Volume 33, Issue 4

Recent publications have shown that primary immunodeficiency diseases must be expected at a prevalence rate higher than previously believed. Early recognition of such disorders is of tremendous relevance to patients, because therapeutic measures may only be effective in reducing morbidity and mortality if introduced early. This review describes a laboratory approach based on clinical problems of the individual patient and, if known, the microorganisms causing severe infections. A stepwise approach will finally result in a molecular diagnosis.

Recent publications have shown that primary immunodeficiency diseases must be expected at a prevalence rate higher than previously believed. Early recognition of such disorders is of tremendous relevance to patients, because therapeutic measures may only be effective in reducing morbidity and mortality if introduced early. This review describes a laboratory approach based on clinical problems of the individual patient and, if known, the microorganisms causing severe infections. A stepwise approach will finally result in a molecular diagnosis.

Mandatory are short periods of sample storage, including transport time. Generally, 2 to a maximum of 4 h is a prerequisite for microbiological rapid diagnostics of acceptable quality (s. all Microbiological – Infectiological Quality Standards – MIQ). This requirement is 1) to prevent overlooking potentially pathogenic microorganisms by extended storage times resulting in their death after 2 h, or overgrowth by colonisation of other bacteria or fungi, 2) to avoid economic losses, e.g., because of the combination with the German Diagnostic Related group (G-DRG) with short times of stay in the hospital or to achieve so-called complication points by detecting resistant microorganisms in time and therefore optimising reimbursement, 3) to fulfil the demand for a specific therapy aimed at the causative infectious agent which may not be detected after a longer storage time and to avoid higher mortality rates by failure or delayed times of specific therapy because of delayed isolation of resistant bacteria.

Mandatory are short periods of sample storage, including transport time. Generally, 2 to a maximum of 4 h is a prerequisite for microbiological rapid diagnostics of acceptable quality (s. all Microbiological – Infectiological Quality Standards – MIQ). This requirement is 1) to prevent overlooking potentially pathogenic microorganisms by extended storage times resulting in their death after 2 h, or overgrowth by colonisation of other bacteria or fungi, 2) to avoid economic losses, e.g., because of the combination with the German Diagnostic Related Group (G-DRG) with short times of stay in the hospital or to achieve so-called complication points by detecting resistant microorganisms in time and therefore optimising reimbursement, 3) to fulfil the demand for a specific therapy aimed at the causative infectious agent which may not be detected after a longer storage time and to avoid higher mortality rates by failure or delayed times of specific therapy because of delayed isolation of resistant bacteria.

Since the last decades of the past century the frequency of rickettsial tick-bite fever has extremely increased. Patients often do not feel or realize a tick-bite. The most important diagnostic criterion after a tick-bite is a blue-red, later black skin ulcer with a black rim, called eschar or tache noir, and local lymph adenopathy. Clinical diagnosis can already be based on these symptoms. In some cases, the typical dark skin discoloration is absent or the color change may be misinterpreted as a minor skin injury. Also, exanthems occur and are of a maculo-papulous appearance. They show different variations and are often absent. In serological diagnosis, nearly half of the patients show antibodies against rickettsiae. Doxycycline as the drug of choice for therapy with one-day treatment (2×200 mg) has proven very effective. Alternative antibiotics include josamycin and ciprofloxacin. Whenever fever, head and/or muscle pain occur combined with typical skin involvement, particularly an exanthem or an eschar, tick-bite fever should be taken into consideration as a differential diagnosis when travelers have returned from a region where rickettsiae are known to be transferred by tick-bites. In returning travelers from defined areas of the American continent, Rocky Mountain spotted fever should be considered because of its complicated course with a mortality rate up to 5%. That is why in all cases, immediate antibiotic therapy should be initiated. Only then a quick defervescence can be accomplished after a one-day antibiotics therapy.

