Automatic oral fluid-based HIV testing in HIV screening programmes: automatic for the people.

OBJECTIVES: UK guidelines recommend routine HIV testing in general clinical settings when the local HIV prevalence is > 0.2%. During pilot programmes evaluating the guidelines, we used laboratory-based testing of oral fluid from patients accepting tests. Samples (n = 3721) were tested manually using the Bio-Rad Genscreen Ultra HIV Ag-Ab test (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK). This was a methodologically robust method, but handling of samples was labour intensive. We performed a validation study to ascertain whether automation of oral fluid HIV testing using the fourth-generation HIV test on the Abbott Architect (Abbott Diagnostics, Maidenhead, UK) platform was possible. METHODS: Oral fluid was collected from 143 patients (56 known HIV-positive volunteers and 87 others having contemporaneous HIV serological tests) using the Oracol+ device (Malvern Medicals, Worcester, UK). Samples were tested concurrently: manually using the Genscreen Ultra test and automatically on the Abbott Architect. RESULTS: For oral fluid, the level of agreement of results between the platforms was 100%. All results agreed with HIV serology. The use of the Oracol+ device produced high-quality samples. Subsequent field use of the test has shown a specificity of 99.97% after nearly 3000 tests. CONCLUSIONS: Laboratory-based HIV testing of oral fluid requires less training of local staff, with fewer demands on clinical time and space than near-patient testing. It is acceptable to patients. The validation exercise and subsequent clinical experience support automation, with test performance preserved. Automation reduces laboratory workload and speeds up the release of results. Automated oral fluid testing is thus a viable option for large-scale HIV screening programmes.

First Report of Resistance to QoI Fungicides in North American Populations of Zymoseptoria tritici, Causal Agent of Septoria Tritici Blotch of Wheat.

The G143A mutation in cytb (cytochrome b gene) is associated with high levels of resistance to quinone outside inhibitor (QoI or strobilurin) fungicides that disrupt electron transport during cellular respiration (1). The G143A mutation in Zymoseptoria tritici (synonyms: Mycosphaerella graminicola and Septoria tritici), the causal agent of septoria tritici blotch of wheat (Triticum aestivum), was first reported in Europe in 2001 (1). Although Z. tritici has a global distribution (3), G143A mutants of Z. tritici have not been reported outside of Europe. We used PCR-RFLP (4) to estimate the frequencies of G143A mutants in Z. tritici populations at two locations in the Willamette Valley of western Oregon: the Hyslop Crop Science Field Research Laboratory (Hyslop Farm, HF), Benton County (44°37'52.85″ N, 123°11'55.19″ W) and research plots planted in a commercial wheat field in Washington County (45°33'58.53″ N, 123°00'11.78″ W) (North Valley Farm, NVF). Isolates originated from flag leaf collections from two cultivars ('Bobtail' and 'Tubbs 06') made in April and June of 2012 from plants in a replicated fungicide-treatment experiment, with isolates collected from both sprayed and unsprayed plots. Sixteen of the 169 isolates (9.5%) from HF possessed the G143A mutation (7 of 132 isolates from plots not receiving a QoI fungicide and 9 of 37 isolates collected from plots receiving two applications of the QoI azoxystrobin). One hundred forty six of the 175 isolates (83.4%) from NVF were G143A mutants (101 of 129 isolates from plots receiving no QoI fungicide and 45 of 46 isolates from plots receiving two applications of azoxystrobin). Results of phenotypic assays of a subset of 10 isolates from each location (5 mutants, 5 wild types from each location; 20 isolates altogether) supported a high level of resistance to azoxystrobin only in the G143A mutants. All 10 G143A mutants developed colonies after 8 days of growth on YMA plates amended with SHAM (2) and 1 ppm or 10 ppm azoxystrobin, with nine and eight G143A mutant isolates developing colonies on plates amended with 1 ppm and 10 ppm azoxystrobin, respectively. None of the wild-type isolates developed colonies on plates amended with SHAM and 1 ppm azoxystrobin, nor on plates amended with SHAM and 10 ppm azoxystrobin. All 20 isolates developed colonies on YMA plates lacking azoxystrobin, and treatments produced identical results across three replicates. These results are consistent with findings of higher levels of azoxystrobin resistance in G143A mutants compared to wild types in European populations (1). Isolates from HF and NVF differ in their previous exposure to QoI fungicides. The majority of the wheat area at HF is planted to breeding plots that are not sprayed with fungicide. Plots at NVF were planted in a commercial wheat field in a county where most wheat fields were treated with two to three applications of strobilurins each year over the past 4 years. Future monitoring for G143A mutants of Z. tritici throughout its range in North America will be necessary to assess whether strobilurin resistance will spread via wind-dispersal of ascospores or emerge de novo in treated fields. In Europe, stobilurins were first applied to wheat in 1996. G143A mutants of Z. tritici emerged de novo several times (4) and were widespread by 2007. References: (1) B. A. Fraaje et al. Phytopathology 95:933, 2005. (2) J. A. LaMondia. Tob. Sci. 49:1, 2012. (3) E. S. Orton et al. Mol. Plant Pathol. 12:413, 2011. (4) S. F. F. Torriani et al. Pest Manag. Sci. 65:155, 2008.

