Ella Mendelson

Vaccination against influenza in patients with rheumatoid arthritis: the effect of rituximab on the humoral response

Objective: To assess the effect of rituximab on the efficacy and safety of influenza virus vaccine in patients with rheumatoid arthritis (RA). Methods: The study group comprised patients with RA treated with conventional disease-modifying drugs with or without rituximab. Split-virion inactivated vaccine containing 15 μg haemagglutinin/dose of B/Shanghai/361/02 (SHAN), A/New Caledonian/20/99 (NC) (H1N1) and A/California/7/04 (CAL) (H3N2) was used. Disease activity was assessed by the number of tender and swollen joints, duration of morning stiffness and evaluation of pain on the day of vaccination and 4 weeks later. CD19-positive cell levels were assessed in rituximab-treated patients. Haemagglutination inhibition (HI) antibodies were tested and response was defined as a greater than fourfold rise 4 weeks after vaccination or seroconversion in patients with a non-protective baseline level of antibodies (<1/40). Geometric mean titres (GMT) were calculated in all subjects. Results: The participants were divided into three groups: RA (n = 29, aged 64 (12) years), rituximab-treated RA (n = 14, aged 53 (15) years) and healthy controls (n = 21, aged 58 (15) years). All baseline protective levels of HI antibodies and GMT were similar. Four weeks after vaccination, there was a significant increase in GMT for NC and CAL antigens in all subjects, but not for the SHAN antigen in the rituximab group. In rituximab-treated patients, the percentage of responders was low for all three antigens tested, achieving statistical significance for the CAL antigen. Measures of disease activity remained unchanged. Conclusion: Influenza virus vaccine generated a humoral response in all study patients with RA and controls. Although the response was significantly lower among rituximab-treated patients, treatment with rituximab does not preclude administration of vaccination against influenza.

Mutation D30N Is Not Preferentially Selected by Human Immunodeficiency Virus Type 1 Subtype C in the Development of Resistance to Nelfinavir

ABSTRACT Differences in baseline polymorphisms between subtypes may result in development of diverse mutational pathways during antiretroviral treatment. We compared drug resistance in patients with human immunodeficiency virus subtype C (referred to herein as “subtype-C-infected patients”) versus subtype-B-infected patients following protease inhibitor (PI) therapy. Genotype, phenotype, and replication capacity (Phenosense; Virologic) were determined. We evaluated 159 subtype-C- and 65 subtype-B-infected patients failing first PI treatment. Following nelfinavir treatment, the unique nelfinavir mutation D30N was substantially less frequent in C (7%) than in B (23%; P = 0.03) while L90M was similar (P < 0.5). Significant differences were found in the rates of M36I (98 and 36%), L63P (35 and 59%), A71V (3 and 32%), V77I (0 and 36%), and I93L (91 and 32%) (0.0001 < P < 0.05) in C and B, respectively. Other mutations were L10I/V, K20R, M46I, V82A/I, I84V, N88D, and N88S. Subtype C samples with mutation D30N showed a 50% inhibitory concentration (IC50) change in susceptibility to nelfinavir only. Other mutations increased IC50 correlates to all PIs. Following accumulation of mutations, replication capacity of the C virus was reduced from 43% ± 22% to 22% ± 15% (P = 0.04). We confirmed the selective nature of the D30N mutation in C, and the broader cross-resistance of other common protease inhibitor mutations. The rates at which these mutational pathways develop differ in C and subtype-B-infected patients failing therapy, possibly due to the differential impact of baseline polymorphisms. Because mutation D30N is not preferentially selected in nelfinavir-treated subtype-C-infected patients, as it is in those infected with subtype B, the consideration of using this drug initially to preserve future protease inhibitor options is less relevant for subtype-C-infected patients.

Rapid Detection of blaKPC Carbapenemase Genes by Real-Time PCR

ABSTRACT Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes using TaqMan chemistry. The q-PCR amplification of blaKPC DNA was linear over 7 log dilutions (r2 = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for blaKPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while blaKPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for blaKPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the blaKPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to blaKPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.

The Effect of Infliximab and Timing of Vaccination on the Humoral Response to Influenza Vaccination in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis

OBJECTIVES To assess the effect of the timing of vaccination in relation to administration of infliximab on the efficacy and safety of influenza vaccine in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS The study population comprised 38 patients treated with infliximab at a mean dosage of 3 mg/kg (20 RA patients; 18 AS patients; 23 RA controls (treated with disease modifying antirheumatic drugs other than anti-tumor necrosis factor-alpha; and 17 healthy controls). Split-virion inactivated vaccine containing 15 mug hemagglutinin/dose of each of A/New Caledionan/20/1999 (H1N1), A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004 (M) was used. Patients treated with infliximab were divided into 2 groups: 22 were vaccinated on the day of administration of infliximab, while 16 received the vaccine 3 weeks after infliximab. Baseline and 4- to 6-week clinical assessment of disease activity included erythrocyte sedimentation rate and C-reactive protein for all patients, the 28-joint disease-activity score for RA patients, and Bath Ankylosing Spondylitis Disease Activity Index for AS patients. Hemagglutination inhibition (HI) antibodies were tested by a standard World Health Organization procedure. Response was defined as >or=4-fold rise in HI antibodies 4 to 6 weeks after vaccination, or seroconversion in patients with a nonprotective baseline level of antibodies (<1/40). Geometric mean titers (GMT) were calculated to assess the immunity of the whole group. RESULTS At baseline, RA patients and controls had similar occurrence of protective levels of HI antibodies and GMT, while AS patients had lower levels reflecting lower rates of previous vaccination. Four weeks after vaccination, a significant and similar increase in GMT for each antigen was observed in all groups (P < 0.004) except in the RA-infliximab group, vaccinated 3 weeks after administration of infliximab, in whom the increase in GMT was not significant for H1N1 (P = 0.12) and H3 (P = 0.06). AS patients demonstrated an increase in GMT, independently of the time of vaccination. The percentage of responders was similar in all groups. The response was not affected by variables such as age, gender, methotrexate, or prednisone use. Parameters of disease activity remained unchanged. No adverse effects other than injection site pain were recorded. CONCLUSIONS Influenza virus vaccine generated a good humoral response in RA and AS patients treated with infliximab.