During the last decades of the past century the frequency of rickettsial tick-bite fever has extremely increased. Patients often do not feel or realize a tick-bite. The most important diagnostic criterion after a tick-bite is a blue-red, later black skin ulcer with a black rim, called eschar or tache noir, and local lymph adenopathy. Clinical diagnosis can already be based on these symptoms. In some cases, the typical dark skin discoloration is absent or the color change may be misinterpreted as a minor skin injury. Also, exanthems occur and are of a maculo-papulous appearance. They show different variations and are often absent. In serological diagnosis, nearly half of the patients show antibodies against rickettsiae. Doxycycline as the drug of choice for therapy with one-day treatment (2×200 mg) has proven very effective. Alternative antibiotics include josamycin and ciprofloxacin. Whenever fever, head and/or muscle pain occur combined with typical skin involvement, particularly an exanthem or an eschar, tick-bite fever should be taken into consideration as a differential diagnosis when travelers have returned from a region where rickettsiae are known to be transferred by tick-bites. In returning travelers from defined areas of the American continent, Rocky Mountain spotted fever should be considered because of its complicated course with a mortality rate up to 5%. That is why in all cases, immediate antibiotic therapy should be initiated. Only then a quick defervescence can be accomplished after a one-day antibiotics therapy.

Primary aldosteronism (PA) affects 5% to 10% of all patients with arterial hypertension and is mainly caused by either aldosterone producing adenoma or by idiopathic hyperaldosteronism due to bilateral adrenal hyperplasia. Patients with PA demonstrate a significantly increased risk of cardiovascular and renal disease when compared to patients with essential hypertension. However, PA can effectively be treated underlining the importance of screening for and diagnosing of PA. Measurement of the aldosterone to renin ratio (ARR) should be performed as a screening test. Patients with an elevated ARR should undergo confirmatory testing for PA, which generally aims to evaluate whether elevated aldosterone levels are suppressible by salt and volume loading or ACE inhibitor therapy. In patients with PA, subtype classification is important to identify patients who are eligible for surgical adrenalectomy (unilateral disease) and to differentiate them from patients with bilateral disease who should be treated with mineralocorticoid receptor antagonists. Adrenal imaging using computed tomography is the first step in subtype classification. Adrenal venous sampling is the preferred method to differentiate between unilateral and bilateral disease, but the posture test can also be useful in selected patients. The present review provides a brief overview of the diagnostic procedures for PA. Nevertheless, we acknowledge that the diagnostic accuracy of screening and confirmation tests for PA is at present insufficiently validated and further studies are required. Zusammenfassung Ein primärer Hyperaldosteronismus (PHA) liegt bei 5% bis 10% aller Patienten mit arterieller Hypertonie vor und wird in den meisten Fällen entweder durch ein Aldosteron produzierendes Nebennierenadenom oder durch einen idiopathischen Hyperaldosteronismus aufgrund einer bilateralen adrenalen Hyperplasie verursacht. Patienten mit PHA haben, verglichen mit Patienten mit essentieller Hypertonie, ein signifikant erhöhtes Risiko für kardiovaskuläre und renale Erkrankungen, wobei der PHA sehr effektiv behandelt werden kann. Dies unterstreicht die Wichtigkeit des Screenings und der Diagnosestellung eines PHA. Die Messung der Aldosteron-Renin-Ratio (ARR) sollte als Screeningtest durchgeführt werden. Patienten mit einer erhöhten ARR sollten einem Bestätigungstest zugeführt werden, der darauf abzielt, zu evaluieren, ob die erhöhten Aldosteronwerte durch Salz- und Volumenbelastung bzw. durch eine ACE-Hemmertherapie supprimierbar sind. Eine Subtypenklassifizierung des PHA ist wichtig, um Patienten zu identifizieren, die mittels Adrenalektomie behandelt werden können (unilaterale Erkrankung) und sie von Patienten mit einer bilateralen Erkrankung zu unterscheiden, die mit Mineralokortikoid-Rezeptor-Antagonisten therapiert werden sollten. Eine radiologische Bildgebung mittels Computertomographie ist der erste Schritt in der Subtypenklassifizierung. Das Nebennierenvenensampling ist die beste Methodik, um zwischen unilateraler und bilateraler Erkrankung zu unterscheiden, wobei der Lagerungstest bei ausgewählten Patienten ebenfalls seine Berechtigung hat. In der vorliegenden Übersicht geben wir einen Überblick über die diagnostischen Methoden für den PHA, wobei wir anmerken, dass die diagnostische Genauigkeit der Screening- und Bestätigungsteste für den PHA derzeit nicht ausreichend gut validiert ist und weitere Studien notwendig sind.

