Gynaecologists' view on diagnostic delay and care performance in endometriosis in the Netherlands.

RESEARCH QUESTION: To evaluate implementation of the key recommendations of the European Society of Human Reproduction and Embryology (ESHRE) guidelines on endometriosis, and to assess factors influencing diagnostic delay of endometriosis from Dutch gynaecologists' point of view. DESIGN: Questionnaire study among gynaecologists from all hospitals in the Netherlands. The questionnaire consisted of 56 questions relating to implementation of the ESHRE guidelines, organization of endometriosis care and diagnostic delay. RESULTS: Gynaecologists from 67 out of 85 hospitals completed the questionnaire. A total of 99-100% of respondents agree with, and 91-100% adhere to, the diagnosis-related recommendations in the guidelines. Diagnostic delay is estimated at 42 months. Main factors contributing to diagnostic delay according to gynaecologists are lack of knowledge and awareness of endometriosis in both patients and medical professionals, as well as limitations in diagnostics and late referral. Suggested interventions to reduce diagnostic delay are aimed at improving knowledge and awareness in both patients and medical professionals, as well as improving collaborations between medical professionals. CONCLUSIONS: Overall familiarity with, and use of, the 2014 ESHRE guidelines among Dutch gynaecologists is high. Dutch gynaecologists agree with the recommendations relating to diagnosis and adhere to them closely. Diagnostic delay, however, is still considerable; therefore, efforts to reduce diagnostic delay of endometriosis should be aimed at improving knowledge and awareness in both patients and medical professionals, as well as improving collaboration.

[Developmental neurology - networked medicine and new perspectives]. / Entwicklungsneurologie ­ vernetzte Medizin und neue Perspektiven.

Developmental neurology is one of the major areas of neuropediatrics and is among other things (legally) responsible for monitoring the motor, cognitive and psychosocial development of all infants using standardized monitoring investigations. The special focus is on infants born at risk and/or due to premature birth before 32 weeks of gestation or a birth weight less than 1500 g. Early diagnosis of deviations from normal, age-related development is a prerequisite for early interventions, which may positively influence development and the long-term biopsychosocial prognosis of the patients. This article illustrates the available methods in developmental neurology with a focus on recent developments. Particular attention is paid to the predictive value of general movements (GM). The current development of markerless automated detection of spontaneous movements using conventional depth imaging cameras is demonstrated. Differences in spontaneous movements in infants at the age of 12 weeks are illustrated and discussed exemplified by three patients (healthy versus genetic syndrome versus cerebral palsy).

Effects of active HCV replication on neurologic status in HIV RNA virally suppressed patients.

BACKGROUND: Hepatitis C virus (HCV) is a frequent copathogen with HIV. Both viruses appear to replicate in the brain and both are implicated in neurocognitive and peripheral neuropathy syndromes. Interaction of the viruses is likely to be complicated and better understanding of the contributions of each virus will be necessary to make evidence-based therapeutic decisions. METHODS: This study was designed to determine if active HCV infection, identified by quantitative HCV RNA determination, is associated with increased neurocognitive deficits or excess development of distal sensory peripheral neuropathy in HIV coinfected patients with stable HIV viral suppression. The AIDS Clinical Trials Group Longitudinal Linked Randomized Trials (ALLRT) study was the source of subjects with known HIV treatment status, neurocognitive and neuropathy evaluations, and HCV status. Subjects were selected based on HCV antibody status (249 positive; 310 negative). RESULTS: HCV RNA viral loads were detectable in 172 participants with controlled HIV infection and available neurologic evaluations in the ALLRT. These participants were compared with 345 participants with undetectable HCV viral load and the same inclusion criteria from the same cohort. Neurocognitive performance measured by Trail-Making A or B and digit symbol testing was not dissimilar between the 2 groups. In addition, there was no significant association between active HCV replication and distal sensory neuropathy. CONCLUSION: Clinically significant neurocognitive dysfunction and peripheral neuropathy were not exacerbated by active hepatitis C virus infection in the setting of optimally treated HIV infection.

Diagnosis of dual human immunodeficiency virus types 1 and 2 infections in a resource-limited setting.

