Development of a panel of well-characterized human immunodeficiency virus type 1 isolates from newly diagnosed patients including acute and recent infections.

The aim of this study was the development of a panel constituted by well-defined HIV-1 strains of different genetic forms, with a particular focus on isolates from acute and recent infections. Fourteen HIV-1 isolates, including four from acute and five from recent infections, were expanded in peripheral blood mononuclear cells. SI phenotype, coreceptors use, and TCID(50)/ml were determined. V3 net charge was calculated. Near full-length genomes were amplified by RT-nested PCR in four overlapping segments. Phylogenetic analyses were performed with neighbor-joining trees and bootscanning. Analysis of cysteine residues, lengths of variable regions, and potential N-linked glycosylation sites in gp120 and gp41 was performed. Viral stocks were produced. Thirteen strains were NSI/R5 and one SI/R5,X4. TCID(50)/ml ranged between 10(4.6) and 10(6). V3 net charge was <+5 in 12 sequences and +5 in two sequences. Near full-length HIV-1 genomes analysis identified viruses of the following genetic forms: eight subtype B, three subtype C, two CRF02_AG, and one subtype G. Cysteine residues that form the V1,V2,V3, and V4 loops were highly conserved. The number of potential N-linked glycosylation sites in gp120 and gp41 ranged between 24-29 and 4-6, respectively. Seven potential N-linked glycosylation sites in gp120 and three in gp41 were conserved. V1, V2, V4, and V5 variable regions exhibited substantial length variation. In addition, an analysis of transmitted and natural resistance to current antiretroviral drugs in these strains was performed. It is worth mentioning that the 13S mutation in the V3 sequence, associated with resistance to maraviroc, was observed in a subtype B strain that harbored resistance mutations to nucleoside reverse transcriptase inhibitors and to T20. The availability of a panel including strains from acute and recent infections should be a valuable resource for optimizing and standardizing vaccine candidate assessment. Near full-length genome characterization may be necessary for evaluating clade-specific reactivities.

An idiopathic case of macroglossia.

SUMMARY: A case of successful reduction glossectomy for idiopathic macroglossia is presented. Macroglossia varies in its degree of severity. With pronounced macroglossia, respiratory and feeding difficulties may ensue. Persistent macroglossia leads to speech problems and anterior open bite. The cause of macroglossia must be clearly defined, and true macroglossia separated from pseudomacroglossia. There are many congenital and acquired causes of true macroglossia. By far, the most common cause of macroglossia is muscular hypertrophy. No similar case of idiopathic macroglossia is encountered in the literature. Corrective reduction glossectomy for this reason is scarcely described in the literature.

High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment.

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.

[Dupuytren's disease in a 12-year-old child]. / Enfermedad de Dupuytren en una niña de 12 años.

A 12-year-old child presented an indurate tumour in the volar aspect of her carpus. After proceeding to incisional biopsy and histological examination, it was concluded that it was a case of Dupuytren's disease. In view of these results, we proceeded to excise this lesion, confirming the previous diagnosis after a new histological examination. There was no sign of recurrence 13 months after surgery in this case of Dupuytren disease in a child. Dupuytren's disease is a very rare entity in childhood, whose incidence increases with age. It is not usually observed in patients younger than 16 years. There are few papers mentioning cases of Dupuytren's disease in childhood. We report a case, histologically confirmed, of Dupuytren's disease in childhood.

Role of alternative sigma factor algU in encystment of Azotobacter vinelandii.

