal fluid (CSF) specimens for the rapid laboratory diagnosis of tuberculous meningitis (TBM). Specific CSF-IgG antibody to Mycobacterium tuberculosis from a culture proven patient with TBM was isolated and coupled with activated Cynogen bromide-Sepharose 4B. Using an immunoabsorbent affinity chromatography, 14 kDa antigen present in the culture filtrates of M. tuberculosis was isolated and this antigen was used in the Dot-Iba, to quantitate specific antimycobacterial antibodies in CSF specimens. The Dot-Iba gave positive results in all the 5 culture proven patients with TBM and gave no false positive results in CSFs from patients with partially treated pyogenic meningitis. Dot-Iba developed in our laboratory is a simple, rapid and specific method and more importantly suited for the routine application in laboratories with limited resources.

Evaluation of two rapid assays for detection of Clostridium difficile toxin A in stool specimens.

Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment. We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B. A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays. With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods. Total agreement for all assays was 90.5% (592 of 654). The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA. While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.

Usefulness of rapid antigen detection for the diagnosis of systemic infections due to Haemophilus influenzae

Twenty-eight cases of systemic infections due to Haemophilus influenzae diagnosed from October 1988 to December 1998 were analyzed retrospectively. The clinical manifestations were 13 meningitis (15 episodes), 9 septic arthritis, 4 acute epiglottitis, 1 septicemia and 1 lung abscess. In the 15 meningitis episodes, 13 had positive CSF culture results, and the other 2 episodes of pretreated with antibiotics were diagnosed by H. influenzae type b (Hib) antigen detection by using concentrated urine specimens. In the 9 septic arthritis cases, 6 had positive synovial fluid culture results. Of the 3 cases with negative results on Gram stain and on synovial fluid and blood cultures, etiological diagnosis was established by Hib antigen detection in synovial fluid. Results of Hib antigen detection were positive in all 8 cases (100%). In 6 of these 8 cases, antimicrobial therapy was started by the results of antigen detection. In the 4 acute epiglottitis, 2 had positive blood culture results, and the other 1 case was diagnosed by Hib antigen detection by using concentrated urine specimen. In 3 of these 4 cases, H. influenzae strains isolated from nasopharyngeal swab or aspirated sputum were serotyped as type b. In this study, rapid antigen detection has several advantages in the rapid laboratory diagnosis of systemic infections due to Haemophilus influenzae. 1. The detection of Hib antigen is the only way to diagnose bacterial etiology of infection in patients who had received partially treatment with antimicrobials. Urine is as an appropriate specimen for antigen testing as CSF in patients with suspected Hib meningitis. Moreover, to detect Hib antigen in synovial fluid is clinically useful in septic arthritis. 2. Both the antigen detection and Gram stain made the rapid presumptive identifications and effected therapeutic decision making. 3. Antigen detection methods have also been used in serotyping of clinical isolates. We conclude that rapid antigen detection is a very useful tool for the rapid etiological diagnosis and guideline for the choice of antimicrobials in systemic infections due to Hib. It is necessary to diagnose bacterial etiology as a routine procedure using not only Gram stain and culture but also rapid antigen detection technique in patients with suspected Hib systemic infection.

MALARIA IN TRAVELERS: Epidemiology, Disease, and Prevention

The combination of increases in international travel and escalating drug resistance has resulted in a growing number of travelers contracting malaria. Preventing malaria-associated morbidity and mortality will require improved health information for travelers about the risk of malaria and appropriate preventive measures, improved recognition of infection by physicians, rapid and accurate laboratory diagnosis, and prompt initiation of effective therapy.

