Mel Krajden

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.

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.

Long-term follow-up of a randomized trial of interferon therapy for chronic hepatitis B in a predominantly homosexual male population

BACKGROUND/AIMSnExtended follow-up of a previously published therapeutic trial with interferon alfa is now available to further clarify the long-term outcome of HIV-negative and HIV-positive subjects with chronic hepatitis B virus infection after interferon alfa therapy.nnnMETHODSnForty-five subjects with compensated liver disease and chronic hepatitis B infection with evidence of active hepatitis B replication were studied. These subjects were enrolled between 1986 and 1991 and had been randomized, stratified by HIV status, to either receive interferon therapy (10 MU/m2 of lymphoblastoid interferon alfa 3 times per week for 12 weeks) or no treatment. Hepatitis B serology, serum hepatitis B viral DNA and alanine aminotransferase were measured on an annual to biannual basis. CD4-positive T lymphocyte counts and HIV RNA concentration were also obtained.nnnRESULTSnFrom 9 months post-interferon alfa treatment to the end of the extended follow-up (4 to 9 years), the relative risk of seroconverting to anti-HBe positive for subjects who had received interferon alfa therapy compared to those who did not was not significant in either HIV-negative (p = 0.80) or HIV-positive (p = 0.62) subjects.nnnCONCLUSIONSnUnlike the first 9 months following interferon alfa therapy, the rate of elimination of markers of hepatitis B virus replication, regardless of HIV status, was not increased above the natural rate beyond 9 months following interferon alfa therapy.

Polymerase chain reaction based assessment of leukoreduction efficacy using a cytomegalovirus DNA transfected human T-cell line

BACKGROUND AND OBJECTIVESnDetection of CMV DNA by PCR in seropositive blood donors is affected by nucleic acid extraction, amplification conditions and PCR product detection sensitivity. Clinical studies have shown that leukoreduced blood products are as effective as CMV-seronegative blood products in minimizing transfusion-associated CMV infection. We developed a PCR-based test model to assess the efficacy of leukoreduction on the removal of CMV DNA from blood donations.nnnMATERIALS AND METHODSnWhole blood units were spiked with a human Jurkat T-lymphocyte cell line which had been stably transfected with a CMV DNA immediate early sequence. All blood units were either CMV seronegative or shown to be CMV PCR negative. The amount of CMV DNA by PCR before, during and after filtration of blood units with third-generation leukocyte reduction filters were determined using a semi-quantitative adaptation of the Digene SHARP Signal System Assay (DSSSA).nnnRESULTSnWhole blood units spiked with 1-2 x 10(6) CMV transfected Jurkat cells contained no detectable CMV DNA after leukoreduction. With a higher inoculum of 2.9 x 10(8) transfected Jurkat cells per unit, CMV DNA was detected after leukoreduction; however, there was an approximate 3 log decrease in the amount of CMV DNA detected.nnnCONCLUSIONnThis CMV DNA transfected human Jurkat cell line in conjunction with semi-quantitative CMV PCR may be used to model leukoreduction efficacy and provides evidence that leukoreduction of blood products can decrease the amount of cellular CMV DNA by approximately 3 logs.

Durability of serological remission in chronic hepatitis C treated with interferon-Alpha-2B

Objective:We assessed the long-term effect of a course of interferon therapy on the biochemical and virological markers of Canadian patients with chronic hepatitis C.Methods:Thirty-six patients with chronic hepatitis C were treated with a median total dose of interferon-α-2B of 181.0 million U (range 109.0–384.0 million U) and were followed for a median of 37.2 months (range 12.0–94.2 months) after completing treatment. All patients received an initial 16 wk of interferon at a dose of 3 million U three times weekly; this was followed by either no further interferon or by 8 wk more at doses ranging from 1.5 to 10.0 million U three times weekly. Serum alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were measured before interferon therapy, 6 months after treatment, and at the end of follow-up for each patient. HCV RNA was analyzed by branched DNA 1.0 assay and, if undetectable, by polymerase chain reaction. HCV genotyping was performed on serum samples.Results:Five (13.6%) of the 36 patients had a sustained treatment response, defined as normal ALT and undetectable viremia 6 months after treatment. All five patients remained in serological remission to the end of their follow-up, a median of 48.2 months (range 23.0–66.2 months) after interferon therapy. Responders were similar to nonresponders in age, gender, initial ALT and serum HCV RNA levels, pretreatment histology, and total dose of interferon received.Conclusions:In patients with chronic hepatitis C, 13.6% had normal ALT and undetectable serum HCV RNA 6 months after finishing interferon therapy. These patients remained in serological remission to the end of their follow-up, 48.2 months after interferon therapy.

