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M Krajden

Publications and source records attributed to M Krajden.

53 records · Page 3Linked to original sources
PubMed1995

Molecular detection of hepatitis C virus: impact of detection methodology on clinical and laboratory correlations.

The clinical manifestations of hepatitis C virus (HCV) infection are generally indistinguishable from other causes of viral hepatitis. HCV infections are usually anicteric, asymptomatic, and rarely cause acute fulminant liver failure. Serological testing for HCV in conjunction with epidemiological studies have verified that HCV was the major cause of parenterally transmitted non-A, non-B hepatitis (NANBH). With the widespread introduction of serological screening of blood products for HCV antibody, the risk of transfusion-associated HCV infection has been dramatically reduced (to < 3 cases per 10,000 units transfused). Despite the virtual elimination of transfusion-associated infections, the diagnosis of HCV remains important because > 50% of infections are sporadic in origin, 50 to 70% of infected individuals develop chronic hepatitis, and these individuals are at risk of developing cirrhosis (> 20%) as well as hepatocellular carcinoma. Although currently available anti-HCV immunoassays function well as blood-donor screening assays, they are poor at detecting acute infection because of the prolonged lag time between infection and detection of seroconversion (approximately 10 to 26 weeks for second-generation immunoassays). In contrast, polymerase chain reaction (PCR)-based detection of HCV RNA in serum can detect infection in as little as 1 to 2 weeks after exposure. This review focuses on the impact of modern serologic and nucleic acid-based HCV detection methodology on the clinical understanding of HCV infection, its associated illnesses, and its transmissability. Quantitative and reproducible nucleic acid-based detection assays will be required to provide additional insights into the clinical spectrum of HCV infections as well as to assess the efficacy of antiviral agents.

Hepacivirus↗
PubMed1994

Polymerase chain reaction kinetics when using a positive internal control target to quantitatively detect cytomegalovirus target sequences.

High-performance liquid chromatography (HPLC) was used to detect and quantify cytomegalovirus (CMV) specific polymerase chain reaction (PCR) products generated during PCR co-amplification. PCR of CMV AD 169 or a plasmid which contains the CMV AD 169 native target sequence using the CMV primer set of Hsia et al. (J. Clin. Microbiol. 27, 1802-1809) generates a 152 bp PCR product. A CMV control sequence plasmid which shared the primer sequence of native CMV AD 169 but when amplified produces a larger 362 bp product was constructed. Under co-amplification conditions there was a linear relationship (over 3 logs) between the molar ratio of input CMV native and control target sequence and the molar ratio of the output PCR products as detected by HPLC despite differences between the two PCR target and product sizes. Co-amplifying known amounts of CMV control sequence plasmid as an internal standard allowed accurate quantitation of the amount of CMV native target sequence in a sample when the two PCR targets were present in approximately equimolar amounts +/- 1.5 log (coefficient of variation (CV) < 12%). By modifying the amount of CMV control target sequence plasmid used for co-amplification, accurate detection of the amount of CMV native sequence in samples could be extended to 5 logs, standard error (S.E.) < or = 16%. Precise quantitation of PCR targets using co-amplification PCR requires multiple sample dilutions to ensure that the CMV native target sequence was in a close equimolar relationship with the CMV control sequence at the time of PCR amplification.

Base Sequence↗
PubMed1994

Evaluation of the Merifluor immunofluorescent assay for the detection of Cryptosporidium and Giardia in sodium acetate formalin-fixed stools.

The Merifluor Cryptosporidium/Giardia direct immunofluorescent assay (MFDFA) was used for the blinded detection of Cryptosporidium and Giardia in 100 previously tested sodium acetate formalin (SAF)-preserved fecal specimens. Examination for ova and parasites by formalin-ether concentration followed by iron-hematoxylin and Kinyon staining had demonstrated Cryptosporidium (n = 32), Giardia (n = 20) and both Giardia and Cryptosporidium (n = 5). The MFDFA detected one additional specimen with Cryptosporidium and another with Giardia and was rapid, accurate, and simple to perform.

