Human immunodeficiency virus (HIV) infection in País Vasco, Spain

An analysis of the epidemiology of the human immunodeficiency virus infection in the Basque Country, Spain is presented. The infection by this virus is mainly detected in intravenous drug addicts. In homosexuals the seropositivity frequency detected was moderate, and infection was also detected in sexual partners of drug addicts.     

Detection of human cytomegalovirus DNA in perilymph of patients with sensorineural hearing loss using real-time PCR

Although cytomegalovirus (CMV) has been detected in the inner ear fluid of patients who succumbed to the complications of symptomatic congenital CMV infection, it has not been detected in the inner ear fluid of living patients. In this study, real-time polymerase chain reaction (PCR) was used to measure CMV DNA in clinical samples (including perilymph) collected from five patients with deafness. In case 1, diagnosed as a symptomatic congenital CMV infection, 3 copies/microl of CMV DNA were detected in perilymph, although no viral DNA was detected in peripheral blood mononuclear cells (PBMCs) or urine samples. In case 4, a suspected asymptomatic congenital CMV infection, 36 copies/microg of CMV DNA were detected in PBMCs, but neither perilymph nor urine contained viral DNA. Likewise, in case 5, a case of deafness of unknown origin, 48 copies/microg of CMV DNA were detected in the PBMCs, but none in the perilymph or urine. CMV DNA was not detected in the samples obtained from the remaining two cases with deafness of unknown etiology. To our knowledge, this is the first report to detect CMV DNA in an inner ear sample obtained from a living human subject.     

Early testing of cell cultures for detection of hemadsorbing viruses.

Hemadsorption of primary monkey kidney cell cultures was commenced at 1 day after inoculation to evaluate how rapidly influenza A virus, influenza B virus, and parainfluenza virus types 1, 2, and 3 could be detected by this method from respiratory specimens. Overall, 38% of all isolates could be detected within 24 h of inoculation, and 69% could be detected within 48 h. All influenza A viruses and all but one influenza B virus were detected by day 3.     

Confirmation of true mosaic trisomy 20 in a phenotypically normal liveborn male

A new case of prenatally detected mosaic trisomy 20 (79% trisomy 20 cells in amniocyte cultures) that was confirmed in newborn tissue is presented. A healthy male infant was delivered at term, with no dysmorphology or apparent malformations; this baby is developing normally. Twenty-five percent of foreskin and 17% of fetal cord cells also showed trisomy 20, while no trisomic cells were detected in newborn blood. High frequency mosaicism for trisomy 20 in this case was thus due to true embryonic origin. Extensive counseling and prenatal follow-up in this case led to an unaffected liveborn, and guarded optimism may be warranted for future cases of mosaic trisomy 20 detected prenatally.     

Hairbulb tyrosinase activity in oculocutaneous albinism: suggestions for pathway control and block location

Hairbulb tyrosinase activity was determined in 72 individuals with five types of oculocutaneous albinism (OCA) and 64 obligate heterozygotes. Type IA (tyrosinase-negative) and type IB (yellow mutant) individuals had low or no measurable tyrosinase activity, and heterozygotes for these two types could be detected with this assay. Type II (tyrosinase-positive) individuals had moderate to high activity, and the heterozygotes for this type could not be detected. Type III (minimal pigment) individuals had low activity, and heterozygote levels were useful in detecting this type of OCA. Type VI (Hermansky-Pudlak syndrome) individuals had moderate to no measurable activity, and heterozygotes for this type could not be detected. The implication of the tyrosinase assay results in light of the phenotype and the possible location of the pigment block in these forms of OCA are discussed.     

Comparison of pathological and biological features of symptomatic and mammographically detected ductal carcinoma in situ of the breast.

