Roland Sahli

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays

ABSTRACT There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the “gold standard” in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Contribution of 20 single nucleotide polymorphisms of 13 genes to dyslipidemia associated with antiretroviral therapy

Background HIV-1 infected individuals have an increased cardiovascular risk which is partially mediated by dyslipidemia. Single nucleotide polymorphisms in multiple genes involved in lipid transport and metabolism are presumed to modulate the risk of dyslipidemia in response to antiretroviral therapy. Methods The contribution to dyslipidemia of 20 selected single nucleotide polymorphisms of 13 genes reported in the literature to be associated with plasma lipid levels (ABCA1, ADRB2, APOA5, APOC3, APOE, CETP, LIPC, LIPG, LPL, MDR1, MTP, SCARB1, and TNF) was assessed by longitudinally modeling more than 4400 plasma lipid determinations in 438 antiretroviral therapy-treated participants during a median period of 4.8 years. An exploratory genetic score was tested that takes into account the cumulative contribution of multiple gene variants to plasma lipids. Results Variants of ABCA1, APOA5, APOC3, APOE, and CETP contributed to plasma triglyceride levels, particularly in the setting of ritonavir-containing antiretroviral therapy. Variants of APOA5 and CETP contributed to high-density lipoprotein-cholesterol levels. Variants of CETP and LIPG contributed to non-high-density lipoprotein-cholesterol levels, a finding not reported previously. Sustained hypertriglyceridemia and low high-density lipoprotein-cholesterol during the study period was significantly associated with the genetic score. Conclusions Single nucleotide polymorphisms of ABCA1, APOA5, APOC3, APOE, and CETP contribute to plasma triglyceride and high-density lipoprotein-cholesterol levels during antiretroviral therapy exposure. Genetic profiling may contribute to the identification of patients at risk for antiretroviral therapy-related dyslipidemia.

Seroepidemiology of Herpes Simplex virus type 1 and 2 in Western and Southern Switzerland in adults aged 25–74 in 1992–93 : a population-based study

BackgroundGenital herpes is one of the most prevalent sexually-transmitted diseases, and accounts for a substantial morbidity. Genital herpes puts newborns at risk for very severe disease and also increases the risk of horizontal HIV transmission. It thus stands as an important public health problem. The recent availability of type-specific gG-based assays detecting IgG against HSV-1 and HSV-2 allows to establish the prevalence of each subtype. Worldwide, few data have been published regarding the seroprevalence in general populations of HSV-2, the major causative agent for genital herpes, while no data exist regarding the Swiss population.MethodsTo evaluate the prevalence of IgG antibodies against HSV-1 and HSV-2 in Switzerland, we used a population-based serum repository from a health examination survey conducted in the Western and Southern area of Switzerland in 1992–93. A total of 3,120 sera were analysed by type-specific gG-based ELISA and seroprevalence was correlated with available volunteers characteristics by logistic regression.ResultsOverall, seroprevalence rates were 80.0 ± 0.9% (SE, 95% CI: 78.1–81.8) for HSV-1 and 19.3 ± 0.9% (SE, 95% CI: 17.6–21.1) for HSV-2 in adults 35–64 year old. HSV-1 and HSV-2 seroprevalence increased with age, with a peak HSV-2 seroprevalence in elderly gentlemen, possibly a seroarcheological evidence of sexually transmitted disease epidemics during World War II. Risk factors for HSV-2 infection included female sex, marital status other than married, and size of town of residence larger than 1500 inhabitants. Unexpectedly and conversely to HSV-1, HSV-2 seroprevalence increased with educational level. HSV-2 infection was less prevalent among HSV-1 infected individuals when compared to HSV-1 uninfected individuals. This effect was most apparent among women at high risk for HSV-2 infection.ConclusionsOur data demonstrate that by the early nineties, HSV-2 had spread quite largely in the Swiss population. However, the epidemiology of HSV-2 in Switzerland presents paradoxical characteristics, e.g. positive correlation with education level, that have not been observed elsewhere.

Partial deletion of CYP2B6 owing to unequal crossover with CYP2B7

Objective To evaluate the possibility of copy number variation (CNV) of CYP2B6. Methods We investigated CNV in 226 HIV-1-infected individuals by quantitative PCR. Identification of a candidate CNV prompted characterization of the size of deletion by assessment of absence of exons, mapping of the recombination site by sequencing, and by southern blot. The functional consequences of CNV were assessed in silico (predicted protein), and in vivo, by evaluation of plasma drug levels of the CYP2B6 substrate efavirenz. Results Analyses identified one white individual carrying a heterozygous deletion of exons 1–4 of CYP2B6. We identified a ∼68 kb deletion between CYP2B7 and CYP2B6, and mapped the crossover to a homologous region in intron 4 of both genes. The new hybrid allele, named CYP2B6*29, carries two amino acid substitutions, Q172H and M198T, previously associated with impaired enzyme function. Consistent with the functional prediction, the average of efavirenz area under the curve values of the patient was mean±SD, 81.64±23.62, versus 47.75±19.73 μg h/ml for individuals with an extensive metabolizer phenotype. Conclusion CYP2B6*29 represents a new mechanism of genetic variation at the CYP2B6 locus, underscoring the highly polymorphic nature of this isoenzyme.

