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PubMed2002

[Mode of sample preparation for pseudotuberculosis laboratory diagnosis by polymer chain reaction method].

A mode of feces sample preparation was developed for polymerase chain reaction (PCR) assay. It was based on alkaline treatment of the material. This treatment killed the most part of indigenous microflora, whereas Yersinia survived, because it was relatively resistant to alkaline. The mode was tested using human feces artificially contaminated with Yersinia pseudotuberculosis. Positive responses in samples containing 10(3)-10(8) microbial cells per ml were obtained by PCR assay with Yersi and Yers2, Invl and Inv2, YP3 and YP4 primers. Diagnostic efficiency of PCR for patients, small mammals, and washings from environmental objects was 4.75, 1.66, and 2.12 times higher than diagnostic efficiency of bacteriological analysis of these samples, respectively. Positive results in PCR were obtained at the day of the material collection and treatment, whereas Y. pseudotuberculosis was isolated only after 8-20 days. Positive samples in PCR and in bacteriological analysis were found to coincide. A brief scheme of the Y. pseudotuberculosis laboratory diagnosis is suggested. According to this scheme, target-oriented bacteriological assay is performed only in those samples, in which preliminary PCR assay after 1-3 days of incubation gave positive results of Y. pseudotuberculosis DNA detection.

Animals↗
PubMed1992

Evaluation of purified protein derivative in the laboratory diagnosis of tuberculous meningitis.

An enzyme-linked immunosorbent assay (ELISA) was standardised for the quantitation of IgG antibody in cerebrospinal fluid (CSF) specimens of patients with tuberculous meningitis (TBM). Purified protein derivative (PPD1) from H37Ra M tuberculosis was used as the antigen in the assay. The sensitivity of the ELISA with this antigen was evaluated in the CSF of 10 culture positive and 40 culture negative patients with TBM. The specificity of the assay was evaluated in the CSF of 50 patients with non-tuberculous neurological diseases (control group). The results obtained with this antigen were compared with commercially available tuberculin purified protein derivative (PPD2) and BCG antigens. PPD2 gave false negative results (50%) in culture positive patients with TBM, and BCG antigen gave false positive results in 32% of non-tuberculous subjects. PPD1 gave a sensitivity of 60% in culture negative patients with TBM and no false positive reactions in the non-tuberculous group. PPD1 antigen, in contrast to other mycobacterial antigens, can be very easily prepared in any routine laboratory, and this antigen is recommended for use as an aid in the laboratory diagnosis of TBM, particularly in culture negative patients with TBM.

Antibodies, Bacterial↗
PubMed2000

Indirect measurements of sweat electrolyte concentration in the laboratory diagnosis of cystic fibrosis.

AIM: To investigate whether analytical methods based on the colligative physical chemical properties of ions or solutes in sweat are less effective than the specific measurement of electrolytes in the diagnosis of cystic fibrosis (CF). METHODS: A single sweat sample was collected (Macroduct) from each of 211 infants and children, of whom 57 had CF, for the measurement of sodium, chloride, osmolality, and conductivity. RESULTS: The ranges within which CF and non-CF individual values overlapped (equivocal ranges), were wider for sodium and osmolality measurement than for chloride or conductivity. Neither of the latter two measurements provided a discriminatory advantage over the other. The utilisation of broadly based age related ranges for non-CF control subjects served to improve the discriminatory power of all four measurements to an extent that, in this cohort, both chloride and conductivity provided complete discrimination. CONCLUSION: Sweat conductivity is as effective as chloride measurement in the laboratory diagnosis of CF.

Adolescent↗
PubMed1992

Laboratory diagnosis of Mycoplasma pneumoniae infection. 4. Antigen capture and PCR-gene amplification for detection of the Mycoplasma: problems of clinical correlation.