To determine whether the ductal carcinoma in situ (DCIS) detected mammographically or presenting clinically is the same or differs, pathological and biological (c-erbB-2 and p53 detection) features of 79 cases of pure DCIS, 5 cases with microinvasion and 8 cases with 1 to 2 mm of invasion, all detected by mammography, have been compared with 59 cases of pure DCIS, 8 cases with microinvasion and 7 cases with 1 to 2 mm invasion, all of which presented clinically. Half of the mammographically detected group were smaller than 20 mm, and there was a higher incidence of these being low grade, whereas 30% of the symptomatic cases were smaller than 20 mm, and more of this group were larger than 50 mm. For the pure DCIS, there were less high-grade and more intermediate-grade cases in the mammographically detected group, although the incidence of low grade was similar between the two groups. There were more cases with a micropapillary pattern in the symptomatic group. C-erbB-2 protein was detected in 42% of the mammographically detected cases, whereas 59% of the symptomatic cases had c-erbB-2 reactivity. P53 detection was similar for both groups (33.0% and 37.0%). There were more symptomatic cases with invasion, and these were predominantly high grade, whereas the mammographically detected cases were both high and intermediate grade. Twelve of the 15 symptomatic cases with invasion expressed c-erbB-2, in comparison with 4 of the 13 mammographically detected cases, with half of the high-grade lesions in the latter group being negative. This study has shown that although there is overlap of pathological and biological features between DCIS presenting clinically and that detected mammographically, there can be differences in extent, grade, and invasion. The impact of this, however, can be determined only by clinical follow-up.     

Monoclonal antibodies against African swine fever viral antigens

Monoclonal antibodies specific for African swine fever (ASF) viral proteins of 14, 32, 73, 174, and 240 kDa were produced and characterized. Immunoelectron microscopy detected the 73 kDa but not the 14-, 32-, or 240-kDa proteins at the surface of the virion. The 32-kDa protein was detected by radioimmunoassay 2 hr after infection of porcine monocytes and Vero cells, was detected in the seven widely divergent ASFV isolates tested, and stained brilliantly virus-infected cells in indirect immunofluorescence suggesting that monoclonal antibodies directed against this protein may be useful in ASFV diagnosis. Two monoclonal antibodies detected heterogeneity between ASF viruses.     

Enzyme content of hepatocellular lysosomes in the liver of the tumour-bearing mouse

An increase in the number of hepatocellular lysosomes was not detected in mice bearing the solid or ascites forms of Sarcoma 180 or Ehrlich's tumour. Despite this, biochemical studies showed significant increases in acid phosphatase, cathepsin-D and beta-galactosidase. This difference between morphological and biochemical observations is explained by the fact that only gross increases in lysosome population are likely to be detected in the small samples examined with the electron microscope, but by biochemical methods whereby many grams of tissue are analysed finer changes may be detected. As far as is known at present there is an increase in hepatocellular lysosomes and/or lysosomal enzymes in tumour-bearing mice, rats and man and no exception to this exists.     

Chromosome abnormalities in chronic lymphocytic leukemia revealed by TPA as a mitogen

Bone marrow and peripheral blood cultures of chronic lymphocytic leukemia patients were mitogenically stimulated with TPA (12-0-tetradecanylphorbol-13-acetate). Clonal cytogenetic abnormalities were detected in frequencies varying from 15% to 100%, in five of the six patients studied. Parallel studies with pokeweek mitogen showed a much lower level of stimulation and only two abnormal clones were detected. The chromosome abnormalities described in this study are similar to those reported in CLL by other authors, particularly with respect to trisomy 12 and deletion 11q. A significant frequency of hypodiploidy and chromosome deletion was also detected in this study, and further studies are underway to determine the significance of these findings.     

Trisomy 5 mosaicism in amniotic fluid with normal outcome

A case of prenatally diagnosed true mosaicism for trisomy 5 with a clinically normal outcome is presented. Trisomy 5 was detected in 23% of cells obtained by amniocentesis, but it was not detected from cells obtained by fetal blood sampling. While in this case the finding at amniocentesis did not reflect the status of the fetus, care must be exercised in reaching this conclusion in all cases.     

