Types Of Clinical Specimens Research Articles

Enterovirus molecular detection and typing

Cell culture and neutralization have been considered the gold standard for enterovirus detection and identification for more than 50 years, but molecular amplification technologies are rapidly replacing the traditional methods in clinical and public health laboratories. Assays based on reverse transcription–polymerase chain reaction or nucleic acid sequence-based amplification may be used to detect enterovirus genome in all types of clinical specimens. More recently, nucleotide sequence has been used as a surrogate for antigenic typing (determination of serotype) by targeting parts of the enterovirus capsid-coding region that contain serotype-specific neutralization epitopes. This review will describe the molecular methods currently being used to diagnose enterovirus infection and disease, starting with the broadest level (family/genus detection) and proceeding through species/serotype identification to genotyping and molecular epidemiology. The commonly used molecular assays are usually more sensitive and more specific than cell culture and antigenic typing. They can reduce the turnaround time for testing of clinical diagnostic specimens to a clinically relevant timeframe and should supplant culture/neutralization as the gold standard in the near future. However, further evaluation and, in particular, more rigorous validation are required before a molecular diagnostic standard can be established.

Quantitative Detection of Lung Cancer Cells by Fluorescence In Situ Hybridization: Comparison With Conventional Cytology

The aim of this study was to clarify whether fluorescence in situ hybridization (FISH) can diagnose lung cancer in various clinical specimens in comparison with conventional cytology.Prospective study.University hospital in a metropolitan area.Fifty consecutive patients with abnormal chest radiography or CT scan findings were enrolled. The patients included 32 men and 18 women, with an average age of 64 years. The final definitive diagnosis was made by histologic examination, as follows: 38 primary lung cancers (24 adenocarcinomas, 8 squamous cell carcinomas, 2 large cell carcinomas, and 4 small cell carcinomas); 1 metastatic renal cell carcinoma; and 11 benign lesions.Four types of clinical specimens were analyzed. Cells obtained by transbronchial brushing and transbronchial fine-needle aspiration using a fiberoptic bronchoscope under fluoroscopy, CT scan-guided percutaneous needle biopsy, and bronchial washings. On every examination, duplicate slides were made for analyses of conventional cytology and FISH.Classifications according to conventional cytology were as follows: class I, 4 patients; class II, 15 patients; class IIIa, 3 patients; class IIIb, 5 patients; and class V, 23 patients. A classification higher than class IIIb was considered to be positive for cancer. For cytology, we found no false-positive cases and 11 false-negative cases. The specificity was 100%, and the sensitivity was 71.8%. By FISH, 34 cases showed aberrant copy numbers in either chromosome 3 or 17. We found no false-positive cases and five false-negative cases. The specificity was 100%, and the sensitivity was 87.1%.The ability of FISH to detect aneusomic lung cancer cells is superior to conventional cytology for the diagnosis of lung cancer.

Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR

A novel, robust, reproducible, and multipurpose universal sample processing (USP) methodology for highly sensitive smear microscopy, culturing on solid and liquid media, and inhibition-free PCR which is suitable for the laboratory diagnosis of both pulmonary and extrapulmonary tuberculosis (TB) has been developed. This method exploits the chaotropic properties of guanidinium hydrochloride for sample processing and involves incubating the specimen with USP solution, concentrating bacilli by centrifugation, and using the processed specimen for smear microscopy, culture, and PCR. The detection limit for acid-fast bacilli in spiked sputum by smear microscopy is approximately 300 bacilli per ml of specimen. USP solution-treated specimens are fully compatible with culturing on solid and liquid media. High-quality, PCR-amplifiable mycobacterial DNA can be isolated from all types of clinical specimens processed with USP solution. The method has been extensively validated with both pulmonary and extrapulmonary specimens. Furthermore, the USP method is also compatible with smear microscopy, culture, and PCR of mycobacteria other than tubercle bacilli. In summary, the USP method provides smear microscopy, culture, and nucleic acid amplification technologies with a single sample-processing platform and, to the best of our knowledge, is the only method of its kind described to date. It is expected to be useful for the laboratory diagnosis of TB and other mycobacterial diseases by conventional and modern methods.

