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A case of highly invasive native valve endocarditis, occurring in a young man with a preexisting cardiac anomaly, and caused by Staphylococcus lugdunensis is described. Despite treatment with antibiotics the disease progressed with further growth of the bacterial vegetations and invasion of the myocard. The patient was successfully treated by surgery. Twenty three cases of endocarditis by S. lugdunensis have now been described. This organism is a major cause of destructive endocarditis accompanied by high mortality. As the outcome usually is more favourable in patients who underwent valve replacement surgery, rapid identification to the species level of coagulasenegative staphylococci is required. S. lugdunensis endocarditis should be treated preferably by surgical intervention.

Nucleic acid sequence-based amplification, NASBA, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24 Mycoplasma pneumoniae strains. A set of primers was chosen from the 16S rRNA sequence alignment of Mycoplasma species. The nucleotide sequences of the (-)RNA amplicons were determined for M. pneumoniae strains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24 M. pneumoniae strains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24 M. pneumoniae strains into two types by other typing methods, confirming the reliability of this technique.

A woman with systemic lupus erythematosus (SLE) presented with a zoster eruption. Transverse myelitis developed at the site of the dermatomal distribution of the rash. SLE and varicella zoster virus (VZV) can both cause myelitis, and are difficult to differentiate. The topographic association between the cutaneous and the neurological involvement suggesting VZV myelitis was confirmed by polymerase chain reaction (PCR) for VZV in the cerebrospinal fluid. This case illustrates the potential role of the selective amplification of VZV DNA in cerebrospinal fluid to diagnose central neurological complications associated with VZV. The value of magnetic resonance imaging of the spinal cord in the evaluation of patients with myelitis is emphasized.

A case of probable vertical transmission of Mycoplasma pneumoniae is presented. The presence of M pneumoniae was demonstrated by the polymerase chain reaction (PCR) in the nasopharyngeal aspirate of a newborn who developed pneumonia shortly after birth. This result was confirmed by performing a second PCR, amplifying another part of the genome of M pneumoniae. It is concluded that M pneumoniae can be added to the long list of pathogens known to cause congenital pneumonia.

A 60-year-old heterosexual man with AIDS was admitted to hospital with dyspnea, a severe paroxysmal non-productive cough of two months' duration, low-grade fever and exhaustion. Bordetella pertussis was cultured from a bronchoalveolar lavage specimen. After erythromycin therapy (500 mg q.i.d. for two weeks) all respiratory symptoms resolved progressively over a four-week period. Bordetella pertussis should be added to the long list of pathogens that may cause respiratory disease in persons with HIV infection.

An overview is presented of different nucleic acid amplification techniques, particularly the polymerase chain reaction (PCR). The possible applications of the PCR in medical microbiology and the different phases of the PCR are discussed. Four PCR applications, as elaborated and used in the authors' laboratory are presented: the diagnoses of Mycoplasma pneumoniae infections, herpes simplex virus encephalitis and Toxoplasma gondii encephalitis, and the detection of Mycobacterium leprae. For the first and the last of these, original internal controls to detect the presence of inhibitors were elaborated. The target amplicon was inserted in a plasmid and the central part modified, either by insertion of a sequence of foreign DNA (Mycoplasma pneumoniae) or excision of a sequence (Mycobacterium leprae). The addition of a small amount (in order to avoid inhibition of the diagnostic reaction) of these plasmids to the reaction mixture, results in coamplification of this modified DNA with the same primers as the target DNA. During three successive winter periods, nasopharyngeal aspirates from children presenting with respiratory infections at the Universitair Ziekenhuis Antwerpen, were examined for viruses and for M.pneumoniae by the PCR. The PCR for herpes virus in cerebrospinal fluid is a powerful diagnostic technique. Five cases could be documented, one of them being the second known case of herpes virus encephalitis of the brain stem. The sensitivity of the PCR for Toxoplasma gondii encephalitis as performed in our laboratory on cerebro-spinal fluid probably needs to be improved. The detection of M. leprae by a PCR for the 16S rRNA can detect as little as 30 organisms, which is insufficiently sensitive for the diagnosis of paucibacillary leprosy in skin biopsy specimens. The technique allowed for the first time the detection of M. leprae DNA in nose mucosae of patients and some of their contacts. The PCR is very promising to elucidate the etiology of diseases the responsible agent of which is present in too small numbers to be detected by traditional techniques, as well as for the diagnosis of agents difficult or impossible to cultivate or to identify still unknown agents.