Current aspects of diagnosis and treatment of cytomegalovirus infections in infants

Background: Human cytomegalovirus (CMV) is the most common cause of congenital and perinatal infections throughout the world. High prevalence of CMV infection is associated mainly with universal breast feeding practices rather than with crowding or poverty. Perinatal CMV infection usually follows a benign course in immunocompetent individuals, but the virus remains latent or persistent in the host cell thereafter. Objective: As a clinical diagnosis of CMV infection is generally not easy, rapid and accurate laboratory diagnosis of CMV infection is required for appropriate patient management. Numerous anti-CMV compounds including ganciclovir, foscarnet and cidofovir have been reported, most of them are unlikely in a clinical trial for perinatal CMV infection in immunocompetent individuals. Understanding the epidemiology of CMV is a key element in the development of strategies for prevention and treatment of infection. Study design: This review focuses on recent advances in the diagnosis and treatment of perinatal CMV infections in infants. Conclusions: Entirely new approaches to prevention and treatment of CMV infections in infants are necessary, including antiviral interventions and the development of a vaccine strategy.

Polymerase chain reaction for detection of Chlamydia trachomatis in conjunctival swabs

AIMS/BACKGROUNDOcular Chlamydia trachomatis infection in the west occurs as ophthalmia neonatorum, acquired from the mother, or adult paratrachoma which is also associated with current genital tract infection. Accurate rapid laboratory...

Critical Assessment of Gene Amplification Approaches on the Diagnosis of Tuberculosis

The resurgence of tuberculosis prompted the development of a number of new options for the rapid laboratory diagnosis of Mycobacteriun tuberculosis (MTB). One of the most promising and exciting methodologies has been the introduction of assays employing amplification technology to detect MTB directly in clinical specimens. Although amplification assays hold significant promise to improve the laboratory diagnosis of tuberculosis, the decision to perform or not perform these assays is complicated. The performance of in-house polymerase chain reaction (PCR) assays and two commercially-prepared assays, GenProbe's AMTD test and Roche's AMPLICOR PCR assay are reviewed. Regardless of the amplification format, all assays have decreased sensitivity with specimens that are acidfast bacilli (AFB) stain-negative. Data from these studies and others indicate possible potential pitfalls of amplification assays, those being sampling errors, the presence of substances in clinical specimens that inhibit the amplification assay, and clinical utility.In light of these findings, the possible roles for these assays in the clinical microbiology laboratory are reviewed. In addition, factors such as cost, assay performance, etc. are discussed in order to facilitate the decision-making process concerning whether an amplification assay would be appropriate in a particular laboratory setting.

Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA.

We developed a PCR-based assay for the rapid and specific laboratory diagnosis of human brucellosis directly from whole blood. Specimens were collected in EDTA tubes from 17 patients with acute serologic brucellosis and 3 patients with chronic relapsing brucellosis as determined by serologic tests and the patient's clinical picture. DNA was extracted from peripheral mononuclear cells obtained from the blood of patients with brucellosis and control individuals. Specific primers for the PCR amplification of a 223-bp region on the sequence encoding the 31-kDa immunogenic Brucella abortus protein (BCSP 31) were used. All amplicons had the expected size of 223 bp. The specificity of amplification was determined by Southern hybridization and restriction endonuclease analysis. DNA extracted from blood taken from 30 healthy individuals as well as from 9 patients with typhoid fever did not show any amplification with the primers used. The test proved to be rapid and specific for the laboratory confirmation of acute human brucellosis. Further studies must be conducted to assess the utility of this test on additional patients with chronic relapsing brucellosis as well as patients under treatment.

Prospective study of adenovirus antigen detection in eye swabs by radioimmune dot-blot.

Rapid laboratory diagnosis of ocular adenovirus infection is crucial in the containment of nosocomial transmission of the virus. In a large prospective study of adenovirus assay in eye swabs, antigen detection by radioimmune dot-blot (turnaround time 72 hours) achieved a sensitivity of 67% (239/355) and a specificity of 93% (3065/3285) in comparison with virus culture (median turnaround time 14 days). When specimens weakly reactive for adenovirus antigen, or equally reactive for both adenovirus antigen and Chlamydia trachomatis antigen, were considered falsely reactive in the adenovirus test, the sensitivity of the latter was reduced and false positive reactions were only marginally less frequent. The radioimmune dot-blot provides a more rapid diagnosis of ocular adenovirus infection than virus culture, but the high risk of false negative and in particular false positive results limits its clinical utility.