The Laboratory Diagnosis of Neisseria gonorrhoeae

The present article describes the laboratory diagnosis of Neisseria gonorrhoeae by culturing of the organism from different types of clinical specimens followed by confirmatory tests. The success of culture methods requires good quality collection and transport of clinical specimens. The present guide describes the media requirements and cultural conditions for N gonorrhoeae growth and the characteristics for a presumptive identification of N gonorrhoeae. Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods. Nucleic acid detection methods include either amplification-based methods or nonamplification tests, and are increasingly used in clinical laboratories where a viable culture is not possible to obtain. Nucleic acid methods can also be used to detect the presence of low numbers in a specimen. Nucleic acid detection methods need confirmation with another amplification method or gene target. Controls must be included to ensure true positive and negative results, and to rule out nucleic acid contamination. Monitoring of antimicrobial susceptibilities of N gonorrhoeae is important to investigate treatment failure and to evaluate the efficacy of currently recommended therapies. Many methods for the characterization of N gonorrhoeae require cultures. The useful typing methods for determining strain relatedness include auxotyping, serotyping, plasmid profile analysis, DNA sequencing of the porB gene and pulsed-field gel electrophoresis. Quality assurance programs for diagnostic testing and antimicrobial susceptibility testing is reviewed.

Advances in diagnostic tests for bacterial STDs.

Because of their asymptomatic nature and nonspecific symptoms, laboratory tests are often required to diagnose a sexually transmitted infection. Over the past few years, there have been advances in technology, such as the development of nucleic acid amplification assays, which have improved our ability to diagnose infections caused by Chlamydia trachomatis. The finding that nucleic acid amplification tests can detect more infected individuals and are useful in screening low prevalence populations, has led to the development of strategies designed to reduce the cost of these assays without significantly impacting their sensitivity. The development of new tests for the diagnosis of syphilis has gained momentum from the report of a synthetic VDRL antigen, which will result in better nontreponemal antibody tests for syphilis. In spite of the completion of the genome sequence of Treponema pallidum and its annotation, we are still unable to cultivate this microorganism in vitro. However, the molecular revolution has resulted in the development of PCR assays for detecting Treponema pallidum in various types of clinical specimens, and to the production of recombinant antigens for use in tests that detect treponemal-specific antibodies. Further research will improve the availability of low cost, sensitive tests for the diagnosis of sexually transmitted infections. The English version of this paper is available too at:http://www.insp.mx/salud/index.html.

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram’s stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Role of Epstein-Barr Virus DNA Load Monitoring in Prevention and Early Detection of Post-transplant Lymphoproliferative Disease

Posttransplant lymphoproliferative disease (PTLD) is a severe and life-threatening complication after stem cell or solid-organ transplantation, virtually always associated with presence of Epstein-Barr virus (EBV) in the proliferating cells. PTLD is probably caused by the iatrogenically impaired T-cell response allowing outgrowth of EBV-positive B-cells. Quantitative EBV DNA load monitoring is a minimally invasive technique increasingly recognized as a valuable tool in posttransplant patient management. In this review, we focus on the clinical utility of EBV DNA load monitoring in the peripheral blood of transplant recipients using PCR and we discuss the currently most-widely used techniques and their value and limitations in predicting and diagnosing PTLD. Options for EBV DNA load-guided pre-emptive therapy and application of monitoring EBV DNA load dynamics in the prediction of clinical response after therapy are described. Origins of elevated EBV DNA loads in immunosuppressed patients and recent insights in the EBV life cycle in the immuncompromised host are discussed. Finally, a standardization of methodology, clinical specimen type, and cut-off values is proposed. This is essential for comparisons between different institutes and more adequate patient management.

Epidemiology of Streptococcus pneumoniae infection in Malaysia.

During a 1-year period from October 1995 to September 1996, 273 isolations of Streptococcus pneumoniae were made from various types of clinical specimens. The majority of the isolates (39.2%) were from sputum whilst 27.5% were from blood, CSF and other body fluids. The organism was isolated from patients of all age groups, 31.1% from children aged 10 years and below, 64.7% of which come from children aged 2 years or below. The majority of the isolates belong to serotypes 1, 6B, 19B, 19F and 23F. Serotypes 1 and 19B were the most common serotypes associated with invasive infection. About 71.9% of the invasive infections were due to serotypes included in the available 23 valent polysaccharide vaccine. The rates of resistance to penicillin and erythromycin were 7.0 and 1.1% respectively. Our findings show that the serotypes of S. pneumoniae causing most invasive infections in Malaysia are similar to those in other parts of the world and the available vaccine may have a useful role in this population.

Antifungal therapy in 'bone marrow failure'.

Antifungal therapy in 'bone marrow failure'.