Acetates↗
PubMed1993

Quantification of polymerase chain reaction products in agarose gels with a fluorescent europium chelate as label and time-resolved fluorescence spectroscopy.

We have 5'-end-labeled one polymerase chain reaction (PCR) primer with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). After performing PCR in the presence of another unlabeled primer, we separated the 362 bp PCR product with 2% low melting point agarose gel electrophoresis. The gel was then immersed into a Eu3+ solution. During soaking, Eu3+ diffuses into the gel and associates with BCPDA to form a fluorescent complex of long fluorescence lifetime. This complex can be quantified by scanning the gel with a time-resolved fluorometric reader. Because BCPDA and Eu3+ are not fluorescent by themselves, background signals are very low. The detection limit was about 5 ng of DNA. We have also shown that the BCPDA-labeled product could be blotted and detected on the membrane by using an anti-BCPDA antibody. These two technologies may find applications other than in PCR, e.g. in fluorescence-based DNA sequencing and in solution hybridization.

Blotting, Southern↗
PubMed1993

High-performance liquid chromatography to assess the effect of serum storage conditions on the detection of hepatitis C virus by the polymerase chain reaction.

The effect of various serum storage conditions on the detection of hepatitis C virus (HCV) by the polymerase chain reaction (PCR) was assessed. 50 microliters aliquots of serum from four HCV PCR positive patients were subjected, in triplicate, to: (a) storage at -20 degrees C for 1, 5, 10 days; (b) storage at room temperature (RT) for 1, 2, 7 days; (c) 1, 3, 5, and 10 successive freeze-thaw cycles; (d) incubation at 37 degrees C, and 56 degrees C for 1, 3, 24 h; and (e) storage in guanidium-thiocyanate extraction buffer at RT, and 4 degrees C for 1, 5, 10 days. PCR products were detected by agarose gel electrophoresis (AGE) and quantitatively by high-performance liquid chromatography (HPLC). Only storage in extraction buffer at RT for 5-10 days and incubation at 56 degrees C for 24 h appeared to result in a loss of > or = 50% of detectable HCV PCR product. Up to 10 successive freeze-thaw cycles, storage at -20 degrees C for up to 10 days or at RT for up to 7 days, storage in extraction buffer at RT for 1 day or at 4 degrees C for up to 10 days, and incubation at 37 degrees C for up to 24 h resulted in minimal PCR signal loss. HPLC was a reproducible method of detecting and quantitating HCV PCR products, and was more sensitive than AGE.

Blood Preservation↗
PubMed1993

Continuous flow perfusion of gentamicin with a scleral shell reduces bacterial colony counts in experimental Pseudomonas keratitis.

We have previously shown the pharmacokinetic value of delivering gentamicin to the rabbit anterior segment using the Morgan Therapeutic Lens. The present study utilized an intrastromal injection model of Pseudomonas keratitis to test the therapeutic efficacy of continuous flow delivery of gentamicin with the Morgan therapeutic lens. All eyes (n = 52) received an intrastromal injection of approximately 1800 colony forming units (CFU) of Pseudomonas aeruginosa. At 22 hours after injection, eyes were perfused for 6 hours with saline or gentamicin (1, 2.5 or 5 mg/ml), or received gentamicin drops (13.6 mg/ml) at 15 minutes for four doses, then hourly for 6 hours. Corneas were homogenized and plated to determine bacterial survival, and expressed as log colonies (CFU). Log CFU recovered were 7.37 +/- 0.04, 6.64 +/- 0.20, 5.64 +/- 0.31, and 3.56 +/- 0.50 log CFU for saline perfusion, 1, 2.5, 5 mg/ml gentamicin perfusion respectively. Following six hours of treatment with topical fortified gentamicin drops, 5.93 +/- 0.34 log CFU were recovered. Gentamicin perfusion (5 mg/ml) was significantly different from saline or the other treatment groups (P < 0.05). Continuous corneal perfusion with the Morgan Therapeutic Lens demonstrated an increasing dose response curve with increasing perfusate concentration. It was effective in the treatment of experimental Pseudomonas keratitis.