Alginate is essential for encystment in Azotobacter vinelandii. Transcription of the algD gene, which codes for GDP-mannose dehydrogenase, a key enzyme in the alginate biosynthetic pathway, is initiated at two promoters, one of which, p2, has sigmaE consensus sequences. AlgU is the A. vinelandii alternative sigmaE factor. In this study, we constructed an algU mutant (SMU88) which, as expected, is impaired in alginate production, encystment, and transcription of the algD gene from the p2 promoter. Plasmid pJMSAT1, carrying the A. vinelandii algU gene, restored alginate production and encystment to SMU88 and to strain UW136, a naturally occurring algU mutant. Plasmid pSMU865, carrying the A. vinelandii mucABCD genes coding for negative regulators of AlgU activity and previously shown to diminish alginate production in the wild-type strain, ATCC 9046, was shown here to impair encystment and transcription of the algD gene from the p2 algU-dependent promoter. Since nonencysting strain ATCC 9046/pSMU865 produced more alginate than some encysting strains, such as UW136/pJMSAT1, we propose an AlgU role in encystment, independent of the structural role that alginate plays in mature cysts.

Characterization of genetic variation and 3'-azido-3'-deoxythymidine- resistance mutations of human immunodeficiency virus by the RNase A mismatch cleavage method.

The RNase A mismatch cleavage method has been applied to the characterization of natural genetic variation of human immunodeficiency virus (HIV) from different geographical areas. The approach provides a rapid and simple assay for the analysis of differences in closely related viral isolates and allows the establishment of phylogenetic relationships between epidemiologically distinct viruses. Our results show a broad clustering of circulating viruses according to their geographical distribution. We also have analyzed the temporal appearance of mutations associated with the acquisition of resistance to 3'-azido-3'-deoxythymidine (AZT). The results show that mutations in codon 215 of the viral reverse transcriptase can be detected readily by this method in HIV isolates and also directly in peripheral blood from HIV-infected individuals after in vitro amplification of viral sequences with the polymerase chain reaction. The specific recurrence of identical double-nucleotide substitutions in epidemiologically and geographically distant viruses suggests that the restricted amino acid substitutions at this position selected by drug exposure are a critical, rate-limiting step in the acquisition of drug resistance.

Localization of human immunodeficiency virus antigens in infected cells by scanning/transmission-immunogold techniques.

An application of high resolution scanning/transmission electron microscopy (STEM) and gold-labelling techniques for the rapid detection of human immunodeficiency virus (HIV) in infected cells has been developed. Experimental in vitro studies for detecting two HIV structural proteins, gp41 and p17, were performed following an indirect labeling procedure that uses monoclonal anti-p17 and anti-gp41 antibodies as primary antibodies and 40 nm gold-linked goat antimouse IgG as secondary antibodies. The cells were then studied by STEM in the scanning mode. Unambiguous localization of the viral antigens was possible by combining the three-dimensional image provided by the secondary electron image and the atomic number-dependent backscattered electron image for the identification of the gold marker. This technique combines both the morphological information and the rapid procedures of scanning electron microscopy with the precise and sensitive antigen detection provided by the use of STEM and immunological methods. The preliminary results of its application to the study of peripheral blood mononuclear cells from four anti-HIV-seropositive patients showing the presence of specific labeling in all of them suggest that it might prove useful for early detection of HIV infection before seroconversion, as well as for quantitative studies.

Human immunodeficiency virus and related retroviruses.

This paper summarizes the current knowledge on the human immunodeficiency virus (HIV) and related retroviruses, describing basic characteristics of this new group of viruses such as morphologic and genetic structure, biological and cultural properties, virus growth characteristics, genetic variability and virus replication. The discovery of new human and simian retroviruses has prompted the World Health Organization (WHO) to convene a group of experts to establish criteria for their characterization. This will allow rapid identification of new variants that may arise and allow public health measures to be implemented accordingly. Different approaches are made to nomenclature in view of the evolution of knowledge about these viruses, and a system of nomenclature has been proposed by the WHO working group. This system, inspired by the one developed for the influenza viruses, is practical and descriptive, providing information on the origins of the organism and its type.

Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture.

The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella.