Advances in rapid laboratory diagnosis of infectious endophthalmitis

Infectious endophthalmitis is a serious ocular condition which always requires rapid medical or surgical intervention. At present this, in a number of situations, remains empirical as a consequence of problems inherent in unequivocal identification of the causative organism, should one be present. Aqueous humor smears and culture have limited value in diagnosis. Vitreous samples can often reveal the presence of microbes, but these may not necessarily be detected in all cases, principally due to suboptimal sampling or pretreatment with antimicrobials which render microbes non-culturable on routine media. Use of electron microscopy and/or immunocytochemistry offers an alternative means of identification, but these approaches have drawbacks principally associated with interpretation. Molecular methods which identify conserved sequences from common causative microbes of endophthalmitis, or which can specify whether the organism is a bacterium or a fungus may become especially important as diagnostic tools in this clinical scenario.

The Histoplasma capsulatum antigen assay in disseminated histoplasmosis in children

Progressive disseminated histoplasmosis is often fatal without treatment and requires rapid and accurate laboratory diagnosis. Radioimmunoassay for Histoplasma capsulatum var. capsulatum antigen has been established as a sensitive and accurate diagnostic technique for disseminated histoplasmosis in adults; this study examines the radioimmunoassay in children. The clinical and laboratory records of 26 patients 18 years old or younger in whom H. capsulatum antigen was detected in urine by radioimmunoassay and at least one other positive corroborative standard test were evaluated. Twenty-two (85%) had disseminated disease, and 4 (15%) had self-limited pulmonary disease. Positive corroborative tests included serologic tests in 17 of 22 (77%) patients tested, tissue stains in 5 of 9 (56%) and fungal cultures in 16 of 24 (67%). Patients with disseminated histoplasmosis had a greater degree of antigenuria than those with self-limited infection. In 20 patients with progressive disease treated with amphotericin B, antigen levels declined, and the decrease in antigenuria correlated with clinical improvement. The radioimmunoassay for H. capsulatum antigen in urine is an important test in the diagnosis of disseminated histoplasmosis and is useful for assessing the efficacy of treatment. The presence of urinary antigen is strong evidence for progressive disease that requires treatment.

Influenza: diagnosis, management, and prophylaxis.

Influenza causes enormous morbidity, death, and economic loss. Annual vaccination is strongly recommended for groups at high risk. Amantadine is effective treatment for and prophylaxis against influenza A during epidemics. New developments include rapid laboratory diagnosis, live attenuated vaccines, and antiviral drugs.

Rapid characterization of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5? noncoding region

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the rapid laboratory diagnosis of pestivirus infections. A direct DNA sequencing method was developed for the analysis of the amplified cDNA from the 5' noncoding region of the viral genome. 70 pestivirus strains were compared in this study. Sequence analysis allowed the characterization of each isolate as either classical swine fever virus (CSFV), bovine viral diarrhea virus, or border disease virus, respectively. The 48 CSFV strains could be further classified into several subgroups, which correlated either with the geographical origin or the date of the first isolation of the respective isolate.