Cyclophosphamide-based in vivo T-cell depletion for HLA-haploidentical transplantation in Fanconi anemia.

Allogeneic hematopoietic cell transplantation (HCT) is the only known cure for patients with Fanconi anemia (FA) who develop aplasia or leukemia. However, transplant regimens typically contain high-dose alkylators, which are poorly tolerated in FA patients. Furthermore, as many patients lack human leukocyte antigen (HLA)-matched family donors, alternative donors are used, which can increase the risk of both graft rejection and graft-versus-host disease (GVHD). To improve on these three concerns, we developed a multi-institutional clinical trial using a fludarabine (FLU)-based conditioning regimen with limited alkylators/low-dose radiation, HLA-haploidentical marrow, followed by reduced-dose cyclophosphamide (CY) to treat three FA patients with aplasia. All three patients engrafted with 100% donor CD3 chimerism at 1 month. One patient died early from disseminated toxoplasmosis infection. Of the two survivors, one had significant pretransplant co-morbidities and inadequate immunosuppression, and developed severe acute GVHD. The other patient had only mild acute and no chronic GVHD. With a follow-up of 2 and 3 years, respectively, both patients are doing well, are transfusion-independent, and maintain full donor chimerism. The patient with severe GVHD has resolving oral GVHD and good quality of life. We conclude that using low-intensity conditioning, HLA-haploidentical marrow, and reduced-dose CY for in vivo T-cell depletion can correct life-threatening aplasia in FA patients.

Impact of mother donor, peripheral blood stem cells and measurable residual disease on outcomes after haploidentical hematopoietic cell transplantation with post-transplant cyclophosphamide in children with acute leukaemia.

Haploidentical hematopoietic-cell transplantation using post-transplant cyclophosphamide(Haplo-PTCy) is a feasible procedure in children with haematologic malignancies. However, data of a large series of children with acute leukaemia(AL) in this setting is missing. We analysed 144 AL Haplo-PTCy paediatric recipients; median age was 10 years. Patients had acute lymphoblastic(ALL; n = 86) or myeloblastic leukaemia(AML; n = 58) and were transplanted in remission(CR1: n = 40; CR2: n = 57; CR3+: n = 27) or relapse (n = 20). Bone marrow was the graft source in 57%; donors were father (54%), mother (35%), or sibling (11%). Myeloablative conditioning was used in 87%. Median follow-up was 31 months. At day +100, cumulative incidence (CI) of neutrophil recovery and acute GVHD (II-IV) were 94% and 40%, respectively. At 2-years, CI of chronic GVHD and relapse, were 31%, 40%, and estimated 2-year overall survival (OS), leukaemia-free survival (LFS) and graft-versus-host-relapse-free survival (GRFS) were 52%, 44% and 34% respectively. For patients transplanted in remission, positive measurable residual disease (MRD) prior to transplant was associated with decreased LFS (p = 0.05) and GRFS (p = 0.003) and increased risk of relapse (p = 0.02). Mother donor was associated with increased risk of chronic GVHD (p = 0.001), decreased OS (p = 0.03) and GRFS (p = 0.004). Use of PBSC was associated with increased risk of chronic GVHD (p = 0.04). In conclusion, achieving MRD negativity pre-transplant, avoiding use of mother donors and PBSC as graft source may improve outcomes of Haplo-PTCy in children with AL.

