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Infection with agents of human granulocytic ehrlichiosis, lyme disease, and babesiosis in wild white-footed mice (Peromyscus leucopus) in Connecticut.

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichia sp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichia strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic Ehrlichia sp. organisms were detected in 17 of 23 (73. 9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent. Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.

Animals↗

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 subtype B DNA in dried blood samples for diagnosis in infants in the field.

The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 microl of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45 degrees C for 20 h. The primers used for nested PCR of the HIV-1 pol gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.

Adolescent↗

Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family.

Polydnaviruses (PDVs) are double-stranded DNA viruses with segmented genomes that replicate only in the oviducts of some species of parasitic wasps and are required for the successful parasitization of lepidopteran insects. PDV DNA segments are integrated in the genomes of their associated wasp hosts, and some are nested; i.e., smaller segments are produced from and largely colinear with larger segments. To determine the internal structure of nested viral segments, the first complete nucleotide sequence of a PDV genome segment and its integration locus was determined. By restriction mapping, Southern blot, and sequence analyses, we demonstrated that the Campoletis sonorensis PDV segment W is integrated into wasp genomic DNA. DNA sequence analysis revealed that proviral segment W terminates in two 1,185-bp direct long terminal repeats (LTRs) in the wasp chromosome, while only one LTR copy is present in the extrachromosomal (viral) W. The results suggest that terminal direct repeats are a general feature of PDV DNA segment integration but that the homology and size of the repeats can vary extensively. Segment W contains 12 imperfect direct repeats of six different types between 89 bp and 1.9 kbp with 65 to 90% homology. The orientation and structure of the repeats suggest that W itself may have arisen through sequence duplication and subsequent divergence. Mapping, hybridization, and sequence analyses of cloned R and M demonstrated that these segments are nested within segment W and that internal imperfect direct repeats of one type are implicated in the homologous intramolecular recombination events that generate segments R and M. Interestingly, segment nesting differentially increases the copy number of genes encoded by segment W, suggesting that the unusual genomic organization of PDVs may be directly linked to the unique functions of this virus in its obligate mutualistic association with parasitic wasps.

Animals↗

Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study.

BACKGROUND: Streptococcus pneumoniae is the leading cause of community acquired pneumonia; however, only a small proportion of cases can be detected by conventional methods. The ability of the polymerase chain reaction (PCR) test performed on whole blood samples to identify patients with pneumococcal pneumonia was investigated. METHODS: One hundred and fourteen consecutive adult patients with community acquired pneumonia were evaluated by a wide battery of diagnostic tests in order to determine the aetiology. Blood samples from these patients and 50 controls were also tested by the nested PCR test to detect selected pneumolysin gene fragments of S pneumoniae. RESULTS: The patients were divided into four groups: (1) 40 patients with pneumococcal pneumonia in 22 of whom (55%) the PCR was positive (eight of 11 with bacteraemia and 14 of 29 without); (2) 30 with pneumonia due to other pathogens in all of whom the PCR was negative; (3) 44 with pneumonia of unknown aetiology in 14 of whom (32%) PCR was positive, and (4) 50 controls in whom the PCR test was positive in two (4%). Thus, the sensitivity of the test was 55% and the specificity 100% (81% if positive PCR tests among undiagnosed patients are considered as false positive results). CONCLUSION: PCR applied to whole blood samples appears to be a sensitive and very specific diagnostic test for identifying patients with pneumococcal pneumonia with a potential application in clinical practice.

Adolescent↗

Babesia microti-like parasites detected in feral raccoons (Procyon lotor) captured in Hokkaido, Japan.

Raccoons (Procyon lotor), which have recently become feral in Japan, were examined for the presence of Babesia microti-like parasites. Out of 372 raccoons captured in the west-central part of Hokkaido, 24 animals with splenomegaly were selected and tested by nested PCR targeting the babesial 18S rRNA gene. B. microti-like parasites were detected in two of the 24 individuals, and their DNA sequences were identical to that of the B. microti-like parasite reported from raccoons in the United States, suggesting that the parasites were probably imported into Japan and that the life cycle of the parasite has already been established in the country. The potential risk of this B. microti-like parasite spreading among dogs and foxes in Japan will need to be carefully monitored, as parasitization by phylogenetically very close parasites has been reported from such animals.

