Prenatal detection of unbalanced chromosomal rearrangements by array CGH.

BACKGROUND: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. METHODS: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. RESULTS: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. CONCLUSIONS: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Prenatal diagnosis by array-CGH.

Microscopic karyotype analysis of cultured cells has been regarded as the gold standard for prenatal diagnosis for over 30 years. Since the first application of this technique to prenatal testing in the early 1970's, this procedure has proved to be highly reliable for identifying chromosome copy number abnormalities (aneuploidy) and large structural rearrangements in foetal cells obtained invasively by either amniocentesis or chorionic villus sampling (CVS). Recognising the need for more rapid testing methods which do not require cell culture, fluorescence in situ hybridisation (FISH) and quantitative fluorescence PCR (QF-PCR) have been introduced to this field in order to answer specific diagnostic questions. However, both FISH and QF-PCR suffer the disadvantage in that they are difficult to scale to a comprehensive, genome-wide screen. Array-comparative genomic hybridisation (array-CGH) in contrast is a comprehensive, genome-wide screening strategy for detecting DNA copy number imbalances which can be rapid, less labour-intensive than karyotype banding analysis and is highly amenable to automation. Array-CGH has the potential to be used for prenatal diagnosis and may address many of the limitations of both conventional microscopic cytogenetic analyses and the more recently employed rapid-screening strategies.

Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features.

The underlying causes of learning disability and dysmorphic features in many patients remain unidentified despite extensive investigation. Routine karyotype analysis is not sensitive enough to detect subtle chromosome rearrangements (less than 5 Mb). The presence of subtle DNA copy number changes was investigated by array-CGH in 50 patients with learning disability and dysmorphism, employing a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Twelve copy number abnormalities were identified in 12 patients (24% of the total): seven deletions (six apparently de novo and one inherited from a phenotypically normal parent) and five duplications (one de novo and four inherited from phenotypically normal parents). Altered segments ranged in size from those involving a single clone to regions as large as 14 Mb. No recurrent deletion or duplication was identified within this cohort of patients. On the basis of these results, we anticipate that array-CGH will become a routine method of genome-wide screening for imbalanced rearrangements in children with learning disability.

Spectrum of dominant mutations in the desmosomal cadherin desmoglein 1, causing the skin disease striate palmoplantar keratoderma.

The adhesive proteins of the desmosome type of cell junction consist of two types of cadherin found exclusively in that structure, the desmogleins and desmocollins, coded by two closely linked loci on human chromosome 18q12.1. Recently we have identified a mutation in the DSG1 gene coding for desmoglein 1 as the cause of the autosomal dominant skin disease striate palmoplantar keratoderma (SPPK) in which affected individuals have marked hyperkeratotic bands on the palms and soles. In the present study we present the complete exon-intron structure of the DSG1 gene, which occupies approximately 43 kb, and intron primers sufficient to amplify all the exons. Using these we have analysed the mutational changes in this gene in five further cases of SPPK. All were heterozygotic mutations in the extracellular domain leading to a truncated protein, due either to an addition or deletion of a single base, or a base change resulting in a stop codon. Three mutations were in exon 9 and one in exon 11, both of which code for part of the third and fourth extracellular domains, and one was in exon 2 coding for part of the prosequence of this processed protein. This latter mutation thus results in the mutant allele synthesising only 25 amino acid residues of the prosequence of the protein so that this is effectively a null mutation implying that dominance in the case of this mutation was caused by haploinsufficiency. The most severe consequences of SPPK mutations are in regions of the body where pressure and abrasion are greatest and where desmosome function is most necessary. SPPK therefore provides a very sensitive measure of desmosomal function.

Ascites and the acquired immunodeficiency syndrome. Report of 54 cases.