Animals↗
PubMed1993

Hepatitis C virus infection in patients infected with the human immunodeficiency virus.

The prevalence and characteristics of hepatitis C virus (HCV) infection in 226 patients who were seropositive for human immunodeficiency virus (HIV) were determined. Antibody to HCV (anti-HCV) was detected by enzyme immunoassay (EIA), and positive results were confirmed by a neutralization EIA or recombinant immunoblot assay. The prevalence of anti-HCV was 8%. Intravenous drug use was the most common risk factor for HCV infection (61.1% of patients), and 52.4% of intravenous drug users were seropositive for anti-HCV (HCV+). Only 16.7% of HCV+ patients had AIDS, as compared with 37.4% of anti-HCV-seronegative (HCV-) patients (P = .04). The prevalence of hepatitis B virus markers in patients with and without anti-HCV was similar. The CD4+ lymphocyte counts were higher for HCV+ patients than for HCV- patients (P = .001), and the prevalence of anti-HCV decreased in parallel with CD4+ counts. Elevated liver function test values were more common for HCV+ patients than for HCV- patients (61.1% vs. 26.0%; P < .01), but abnormalities were usually slight (< 2-fold elevation in values). HCV viremia was detected by the polymerase chain reaction in 88.2% of HCV+ patients. Despite the coexistence of HIV and HCV infection, liver disease appeared to be mild, and HCV infection did not appear to increase the severity of HIV infection. Serological tests for HCV appear to underestimate the prevalence of HCV infection in patients with advanced HIV infection or AIDS.

Adult↗
PubMed1993

Pseudomonas cepacia in lung transplant recipients with cystic fibrosis.

Twenty-four isolated double lung transplants (LTXs) have been performed in 22 patients with cystic fibrosis, with a follow-up of 4 to 47 months. Prior to LTX, all patients were colonized with Pseudomonas aeruginosa, and ten patients were also colonized with Pseudomonas cepacia. Both organisms were specifically sought before LTX. All patients who grew P cepacia before LTX did so after LTX. Five additional patients only grew this bacterium after LTX. There was no difference between those who grew P cepacia and those who did not in terms of data before LTX for age, weight, pulmonary function, and 6-min walk. After LTX, 7 of the 15 patients who had ever grown P cepacia died. No patient who grew only P aeruginosa died. The median survival in the subgroup with P cepacia was 28 days. Five of the seven died as a direct result of P cepacia pneumonia and sepsis. One died of cyclosporin A (cyclosporine) neurotoxicity with concurrent P cepacia pneumonia, and one died at the time of a retransplant for graft failure (associated with three bouts of P cepacia pneumonia and cytomegalovirus). Four of seven had not grown this bacterium before LTX. There were no perioperative factors, including antibiotic choices, that distinguished survivors and nonsurvivors. Overall 1-year survival is about 70 percent (15/22). Fourteen bouts of P cepacia pneumonia occurred in 12 patients. Four empyemas, one lung abscess, one suppurative pericarditis, and five cases of sinusitis were also due to this bacterium. In conclusion, P cepacia is responsible for excess morbidity and mortality after LTX. This organism is particularly lethal if isolated for the first time after LTX. Factors predicting its acquisition in this setting are unknown. While it is possible that the facial sinuses may act as an unrecognized reservoir or that patients or equipment provide a source, further study into the epidemiology of this organism is necessary to improve the survival of colonized patients undergoing LTX.

Adult↗
PubMed1992

Continuous flow contact lens delivery of gentamicin to rabbit cornea and aqueous humor.