Detection of foot-and-mouth disease virus RNA in clinical samples and cell culture isolates by amplification of the capsid coding region

Foot-and-mouth disease is one of the most economically important virus diseases of livestock. Two important requirements for the control of this disease are rapid laboratory diagnosis and epidemiological investigation. The use of the polymerase chain reaction method (PCR) to amplify specific nucleic acid regions offers the unique possibility of combining swift viral detection with the production of genetic material suitable for sequencing and other methods of molecular epidemiological analysis. The sequencing of the region of foot-and-mouth disease virus (FMDV) genome encoding the capsid proteins of the virus (∼ 2260 bps), provides valuable information that adds to the molecular characterization of an isolate. This paper describes the use of the PCR for the amplification of this region of the FMDV genome from bovine clinical samples and cell culture isolates. Suitable pairs of oligonucleotide primers were selected from the published sequence of FMDV type O 1, Kaufbeuren. One primer set amplified 2091 bps of the capsid coding region of all seven serotypes of FMDV. The other primer set amplified 216 bp from this region of FMDV type O 1, BFS 1860, in nucleic acid extracts from several clinical samples. Nucleic acid extracts from the picornaviruses, bovine enterovirus and swine vesicular disease virus, which affect the same animals, were not amplified. Direct sequencing was carried out on the amplified fragments and showed that the PCR products were >98% homologous to published FMDV sequences.

Prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus

To determine the most effective infection control procedure in preventing nosocomial infection with respiratory syncytial virus (RSV), we did a prospective controlled study of four infection-control strategies in four wards in a large paediatric hospital in the west of Scotland. All children under two years old admitted to four general wards during three winter RSV epidemics (1989-92) were screened for RSV infection (by nasopharyngeal aspirate and direct immunofluorescence) within 18 hours of admission. The main outcome measure was the occurrence of nosocomial infection, defined as the number of children initially RSV negative who became RSV positive 7 days or more after hospital admission (incubation period for RSV infection is 5-8 days). Without special precautions, there was a high rate of nosocomial RSV infection (26%). Nosocomial infection was significantly reduced by the combination of cohort nursing with the wearing of gowns and gloves for all contacts of RSV-infected children (p=0·0022). Neither the use of gowns and gloves alone nor cohort nursing alone produced a significant reduction in cross-infection. In the final year, general clinical use of a policy of cohort nursing with gowns and gloves resulted in a reduction in the cross-infection rate by two-thirds of its original value (9·5% vs 26%). Combined with rapid laboratory diagnosis, cohort nursing and the wearing of gowns and gloves for all contacts with RSV-infected children can significantly reduce the risk of nosocomial RSV infection.

Differentiation of species in human beta-haemolytic group G streptococci using immunoglobulin Fc fragment receptor.

To assess the ability of human immunoglobulin Fc fragment binding activity to differentiate human biotype large colony group G streptococci from the group G "Streptococcus milleri group". Fifty two isolates of large colony group G streptococci and 30 group G "S milleri group" strains were tested for their ability to bind fluorescein conjugated human IgG Fc fragments after acetone fixation. Immunoblotting with peroxidase labelled human Fc fragments after resolution of bacterial polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for six large colony strains. All large colony group G streptococci showed positive Fc fragment binding whereas all "S milleri group" bacteria failed to bind Fc fragments when viewed by fluorescence microscopy. All six large colony strains showed similar immunoblot binding patterns. Immunoglobulin Fc fragment receptor content distinguishes the large colony group G streptococci from the group G "S milleri group" and mayhave a role in the rapid laboratory diagnosis of pharyngeal pathogens.

Comparison of polyclonal antisera and anti-43kDa antigen monoclonal antibodies for the culture-amplified antigenic detection of Mycoplasma pneumoniae by immunoblotting

Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory disease whose rapid laboratory diagnosis is complicated. We developed a culture-amplified immunological antigen detection assay which employs a monoclonal antibody for the recognition of a polypeptide antigen of 43kDa in an immunoblotting technique. Polyclonal antisera to the same antigen were obtained from rabbit and hamster immunization with affinity chromatography-purified antigen. The latter sera, sera from natural human infection and experimental hamster infection, and a pool of anti-43kDa monoclonal antibodies were compared to the single anti-43kDa monoclonal antibody, OC2F5. Among these, only polyclonal hamster anti-43kDa sera were able to enhance antigen detection quantitatively. This enhancement however did not result in a significant benefit to the culture-amplified system.