Role of penile biopsy in the diagnosis of penile dermatoses.

The aim of this study was to review the utility of penile biopsy in genitourinary (GU) medicine clinics, its acceptability and the range of penile dermatoses diagnosed histologically. A retrospective case-notes review of 401 cases attending a dedicated penile dermatoses clinic from 1 June 2001 to 30 November 2007 was carried out. In 115/401 (28.7%) of cases a biopsy was invoked to resolve ambiguities of diagnosis. In 60/401 (15%) of those cases differential diagnosis was resolved, in 26/401 (6.5%) clinically suspected diagnoses were confirmed and in 22/401 (5.5%) clinically unsuspected diagnoses were identified. In 7/401 (1.7%) of cases the biopsy was also the treatment. Targeted penile biopsy has an important role in the diagnosis of penile dermatoses, particularly where there is clinical uncertainty. As a diagnostic tool in GU medicine departments it should be readily available even if not for routine use. There were indications that circumcision may reduce the incidence of penile dermatoses. There was also some indication of a possible ethnic factor in the relative incidences of penile lichen sclerosus and lichen planus in biopsied patients.

Serious acute or chronic graft-versus-host disease after hematopoietic cell transplantation: a comparison of myeloablative and nonmyeloablative conditioning regimens.

We previously reported a 25% incidence of serious graft-versus-host disease (GVHD) (that is, acute or chronic GVHD that caused death, lengthy hospitalization or disability, or resulted in recurrent major infections) among 171 hematopoietic cell transplantation (HCT) recipients after nonmyeloablative (NMA) regimen. Here we present a retrospective study applying the same criteria to 264 recipients of peripheral blood HCT after myeloablative (MA) regimen, and compare the results with the previous study after additional follow-up. The MA group was younger and had lower comorbidity scores at HCT than those in the NMA group. The overall incidence of serious GVHD was 17% (44/264) in the MA group versus 28% (48/171) in the NMA group. The adjusted hazard ratio (HR) of serious GVHD in the MA group compared to the NMA group was 0.65 (95% CI, 0.4-1.1); P=0.13, and if follow-up was censored at the onset of recurrent or progressive malignancy, HR was 0.67 (95% CI, 0.4-1.3), P=0.22. We conclude that the choice between MA and NMA regimens does not greatly affect the risk of serious GVHD as an overall indicator of outcomes related to either acute or chronic GVHD. Serious GVHD may be considered as an endpoint in clinical trials with GVHD-related outcomes.

Two for the price of one.

This case is about an HIV seropositive young woman referred for the treatment of severe menorrhagia causing anaemia due to adenomyosis where the levonorgestrel-releasing intrauterine system (Mirena) proved useful in treating her heavy periods and also provided effective contraception without interference from the liver enzyme-inducing effects of antiretroviral medications.

Awareness of HIV post-exposure prophylaxis after sexual exposure and emergency hormonal contraception in HIV-positive women.

Women attending a dedicated medical gynaecology and family planning referral clinic for women with HIV were surveyed using a standard questionnaire about their knowledge and attitudes to post-exposure prophylaxis after sexual exposure (PEPSE) and emergency hormonal contraception (EHC). Eighty percent of them had not heard of PEPSE, but once informed about it, 86% said they would inform a partner about it. Less than 10% had any idea of the duration of effectiveness. Seventy-three percent of the women knew about EC and 45% of them had used it previously. Ninety-eight percent of them would use it in the future if necessary. Eighty percent of them knew its period of effectiveness. There is a clear need for information about PEPSE, which needs to be delivered around the time of HIV diagnosis and reinforced later. Some women will need help in discussing it with HIV-negative partners.

Contraception and medical gynaecology for HIV-positive women in a one-stop clinic.

HIV-positive women may be reluctant to attend gynaecology or family planning clinics for fear of divulging their condition. Therefore, a referral clinic was opened within the HIV clinic. Retrospective case-note reviews of 197 new patients revealed 109 with a variety of medical gynaecology conditions (menorrhagia being the commonest) and 88 sought contraception. The full range of contraceptives was used, including Mirena for the treatment of menorrhagia as well as contraception and the combined pill adjusted for interaction with liver enzyme-inducing antiretroviral drugs. The acceptance of contraceptive advice and gynaecological evaluation by the patients has resulted in improved reproductive health services for these HIV-positive women. In centres with large cohorts of HIV-positive women, this type of one-stop specialist clinic will be very effective in providing high-quality reproductive health care and hence, this type of clinic is recommended for such centres.

