Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.

Review of diagnostic screening instruments for alcohol and other drug use and other psychiatric disorders

In recent years there has been a growing recognition that many people with drug or alcohol problems are also experiencing a range of other psychiatric and psychological problems. The presence of concurrent psychiatric or psychological problems is likely to impact on the success of treatment services. These problems vary greatly, from undetected major psychiatric illnesses that meet internationally accepted diagnostic criteria such as those outlined in the Diagnostic and Statistical Manual (DSM-IV) of the American Psychiatric Association (1994), to less defined feelings of low mood and anxiety that do not meet diagnostic criteria but nevertheless impact on an individual’s sense of wellbeing and affect their quality of life.

Orofacial granulomatosis: A diagnostic problem for the unwary and a management dilemma. Case reports

Orofacial granulomatosis is a condition that, may be difficult to diagnose for those unfamiliar with the entity. This paper describes two cases and addresses the presentation, pathogenesis and treatment. The clinical recognition of his condition is important as is the subsequent investigation by an appropriate specialist. Management of patients needs to take into account the results of further investigations, the patient's expectations, and the severity of the condition.

Prevalence and diagnostic workup of primary aldosteronism: new knowledge and new approaches

No Abstract

Diagnostic performance of Antineutrophil Cytoplasmic Antibody tests for Idiopathic Vasculitides: Metaanalysis with a focus on antimyeloperoxidase antibodies

Objective. The diagnostic value of tests for antimyeloperoxidase antibodies (anti-MPO) for systemic vasculitis is less established than that for cytoplasmic antineutrophil cytoplasmic antibody (cANCA)/antiproteinase 3 antibodies (anti-PR3). Controversy exists regarding the optimal utilization of indirect immunofluorescence (IIF) ANCA testing versus antigen-specific ANCA testing. To summarize the pertinent data, we conducted a metaanalysis examining the diagnostic value of ANCA testing systems that include assays for anti-MPO. Methods. We performed a structured Medline search and reference list review. Target articles in the search strategy were those reporting the diagnostic value of immunoassays for anti-MPO for the spectrum of systemic necrotizing vasculitides that includes Wegener's granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, and isolated pauci-immune necrotizing or crescentic glomerulonephritis, regardless of other types of ANCA tests. Inclusion criteria required specification of a consecutive or random patient selection method and the use of acceptable criteria for the diagnosis of vasculitis exclusive of ANCA test results. Weighted pooled summary estimates of sensitivity and specificity were calculated for anti-MPO alone, anti-MPO + perinuclear ANCA (pANCA), and anti-MPO/pANCA + anti-PR3/cANCA. Results. Of 457 articles reviewed, only 7 met the selection criteria. Summary estimates of sensitivity and specificity (against disease controls only) of assays for anti-MPO for the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval 26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively. When the pANCA pattern by IIF was combined with anti-MPO testing, the specificity improved to 99.4%, with a lower sensitivity, 31.5%. The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA) increased the sensitivity to 85.5% with a specificity of 98.6%. Conclusion. These results suggest that while anti-MPO is relatively specific for the diagnosis of systemic vasculitis, the combination system of immunoassays for anti-MPO and IIF for pANCA is highly specific and both tests should be used together given the high diagnostic precision required for these conditions. Because patients with ANCA associated vasculitis have either anti-MPO with pANCA or anti-PR3 with cANCA, and rarely both, a combined ANCA testing system including anti-PR3/cANCA and anti-MPO/pANCA is recommended to optimize the diagnostic performance of ANCA testing. (J Rheumatol 2001;28:1584-90)

Some factors affecting diagnostic accuracy

Diagnosis involves a complex and overlapping series of steps, each of which may be a source of error and of variability between clinicians. This variation may involve the ability to elicit relevant information from the client or animal, in the accuracy, objectivity and completeness of relevant memory stores, and in psychological attributes including tolerance for uncertainty and willingness to engage in constructive self-criticism. The diagnostic acumen of an individual clinician may not be constant, varying with external and personal factors, with different clients and cases, and with the use made of tests. In relation to clients, variations may occur in the ability to gain their confidence, to ask appropriate questions and to evaluate accurately both verbal and nonverbal responses. Tests may introduce problems of accuracy, validity, sensitivity, specificity, interpretation and general appropriateness for the case. Continuing effectiveness as a diagnostician therefore requires constant attention to the maintenance of adequate and up-to-date skills and knowledge relating to the animals and their diseases and to tests, and of sensitive interpersonal skills.

Diagnostic accuracy of paediatric echocardiograms interpreted by individuals other than paediatric cardiologists

Objective: To assess the diagnostic error rate among echocardiograms undertaken by individuals other than paediatric cardiologists in our referral area. Methodology: External group: The charts and echocardiographic results of all patients who had undergone outside echocardiograms between January 1996 and December 1999 were reviewed (110). Age at echocardiography, diagnostic complexity, presence of any diagnostic errors and the severity of any diagnostic errors were identified. Internal group: To assess our own error rate, the initial echocardiographic diagnoses of 100 patients undergoing cardiac catheterisation or corrective surgery were compared with the post-catheterisation or postoperative diagnoses. Age and diagnostic complexity were also assessed in the control group. Results: Diagnostic errors occurred in 47/110 patients (44%) of the externally studied group (of which 24% were either major or life threatening) as opposed to 3/100 of the internally studied group, despite the internally studied group being of increased diagnostic complexity. Errors were more common and of increased severity in infants less than 1 month of age but extended throughout all age groups. Major and life threatening errors increased with increasing diagnostic complexity. In the externally studied group, 8/47 errors were patients inappropriately designated as normal. Four of these patients required cardiac surgery or interventional cardiac catheterisation. Conclusions: This study suggests an unacceptably high error rate in paediatric echocardiographic diagnoses by non-paediatric cardiologists throughout all age groups. Such errors are more likely in younger infants and with increasing diagnostic complexity.

Rapid diagnosis in pediatric infectious diseases: The past, the present and the future

The focus of rapid diagnosis of infectious diseases of children in the last decade has shifted from variations of the conventional laboratory techniques of antigen detection, microscopy and culture to that of molecular diagnosis of infectious agents. Pediatricians will need to be able to interpret the use, limitations and results of molecular diagnostic techniques as they are increasingly integrated into routine clinical microbiology laboratory protocols. PCR is the best known and most successfully implemented diagnostic molecular technology to date. It can detect specific infectious agents and determine their virulence and antimicrobial genotypes with greater speed, sensitivity and specificity than conventional microbiology methods. Inherent technical limitations of PCR are present, although they are reduced in laboratories that follow suitable validation and quality control procedures. Variations of PCR together with advances in nucleic acid amplification technology have broadened its diagnostic capabilities in clinical infectious disease to now rival and even surpass traditional methods in some situations. Automation of all components of PCR is now possible. The completion of the genome sequencing projects for significant microbial pathogens, in combination with PCR and DNA chip technology, will revolutionize the diagnosis and management of infectious diseases.