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergence in the entire genome of >8%, HBV genomes have been classified into nine groups designated A to I. The genotypes of HBV have distinct geographic distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and the natural history of infection and response to antiviral therapy, further evaluations employing larger cohorts of patients are necessary to provide conclusive evidence. The analytical sensitivity of HBV surface antigen (HBsAg) and antibodies to the HBV surface antigen (anti-HBs) assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NATs) has so far been poorly investigated. Preliminary results show that new real-time NATs detect genotypes A to G with equal sensitivity. Different mechanisms intervening at the translational or post-translational level, including conformational changes, hydrophobic changes, insertion of basic residues, and reduced synthesis or secretion of HBsAg may account solely or in combination for escape mutations to the immune response and to detection in HBsAg immunoassays. The clinical significance of S gene mutants needs, similarly to that of HBV genotypes, to be investigated further. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is being continuously improved. Immunoassays based on polyclonal capture antibodies demonstrate the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants in Western Europe and assays for the (differential) screening of mutants need to be developed and evaluated. Zusammenfassung Die genetische Variabilität des Hepatitis B-Virus (HBV) stellt eine Herausforderung an die Sensitivität immunologischer und molekularer Diagnostikverfahren dar. Basierend auf einem Sequenzunterschied von mehr als 8% auf dem kompletten Genom, wurden neun unterschiedliche HBV-Genotypen A bis I definiert. Jeder dieser Genotypen besitzt eine spezifische geografische Verbreitung. Obwohl vorläufige klinische Studien eine Auswirkung des Genotyps auf den klinischen Verlauf einer HBV-Infektion sowie das Ansprechen auf antivirale Therapien zeigen, fehlt es noch an ausreichend Studien mit größeren Patientenzahlen, bevor sich ein vollständiges Bild zu diesem Thema ergibt. Eine Auswirkung des Genotyps auf die analytische Sensitivität von HBsAg- und anti-HBs-Assays ist bekannt, bedarf aber ebenfalls weiterer Studien. Der Zusammenhang zwischen HBV-Genotyp und Nukleinsäureamplifikationstests (NATs) wurde nur unzureichend untersucht. Jedoch zeigten vorläufige Resultate mit Echtzeit-NATs keinen Einfluss der Genotypen A bis H auf die Sensitivität dieser Tests. Unterschiedliche Mechanismen auf translationaler und post-translationaler Ebene, einschließlich koformationeller und hydrophober Veränderungen, Insertionen basischer Aminosäuren, sowie reduzierte Synthese und Ausschüttung des HBsAg, könnten einzeln oder im Zusammenspiel für „Escape“ – Mutationen bei Immunantwort und Nachweis durch HBsAg-Immunoassays verantwortlich sein. Die klinische Bedeutung von S-Gen-Mutation erfordert, analog zu den HBV-Genotypen, weitere Untersuchungen. HBV-Mutationen bleiben zeitlich stabil und können sowohl horizontal als auch vertikal übertragen werden. Die Sensitivität von HBsAg-Immunoassays auf diese Mutationen wird kontinuierlich verbessert. Immunoassays, die auf polyklonalen Antikörpern basieren, zeigen die höchste Sensitivität beim Nachweis von rekombinanten HBV-Strängen oder mutierten HBsAg-Formen. Auf der anderen Seite ist eine optimale Spezifität nicht gewährleistet. Die momentan angewandten Immunoassays zum HBsAg-Nachweis müssen verbessert werden, um bekannte Mutationen sowie niedrige HBsAg-Lasten zu detektieren. Außerdem werden zusätzliche epidemiologische Daten über die Ausbreitung von HBsAg-Mutanten in Westeuropa benötigt und neue Immunoassays zum (differentiellen) Nachweis von Mutationen müssen entwickelt und bewertet werden.