We identified 54 patients with AIDS and ascites seen over a 4.5-year period at a university hospital. This retrospective study is the largest reported series of patients with AIDS and ascites. Patients with AIDS who are evaluated for ascites should be stratified by the CD4 + cell count and the presence or absence of portal hypertension based upon the serum-ascites albumin gradient and clinical presentation. Awareness of possible surgery-related causes of ascites is crucial, as these patients may not manifest the usual signs and symptoms of peritonitis or abdominal catastrophes seen in immunocompetent hosts. Patients with evidence of portal hypertension due to hepatic cirrhosis and an elevated ascitic neutrophil count should be suspected to be infected with common bacterial pathogens associated with peritonitis unless the CD4 + cell count is below 50 cells/mm3. When the CD4 + cell count declines below this threshold, infections due to Mycobacterium avium complex, cytomegalovirus, and other opportunistic infections should be considered.

The potential for transmission of human immunodeficiency virus through human bites.

We review the potential for human immunodeficiency virus (HIV) transmission by human bites. HIV may be present in saliva, although infrequently and at low levels. In prospective studies, 13 people bitten by HIV-infected individuals have remained HIV seronegative. Only two cases have been published in which HIV transmission through bites may have occurred. Both blood-contaminated and cell-free saliva may contain HIV. The presence of blood in the saliva may potentially heighten the theoretical risk of HIV transmission through human bites. We have estimated the risk of HIV transmission through human bites and have compared it with the known risks of HIV seroconversion by needle stick (0.3-0.5%). Needle sticks, on average, could transmit 20 times more HIV-infected cells than would a human bite. We conclude that the transmission of HIV through human bites is biologically possible but remains unlikely, epidemiologically insignificant, and, as yet, not well documented.

Rapid clinical diagnosis of pulmonary abnormalities in HIV-seropositive patients by auscultatory percussion.

A prospective, blinded study of pulmonary findings in hospitalized patients with HIV infection compared auscultatory percussion (AusP) with conventional percussion (ConP) and conventional auscultation (ConA) using chest radiographs as the gold standard. Sixty-three patients had chest radiographs and were examined by one to three examiners. Seventy of the 126 lungs had radiographic abnormalities (55.6 percent). Auscultatory percussion proved to be the most sensitive of all techniques for each examiner (range, 51.0 to 69.6 percent) for detecting radiographic abnormalities and also had higher likelihood ratios for two of the three examiners; AusP also had the highest likelihood ratio pooled across examiners. Of the 166 abnormal results of lung examinations, the combination of AusP and ConA detected 31 more abnormalities than ConP and ConA combined, with 14 of these being diagnosed with Pneumocystis carinii pneumonia. No abnormalities were detected by ConP that were not detected by AusP. These findings suggest that AusP, a rapid clinical maneuver, is more sensitive and specific than ConA and ConP in determining pulmonary abnormalities in HIV-infected inpatients.

Initial worker evaluation of a new safety syringe.

A prospective evaluation of a new safety syringe requiring a one-step activation was carried out at the University of California, San Diego Medical Center. Only 59.5% of 390 syringes were activated, and user acceptance and satisfaction were unfavorable. The development of safety devices should incorporate passive activation and take end-user satisfaction into consideration.

Combination ganciclovir and foscarnet in the treatment of clinically resistant cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome.