Diagnosis of enteric pathogens in children with gastroenteritis

The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12-month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Children's Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1 %). Giardia-specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients (P = 0.23). Amongst this subset, Norwalk-like viruses (NLVs) were detected by RT-PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral-like particles (SVLPs), and three of 126 EM-negative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary. The cost-effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.     

Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.     

Detection of Rickettsia, Borrelia, and Bartonella in Carios kelleyi (Acari: Argasidae)

Carios kelleyi (Colley & Kohls 1941), a tick associated with bats and bat habitats, has been reported to feed on humans, but there is little published data regarding the presence of vector-borne pathogens in these ticks. C. kelleyi nymphs and adults were collected from residential and community buildings in Jackson County, Iowa, and tested by polymerase chain reaction for Rickettsia, Borrelia, Bartonella, Coxiella, and Anaplasma. Rickettsia DNA was detected in 28 of 31 live ticks. Sequences of the 17-kDa and rOmpA genes suggest that this agent is a novel spotted fever group Rickettsia. Transstadial and transovarial transmission of this Rickettsia were demonstrated. The flagellin gene of a Borrelia, closely related to B. turicatae, was detected in one of 31 live ticks. The 16S-23S intergenic spacer region of Bartonella henselae also was detected in one of 31 live ticks. Coxiella or A. phagocytophilum DNA were not detected in these ticks.     

Cross-reactivity of goat alloantisera lymphocyte antigens with ovine peripheral blood lymphocytes and erythrocytes

Twenty-two alloantisera that detected eleven specificities in two loci of the goat (SD1 and SD2) were studied on ovine lymphocytes from 55 Spanish Merino sheep by microlymphocytotoxicity assay. All the sera detected antigenic variability in sheep although this variability was not comparable to that described in the goat. No pancytotoxic sera were found. Agglutinating and hemolytic antibodies present in the sera were not related to any antigen present in erythrocytes and lymphocytes. Furthermore these samples were typed with sixteen ovine alloantisera and no relationship between specificities detected by ovine sera and the antigenic variability detected by goat alloreactives was found.     

Development of a hypersensitive detection method for human parvovirus B19 DNA

A new detection method for human parvovirus B19 DNA was established using PCR coupled with a hybridization protection assay. The amplified product was detected using acridinium ester-labeled DNA probes. By this method, a few copies of B19 DNA were detected in human serum albumin.     

Effects of local geographic barriers and latitude on population structure in Anopheles punctipennis (Diptera: Culicidae)

We sampled Anopheles punctipennis (Say) from 11 localities throughout Vermont to examine the effects of latitude and two local geographical boundaries, Lake Champlain and the Green Mountains, on the population genetic structure of this species. Thirty-five mitochondrial haplotypes were detected in 104 individuals using a variable region of the COI gene. When latitude was examined, we detected significant structure within localities and among localities within latitudinal regions. For geographic analysis, significant genetic structure was detected only within localities. Estimates of gene flow across geographic regions indicate that the Green Mountains, but not Lake Champlain, is a barrier to dispersal for this species. We found no correlation between genetic and geographic distances for An. punctipennis.     

Enzyme content of hepatocellular lysosomes in the liver of the tumour-bearing mouse.

An increase in the number of hepatocellular lysosomes was not detected in mice bearing the solid or ascites forms of Sarcoma 180 or Ehrlich''s tumour. Despite this, biochemical studies showed significant increases in acid phosphatase, cathepsin-D and beta-galactosidase. This difference between morphological and biochemical observations is explained by the fact that only gross increases in lysosome population are likely to be detected in the small samples examined with the electron microscope, but by biochemical methods whereby many grams of tissue are analysed finer changes may be detected. As far as is known at present there is an increase in hepatocellular lysosomes and/or lysosomal enzymes in tumour-bearing mice, rats and man and no exception to this exists.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.