Multi-organ donor transmission of hepatitis C virus to five solid organ transplant recipients and lack of transmission to corneal transplant recipients

BACKGROUNDnA multi-organ donor seronegative for hepatitis C virus (HCV) by 1st generation enzyme immunoassay (EIA) supplied 5 solid organs and 2 corneas to 7 recipients. This donor was retrospectively shown to be 2nd generation HCV EIA-positive and polymerase chain reaction (PCR)-positive. All 5 solid organ recipients but none of the corneal recipients developed HCV infection.nnnOBJECTIVESnTo demonstrate the discordance between serological and PCR-based HCV detection in solid organ transplant recipients and the lack of transmission of HCV to the two corneal transplant recipients.nnnSTUDY DESIGNnAll 5 solid organ recipients were retrospectively shown to be HCV PCR-negative and -seronegative pre-transplant and HCV PCR-positive post-transplant. Serial serum samples on 3 recipients were evaluated by 2nd generation EIA, a prototypic structural and non-structural dual bead assay (EIA-SA, Abbott), the Chiron Recombinant Immunoblot Assay, 2nd generation (RIBA-2), and the Chiron RIBA HCV Test System Strip Immunoblot Assay 3.0 (RIBA-3, Chiron).nnnRESULTSnThe dual bead EIA-SA and RIBA-3 were able to detect HCV seroconversion approximately 6 months earlier than the 2nd generation EIA in 2 recipients, and in 1 recipient only PCR detected infection within the first 10 months. There was no evidence of HCV transmission to the corneal recipients.nnnCONCLUSIONSnAlthough third generation assays such as the RIBA-3 and EIA-SA narrowed the window of HCV seronegativity in transplant recipients compared with the 2nd generation EIA, PCR was the most sensitive method of detecting acute HCV infection. Despite transmission of HCV to all of the solid organ recipients HCV was not transmitted to the corneal transplant recipients.

HIV viral suppression results in higher antibody responses in HIV-positive women vaccinated with the quadrivalent human papillomavirus vaccine.

OBJECTIVEnTo evaluate the immunogenicity and safety of the quadrivalent HPV (qHPV) vaccine in HIV-positive women over 24months.nnnDESIGNnBetween November 2008 and December 2012, 372 women aged 15 and older were enrolled from 14 Canadian HIV outpatient clinics in an open label cohort study. The qHPV vaccine (0.5mL) was administered intramuscularly at months 0, 2 and 6. The primary study endpoint was seroconversion to any of the HPV types targeted by the qHPV vaccine. Antibody levels were measured at 0, 2, 7, 12, 18, and 24months. Adverse events were recorded throughout.nnnRESULTSnOf 372 participants enrolled, 310 (83%) received at least one dose of the qHPV vaccine and 277 (74%) received all three doses. Ninety-five percent (293/308) were seronegative for at least one vaccine type at baseline. The median age was 38years (IQR 32-45, range 15-66), 36% were white, 44% black and 13% were of Indigenous origin. Seventy-two percent of participants had a suppressed HIV viral load (VL<40c/ml) at baseline, with a median CD4 count of 510cells/mm(3) (376-695). Month 7 HPV type-specific seroconversion rates were 99.0%, 98.7%, 98.1% and 93.6% for HPV types 6, 11, 16 and 18 respectively in the per-protocol population. Participants with suppressed HIV VL at first vaccine had a 1.74-3.05fold higher peak antibody response compared to those without (p from 0.006 to <0.0001).nnnCONCLUSIONSnThis study is the first to examine the qHPV vaccine in HIV-positive women out to 24months and the first to include HIV-positive women through to age 66. The qHPV vaccine was well tolerated, and highly immunogenic. As women with suppressed viral load had higher antibody responses, planning HPV vaccination to occur when persons are virologically suppressed would be optimal for maximizing immune response. Findings provide strong evidence that older HIV-positive women can still benefit from HPV vaccination.nnnCLINICAL TRIAL REGISTRATIONnhttp://www.isrctn.com/ISRCTN33674451.

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.

Prevalence and risk factors for neutralizing antibodies to human papillomavirus types 16 and 18 in HIV-positive men who have sex with men.

Objective:Human papillomavirus (HPV) vaccination is routinely recommended in HIV-positive men who have sex with men (MSM) aged ⩽26 years. Levels of previous HPV exposure in older HIV-positive MSM are assumed to be too high to warrant routine HPV vaccination. However, little is known about the prevalence of and risk factors for neutralizing antibody seropositivity to HPV-16 or HPV-18, a key measure of previous exposure to these types. Methods:Cross-sectional analysis of baseline visit for 296 HIV-positive MSM participating in a prospective cohort study of anal squamous intraepithelial lesions at a university-based research clinic. Participants completed a questionnaire detailing behaviors and medical history. Phlebotomy, anal cytology, HPV DNA testing with quantitation, and high-resolution anoscopy with biopsy were performed. A pseudovirion-based neutralizing antibody assay was used to measure HPV-16 and HPV-18 neutralizing antibodies. Results:One hundred thirty-two of 296 (45%) men were HPV-16 seropositive and 141 of 296 (48%) were HPV-18 seropositive. One hundred seventy-five of 296 (59%) of the men were positive for HPV-16 antibodies or DNA and 167 of 296 (56%) were positive for HPV-18 antibodies or DNA. In multivariable analysis, HPV-16 seropositivity did not correlate with age, years of HIV positivity, CD4+ level, or HIV viral load. Significant risk factors included HPV-16 DNA positivity with higher DNA levels (ptrend < 0.001) and higher number of receptive sexual partners in the last year (ptrend = 0.012). Conclusions:A high proportion of HIV-positive MSM aged >26 years are DNA negative and seronegative to HPV-16 and HPV-18 even when using a sensitive pseudovirion-based neutralizing antibody assay. Prospective studies are needed to determine the clinical- and cost-effectiveness of HPV vaccination in HIV-positive MSM aged >26 years.