OBJECTIVE: To assess the clinical response and patient tolerance to daily infusions of both ganciclovir sodium and foscarnet sodium for the treatment of clinically resistant cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome. DESIGN AND PATIENTS: Nine patients with clinically resistant cytomegalovirus retinitis who had shown progression of retinitis despite extended intravenous induction single-drug therapy or alternating therapy with induction doses of ganciclovir or foscarnet at 6 weeks were subsequently treated with a combination of ganciclovir and foscarnet. The dosing regimen for induction combination therapy was ganciclovir at 5 mg/kg every 12 hours and foscarnet at 60 mg/kg every 8 hours. Maintenance combination therapy was ganciclovir at 5 mg/kg every 12 to 24 hours and foscarnet at 90 to 120 mg/kg every day. Patients were observed closely for signs of a toxic effect or intolerance to the drug regimen. RESULTS: All patients exhibited a favorable response to combination therapy, with complete healing of retinitis in 12 of 14 eyes and partial healing of retinitis with decreased border activity and a cessation of border advancement in two of 14 eyes. Two of the nine patients stopped receiving combination therapy before completion of the study owing to their dissatisfaction with the time commitment. The regimen was otherwise well tolerated, with no significant medical toxic effects attributable to the drugs requiring cessation of therapy. CONCLUSIONS: Combination anticytomegalovirus therapy should be considered in those patients who have shown a poor clinical response to sustained single-drug induction therapy and alternating drug therapy. As survival time for patients with cytomegalovirus retinitis continues to improve, clinical resistance may become more common. Further work to delineate the optimal dosing and indications for combination therapy will be important.

Clinicopathologic correlations in acute retinal necrosis caused by herpes simplex virus type 2.

The acute retinal necrosis syndrome is a rapidly progressive and potentially devastating disease. A case of acute retinal necrosis developed in an immunocompetent man, Presumably due to the stress, trauma, or immunomodulation related to a craniotomy for a parasellar craniopharyngioma. Vitrectomy and endoretinal biopsy were performed. Polymerase chain reaction studies of the vitreous revealed herpes simplex virus type 2 as the cause, which has not been previously well documented. Results of cerebrospinal fluid antibody studies were also consistent with the diagnosis. Results of cytology and histopathologic examination demonstrated extensive retinal destruction and mononuclear cell infiltration. Sloughing of the inner retina was evidenced by the presence of retinal vascular remnants in the vitreous cytology specimen. As is characteristic of this disease, the visual outcome of this patient was poor.

Expanding the role of the infection control professional in the cost-effective use of antibiotics.

There is a growing demand that health care expenses be contained and that excessive and inappropriate use of antibiotics be eliminated. At the University of California, San Diego Medical Center, strategies aimed at controlling drug usage and subsequently reducing costs have been implemented and found to be effective. Mechanisms designed to achieve such goals without diminishing quality of care involve expanding the role of the infection control professional (ICP) while implementing antibiotic control stratagems such as antimicrobial utilization teams, antibiotic order sheets, audits of use, automatic stop orders, computer-assisted management, drug use reviews, educational efforts, formulary practice, restricted drug policies, and target drug monitoring. The infection control professional, as well as other members of the antimicrobial utilization team, contributes to the promotion of the appropriate use of antibiotics in part by identifying individual cases in which antibiotics might be used inappropriately, such as for the treatment of colonization rather than infection or when appropriate microbiologic testing has not been carried out.

Novel uses of the aromagram in infection control and epidemiology.

BACKGROUND: To facilitate the interpretation of data used in infection control and epidemiology, a novel data presentation format (the aromagram) has been developed and modified. METHODS: Aromagrams were developed with a personal computer-based graphics application. Aromagrams were based on antimicrobial susceptibility data from all specimen submitted to the University of California San Diego Medical Center's clinical microbiology laboratory between July 1992 and December 1994. RESULTS: The aromagrams created displayed both bacterial species-specific and antimicrobial agent-specific susceptibilities. Additional modified aromagrams incorporated costs of antimicrobial agents and temporal trends in susceptibility of individual species to selected antibiotics. CONCLUSIONS: The aromagram is a unique format for data presentation that can be used to illustrate antimicrobial susceptibilities (specific to both organisms and antimicrobial agents), temporal trends in susceptibility data, and antimicrobial costs. Aromagrams may be used to display data useful to infection control and epidemiology professionals and to clinicians.

Streptococcus pneumoniae: bacteremia in an era of penicillin resistance.