Animals↗

The application of molecular analyses for primary granulocytic sarcoma with a specific chromosomal translocation.

Primary granulocytic sarcoma (GS) is a rare disease defined by the absence of antecedent or concomitant leukemic cells in the bone marrow and the peripheral blood. Immunohistochemical staining for myeloperoxidase is necessary for a definite diagnosis. Otherwise, primary GS is often misdiagnosed as a malignant lymphoma or other malignancies. Primary GS is well known to frequently develop into acute myeloid leukemia (AML). Here we describe a 28-year-old woman with primary GS manifesting as an epidural tumor in the sacral region accompanied by meningeal dissemination. Fluorescence in situ hybridization analysis detected the AML1/MTG8 fusion gene in neoplastic cells obtained from her cerebrospinal fluid specimen and the epidural mass. The AML1/MTG8 fusion gene transcript was also detected by a nested reverse transcriptase-polymerase chain reaction analysis of mononuclear cells from the bone marrow, although leukemic cells were not recognized in a microscopical examination of the patient's bone marrow. Systemic chemotherapy with high-dose cytarabine followed by local radiotherapy was performed, and the patient clinically achieved a complete response. These molecular analyses provide a precise method of diagnosis, especially with respect to the French-American-British AML classification, according to the characteristic karyotypic alterations, and a patient consequently can quickly be given appropriate systemic chemotherapy as induction therapy.

Adult↗

Population genetics and colony structure of the Argentine ant (Linepithema humile) in its native and introduced ranges.

Introduced species often possess low levels of genetic diversity relative to source populations as a consequence of the small population sizes associated with founder events. Additionally, native and introduced populations of the same species can possess divergent genetic structuring at both large and small geographic scales. Thus, genetic systems that have evolved in the context of high diversity may function quite differently in genetically homogeneous introduced populations. Here we conduct a genetic analysis of native and introduced populations of the Argentine ant (Linepithema humile) in which we show that the population-level changes that have occurred during introduction have produced marked changes in the social structure of this species. Native populations of the Argentine ant are characterized by a pattern of genetic isolation by distance, whereas this pattern is absent in introduced populations. These differences appear to arise both from the effects of recent range expansion in the introduced range as well as from differences in gene flow within each range. Relatedness within nests and colonies is lower in the introduced range than in the native range as a consequence of the widespread genetic similarity that typifies introduced populations. In contrast, nestmates and colony-mates in the native range are more closely related, and local genetic differentiation is evident. Our results shed light on the problem posed for kin selection theory by the low levels of relatedness that are characteristic of many unicolonial species and suggest that the loss of genetic variation may be a common mechanism for the transition to a unicolonial colony structure.

Animals↗

New protocol for DNA extraction of stool.

Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.

Base Sequence↗

Clinical correlations of peripheral blood microchimerism after liver transplantation.

The aim of this study was to evaluate microchimerism after human liver transplantation (LT). This study included 13 female recipients who received hepatic allograft from male donors at Asan Medical Center. A nested PCR specific for Y-chromosome gene (DYZ3) was used to analyze the small number of male cells in the peripheral blood mononuclear cells of the female recipients. Microchimerism was observed in 6 of 13 recipients and 16 out of 35 samples. Only 3 patients showed microchimerism 3 months after LT. There was no statistical difference between the presence of microchimerism and clinical findings such as type of donor, type of immunosuppression, episode of rejection and age of recipient. This study did not show any clinical relevance of microchimerism and further larger study are needed to confirm the results.

Adolescent↗

[Molecular cytogenetics of bone tumors].