BACKGROUND: The presence of dual HIV-1/HIV-2 infection in Ghana and the different drug requirements for the treatment of HIV-1 and HIV-2 presents difficulties for the treatment of dual infections with both viruses. OBJECTIVES: To determine the prevalence of the dual sero-positive profile in treatment naive patients at a principal ART Clinic in Accra, Ghana and to investigate if rapid screening assays could be useful for diagnosis. DESIGN: A cross-sectional study. SETTING: A principal antiretroviral treatment centre in Accra, Ghana. SUBJECTS: Three hundred and twenty eight antiretroviral treatment naive patients. RESULTS: A total of 12 (3.7%) of patients seen were dual seropositive. There was a slight tendency of dual seropositive females being older than their HIV-1 counterparts (p = 0.088, CI = -10.833 to 0.753). Eight of the 12 of the dual seropositives were reactive for Genie II and were considered as possibly infected with both HIV-I and HIV-2. Seven (87.5%) of Genie II dual seropositives had strong intensities (> 1+) on both HIV-2 specific bands (sgp105 and gp36) on Innolia. CD4 counts were not significantly different in dual seropositives as compared to HIV-1 infected patients. CONCLUSIONS: Dual HIV-1/HIV-2 seropositives (and possibly infections) maybe common especially in older women. The Genie II will be useful as a supplemental rapid test for rapid and accurate differentiation of HIV-1 and HIV-2 antibodies at treatment centres.

Increasing prevalence of resistance mutations in antiretroviral-naïve individuals with established HIV-1 infection from 1996-2001 in St. Louis.

BACKGROUND: Transmission of drug-resistant virus in HIV-1 infected individuals is well documented, particularly in patients with primary infection. Prevalence in chronically infected antiretroviral-naïve patients is reportedly low. Routine genotyping in this population is not recommended. PURPOSE: The purpose of this study was to evaluate resistance profiles in patients with established HIV infection in St. Louis. METHOD: We selected specimens from drug-naïve individuals (CD4 >300 cells/mL and VL >1000 copies/mL) with established HIV infection between 1996-2001. 62 of 75 specimens were available for genotyping. We excluded patients with evidence of acute HIV infection and long-term nonprogressors. RESULTS: The overall prevalence of resistance was 11% (7/62). From 1996 to 1998, a prevalence of 4% was observed (1/27 individuals). During the subsequent period from 1999 to 2001, the frequency increased to 17% (6/35 participants; p =.08; 95% CI 5-29%). CONCLUSION: The results suggest that the prevalence of primary resistance is increasing in our region to the point that it justifies genotypic testing in all individuals before the initiation of antiretroviral therapy. This has to be considered when designing antiretroviral clinical trials.

Clinically relevant sequence-based genotyping of HBV, HCV, CMV, and HIV.

The term 'genotyping' describes the genetic characterization of a genome. The genotype analysis is performed to identify mutations that differentiate one individual or strain from another. The mutations may confer resistance to specific antiviral drugs or they may simply allow classification of a strain as to 'type' and 'subtype'. There are four human viruses for which genotype information is clinically useful. Hepatitis B virus (HBV) infections are being treated with antiretroviral drugs and resistance after prolonged treatment is common. Since HBV cannot be cultured, the only method of detecting resistance-conferring mutations in the genome is a genotypic analysis. Hepatitis C virus (HCV) infection can be cured by treatment with the combination of interferon and ribavirin but certain strains of virus are more resistant to treatment than others. The current recommendations are that all HCV type 1 infections be treated for 12 months whereas other types may be successfully treated in 6 months. Since interferon treatment may have significant side effects, the determination of HCV genotype is an important aspect of this therapeutic regimen. Treatment of cytomegalovirus (CMV) disease with nucleoside analogues occasionally results in resistant virus with mutations in the phosphotransferase gene (UL97) and/or the DNA polymerase gene (UL54) that can be tested with phenotypic or genotypic assays. Since CMV grows very slowly, it may be more clinically useful to perform a rapid genotypic assay although only the UL97 gene can be efficiently genotyped. Finally, the virus for which genotyping has become the standard of care, human immunodeficiency virus type 1 (HIV-1) can now be genotyped routinely by many clinical virology labs experienced with molecular amplification methods and automated DNA sequencing technology. All currently-available antiretroviral drugs are directed against either the protease or reverse transcriptase genes of HIV-1 and the mutations within these genes that confer resistance have been well described. Sequence-based genotyping methods are not necessarily the best approach for routine genotyping of these four viruses, but sequencing is the gold standard from which other methods are developed and against which they are compared.