Preemptive treatment approach to cytomegalovirus (CMV) infection in solid organ transplant patients: relationship between compliance with the guidelines and prevention of CMV morbidity.

Cytomegalovirus (CMV) remains a major cause of morbidity in solid organ transplant patients. In order to reduce CMV morbidity, we designed a program of routine virological monitoring that included throat and urine CMV shell vial culture, along with peripheral blood leukocyte (PBL) shell vial quantitative culture for 12 weeks post‐transplantation, as well as 8 weeks after treatment for acute rejection. The program also included preemptive ganciclovir treatment for those patients with the highest risk of developing CMV disease, i.e., with either high‐level viremia (>10 infectious units [IU]/106 PBL) or low‐level viremia (<10 IU/106 PBL) and either D+/R− CMV serostatus or treatment for graft rejection.

Determinants of protracted cytomegalovirus infection in solid-organ transplant patients.

Background. Recurrent infection frequently follows the response to the initial treatment of cytomegalovirus (CMV) infection in solid-organ transplant (SOT) recipients. The objective of this study was to describe the course of CMV infection in SOT patients and to identify factors that would predict protracted CMV infection with recurrences. Methods. Quantitative polymerase chain reaction (PCR) assay for CMV DNA in leukocytes and in plasma were used to assess viral load changes retrospectively in consecutive SOT patients, whose CMV infection episodes had been attended therapeutically or preemptively using quantitative blood culture. Results. Among 101 SOT patients, CMV infection occurred in 63, of whom 32 developed recurrent infection after the initial episode. In patients with recurrent infection, PCR indicated that a majority (27) of recipients had high level of CMV DNA in peripheral blood leukocytes and plasma throughout a protracted (≥1 month) period including after preemptive or therapeutic ganciclovir courses. Predictors of protracted high-level infection were increasing age, CMV donor seropositivity, and all measures of viral load during the initial episode. CMV recipient seropositivity protected strongly against protracted infection. End of treatment plasma CMV DNA best discriminated between patients who did or did not develop protracted infection. Conclusions. In SOT patients, protracted CMV infection is associated with increasing age, donor seropositivity, recipient seronegativity, and high viral load during the first episode. End of therapy plasma CMV DNA level best predicts the occurrence of protracted infection. In patients with a high risk of protracted infection, prophylaxis is likely to be particularly cost effective.

Chronic norovirus gastroenteritis in a double hematopoietic stem cell and lung transplant recipient

N. Boillat Blanco, R. Kuonen, C. Bellini, O. Manuel, C. Estrade, J. Mazza‐Stalder, J.D. Aubert, R. Sahli, P. Meylan. Chronic norovirus gastroenteritis in a double hematopoietic stem cell and lung transplant recipient.
Transpl Infect Dis 2011: 13: 213–215. All rights reserved

Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV Assay for Human Papillomavirus Genotyping

ABSTRACT The Anyplex II HPV28 (H28; Seegene) is a new semiquantitative real-time multiplex PCR assay for screening and genotyping 28 human papillomaviruses (HPV) in only 2 reaction wells. H28 was compared to the PGMY-CHUV assay (PG) with 309 archival DNA samples from cervical smears collected over 8 years in our laboratory. H28 and PG were fully concordant at the genotypic level on 228 (73.8%) out of 309 samples: 27 HPV negative and 201 HPV positive. The 201 fully concordant positive samples corresponded to single infections (n = 145) and to multiple infections (2 genotypes, n = 38; 3 to 5 genotypes, n = 18). The remaining 81 samples (26.2%) were either partially concordant (n = 64, 20.7%) or fully discordant (n = 17, 5.5%). While genotype-specific agreement was nearly perfect (κ = 0.877), HPV51 was significantly less well detected by H28 and the converse was observed for HPV40, -42, -54, and -68. Sequencing of PG amplicons confirmed HPV51 discordants and suggested the involvement of a possibly local HPV51 subtype. Mismatches in the PGMY09 primers to HPV68a explained most of the HPV68 discordants, confirming the specificity of H28 toward HPV68. With PG as a reference, the sensitivity and specificity of H28 were 93.4% and 99.0%, respectively. Considering H28 as a reference, the sensitivity and specificity of PG were 83.8% and 99.6%, respectively. H28 is a very sensitive and specific HPV genotyping assay suitable for research and clinical use as an adjunct to a clinically validated test. H28 semiquantitative readout ought to be evaluated for primary cervical cancer screening.

Validation of a Low-Cost Human Papillomavirus Genotyping Assay Based on PGMY PCR and Reverse Blotting Hybridization with Reusable Membranes

ABSTRACT The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n = 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n = 182) and negative samples (n = 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; κ of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P = 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n = 275 samples; 392 genotype-sample combinations; κ of 0.933) involving different reagents lots and different technicians. Discordant results (n = 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at

Expression of human papillomavirus 16 E2 protein in Schizosaccharomyces pombe delays the initiation of mitosis

2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.