The Growing Importance of Urinary Cytologic Testing

Urinary cytology is an accurate and indispensable diagnostic test for numerous urinary system disorders. It is reliable in detecting high-grade urothelial neoplasia and carcinoma in situ. Recent developments have expanded the role of urinary cytology to include the detection and monitoring of renal disease. This article reviews the clinical indications, specimen types, and diagnostic categories, and describes briefly common cytologic pitfalls. The importance of cytodiagnostic urinalysis and the usefulness of image cytometry and DNA analysis is discussed. Clinical implications and follow-up will be suggested. This is the third and final article in a continuing education update series on urinalysis. Other articles in this series focused on urine sediment diagnosis and the identification of urine crystals.

Molecular probes for diagnosis of fungal infections

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human β-globin DNA

Human cytomegalovirus and human herpesvirus-6 are closely related viruses which cause similar diseases, have similar cellular repositories of latent infection, and may be detected largely in the same types of clinical specimens. DNA amplification appears likely to play an increasing role in the diagnosis of recent and remote infection with these agents. A sensitive multiplex polymerase chain reaction was therefore developed for the two viruses and for human beta-globin DNA. Optimization of parameters such as the primers, primer concentrations, magnesium concentration, and buffer constituents was crucial in achieving a sensitive assay. Preliminary results indicated that the assay could simultaneously monitor DNA extraction from clinical specimens and allow detection of HCMV or HHV-6 in patients with diseases possibly caused by either pathogen.

The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes

A plasmid DNA library was constructed from restriction endonuclease digested genomic deoxyribonucleic acid (DNA) of a virulent strain of Mycobacterium tuberculosis isolated from sputum of a patient. The sensitivity and specificity of two of the cloned DNA fragments in detecting M. tuberculosis and its related DNA sequences were analysed by DNA-to-DNA hybridization. The level of detection was determined to be 50 picograms of M. tuberculosis DNA, which is approximately equivalent to 10 000 mycobacterial genomes. These two M. tuberculosis DNA probes did not cross-hybridize to DNA of non-mycobacterial origin, nor with DNA from 9 out of 11 other mycobacterial species. Mycobacterial DNA sequences could be detected in 134 of 441, or 30.4%, of various types of uncultured clinical specimens from 365 patients by the DNA probes, whereas traditional culture method showed only a 19.0% positivity rate for the same specimens (p<0.001). The overall sensitivity and specificity of the DNA probes in detecting M. tuberculosis are 90.5% and 83.8% respectively. The DNA hybridization test may become a useful tool for the early and rapid determination of mycobacterial infection in uncultured clinical specimens.

Distribution by serological type of group B streptococci isolated from clinical specimens at hospitals; Japan, 1977-1983

Group B streptococci is becoming a problem as a cause of neonatal sepsis and meningitis in recent year.The authors have recently studied the distribution of age and sex of patient, suorces of isolates, type of clinical specimen and serological type among 1, 944 strains of group B streptococci (GBS) isolated from clinical specimens at hospitals in Japan during the period of 1977-1983.The results were as fillows;1) Distribution of age of patients from whom GBS was isolated; Among 1, 233 cases, who had been well documented, the rates of each age group were 0 year, 3.9%; 1-5 years6.9%; 6-10years, 6.9%; 11-20 years, 9.9%; 21-30 years, 16.5; 31-40 years, 18.2%; 41-50 years, 14.6%; 51-60 years, 8.0%; 61-70 years, 5.7%;and over 70 years, 9.3%.2) Isolation rates of GBS arranged according to sex; Among 1, 694 cases, who had been well documented, the 547 cases (32.3%) were from male and the 1, 147 (67.7%) cases were from female.3) Type of clinical specimen from which GBS was isolated; Among.1, 850 cases, who had been well documented, the isolation rates by type of specimen were urine, 36.5%;throat swab, 31.4%; Vaginal swab, 17%;sputum, 4.2%; pus, 3.6%; semen, 1.8;cerebrospiral fluid, 1.2%; blood, 1.1%;and others, 3.3%.4) Distribution of serological type of GBS isolrated; The rates of serotype of 1944 strains were type Ia, 28%;type Ib, 11%;type Ic, 15%;type II, 4%;type III, 24%;type IIIR, 9%;type R, 3%;and untypable, 7%.Of 14 cases isolated from clinical specimens (blood or cerebrospinal fluid) with neonatal sepsis and/or meningitis, 13 cases (93%) were type III and tyee IIIR (type III 11 strains, type IIIR 2 strains).