OBJECTIVE: To study alterations of molecular cytogenetics of bone tumor serially. METHODS: Chromosomal analysis, immunohistochemistry, molecular hybridization, nested-PCR/SSCP, DNA sequence analysis, and gene transfection techniques were used to investigate the common bone tumors. RESULTS: Chromosomal aberrations were found in 50 cases of aggressive bone tumors, with a chromosome number aberration rate of 86.96% and a structure aberration rate of 93.94%. However, there were differences among different kinds of bone tumors. The incidence of chromosomal instability in 53 patients with bone tumors was higher than that in normal controls. Meanwhile, the fragile sites of chromosomes of the patients were closely correlated with the high nonrandom chromosome breakpoints of tumor cells and related oncogene loci. The data on p53 and Rb from 305 cases and 89 cases, respectively, indicated that abnormalities of p53 and Rb were common events in bone tumors. The accumulation of p53 protein and loss of Rb protein were more common in aggressive bone tumors. Structures and their products of related proto-oncogenes (ras, myc, fos, bcl-2, met) were studied in 124 cases. Bone tumors presented multi-oncogenes abnormalities with differences in different pathological kinds. Exogenous wild-type p53 gene transfected into osteosarcoma cell line induced growth inhibition and apoptosis of tumor cells. CONCLUSIONS: Abnormalities of chromosomes, proto-oncogenes, and tumor suppressor genes frequently exist in bone tumor cells and are correlated with oncogenesis.

Bone Neoplasms↗

[Genotype and sequence analysis of merozoite surface protein 1 of Plasmodium falciparum isolates in Yunnan province].

AIM: To identify the genotype of merozoite surface protein 1 of Plasmodium falciparum in Yunnan Province and explore the polymorphism of MSP1 genes in geographical characteristics and genetics. METHODS: Nested polymerase chain reaction was applied to genotyping of P. falciparum isolated in Yunnan Province. Some of parasite alleles were sequenced by dye primer cycle sequencing. RESULTS: In 30 P. falciparum infections, 38 different alleles were found. Of them, the dominant allele was a variant of MAD20, while was K1 less and the RO33 was few. In addition, incidences of mixed allele infections were observed. Sequence analysis showed that DNA sequences of MAD20, K1 and RO33 alleles from Yunnan were highly homologous with those of international standard strains, respectively. CONCLUSION: Composition and sequence characteristics of P. falciparum arasite population in the endemic area can be detected by genotyping with MSP1 as genetic marker, which would be useful for the prevention and treatment of malaria.

Alleles↗

A simplified method for detecting cytomegalovirus by polymerase chain reaction from histologic sections of small biopsies.

Using a simplified procedure, we have extracted DNA from unstained paraffin sections of needle biopsies of kidney and liver transplants and identified the presence of CMV using the polymerase chain reaction. This method utilizes oligonucleotide primers for two genes shown to be specific for cytomegalovirus (CMV) as well as an internal control gene (hemoglobin) in a single reaction. Utilizing nested PCR amplification with agarose gel electrophoresis, CMV can be detected without radioisotopes to a level of sensitivity equivalent to one one-hundredth of a cytomegalic virocyte per cm2 of a 3-microM paraffin section. This method is applicable to situations where only scarce paraffin-embedded tissue is available.

Base Sequence↗

Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital of the Chinese People's Liberation Army.

A worldwide outbreak of severe acute respiratory syndrome (SARS) had been reported. Over 8439 SARS cases and 812 SARS-related deaths were reported to the World Health Organization from 32 countries around the world up to 5 July 2003. The mechanism of transmission of SARS-CoV has been limited only to close contacts with patients. Attention was focused on possible transmission by the sewage system because laboratory studies showed that patients excreted coronavirus RNA in their stools in Amoy Gardens in Hong Kong. To explore whether the stool of SARS patients or the sewage containing the stool of patients would transmit SARS-CoV or not, we used a style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing, as well as cell culture, semi-nested RT-PCR and sequencing of genes to detect and identify the viruses from sewage. There was no live SARS-CoV detected in the sewage in these assays. The nucleic acid of SARS-CoV was found in the sewage before disinfection from both hospitals by PCR. After disinfection, SARS-CoV RNA could be detected from some samples from the 309th Hospital of the Chinese People's Liberation Army, but not from Xiao Tang Shan Hospital after disinfection. In this study, we found that the virus can survive for 14 days in sewage at 4 degrees C, 2 days at 20 degrees C, and its RNA can be detected for 8 days though the virus had been inactivated. In conclusion, this study demonstrates that the RNA of SARS-CoV could be detected from the concentrates of sewage of both hospitals receiving SARS patients before disinfection and occasionally after disinfection though there was no live SARS-CoV; thus much attention should be paid to the treatment of stools of patients and the sewage of hospitals receiving SARS patients.