Disinfection of Goldmann tonometers after contamination with hepatitis C virus.

PURPOSE: To compare methods of disinfecting Goldmann tonometer tips inoculated with hepatitis C virus. METHODS: Hepatitis C virus was placed on Goldmann tonometer tips, air dried, and then disinfected by dry gauze wipes, isopropyl alcohol wipes, cold water washes, povidone iodine 10% wipes, and hydrogen peroxide or isopropyl alcohol soaks followed by a cold water wash and dry. Hydrogen peroxide and isopropyl alcohol disinfection techniques followed the Centers for Disease Control and Prevention guidelines for prevention of possible transmission of human immunodeficiency virus (HIV). After disinfection, samples from tonometer tips were amplified by polymerase chain reaction to quantitate the amount of hepatitis C virus RNA remaining. RESULTS: Percentage of hepatitis C virus RNA remaining after disinfection: dry gauze wipes 95.65%, isopropyl alcohol 5-second wipes 88.91%, cold water wash 4.78%, povidone iodine 10% 5-second wipes 0.72%, hydrogen peroxide soak with cold water wash 0.07%, and isopropyl alcohol soak and cold water wash 0.02%. CONCLUSIONS: After inoculation of Goldmann tonometer tips with hepatitis C virus, a 5-minute soak in 3% hydrogen peroxide or 70% isopropyl alcohol followed by washing in cold water resulted in the greatest reduction in hepatitis C virus RNA.

Long-term persistence of Coxiella burnetii in the host after primary Q fever.

After a primary infection Coxiella burnetii may persist covertly in animals and recrudesce at parturition to be shed in the products of conception and the milk. Similar latent persistence and recrudescence occurs in man: namely, infection of placenta, heart valve or mural endocardium, bone or liver. The numbers of organisms, their viability and cellular form, and the underlying organ sites of latent infection for the coxiella are obscure. During investigations of 29 patients with a chronic sequel to acute Q fever, the post-Q fever fatigue syndrome (QFS) [1-3], sensitive conventional and TaqMan-based PCR revealed low levels of C. burnetii DNA in blood mononuclear cells (5/29; 17%), thin needle liver biopsies (2/14; 14%) and, notably, in bone marrow aspirates (13/20; 65%). Irrespective of the ultimate significance of coxiella persistence for QFS, the detection of C. burnetii genomic DNA in bone marrow several years after a primary infection unveils a new pathological dimension for Q fever.

Zinc salts inactivate clinical isolates of herpes simplex virus in vitro.

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37 degrees C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.

Rapid phenotypic drug susceptibility assay for HIV-1 with a CCR5 expressing indicator cell line.

Phenotypic drug susceptibility assays of human immunodeficiency virus type 1 (HIV-1) isolates generally use time-consuming, expensive assays with peripheral blood mononuclear cells. A new HIV-1 indicator cell line, MAGI-CCR5, has been developed and applied for this purpose. This cell line expresses human CD4, the two major HIV-1 coreceptors, CCR5 and CXCR4, the reporter gene beta-galactosidase driven by the HIV-1 LTR, and quantitates infection within 48 h. A panel of reference strains and primary HIV-1 isolates were all found to infect this cell line. Susceptibility assays with a nucleoside (zidovudine, ZDV) and a non-nucleoside reverse transcriptase inhibitor (nevirapine, NVP) were performed with reference and primary isolates. The assay was modified into two steps for protease inhibitor (indivinavir, IDV and ritonavir, RTV) susceptibility assays. Primary isolates derived from drug naive patients displayed mean baseline 50% effective concentrations (EC50) of 0.14 microM for ZDV, 0.33 microM for NVP, and 0.02 microM for IDV. Isolates derived from patients under treatment displayed increased EC50 concentrations. The MAGI-CCR5 cell line offers a rapid, efficient, and reproducible method of testing a wide range of HIV-1 isolates for drug susceptibility.

Diagnostic testing for HIV type 1 RNA in seronegative blood.

