Same-day or next-day sentinel node biopsy after lymphoscintigraphy for melanoma using 99m Tc-labelled antimony sulphide colloid.

BACKGROUND: Two recent publications have reported that a shorter interval between preoperative lymphoscintigraphy and sentinel node biopsy (SNB) is associated with improved survival of patients with primary cutaneous melanoma. The aims of this study were to analyse prospectively collected survival data for patients who had SNB on the same day as lymphoscintigraphy or the day after; and to assess tracer migration from sentinel nodes to second-tier nodes after lymphoscintigraphy on the previous day. METHODS: Outcome data were obtained for patients who had lymphoscintigraphy and SNB on the same day (time interval less than 8 h) or the next day (interval more than 16 h). In a separate prospective cohort, same-day and next-day lymphoscintigraphic images of sentinel nodes and second-tier nodes were compared. RESULTS: Following lymphoscintigraphy, 2848 patients had same-day and 3328 had next-day SNB. Survival outcomes did not differ between these groups. In a prospectively studied cohort of 30 patients, none had significant tracer migration from sentinel nodes to second-tier nodes on imaging the following day. CONCLUSION: No difference in survival after same- or next-day sentinel node biopsy is seen when 99m Tc-labelled antimony sulphide colloid is used. This may be because of less tracer migration to second-tier nodes.

ANTECEDENTES: Dos publicaciones recientes han señalado que reducir el intervalo entre la linfografía isotópica preoperatoria y la biopsia del ganglio centinela (sentinel node biopsy, SNB) se asocia con una mejor supervivencia en pacientes con melanoma maligno primario. Los objetivos de este estudio fueron los siguientes: (1) analizar los datos de supervivencia recogidos prospectivamente en pacientes en los que se realiza la SNB el mismo día o al día siguiente de la linfografía isotópica, y (2) evaluar la migración del marcador desde los ganglios centinela a los ganglios de segundo nivel a partir de la linfografía del día anterior. MÉTODOS: Se analizaron los resultados de los pacientes a los que se realizó una linfografía isotópica y la SNB el mismo día (intervalo de tiempo < 8 h) o al día siguiente (intervalo > 16 h). En una cohorte prospectiva diferente, se compararon las imágenes de los ganglios centinela y de los ganglios de segundo nivel en linfografías isotópicas realizadas el mismo día o al día siguiente. RESULTADOS: Tras la linfografía isotópica, se realizó la SNB el mismo día en 2.848 pacientes y al día siguiente en 3.328 pacientes. No hubo diferencias en la supervivencia entre ambos grupos. En una cohorte de 30 pacientes estudiada de forma prospectiva, no hubo migración significativa del trazador de los ganglios centinela a los ganglios de segundo nivel en las imágenes obtenidas al día siguiente. CONCLUSIÓN: La mejor supervivencia que se obtiene cuando se realiza la SNB poco después de la linfografía isotópica podría explicarse por una diferencia en la migración del trazador a los ganglios de segundo nivel entre los dos marcadores utilizados: nanocoloide de albúmina humana o coloide de sulfuro de antimonio, ambos marcados con 99mTc. En este estudio y con este último marcador, no se observó migración a ganglios de segundo nivel durante la noche.

Rapid diversification of a species-rich genus of neotropical rain forest trees.

Species richness in the tropics has been attributed to the gradual accumulation of species over a long geological period in stable equatorial climates or, conversely, to speciation in response to late Tertiary geological events and unstable Pleistocene climates. DNA sequence data are consistent with recent diversification in Inga, a species-rich neotropical tree genus. We estimate that speciation was concentrated in the past 10 million years, with many species arising as recently as 2 million years ago. This coincides with the more recent major uplifts of the Andes, the bridging of the Isthmus of Panama, and Quaternary glacial cycles. Inga may be representative of other species-rich neotropical genera with rapid growth and reproduction, which contribute substantially to species numbers in the world's most diverse flora.

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis.

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.

Manufacturing of a fiber optic Young's double pinhole interferometer for use as a 3-D profilometer.

The method used to manufacture a Young's double pinhole interferometer is dis ussed. This interferometer is destined to be used in a surface profilometer using two wavelengths so that the zero order fringe can be determined. Hence stringent requirements are placed on the absolute length difference between the two output fibers of a single mode coupler. These requirements are discussed along with the manufacturing process. The interferometer is shown along with measurements showing a length difference on the order of 6microm.

Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis.

AIMS: To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS: Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS: IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS: IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.