BACKGROUND: The proportion of penicillin-resistant Streptococcus pneumoniae isolates and associated risk factors varies by geographic area in the United States. We conducted a retrospective study to determine the extent of penicillin-nonsusceptible S pneumoniae bacteremia and associated risk factors in a tertiary care medical center in San Diego. METHODS: Patients with S pneumoniae bacteremia at the University of California, San Diego Medical Center from September 15, 1991, through July 31, 1998, were identified by hospital-based computerized microbiology records. Hospital records included demographic information, patient data, and antibiotic prescription records for patients with bacteremia as a result of S pneumoniae. Univariate and multivariate analyses were used to determine risk factors for penicillin-nonsusceptible S pneumoniae bacteremia. RESULTS: Of 281 isolates of S pneumoniae identified, 192 (68%) were from hospitalized patients. After controlling for other factors, patients from 1 to 5 years of age (P = .01; odds ratio [OR] = 3.96; 95% CI, 1.50 to 10.44), 6 to 18 years of age (P =.04; OR = 6.42; 95% CI, 1.13 to 36.51), and HIV seropositive patients (P =.002; OR = 5.12; 95% CI, 1.83 to 14.32) were more likely to have penicillin-nonsusceptible S pneumoniae bacteremia. There was a significant increasing trend of penicillin-nonsusceptible S pneumoniae bacteremia from 14% in 1991 to 42% in 1998 (P = .001; OR = 1.42; 95% CI, 1.16 to 1.73); this included only 2 isolates that were highly resistant to penicillin. There was no increase in mortality in patients who had penicillin-nonsusceptible S pneumoniae bacteremia. CONCLUSION: With the increase in S pneumoniae resistance to penicillin, it is important to continue surveillance of infections caused by S pneumoniae. Hospital-based studies are useful for tracking epidemiologically important pathogens.

Acridine orange detection of Plasmodium falciparum malaria: relationship between sensitivity and optical configuration.

Blood samples collected from five volunteers participating in a P. falciparum infectivity trial were examined to determine the efficacy of the acridine orange technique. Several lens configurations were tested for efficiency in the diagnosis of malaria using this system. There was no significant difference in the sensitivity for detecting positive specimens or number of parasites among three lens configurations: a 50x long working distance objective (0.34 mm) with either a 10x ocular (total magnification 500x) or a 12.5x ocular (625x) and a 750x configuration using a 50x objective with a shorter working distance (0.24 mm). All three lens configurations were significantly better than the 1,000x configuration using a commonly available 100x oil immersion objective. The results achieved using this lens still exceeded the sensitivity of the thick blood film.

Acridine orange diagnosis of Plasmodium falciparum: evaluation after experimental infection.

The value and role of the acridine orange/microhematocrit tube method (quantitative buffy coat [QBC] analysis) in the diagnosis of malaria remains controversial. To establish the true sensitivity of this test in comparison with the thick blood film, we studied 49 subjects who were experimentally infected with Plasmodium falciparum in 10 malaria vaccine and infectivity trials. Diagnosis was made by the acridine orange staining method 1-3 days earlier than by the thick blood film in 23 subjects (47%) and at the same time as the thick blood film in 20. On the other hand, diagnosis was made by thick blood film earlier than by the acridine orange staining method in six individuals. There were no false positive results using acridine orange among 584 specimens studied. Diagnosis was made using acridine orange at a parasitemia of less than 11 parasites/microliters of blood in 65% of cases. Where available, the acridine orange assay is clearly preferable in terms of speed and accuracy to the thick blood film for diagnosis with parasitemias of less than 150/microliters of blood, and perhaps as important, for ruling out infection with P. falciparum in a symptomatic patient.

Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans.

Malaria was transmitted to only 5 of 10 volunteers bitten by 1-2 Anopheles stephensi carrying sporozoites of the 3D7 clone of the NF54 strain of Plasmodium falciparum in their salivary glands. Parasites were detectable by culture in blood taken 7-10 days following exposure and by thick blood film 14-16.5 days after exposure. Infectivity did not correlate with the numbers of sporozoites in the salivary glands.