We studied the feasibility of routine diagnostic testing for HIV-1 RNA at a publicly funded testing site. HIV-1 RNA was determined with a commercial polymerase chain reaction assay in pooled seronegative blood samples submitted for HIV testing to a public health laboratory. Recovery of HIV-1 RNA from the samples was estimated as at least 8% of viral RNA that was found in freshly prepared plasma. We estimated that screening for HIV-1 RNA in serum pools would result in the identification of blood specimens from more than 95% of acutely infected patients. The frequency of HIV-1 RNA in seronegative blood samples was estimated to be between 19 and 601 per 10(6) submitted specimens. The ratio of HIV-1 RNA positive and seronegative samples to specimens with HIV-1 antibodies confirmed by Western blot was estimated to be between 0.2% and 6.6%. The reagent costs for identifying 1 HIV-infected blood sample were 10-fold higher with the commercially available HIV-1 RNA assay compared with the HIV antibody enzyme-linked immunosorbent assay. Diagnostic testing for HIV-1 RNA may be warranted in high-risk populations since acutely infected patients may benefit most from anti-retroviral therapy and are thought to contribute disproportionately to the HIV epidemic.

The impact of ganciclovir-resistant cytomegalovirus infection after lung transplantation.

BACKGROUND: Cytomegalovirus (CMV) resistance to ganciclovir has become increasingly common in acquired immunodeficiency syndrome patients but has only rarely been reported in recipients of solid organ transplants. METHODS: A retrospective study of ganciclovir susceptibility testing of CMV isolates recovered from lung transplant recipients was performed. Patients with CMV isolates having partial (1 or =3 microg/ml) to ganciclovir determined by plaque reduction assay were included in a case-control study to identify risk factors for ganciclovir resistance. RESULTS: Between 2/91 and 5/98, 18 patients (5.2% of patients transplanted) were found to have CMV infections with some degree of ganciclovir resistance (4 partially, 14 fully resistant). More positive viral blood cultures (3.2+/-2.5 vs. 1.6+/-1.4 CMV positive cultures, P=0.02) and more episodes of CMV pneumonitis (0.24+/-0.23 vs. 0.10+/-0.17 episodes/bronchoscopy, P=0.02) occurring before the detection of resistance were seen among resistant patients than controls. Ganciclovir-resistant patients received more antithymocyte globulin during induction (70+/-44 vs. 45+/-39 mg/kg, P=0.03) and received ganciclovir for a greater number of days (79+/-52 vs. 64+/-53 days, P=0.005) before the detection of resistance than controls. Ganciclovir-resistant patients had a shorter survival and an earlier onset of bronchiolitis obliterans syndrome compared with patients in the transplant database at Washington University. CONCLUSIONS: Ganciclovir-resistant CMV infection is a serious complication of solid organ transplantation associated with more episodes of viremia, more frequent disease, earlier onset of bronchiolitis obliterans and shorter survival. The use of antithymocyte globulin and prolonged exposure to ganciclovir are risk factors for the development of ganciclovir resistance.

Methods for subtyping and molecular comparison of human viral genomes.

The development over the past two decades of molecular methods for manipulation of RNA and DNA has afforded molecular virologists the ability to study viral genomes in detail that has heretofore not been possible. There are many molecular techniques now available for typing and subtyping of viruses. The available methods include restriction fragment length polymorphism analysis, Southern blot analysis, oligonucleotide fingerprint analysis, reverse hybridization, DNA enzyme immunoassay, RNase protection analysis, single-strand conformation polymorphism analysis, heteroduplex mobility assay, nucleotide sequencing, and genome segment length polymorphism analysis. The methods have certain advantages and disadvantages which should be considered in their application to specific viruses or for specific purposes. These techniques are likely to become more widely used in the future for epidemiologic studies and for investigations into the pathophysiology of virus infections.

Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis.