Direct detection assays for Mycoplasma pneumoniae were established by PCR amplification of short sequences within the foot protein/adhesin (P1) gene and the 16S ribosomal RNA gene. Specificity and sensitivity was excellent, no hybridization was observed with M. genitalium and other human Mycoplasma species. In nose and throat washings from subjects with respiratory infection a pattern of high counts (c.f.u./ml) of M. pneumoniae (deduced from the amount of amplified PCR product), and a positive antigen capture assay, was found in 83% of subjects with serological evidence of current infection with M. pneumoniae. A small proportion of subjects with serological patterns suggesting infection in the more distant past had positive PCR assays. This was considered to represent either persistence of the organism from a previous infection or perhaps transient carriage during a reinfection, without substantial change in antibody response. PCR-based assay of M. pneumoniae offers a powerful, rapid, and sensitive substitute for culture of the mycoplasma. Antigen capture, while less sensitive than PCR, offers the advantage that it is more often positive with samples from current infection and requires less stringent laboratory organization to contain false positive results. We conclude however that the laboratory diagnosis of a chosen clinical episode should not rest on the PCR or Ag-EIA assays alone, but must also include antibody assays to confirm whether infection is current or represents persistence from past exposure.

Antigens, Bacterial↗
PubMed1993

Urine and the laboratory diagnosis of Chlamydia trachomatis in males.

OBJECTIVE: To determine whether the use of urine samples from male patients can replace urethral swabs for the rapid detection of Chlamydia trachomatis by the Pharmacia EIA. SETTING: The STD clinic, Adelaide, South Australia. PATIENTS: There were two separate groups of male patients. Group A (398) patients provided urethral specimens for the EIA and culture tests. The patients in Group B (356) provided an urethral swab and a urine sample for the EIA test. METHODS: The urine samples and urethral swabs were tested for the presence of C trachomatis by the Pharmacia Chlamydia EIA. In addition, the urethral swabs from Group A patients were cultured for the organism by standard cell cultures. The infected cell cultures were identified by an immunofluorescence test using a FITC-monoclonal antibody to C trachomatis (Kallestad). RESULTS: When the EIA was validated against culture, it showed a sensitivity of 100% and a specificity of 95% with the urethral swabs from Group A patients. The urine specimens were positive in 24% of those patients who yielded a positive EIA result in the urethral swabs. CONCLUSIONS: Although the EIA test on urethral swabs showed high sensitivity and specificity when validated against culture, our results showed that the use of urine samples cannot replace urethral swabs for the laboratory diagnosis of this sexually transmitted disease.

Adult↗
PubMed2002

Laboratory diagnosis of central nervous system infections caused by herpesviruses.

BACKGROUND: Herpesviruses may be associated with various types of central nervous system (CNS) infections. Herpes simplex encephalitis (HSE) has to be considered one of the most severe diseases. As effective antiviral drugs are available, rapid and reliable diagnosis has become important. OBJECTIVES: To describe polymerase chain reaction (PCR) and serological methods for the detection of herpesvirus-induced CNS infections by the example of HSE. STUDY DESIGN: 620 cerebrospinal fluid (CSF) and 2400 serum samples from 2700 selected hospitalized patients with clinical suspicion of encephalitis were tested for herpes simplex virus (HSV) as well as varicella-zoster virus (VZV) DNA and HSV-specific antibodies, respectively. RESULTS: HSV-1 DNA could be detected in eight and HSV-2 in three patients with focal encephalitis. In addition, HSV-2 DNA was found in two newborns with encephalitis and two adults suffered from transverse lumbar myelitis. One VZV DNA-positive patient had developed herpes zoster accompanied by meningoencephalitis, and in the other an encephalitis without cutaneous rash was diagnosed. Intrathecal antibody synthesis could be measured when CSF was cleared from viral DNA. CONCLUSIONS: The detection of viral DNA by PCR technique has become the "gold standard" method for laboratory diagnosis of herpesvirus infections of CNS. Serodiagnosis may be useful to confirm the diagnosis retrospectively.