Bacteriophages↗

International collaborative survey on epidemiology of hepatitis E virus in 11 countries.

We conducted seroepidemiological studies on antibody prevalence to hepatitis E virus (HEV) in 5,233 sera from 11 countries to ascertain the present state of HEV infection on a global basis. The prevalence of anti-HEV IgG increased with age in these tested countries, but the rate of antibody positivity was over 20% in the 16-30 year-old group in most of the participating countries, except for Japan, the USA, and Spain. Of patients with acute hepatitis of unknown etiology from Nepal, 56% (14/25) were positive for the IgM class of anti-HEV antibody. In addition, HEV RNAs in the serum from 3 Nepali patients who had the IgM antibody were detected by nested PCR and all of the HEV genes isolated belonged to genotype 1. Our results indicate that HEV is spreading worldwide, not only in developing countries, but also in more industrialized countries than previously thought.

Adolescent↗

Long-term follow-up of hepatitis C virus (HCV) infection in liver transplant patients.

Chronic hepatitis represents a frequent event after orthotopic liver transplantation (OLT). To ascertain the influence of HCV infection on the clinical and histological outcome of these patients, we have investigated the long-term outcome of 22 patients with end-stage chronic liver disease undergoing liver transplant focusing the attention on the role of different HCV genotypes in determining recurrence and severity of post-OLT liver disease. For all patients blood samples taken before OLT and 3 months, 1, 2 and 3 years after OLT were tested for antiHCV antibodies by two different enzyme-linked immuno-assays and by recombinant immuno-blot II and for the presence and type of HCV RNA by nested PCR (5' untranslated region and core gene primers). Of the 16 pre-OLT antiHCV-positive patients, 14 (87.5%) had recurrence of HCV infection while 2 cleared HCV. Pre-OLT genotype recurred in 11 of these 14 patients (2 genotype I) 8 genotype II - in 1 case associated with genotype III - and 1 genotype IV). Of the 6 pre-OLT antiHCV-negative patients, only 1 (16.6%) became persistently HCV-infected, with genotypes I and II. The recurrence of genotype II strictly related with development of severe chronic hepatitis while genotype I and IV were associated with milder forms of liver disease and were more easily cleared.

Chronic Disease↗

Detection of pseudorabies viral DNA in tonsillar epithelial cells of latently infected pigs.

The Rice strain of pseudorabies virus (PRV) was intranasally instilled in pigs that were seronegative to PRV. Cells were scraped or brushed from tonsillar surfaces biweekly until pigs were euthanatized at either 10 or 16 weeks after infection. The DNA extracted from tonsillar cells or parenchyma were subjected to polymerase chain reaction analysis, using either a single set of oligonucleotide primers or nested primers from the PRV gII glycoprotein gene. Pigs became seropositive to PRV by 3 weeks after infection. The virus was isolated from the trigeminal ganglia and tonsils of pigs that were euthanatized or died 1 to 2 weeks after infection, but not from pigs that were euthanatized 10 or 16 weeks after infection. The PRV gene products were consistently detected in trigeminal ganglia and tonsils of all pigs at 1, 10, and 16 weeks after infection, and sporadically in the nasal mucosa, lymph nodes, and lungs of pigs that were euthanatized or died during the first 2 weeks after infection. Cells collected biweekly from tonsillar surfaces were mostly nucleated, squamous epithelial cells with fewer lymphocytes and neutrophils. Polymerase chain reaction analysis of DNA extracted from these cells revealed PRV DNA in a large proportion of the samples when sufficient cells were collected to provide 1 microgram of extracted DNA for use in the reaction mixtures. A second group of pigs had PRV strain 4892 intranasally instilled. The virus was isolated from tonsillar swab specimens until 3 weeks after infection. Tonsillar brushing specimens were collected biweekly until 14 weeks after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