Medical students are exposed to infectious diseases during the course of their clinical training. Unfortunately, vaccination rates among medical students remain insufficient. However, immunizations against vaccine-preventable diseases should be carried out before the students enter clinical courses. This is vital in order to prevent nosocomial infections. We screened 366 medical students in their first clinical year for hospital-related viral diseases. Serum samples were collected between April and May 2007. Antibody testing was carried out using commercial ELISA systems against measles, mumps, rubella, varicella, hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV). Overall, 63.9% (n=234) of the students were sufficiently vaccinated against HBV. In contrast, 31.7% (n=116) had not received any HBV vaccine dosage, and 4.4% (n=16) had not completed the full vaccine cycle (<3 dosage). Remarkably, two students showed serological markers of resolved HBV infection. In addition, one student was HCV-positive and one was HIV-positive, respectively. The following seronegative rates were found: measles (7.9%), mumps (17.5%), rubella (6.5%), and varicella (2.2%). Further work is needed to identify optimal strategies for improving vaccination rates among medical students. It is imperative to identify and limit possible disparities in immunity of vaccine-preventable diseases before initial patient contact. With regard to the primary diagnosis of serious virus diseases including HBV, HCV and HIV, medical students should be screened for these blood borne pathogens.

Medical students are exposed to infectious diseases during the course of their clinical training. Unfortunately, vaccination rates among medical students remain insufficient. However, immunizations against vaccine-preventable diseases should be carried out before the students enter clinical courses. This is vital in order to prevent nosocomial infections. We screened 366 medical students in their first clinical year for hospital-related viral diseases. Serum samples were collected between April and May 2007. Antibody testing was carried out using commercial ELISA systems against measles, mumps, rubella, varicella, hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV). Overall, 63.9% (n=234) of the students were sufficiently vaccinated against HBV. In contrast, 31.7% (n=116) had not received any HBV vaccine dosage, and 4.4% (n=16) had not completed the full vaccine cycle (<3 dosage). Remarkably, two students showed serological markers of resolved HBV infection. In addition, one student was HCV-positive and one was HIV-positive, respectively. The following seronegative rates were found: measles (7.9%), mumps (17.5%), rubella (6.5%), and varicella (2.2%). Further work is needed to identify optimal strategies for improving vaccination rates among medical students. It is imperative to identify and limit possible disparities in immunity of vaccine-preventable diseases before initial patient contact. With regard to the primary diagnosis of serious virus diseases including HBV, HCV and HIV, medical students should be screened for these blood borne pathogens.

Patients' results require reference points, so-called guide values. The quality of guide values was surprisingly neglected in the past, whereas the quality assurance of analytical procedures has made enormous progress in the last decades. Several concepts of guide values are used for the interpretation of analytical results. These values discriminate at least two subgroups of populations, of which samples are sent to medical laboratories (a non-diseased and a diseased subgroup). The concepts are classified into four groups: reference limits, decision limits, action limits, and therapeutic limits. These groups have different benefits and disadvantages which are demonstrated by some practical examples.

Patients' results require reference points, so-called guide values. The quality of guide values was surprisingly neglected in the past, whereas the quality assurance of analytical procedures has made enormous progress in the last decades. Several concepts of guide values are used for the interpretation of analytical results. These values discriminate at least two subgroups of populations, of which samples are sent to medical laboratories (a non-diseased and a diseased subgroup). The concepts are classified into four groups: reference limits, decision limits, action limits, and therapeutic limits. These groups have different benefits and disadvantages which are demonstrated by some practical examples.