Adolescent↗
PubMed1999

Length of time to laboratory diagnosis of Mycobacterium tuberculosis infection: comparison of in-house methods with reference laboratory results.

OBJECTIVES: To audit the time taken to obtain laboratory confirmation of infection with Mycobacterium tuberculosis using in-house methods of polymerase chain reaction (PCR) and culture and referral to a reference laboratory. METHODS: Retrospective collection of data from laboratory records covering a period of 1 year. RESULTS: Median time to microbiological diagnosis of a new infection using the in-house services in addition to the reference laboratory was 22.0 days. Using reference laboratory results alone, median time to diagnosis would have been 61.5 days. CONCLUSIONS: Development of on-site laboratory facilities to identify Mycobacterium tuberculosis can reduce the time to its identification by almost two-thirds.

Clinical Laboratory Techniques↗
PubMed1999

Comparison of two PF4/heparin ELISA assays for the laboratory diagnosis of heparin-induced thrombocytopenia.

Serum samples from 105 patients with suspected heparin-induced thrombocytopenia (HIT) were evaluated using the 14C-serotonin release assay (SRA), considered the "gold standard" for the diagnosis of HIT, and two enzyme-linked immunosorbent assays (ELISA) that measure anti-platelet factor (PF) 4/heparin antibodies to determine the performance characteristics of the newly available ELISA assays. Relative to the SRA, the sensitivity and specificity of the Asserachrom HPIA assay were 73% and 77%, respectively, in this population of patients. The sensitivity and specificity of the GTI-HAT assay were 60% and 93%, respectively. In serum negative by SRA, GTI-HAT and HPIA detected antibodies in 9% and 25%, respectively. Antibodies were detected by HPIA in 18% of the sera negative by both SRA and GTI-HAT. In a second study, samples evaluated from patients (n = 10) treated for established thrombosis with a low-molecular-weight heparin and who had no decrease in platelet counts, showed a weak antibody titer in 50% of the patients after 12 days of therapy by GTI-HAT, whereas the HPIA identified a strong antibody titer in 75% of the patients after 4 days. These data suggest that the currently available ELISA methods for the detection of anti-PF4/heparin antibodies offer a limited sensitivity and specificity in comparison to SRA. The ELISA assays on their own are thus of limited value for the laboratory diagnosis of HIT. SRA-positive sera do not always have positive antibody titers, and antibodies can be present in SRA-negative sera. Furthermore, these data show that ELISA methods differ in their relative sensitivities and specificities for the detection of anti-PF4/heparin antibodies.

Adult↗
PubMed1994

Laboratory diagnosis of infectious diarrhea.

The microbiology laboratory, in conjunction with the medical staff, must determine a reasonable approach to the evaluation of diarrheal stools since the cost to rule out all potential pathogens is prohibitive and control of the use of laboratory services is now a major focus in all institutions. All stool cultures should be examined for Campylobacter, Salmonella, and Shigella, the most common causes of inflammatory bacterial diarrhea in the United States. Special media for other pathogens should be added only if there is high regional endemicity or significant clinical suspicion. If a child has bloody diarrhea, a search for E coli O157:H7 is indicated. For patients with a history of raw seafood ingestion or foreign travel, the laboratory should be asked to screen specimens for Vibrio and Plesiomonas species. The report from the laboratory should specifically state what enteropathogens have been excluded, for example, "No Salmonella, Shigella, or Campylobacter isolated." A report of "negative" or "no enteric pathogens" is not very useful. Diagnosis of viral and parasitic enteritis and antibiotic-associated diarrhea require a variety of additional tests. Clinicians are encouraged to discuss these issues with the pathologist or microbiologist at their local laboratory and be familiar with community microbiology practice, particularly which organisms require a special request for the laboratory to attempt identification.

Bacterial Infections↗
PubMed2002

A brief review on the laboratory diagnosis of leptospirosis.