Hepatitis Bs antibody in alcoholic cirrhosis.

Sera from patients with chronic liver disease were tested for antibody against hepatitis B surface antigen by radioimmunoassay. The antibody was found in 25% of patients with alcoholic cirrhosis and in 52% when alcoholic cirrhosis was associated with portal hypertension, these results being significantly higher than in a matched control population. Other forms of chronic liver disease did not differ from the control population. Hepatitis B virus infection might be a factor in determining which alcoholic patients go on to develop chronic liver disease and cirrhosis.

Do IgA, IgE, and IgG avidity tests have any value in the diagnosis of toxoplasma infection in pregnancy?

AIM: To determine the value of tests for specific IgA, IgE, and IgG avidity in diagnosing Toxoplasma gondii infection during pregnancy. METHODS: In a retrospective study, current serological tests (dye test and three IgM assays with different sensitivities) were compared with immunosorbent agglutination assays (ISAGA) for specific IgA and IgE and an IgG avidity enzyme linked immunosorbent assay (ELISA). Patient group 1 comprised six women with definite or probable infection during pregnancy determined by congenital toxoplasmosis or laboratory results. Group 2 comprised seven women infected during or before 11 pregnancies (two consecutive pregnancies in two patients and three in a third). RESULTS: One patient in group 1 seroconverted during pregnancy. IgA ISAGA and avidity confirmed acute infection when confirmatory IgM ELISA remained negative. In five of six patients from group 1, IgA and IgE ISAGA and avidity confirmed acute infection. In group 2, the dye test titre was raised in seven of 11 pregnancies (six of seven patients). Specific IgM and IgA were positive during all 11 pregnancies. IgE ISAGA was positive in only four of 11 pregnancies (three of seven patients), but negative results in the remainder may exclude acute infection. High avidity antibodies indicative of past infection were found in four of 11 pregnancies (two of seven patients). CONCLUSIONS: Each test improved diagnosis or timing of infection but no single test was ideal. The IgA ISAGA was sensitive and detected seroconversion. Positive IgE ISAGA and low avidity both confirmed infection, whereas negative IgE may exclude acute infection. High avidity diagnosed past infection but persistence of low avidity reduced its value to differentiate acute and past infection. Further studies with larger patient groups are needed to determine the optimum diagnostic strategy. These techniques are valuable in complementing existing tests.

Clonal analysis of non-typable Haemophilus influenzae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell polypeptides.

A collection of 223 strains of non-typable Haemophilus influenzae was assembled. Most strains were isolated from hospital in-patients in North East Scotland between 1984 and 1989. These isolates were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell polypeptides. Variability was assessed in terms of apparent molecular weight differences between the protein profiles. Isolates were grouped on the basis of Dice coefficients of similarity and assigned to clones. Of the 223 strains, 147 were unique; the remaining strains were assigned to multi-member clones of which 13 clones had two members, one clone had three members, three clones had four members, three clones had five members, two clones had six members and one clone had eight members. The longest time interval between isolation of clonally related strains virtually equalled the limits of the study. Isolates from a childrens' hospital were significantly less diverse than those from patients in adult wards.

Staphylococcal whole-cell polypeptide analysis: evaluation as a taxonomic and typing tool.

Whole-cell-polypeptide profiles obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were used in conjunction with the API-Staph technique to identify different strains of Staphylococcus aureus, S. epidermidis, S. saprophyticus and S. capitis. Complete concordance of results from both techniques was achieved with all strains examined. Visual analysis of the polypeptide patterns and comparison by use of the coefficient of Dice showed minor differences in band pattern between strains of the same species but each species produced a pattern distinguishable from that of any other. The results suggest that although SDS-PAGE can be used to identify staphylococcal species, this type of analysis will not readily provide the basis for a typing method.

Typing of strains of Staphylococcus aureus by Western Blot analysis of culture supernates.

Extracellular proteins produced by Staphylococcus aureus strains were examined by Western Blot analysis with blood donor plasma as a source of antibodies. Comparison of epidemiologically related strains showed strong concordance between plot pattern and phage type.

Characterisation of methicillin-resistant isolates of Staphylococcus aureus by analysis of whole-cell and exported proteins.