BACKGROUND: Human ehrlichiosis is a recently recognized tick-borne infection. Four species infect humans: Ehrlichia chaffeensis, E. sennetsu, E. canis, and the agent of human granulocytic ehrlichiosis. METHODS: We tested peripheral-blood leukocytes from 413 patients with possible ehrlichiosis by broad-range and species-specific polymerase-chain-reaction (PCR) assays for ehrlichia. The species present were identified by species-specific PCR assays and nucleotide sequencing of the gene encoding ehrlichia 16S ribosomal RNA. Western blot analysis was used to study serologic responses. RESULTS: In four patients, ehrlichia DNA was detected in leukocytes by a broad-range PCR assay, but not by assays specific for E. chaffeensis or the agent of human granulocytic ehrlichiosis. The nucleotide sequences of these PCR products matched that of E. ewingii, an agent previously reported as a cause of granulocytic ehrlichiosis in dogs. These four patients, all from Missouri, presented between May and August 1996, 1997, or 1998 with fever, headache, and thrombocytopenia, with or without leukopenia. All had been exposed to ticks, and three were receiving immunosuppressive therapy. Serum samples obtained from three of these patients during convalescence contained antibodies that reacted with E. chaffeensis and E. canis antigens in a pattern different from that of humans with E. chaffeensis infection but similar to that of a dog experimentally infected with E. ewingii. Morulae were identified in neutrophils from two patients. All four patients were successfully treated with doxycycline. CONCLUSIONS: These findings provide evidence of E. ewingii infection in humans. The associated disease may be clinically indistinguishable from infection caused by E. chaffeensis or the agent of human granulocytic ehrlichiosis.

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

A rapid assay to screen for drug-resistant herpes simplex virus.

A rapid assay was developed to screen for herpes simplex virus (HSV) isolates that are resistant to acyclovir and other antiviral agents. The assay is a modified plaque reduction assay (PRA) in which the number of plaques seen in the absence of acyclovir was compared with that seen in the presence of a single cutoff concentration of acyclovir (2 microg/mL). This assay utilizes a cell line that expresses beta-galactosidase only after infection with HSV. Since histochemically stained plaques are easily visualized, small plaques can be easily enumerated. This allows the assay to be performed on dilutions of untitered specimens in the small wells of a 24-well plate and allows the results to be read only 2 days after inoculation of the virus. The assay performed well compared with a standard PRA and should be a valuable tool in identifying drug-resistant HSV in a timely manner.

Stabilities of free and complexed human immunodeficiency virus p24 antigens during short- and long-term storage.

By the standard p24 assay there was a 25 to 27% decrease in free p24 antigen in serum after storage at 4 degrees C over 14 days but no loss at -70 degrees C. There was no loss at either temperature by the immune complex dissociation (ICD) procedure. Furthermore, there was no significant loss of detectable p24 in serum by either the ICD or the standard p24 assay after 700 days of storage at -70 degrees C.

Sequence heterogeneity of Nef transcripts in HIV-1-infected subjects at different stages of disease.

Nef transcripts were analyzed from peripheral blood mononuclear cells of 10 HIV-1-infected subjects with 9-822 CD4+ lymphocytes/cu mm, including 4 individuals with a probable common source infection. There was no relationship between the phylogenetic position of the various nef sequences and the disease state of the person from whom they were derived. The nef open reading frame was disrupted in all three clones from only 1 subject. Functional analyses of a representative clone from each of the remaining 9 subjects showed that all nef alleles were capable of CD4 cell surface down-regulation, but only three nef alleles suppressed the induction of IL-2 transcription.

Resistance of microorganisms to disinfection in dental and medical devices.

The U.S. Centers for Disease Control and Prevention (CDC) recommends that only heat sterilization be used for all reusable devices entering the oral cavity. However, chemical disinfection is still employed for reprocessing dental devices in many areas of the world. In an analysis of a Florida dental practice responsible for nosocomial human immunodeficiency virus (HIV) transmissions, the possible role of contaminated devices was deemed unlikely in part because they were subjected to high-level disinfection with 2% glutaraldehyde. Disease transmissions have, however, been documented for endoscopes used in diagnostic and surgical procedures even after this treatment. In some dental devices, lubricants mix with potentially infectious patient materials, and organic debris has been observed in endoscopes after cleaning. We have investigated whether lubricants can render high-level chemical disinfection procedures ineffective and have addressed the role that some common devices may play in disease transmission. We report here that HIV in whole-blood samples and Pseudomonas aeruginosa in blood and plasma survived high-level disinfection when entrapped in lubricants used in dental handpieces and endoscopes. We also found that lubricated dental devices used to clean and polish teeth (prophylaxis angles) have the potential to transfer sufficient amounts of blood to infect human lymphocyte cultures with HIV. These results emphasize the need to subject reusable dental devices to a heat-sterilization protocol that penetrates the lubricant.