Leptospirosis is a ubiquitous zoonotic disease that may be maintained in either wild or domesticated animal species. These bacteria have been classified into serovars based on their antigenic characteristics and, more recently, into species based on genomic studies. They produce both chronic and acute infections. Chronic infections of serovars in the host species to which they have become adapted can result in long term shedding, providing a source of acute infection for other species. As clinical presentation can vary greatly, diagnosis often depends on laboratory methods. In addition to diagnostic testing, herd health monitoring and screening for international trade purposes are performed at veterinary laboratories. The test method selected varies depending on the samples available and the purpose of testing. An increasing variety of laboratory methods are being described for detection of bacteria and antibodies. In addition to classical methods such as culture, dark-field, microscopy and the microscopic agglutination test (MAT), a variety of polymerase chain reaction (PCR), indirect enzyme-linked immunosorbent assay (ELISA), competitive ELISA and other rapid serological tests have been described. This review describes the advantages and limitations of these assays together with other factors that may affect results and their interpretation, such as species variation, vaccination and antibiotic administration.

Journal Article↗
PubMed1979

Differential diagnosis, laboratory tests and histology in 245 patients with cholestasis.

In the course of 4 years, among 11,738 admissions there were 245 (2.08%) patients with cholestasis (106 women and 139 men). Intrahepatic cholestasis (i.c.) was detected in 46.5%, and extrahepatic (e.c.) in 53.5%. The most frequent cause of i.c. were alcoholic and nonalcoholic chr. liver disease (fatty liver, chr. hepatitis, cirrhosis) (37% and 30%), acute viral hepatitis (15%) and toxic liver injury (14%) respectively. The causes of e.c. were: choledocholithiasis (44%), cancer of the pancreatic head (15%), cancer of gallbladder and extrahepatic ducts (12%) and cancer of liver (10%). The causes of c. were benigne, in 78.2%, while malignant neoplasms were present in 21.8%. Out of the multitude of laboratory tests two appeared particularly significant: glut, transpeptidase was pathologic in 81% of alcoholic liver disease, in 62% of the cases with obstructive jaundice and in 27.7% of malignant neoplasms. LX-lipoprotein examined in 52 patients was positive in 24% of i.c., and 60% of e.c. Proliferation of bile ducts was the most frequent finding in surgical liver biopsies in choledocholithiasis cases.

Adolescent↗
PubMed2000

Laboratory diagnosis of nosocomial pneumonia.

The second most common nosocomial infection in the United States is pneumonia, with the highest rates seen in patients requiring mechanical ventilation. Nosocomial pneumonia is a serious disease associated with significant morbidity and mortality; crude mortality rates have been estimated at 20% to 50%. The rapid institution of appropriate antimicrobial therapy has been shown to improve mortality in patients with ventilator associated nosocomial pneumonia. Thus, the identification of nosocomial pneumonia with a timely microbiologic diagnosis is important for the management of these patients. However, the accurate diagnosis of nosocomial pneumonia, along with identification of the responsible organism(s), can be challenging. This task becomes even more difficult in patients who are mechanically ventilated. The presence of new pulmonary infiltrates along with clinical criteria including fever, cough, and purulent secretions are neither sensitive nor specific for the diagnosis of nosocomial pneumonia. The laboratory can enhance the accuracy of pneumonia diagnosis, as well as provide the identification of an etiologic organism(s). There are, however, many challenges which confront the laboratory including: the ability to identify organisms from an extensive microbiologic spectrum; distinguishing colonization from infection of predominately gram-negative oropharyngeal flora; and providing timely results. This article reviews the various diagnostic tests available for nosocomial lung infections, and in particular, ventilator associated pneumonia including: blood cultures; pleural fluid; expectorated sputum; endotracheal aspirates; and respiratory specimens obtained by more invasive techniques using bronchoscopy and transthoracic needle aspiration. Emphasis is placed on optimal specimen collection, the processing of samples in the laboratory, and on the evaluation of potential risks and benefits associated with the varying techniques.

Bacteria, Anaerobic↗