Thirty-four isolates of methicillin-resistant Staphylococcus aureus (MRSA) from patients in Glasgow Royal Infirmary were studied. Whole-cell protein profiles obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were compared with banding patterns produced by immunoblots of exported proteins. Human plasma was used as a source of staphylococcal antibodies for the immunoblots. SDS-PAGE of whole-cell extracts did not usefully distinguish different isolates of MRSA. Reproducible banding patterns were obtained by immunoblots of exported proteins. Analyses of immunoblots by use of the Dice coefficient demonstrated that isolates of MRSA could be divided into two main groups. Immunoblots of exported proteins provided a rapid, reproducible and sensitive method for characterisation of MRSA.

Western blot analysis of staphylococcal antibodies present in human sera during health and disease.

IgG antibodies directed against Staphylococcus aureus were examined by Western blotting in sera from 15 healthy individuals isolated for a year in Antarctica. Sera reacted with many staphylococcal antigens in whole-cell extracts and individuals showed unique and unchanging blot profiles. The IgG and IgM profiles of patients with deep-seated staphylococcal infections were also examined by Western blotting. Anti-staphylococcal IgM antibodies that reacted with an antigen of apparent molecular mass 31 X 10(3) were present in all patients with staphylococcal disease, and were absent from, or detected in much smaller amounts in, control sera.

Molecular typing methods for Neisseria meningitidis.

Neisseria meningitidis is an important pathogen because it causes life-threatening infections. The rapid course of meningococcal disease and the capacity of some serogroups to cause large-scale epidemics necessitates the use of sensitive, reliable and rapid typing methods to characterise strains. Molecular typing techniques for N. meningitidis are used for epidemiological purposes to investigate outbreaks and the spread of organisms and to examine the population genetic structure of the organism to understand better its variation and evolution. Many investigators have employed molecular typing methods and shown that meningococcal disease is associated with a variety of different epidemiological patterns. The choice of a typing method is dependent upon the epidemiological questions to be answered and on the population genetics of the organism under investigation. With highly clonal populations comprising independent non-recombining lineages such as serogroup A meningococci, ribotyping, multilocus enzyme electrophoresis (MLEE), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR with arbitrary primers (RAPD) or with other gene-based primers each provides a constant measure of the relationship between strains. A more restricted portfolio of molecular methods - PFGE, MLEE and MLST - is appropriate for the investigation of less clonal serogroup B and C meningococci from localised outbreaks. If a thorough evaluation of the overall population is sought to determine the relationship between new isolates and members of hyper-endemic clonal complexes then quantitative methods such as MLEE and MLST are necessary. Several PCR-based methods are used for the detection and typing of meningococcal strains, many requiring rigorous standardisation before they can be considered suitable for rapid and reliable differentiation between clones. This review examines strain characterisation by molecular techniques and non-culture-based subtyping of meningococci in clinical specimens. It assesses the importance of these techniques and examines the epidemiological questions that they answer and also their limitations.

Differences exist in the immunoblotting profiles of cyst and trophozoite antigens of Pneumocystis carinii.

The antigenic profiles of Pneumocystis carinii trophozoites and cysts were compared by immunoblotting with hyperimmune rat sera against cyst and trophozoite antigens. Strong bands corresponding to proteins of 50-60 kDa and 104 kDa were demonstrated in cyst and trophozoite antigens by all antisera. Additional prominent proteins of 81 and 63 kDa and less prominent proteins of 88, 73, 69 and 37 kDa were found only in trophozoite antigen. The latter proteins were recognised by anti-trophozoite and anti-cyst antisera but the 81- and 63-kDa proteins were associated specifically with trophozoites. With cyst-rich antigen, antibodies to the 50-60-kDa protein were detected in only two of 14 sera from P. carinii pneumonia (PCP)-positive rats. With trophozoite-rich antigen, 11 of 24 rats with PCP and one of 18 PCP-negative animals had antibodies to both the 50-60 kDa and 104-kDa antigens. Antibodies to the 81- or 63-kDa antigens were demonstrated in 15 of 24 PCP-positive animals and none of the PCP-negative animals. The use of trophozoites rather than cysts increased the sensitivity of immunoblotting. As trophozoites predominate in PCP, antibody to trophozoite-specific antigens rather than common cyst and trophozoite antigens is likely to be a more useful marker of current infection.

The use of a nested polymerase chain reaction for detecting Pneumocystis carinii from lung and blood in rat and human infection.