Nose swabs from 4 paucibacillary (PB) and 8 multibacillary (MB) leprosy patients and their contacts were tested for the presence of Mycobacterium leprae by two polymerase chain reactions (PCR); 30% of the samples contained inhibitors for the PCR, 1 of 52 (1.9%) swabs and 13 of 164 (7.9%) swabs were positive for M. leprae among contacts of PB and MB patients, respectively. Since this difference is not significant, and some positives were found among contacts of MB patients treated and cured of their infection, it is concluded that the observed infections are community acquired.

The polymerase chain reaction (PCR) was used to amplify a 209 base-pair fragment of Mycoplasma pneumoniae DNA. The amplicon was transferred into a plasmid and a 680 base-pair piece of foreign DNA was inserted between the two amplimer sites. Plasmid DNA was added to the reaction mixture as an internal control for the polymerase chain reaction. Since the original hybridization target sites were included in this construction, one pair of amplimers could be used to amplify both the target DNA and the internal control DNA. Separation of internal control from target DNA after amplification was easily obtained on agarose gel electrophoresis. For the analysis of clinical samples with the polymerase chain reaction, the addition of internal control DNA allowed monitoring of the overall effectiveness of the amplification in each tube. With this technique approximately one-third of the tests were shown to be unsatisfactory due to technical errors or contaminating inhibitors. Adequate internal controls are necessary to avoid false-negative results with the polymerase chain reaction.

Manganese has been proven to be an essential trace element in animals since 1931. Today, it is known that manganese deficiency results in a wide variety of structural and physiological defects in animals. For humans, the safe and adequate range of intakes for manganese by enteral nutrition is 2.0 to 5 mg per day; for parenteral nutrition a range of 0.15 to 0.8 mg per day is proposed. The manganese content of components of enteral nutrition (EN) and total parenteral nutrition (TPN) solutions is determined using electrothermal atomic absorption spectrometry. Comparison is made between calculated and measured values. The manganese present in supplementary nutrition and flavor stuffs is also measured. The intake of manganese by EN is calculated based on the daily administration, and is found to vary from 192 to 7373 micrograms per day. In prepared TPN-solutions, the manganese contribution of all components before adding the Oligo Complex is found to be less than 0.2% of the total manganese content. After addition of the Oligo Complex mixture, a mean daily intake of 5.02 +/- 0.16 mg per day is found.

The sensitivity of the polymerase chain reaction (PCR) on the DNA coding for the species-specific fragment of 16S rRNA of Mycobacterium leprae studied on mouse foot pad harvests and human skin biopsies varies widely between 1 and 3 x 10(4) organisms. This is probably the result of variations in the proportions of organisms with sufficiently intact DNA suitable for PCR. Preserving human skin biopsies for 3 weeks at an ambient temperature even after boiling for 6 minutes gives rise to a 10-fold decrease in sensitivity. Fixation of tissues in formol 10% or Lowy fixative or preserving in Dubos OAA broth is very harmful to the PCR, mainly due to the enhancement of an inhibitory effect on the PCR reaction. For preservation, the best choice at the moment seems to be alcohol 70%. Sample preparation of five cycles of freeze-boiling is simple and generally more efficient than proteinase K treatment and DNA extraction.

Extracts prepared from 100 samples of higher plants were evaluated for antiviral activity against several viruses, including Semliki forest, coxsackie, measles, poliomyelitis, herpes, and adeno viruses. The plants were chosen from 73 genera and 43 families on the basis of literature data and medicinal reports on antimicrobial activity. The results of the antiviral testing showed that eight of these extracts exhibited a pronounced activity against one or more of the test viruses. Another three plant extracts showed a moderate level of antiviral activity against some of the test viruses.