Síndrome de inmunodeficiencia adquirida: estudios clínicos, inmunológicos y microbiológicos en pacientes hemofílicos / Acquired immunodeficiency syndrome: clinical, immunolofical and microbiological studies in hemophilic patients

La reciente constatación de que el síndrome de inmunodeficiencia adquirida (SIDA) podría afectar en forma especial a homosexuales, drogadictos por vía intravenosa y hemofílicos motivó el inicio, en marzo de 1983, de un seguimiento clínico, inmunológico y microbiológico en 67 pacientes hemofílicos que reciben atención en la Unidad de Hemofilia de la Ciudad Sanitaria La Paz de Madrid. De ellos, 31 eran asintomáticos y 36 portadores de alguno de los signos o síntomas prodrómicos del SIDA. Los resultados obtenidos demostraron, em primer lugar, que un alto número de pacientes hemofílicos presentaban inversión del cociente de linfocitos T inductores/linfocitos T supresores sin que existiera una relación clara con la presencia de signos y síntomas asociados con el SIDA; en segundo lugar, la presencia de infecciones recientes y pasadas en proporciones similares en pacientes sintomáticos y asintomáticos, así como la mayor frecuencia de infecciones recientes por virus del grupo herpes en los hemofílicos con síntomas clínicos, y, en tercer lugar, que uno de los casos evolucionó hacia un cuadro compatible con SIDA. Estos resultados sugieren la existencia del riesgo de que los enfermos de hemofilia padezcan el SIDA, por lo debe mantenerse un control médico especial sobre ellos

Evaluation of commercial methods of enzyme immunoassay (EIA) for the measurement of rubella-specific IgM.

Four commercial EIA methods for measuring rubella-specific IgM (three indirect tests and one anti-mu capture test) were evaluated, using sucrose gradient centrifugation and hemagglutination inhibition as the reference method. Evaluation was conducted with the aid of four serum panels, including 53 primary rubella cases, 30 healthy pregnant women, 21 sera positive for rheumatoid factor(s) (RF) and 35 sera from 29 cases of heterophil-positive infectious mononucleosis with EBV-specific IgM detected by immunofluorescence. All EIA methods were more sensitive than the reference method when applied to very early samples (1-5 days post-exanthema) and no differences in sensitivity were found between them. On the other hand, we observed a significant incidence of false-positive results if an indirect EIA method is applied to RF-positive samples. False positivity is significantly reduced, but not totally eliminated, when samples are preabsorbed with anti-human IgG serum and, in all cases, the absorbance values obtained were low. In contrast, there were no false-positive results using an anti-mu capture method, even in sera from cases of infectious mononucleosis. The basis for choosing between an indirect method and an anti-mu capture method for the diagnosis of congenital and post-natal rubella virus infection is discussed.

Generalised tuberculosis in a patient with acquired immunodeficiency syndrome.

We describe a heroin addict who presented with cellular immunodeficiency, generalised tuberculosis, and pneumonia caused by Pneumocystis carinii, and discuss the risk of these associations.

[Etiology of acute respiratory infection in hospitalized children (author's transl)]. / Etiología de infecciones respiratorias agudas en iniños hospitalizados

Incidence of respiratory tract infection represents 23% of the total number of admissions between 1-24 months of age, during a period of 18 months. The diagnosis were: bronchiolities, 143 cases; bronchopneumonia, 134 cases; tracheobronchitis, 50 cases; laryngitis, four cases, and bacterial pneumonia, 61 cases. Monthly incidence was maximal in December of each year. From the total group, 144 cases were included in the present study to determine etiology of the infection. In 19% of the cases a serological diagnosis was posible. The adenovirus group was the most frequently found, followed by mycoplasma pneumoniae, parainfluenza 2, RS virus and M. parotiditis. RS virus was associated with a clinical picture of bronchopneumonia, mycoplasma pneumoniae with one of bronchiolitis and adenovirus was indistinctly associated with features either bronchopneumonia or bronchiolitis. In two cases it was detected a mixed infection by two virus: influenza 2 and mycoplasma pneumoniae. In four cases a bacterial surinfection was demonstrated: in two cases with coagulase-positive staphilococus and other two with klebsiella pneumoniae.