A nested polymerase chain reaction (PCR) assay was developed to detect both rat- and human-derived Pneumocystis carinii DNA. The nested PCR product was 125 bp long and was representative of part of the gene coding for the large subunit of mitochondrial ribosomal RNA. Twenty serial blood samples and 24 tissues from six immunosuppressed Sprague-Dawley rats were examined by nested PCR. All lung samples were positive by PCR and Toluidine blue O staining. Buffy coat samples and all the other tissues were PCR-negative during up to 6 weeks of immunosuppression. Thirty-five clinical bronchoalveolar lavage, induced sputum or tracheal aspirate samples from human patients were tested. Twelve of 35 were positive by both PCR and indirect fluorescence assay (IFA) and 19 of 35 were both PCR- and IFA-negative. Four of 35 were IFA-negative but PCR-positive and there were good responses in these patients to specific therapy, indicating that PCR may be more useful than IFA in clinical samples. P. carinii DNA was not detected in three blood samples. The nested PCR is a sensitive and specific DNA amplification method suitable for the routine diagnosis of P. carinii in human respiratory samples.

Progression of Pneumocystis carinii infection in an animal model.

The development of Pneumocystis carinii infection in immunosuppressed rats is important not only in understanding the infection, but also as a source of P. carinii antigen for use in diagnostic serological tests. The aims of this study were to monitor the progression of P. carinii infection in the Sprague Dawley rat model and then determine parameters that indicate the maximum production of P. carinii antigen. Seventeen Sprague Dawley rats were killed at intervals up to 9 weeks after the start of immunosuppressive therapy. The progression of P. carinii lung infection was observed by Giemsa staining of lung imprints and by a hemi-nested polymerase chain reaction (PCR). Body weight, food and water intake and the appearance and activity of the rats were measured daily. Seven control rats were kept under the same conditions. P. carinii infection was detected in the lung 2 weeks after immunosuppression by hemi-nested PCR and after 3 weeks by Giemsa staining. No P. carinii DNA was detected in any of the blood samples. Rats with moderate or severe lung infection had been immunosuppressed for > or = 6 weeks. Body weight was significantly greater in control rats than in the immunosuppressed rats. Six weeks of immunosuppression was used as a cut-off to determine measurements to identify those rats with moderate or severe infections in their lungs. A combination of > 34% body weight loss at 6 weeks after immunosuppression and the condition of the animals with scores < or = 9 used in conjunction with duration of immunosuppression may be useful to maximise the yield of P. carinii infection from individual rats.

Usefulness of rat-derived antigen in the serodiagnosis of Pneumocystis carinii infection.

Sera from patients with likely and possible Pneumocystis carinii pneumonia (PCP) on the basis of clinical information and laboratory investigations were tested by immunoblotting to assess the usefulness of trophozoites in the serodiagnosis of PCP. IgG antibodies to 50-60- kDa proteins were demonstrated with cyst antigen, but antibodies to additional proteins of 61, 70, 82, 95, 99 and 116 kDa were found with trophozoite antigen. These bands were not demonstrated with control sera. IgG antibody to the 116-kDa protein was found in 18 (46%) of 39 sera from patients with possible PCP compared with 5 (17%) of 30 sera from patients with likely PCP. There was no other significant difference between the two patient groups in detection of these proteins. Sera with higher indirect immunofluorescence assay (IFA) IgG titres were more likely to be immunoblot positive. Only 4 of 16 patients with likely PCP were IgG negative in the IFA; three of these were IgG immunoblot positive. In 4 of 10 patients with likely PCP and 6 of 15 patients with possible PCP, demonstration of IgM or IgA, or both, by IFA or immunoblotting provided evidence suggestive of current infection. This study confirms the usefulness of rat-derived antigen, especially trophozoite antigen, in PCP serology. The IgG IFA remains the most useful test, but IgM and IgA testing and immunoblotting can support the diagnosis.

Absorption of IgG does not enhance toxoplasma IgM and IgA immunoblotting.

Total IgG, IgM and IgA levels and toxoplasma IgG, IgM and IgA immunoblotting patterns were assayed in 10 sera before and after IgG absorption with Protein G-Sepharose 4. Removal of IgG (mean reduction 96%) was accompanied by a significant reduction in the level of IgM (mean reduction 56%) and IgA (mean reduction 53%) in nine of the 10 sera. The absorbed supernates showed fewer and weaker IgM bands in five sera, but IgA immunoblotting patterns were unaffected by absorption. There was no benefit in removing IgG in toxoplasma IgM and IgA immunoblotting.

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis.

Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.

Necrotising fasciitis: quantitative characteristics of the 1994 British media outbreak.

A small number of cases of necrotising fasciitis in England caused by group A streptococci became the focus of a massive amount of media attention during May 1994. This report presents quantitative data on the interaction between journalists and a microbiologist during this period, and discusses possible explanations of the event.