The Morgan Therapeutic Lens (MTL) was investigated as a continuous corneal perfusion system in New Zealand white rabbits. Gentamicin concentration in the cornea and aqueous humor delivered by the MTL was compared to gentamicin drops (13.6 mg/ml) administered every 15 or 30 minutes. Gentamicin (1 mg/ml) or 5 mg/ml was perfused at 10 ml/hr for up to 4.0 hours. At each time interval of 0.5, 1, 2 and 4 hours, homogenized corneas and aqueous humour were assayed for gentamicin concentrations. The highest aqueous humour gentamicin concentrations of 57.99 +/- 12.86 micrograms/ml were significantly higher with the MTL and 5 mg/ml of gentamicin compared with the MTL and 1 mg/ml of gentamicin or than drops applied every 30 minutes, but not significantly different than drops every 15 minutes. Highest corneal concentrations of gentamicin of 496.04 +/- 101.16 micrograms/gm cornea were significantly higher with MTL and 5 mg/ml of gentamicin compared with the MTL and 1 mg/ml gentamicin or than drops applied every 30 minutes, but not significantly different than drops every 15 minutes. All mean gentamicin concentrations attained via the MTL exceeded the mean inhibitory concentration for most sensitive bacterial species. The MTL is a reliable drug delivery system with distinct advantages, and may be a useful therapeutic modality in the ocular delivery of gentamicin and other drugs.

Animals↗
PubMed1992

Innovative endpoint determination system for antifungal susceptibility testing of yeasts.

Fungal infections and antifungal resistance are increasingly recognized. Antifungal susceptibility testing remains unstandardized, and a particularly important problem is endpoint determination. In this paper we propose the yeast metabolic reduction of the tetrazolium salt 2,3-bis(2- methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetraz olium hydroxide (XTT) as a colorimetric endpoint which is quantitative and objective. Amphotericin B, fluorocytosine, and fluconazole dose-response curves were obtained, and a metabolic MIC could be determined by using precise criteria.

Antifungal Agents↗
PubMed1991

Cardiovascular manifestations of Lyme disease.

Although the cardiac manifestations of Lyme disease may be diverse, in general they are treatable with currently available therapies. A high index of suspicion is required to make a diagnosis, especially for patients who may lack a suggestive history of tick exposure or residence in an endemic region. Lyme disease-related heart block may require pacemaker insertion and supportive care. The efficacy of antibiotics in the therapy of acute and chronic cardiac Lyme disease will require further study. Serologic testing and cardiac histopathology are the most precise methods of diagnosis. There is a need to develop more sensitive and specific diagnostic tests for Lyme disease and for Lyme carditis in particular.

Administration, Oral↗
PubMed1990

Clinical features of adenovirus enteritis: a review of 127 cases.

We retrospectively analyzed the clinical features of 127 hospitalized pediatric patients whose fecal samples were positive for adenovirus (Ad) by electron microscopy during an 18-month period. Serotyping results obtained by microneutralization tests and restriction endonuclease analysis were available for 105 of 127 cases. There were 69 males and 58 females and 94% of patients were less than 4 years of age. The average body temperature was 38 degrees C rectal (range, 36.2-40.8 degrees C) with an average duration of fever of 1.6 days. The average duration of clinical illness was 8.8 days (range, 1 to 32 days). Although Ad 40 and Ad 41 were isolated in the majority of cases (59 of 105 (56%], Ad 31 was associated with 18 of 105 cases (17%). Of the 18 cases associated with Ad 31, 14 were nosocomial and associated with diarrhea. Our survey confirms the importance of fastidious enteric Ad in infantile diarrhea (Ad 40, Ad 41) and suggests that Ad 31 produces a clinical syndrome indistinguishable from that caused by Ad 40 and Ad 41. The occurrence of Ad enteritis in patients admitted for unrelated illnesses well after initial hospitalization suggests that Ad is also an important cause of nosocomial enteritis in our hospital.