The prevalence of yeasts in clinical specimens from cancer patients.

Yeasts recovered from cancer patients during a 15-month period were speciated, and the prevalence of these isolates in various types of clinical specimens was determined. Five species, including Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, and Torulopsis glabrata, accounted for 97.1% of the isolates. Eighteen different species were recovered. Respiratory and urine specimens yielded 75% of the organisms. C. albicans, C. tropicalis, and C. parapsilosis were recovered in about equal frequency from blood cultures. Certain species usually were recovered from one type of specimen: Candida quilliermondii from urine, Trichosporon cutaneum and Candida pseudotropicalis from respiratory sites, and Cryptococcus neoformans from spinal fluid. Pityrosporum orbiculare was isolated only from ear and urine cultures. Most of the yeasts (95.4%) were identified within 48 hours after isolation.

Use of ethanol for selective isolation of sporeforming microorganisms

When mixed cultures containing sporeforming bacteria were treated with heat or with ethanol, the latter consistently resulted in better recovery of Clostridium and Bacillus species. Both techniques were effective in eliminating vegetative cells. An ethanol concentration greater than 25% and exposure for 45 min or longer were necessary to kill all vegetative cells in mixed-culture samples. Ethanol treatment (50% ethanol for 1 h) was effective for isolating sporeforming bacteria from intestinal specimens. Seven different species of Clostridium were the only bacteria isolated from the ethanol-treated specimen of intestinal contents from the large bowel of a patient. It was concluded that treatment with ethanol for 1 h is an effective technique for selective isolation of sporeforming bacteria from mixed cultures and certain types of clinical specimens.

Automated Microbiological Detection/Identification System

An automated, computerized system, the AutoMicrobic System, has been developed for the detection, enumeration, and identification of bacteria and yeasts in clinical specimens. The biological basis for the system resides in lyophilized, highly selective and specific media enclosed in wells of a disposable plastic cuvette; introduction of a suitable specimen rehydrates and inoculates the media in the wells. An automated optical system monitors, and the computer interprets, changes in the media, with enumeration and identification results automatically obtained in 13 h. Sixteen different selective media were developed and tested with a variety of seeded (simulated) and clinical specimens. The AutoMicrobic System has been extensively tested with urine specimens, using a urine test kit (Identi-Pak) that contains selective media for Escherichia coli, Proteus species, Pseudomonas aeruginosa, Klebsiella-Enterobacter species, Serratia species, Citrobacter freundii, group D enterococci, Staphylococcus aureus, and yeasts (Candida species and Torulopsis glabrata). The system has been tested with 3,370 seeded urine specimens and 1,486 clinical urines. Agreement with simultaneous conventional (manual) cultures, at levels of 70,000 colony-forming units per ml (or more), was 92% or better for seeded specimens; clinical specimens yielded results of 93% or better for all organisms except P. aeruginosa, where agreement was 86%. System expansion in progress includes antibiotic susceptibility testing and compatibility with most types of clinical specimens.

Mycoplasma recovery from the male genitourinary tract: voided urine versus the urethral swab.

A study of Mycoplasma recovery from the genitourinary tract was made on a group of 50 males attending a venereal disease clinic. The purpose of the investigation was to compare recovery rates of mycoplasma from two types of clinical specimens-the urethral swab and voided urine. The total number of positive cultures did not differ significantly when either the swab or urine was used; Mycoplasma hominis type 1 was the only taxonomic species isolated, either alone or mixed with T-strain mycoplasma. Recovery rates of the two types from both the swab and the urine did not differ significantly. Age did not relate to the presence of mycoplasma in the group studied.

Extended study of Serratia in a diagnostic bacteriology laboratory.

The incidence of Serratia in clinical material received in a general hospital diagnostic laboratory was studied. Initially the morphologic features, biochemical characteristics, chromogenesis, and antibiotic susceptibility of 21 isolates were examined. On the basis of these studies certain cultural and biochemical tests were selected for routine identification of Serratia.Serratia isolated from various types of clinical specimens received during a 4-year period (1960–1964) were recorded. Most of the Serratia were nonchromogenic except on a special medium. The cultures were resistant to a number of antibiotic agents, particularly penicillin, polymyxin B, and the tetracyclines. A higher incidence of Serratia in sputum than in throat and nasopharyngeal cultures was observed. There was a noticeable increase in the overall incidence of Serratia isolates over the 4-year period. This may be a reflection of hospital acquired infections.