Treatment of viral infections with 5-aminolevulinic acid and light

When 5‐aminolevulinic acid (ALA) is exogenously supplied, protoporphyrin IX (PpIX) is accumulated in various cells and makes them light sensitive. The possibility of using such an approach for the treatment of viral infections was studied in this work.

Dengue fever among travelers

PURPOSE The present paper describes the epidemiology and clinical manifestations of dengue fever in a nonendemic population of travelers. PATIENTS AND METHODS Clinical manifestations, epidemiologic information, and laboratory findings are described for a series of 18 Israeli travelers who tested serologically positive for dengue. RESULTS All the patients in the series contracted the disease in Southeast Asia, mostly in Thailand; 30% had to be evacuated due to severe morbidity. The clinical symptoms in travelers somewhat differ from the classical description among endogenous populations. High fever, chills, extreme fatigue, and severe headaches were prevalent. Other symptoms considered to be typical of dengue fever, such as myalgia, arthralgia, rash, biphasic fever, were uncommon. Laboratory findings were marked leukopenia, usually accompanied by lymphopenia, thrombocytopenia, liver function impairment, and hyponatremia. Some hemorrhagic phenomena were manifest despite it being the first exposure, without mortality. CONCLUSIONS Dengue fever among the nonimmune has a somewhat different manifestation from that reported for the Southeast Asian population. Although it is a significant cause for morbidity and hospitalization, it is underestimated as a factor affecting travelers health. More efforts should be expended in developing an effective vaccine.

Evaluation of ELISA-based sero-diagnosis of dengue fever in travelers

Diagnosis of dengue virus infection in travelers is often based on commercially available ELISA-based serological assays and not on the more difficult and costly procedures of Hemagglutination inhibition (HI), virus isolation or RT-PCR. These standard assays are not quantitative and are designed to diagnose primary and secondary dengue virus infections by testing for IgG and IgM antibodies. However, cross reactivity between various flaviviruses and the fact that most travelers today are prevaccinated against Japanese encelphalitis (JE) and yellow fever (YF) create a potential problem in such diagnosis. Our study was aimed at measuring the extent of false positive diagnosis in prevaccinated travelers which we have assessed by testing for dengue IgG and IgM antibodies in a group of prevaccinated healthy travelers using the PanBio indirect IgG ELISA and IgM capture ELISA kits. The IgM test was negative in all healthy vaccinees, thus, being highly specific. However, the kit had a disadvantage, which was recognized in other travelers clinically ill with dengue fever (DF), in which the IgM response was detected only 4-8 days after onset of the clinical symptoms. The IgG test yielded 11-17 and 15-44% positives in healthy travelers vaccinated against JE and YF, respectively. We conclude that the specificity of the IgG-ELISA assay in prevaccinated travelers is much lower than in unvaccinated populations. Thus, an IgG-positive results in a vaccinated traveler and IgM negative result during the 1st week of the illness period, are both inconclusive.

Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

ABSTRACT The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories.

Detection of adeno-associated virus type 2 in human peripheral blood cells.

The non-pathogenic human parvovirus, adeno-associated virus (AAV) is helper virus-dependent. However, it integrates into the cellular genome in the absence of its helper viruses. Therefore it could become a useful vector for gene therapy. Previous studies and our own results have shown that 40 to 80% of adults are seropositive for AAV and that seroconversion occurs during the first few years of life, but little is known about the route of natural infection with the virus. We used the polymerase chain reaction to detect the AAV-2 genome and identify AAV sequences within peripheral blood leukocytes (PBLs). We could detect AAV in PBLs of two of 55 healthy blood donors, and two of 16 haemophilic patients. AAV DNA replication and viral protein production in PBLs propagated in tissue culture were also examined. AAV DNA replicated very efficiently in the presence of helper adenovirus, but capsid proteins were produced at a lower level and the yield of infectious virus was very low. Our findings prove that in vivo infection of PBLs occurs, and that PBLs could mediate the spread of AAV infection to different body tissues.

Detection of Zika virus RNA in whole blood of imported Zika virus disease cases up to 2 months after symptom onset, Israel, December 2015 to April 2016

Zika virus RNA presence in serum, whole-blood and urine samples from six Israeli travellers symptomatic for Zika virus disease was examined. Whole-blood samples were positive for as late as 2 months (58 days) post-symptom onset, longer than for urine (26 days) and serum (3 days). These findings suggest the utility of whole blood in Zika infection diagnosis.