Adenovirus Infections, Human↗
PubMed1986

Approaches to the detection of enteric pathogens, including Campylobacter, using nucleic acid hybridization.

A previous study of the detection of Campylobacter jejuni in fecal samples spotted directly onto nitrocellulose filters before hybridization revealed a relatively low sensitivity and some false-positive results. We have investigated two factors that interfere with the detection of Campylobacter jejuni in fecal samples: interfering substances that create false-positive background signals and nonspecificity of the probe. Heterologous deoxyribonucleic acid probes bound nonspecifically to partially extracted fecal samples, indicating that the major basis for false-positive background was not due to homologous sequences. The problem of false-positive results could be reduced, but not eliminated, by extensive deoxyribonucleic acid extraction procedures applied to the clinical sample. The relative concentration of protein in each sample may be an important contributor to nonspecific binding. Other measures, such as sonication, glass bead fragmentation, and column separation, were not helpful. Development of a species-specific deoxyribonucleic acid probe derived from sequences encoding 16S ribonucleic acid is underway.

Campylobacter Infections↗
PubMedDate not supplied

Long term follow-up of patients with chronic hepatitis C treated with interferon alpha.

Interferon alpha (IFN alpha) treatment for chronic hepatitis C induces a sustained biochemical and virological response at six months after completing 24 weeks of therapy in approximately 10% of patients. The long term durability of this 'sustained' response is still controversial. The aim of this multicentre study was to assess the long term virological response in patients considered to have achieved a sustained biochemical response six months after completing IFN treatment. The majority (36 of 41) of the sustained responders identified had been treated for six months with IFN therapy. Twenty-nine of the 41 patients (70%) had undetectable hepatitis C virus (HCV) RNA after a mean follow-up of 38 months after cessation of treatment (range six to 92 months). All but one of those 29 individuals had normal serum alanine aminotransferase (ALT) levels. Of the 16 patients (out of 41) who had been tested for HCV RNA six months after treatment, HCV RNA remained undetectable in 14 (88%) at final follow-up. Serum ALT values in the 11 of 12 patients whose HCV RNA was positive at final follow-up were lower than pretreatment values, and in six cases were within the normal range. The long term sustained virological response in those considered a 'sustained responder' six months after receiving only six months of IFN is high. Measurement of ALT is an unreliable marker of sustained response to therapy.

Adult↗
PubMedDate not supplied

Hepatitis C virus diagnosis and testing.

Development of serological and nucleic acid testing (NAT) has revolutionized hepatitis C virus (HCV) diagnosis. Although third generation anti-HCV enzyme immunoassays (EIAs) are very effective for testing high prevalence populations, confirmatory testing is still necessary when these tests are applied to populations with a low HCV prevalence to exclude false positive results. Limitations of third generation anti-HCV EIAs include: the relatively prolonged time between acute infection and detection of seroconversion (which typically requires at least 5-6 weeks); delayed seroconversions in immunocompromised hosts (requiring months to years); and the inability of serological tests to confirm active HCV infection. In contrast, nucleic acid testing (NAT) can directly detect HCV RNA in serum, plasma or tissue and thereby confirm active infection as well as narrow the window between infection and HCV detection to as little as 1-2 weeks. Commercial NAT assays are now highly sensitive, specific, and reproducible and have largely replaced unreliable home brew nucleic acid amplification assays. Qualitative commercial NAT are typically more sensitive than quantitative assays and therefore the method of choice to confirm active infection. Given the efficacy of combination therapy with interferon/ribavirin and newer antiviral agents under development, HCV infection may become curable, which will likely impact future disease transmission. As the therapeutic costs are currently very high, there is clearly a need to assess the utility of quantitative NAT and to further evaluate the role of HCV genotyping to optimize antiviral therapy. Thus for the foreseeable future, a combination of both serological tests and NAT will be required for cost-effective HCV diagnosis and monitoring.

Hepatitis C↗