Correlation between CD4+ counts and prevalence of cytomegalovirus retinitis and human immunodeficiency virus-related noninfectious retinal vasculopathy in patients with acquired immunodeficiency syndrome.

We prospectively studied 132 patients with acquired immunodeficiency syndrome (AIDS) to determine the cross-sectional prevalence of cytomegalovirus retinitis. All patients had serum CD4+ lymphocyte counts to determine the degree of immune dysfunction. Correlations between CD4+ counts, the presence of cytomegalovirus retinitis or human immunodeficiency virus (HIV)-related noninfectious retinal vasculopathy, and ocular symptoms were made. The study disclosed that 26 of 132 patients with AIDS (20%) had cytomegalovirus retinitis. However, subset analysis according to CD4+ count disclosed that in patients with CD4+ counts of 50 cells/mm3 or less, 26 of 87 (30%) had cytomegalovirus retinitis, whereas in patients with CD4+ counts of 50 cells/mm3 or more, none of 45 was noted to have cytomegalovirus retinitis. Similarly, 46 of 132 patients (35%) were noted to have HIV-related noninfectious retinal vasculopathy, with a trend toward increasing prevalence associated with declining CD4+ count. In patients with CD4+ counts of 50 cells/mm3 or less, 39 of 87 (45%) had HIV-related noninfectious retinal vasculopathy, whereas seven of 45 patients (16%) with CD4+ counts of 50 cells/mm3 or more were noted to have these changes. We confirmed the clinical impression that cytomegalovirus retinitis and HIV-related noninfectious retinal vasculopathy are late manifestations of AIDS, demonstrated an increased risk for patients with low CD4+ counts, and suggested a basis for coherent chemoprophylaxis and screening strategies for cytomegalovirus retinitis.

Eustachian valve endocarditis: a case series and analysis of the literature.

Eustachian valve endocarditis (EVE) is a syndrome characterized by clinical signs and symptoms of right-sided infective endocarditis in association with an infective vegetation on the eustachian valve. EVE usually occurs without associated involvement of any other cardiac valves, and it is difficult to diagnose accurately by transthoracic echocardiography. Transesophageal echocardiography appears to be a more sensitive tool for the diagnosis of EVE, and it is recommended when a patient with typical signs of right-sided endocarditis has normal results on a transthoracic echocardiography study. In general, EVE responds well to conventional antibiotic therapy.

Variation in the sensitivity of successive variable antigen types in a Trypanosoma (Trypanozoon) brucei subspecies clone to some African game animal sera.

A Trypanosoma brucei subspecies clone was passaged in rats at ten-day intervals and the sensitivity/resistance to a variety of mammalian sera, of the successive variable antigen types (VATs) produced, was examined sequentially in a modified version of the blood incubation infectivity test (BIIT). A proven homogeneous VAT was used to initiate this series of tests, in which seven successive and different VATs were each exposed in vitro at 38.5 degrees C for 2 h to standard samples of pooled rat serum (PRS), normal human serum (NHS) and to sera from two different eland and three different hippopotami. Samples were then inoculated into susceptible rats to determine the effects of the individual sera on the subsequent infectivity of the trypanosomes. The seven VATs were found to vary widely in their sensitivity to the different game sera, though all remained strongly resistant to the pooled rat and the normal human serum samples. BII testing of isolates from positive test rats in the sequential study showed that their resistance to normal human serum was unaffected by their exposure in vitro to the different game sera.

Eustachian valve endocarditis: detection with multiplane transesophageal echocardiography.

Right-sided involvement is fairly common in infective endocarditis, but involvement of the eustachian valve is distinctly rare. We present the case of a 36-year-old intravenous drug user with staphylococcal bacteremia and septic pulmonary emboli. Transthoracic echocardiography was normal, but transesophageal echocardiography revealed a large eustachian valve vegetation. This case illustrates the utility of multiplane transesophageal echocardiography in the evaluation of eustachian valve pathology.