Results of donor lymphocyte infusions for relapsed myelodysplastic syndrome after hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) represents a potentially curative approach for patients with myelodysplastic syndromes (MDSs). While a large proportion of HCT recipients become long-term disease-free survivors, recurrence of MDS remains the leading cause of mortality after HCT. The role of donor lymphocyte infusion (DLI) in patients with relapsed MDS after HCT is unclear. We report results among 16 patients treated with DLI for relapsed MDS after HCT at a single institution between March 1993 and February 2004. The cohort contained 10 men and 6 women with a median age of 49 (range, 22-67) years. CR with resolution of cytopenias and prior disease markers occurred in 3 of 14 patients who could be evaluated. Two patients survived without MDS for 68 and 65 months after DLI, respectively, but died with pneumonia. Grades II-IV acute GVHD and chronic GVHD occurred after DLI in 6 (43%) and 5 (36%) patients, respectively. All three responders developed grades III-IV acute GVHD and extensive chronic GVHD after DLI. Our results confirm prior reports that DLI can result in CR in some patients with recurrent MDS after transplant, but long-term survival is infrequent.

Metabolic syndrome and cardiovascular disease following hematopoietic cell transplantation: screening and preventive practice recommendations from CIBMTR and EBMT.

Metabolic syndrome (MetS) is a constellation of cardiovascular risk factors that increases the risk of cardiovascular disease, diabetes mellitus and all cause mortality. Long-term survivors of hematopoietic cell transplantation (HCT) have a substantial risk of developing MetS and cardiovascular disease, with the estimated prevalence of MetS being 31-49% among HCT recipients. Although MetS has not yet been proven to impact cardiovascular risk after HCT, an understanding of the incidence and risk factors for MetS in HCT recipients can provide the foundation to evaluate screening guidelines and develop interventions that may mitigate cardiovascular-related mortality. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal of reviewing literature and recommend practices appropriate to HCT recipients. Here we deliver consensus recommendations to help clinicians provide screening and preventive care for MetS and cardiovascular disease among HCT recipients. All HCT survivors should be advised of the risks of MetS and encouraged to undergo recommended screening based on their predisposition and ongoing risk factors.

Practical experiences of moving molecular diagnostics into routine use at the Veterinary Laboratories Agency.

The practical bench application of molecular tests (here defined as nucleic acid-based tests) in a routine testing situation is not without its challenges. This paper outlines the approaches we take at the Veterinary Laboratories Agency (VLA) and highlights some of the practical issues which we have found to be important for the successful introduction and use of molecular tests in a routine testing environment. The potential advantages of molecular tests, and factors which dictate which tests are adopted for routine testing, are discussed before giving some examples of molecular tests in routine use at the VLA. The instrumentation, reagents and assays we use are outlined, followed by sections of how we approach validation and how we manage and resource the transfer of molecular tests into routine use.

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.

Confounding factors affecting the National Institutes of Health (NIH) chronic Graft-Versus-Host Disease Organ-Specific Score and global severity.

The 2005 NIH chronic GVHD (cGVHD) organ severity is based on the assessment of current status regardless of whether abnormalities are due to GVHD. The score assignment does not require knowledge of past manifestations, attribution or whether cGVHD is still active. The aim of this study is to describe confounding factors affecting organ scores in patients with cGVHD. The study included 189 consecutive cGVHD patients evaluated at our center in 2013. Providers completed the NIH 0-3 organ-specific scoring evaluation with two questions added for each organ to identify abnormalities that were (i) not attributed to cGVHD or (ii) attributed to cGVHD plus other causes. Abnormalities attributed to causes other than GVHD were recorded. Eighty (14%) abnormalities were not attributed to cGVHD in at least one organ, and 41 (7%) abnormalities were attributed to cGVHD plus other causes in at least one organ. A total of 436 (78%) abnormalities were attributed only to cGVHD. Abnormalities not attributed to cGVHD were observed most frequently in the lung, gastrointestinal tract and skin. Most common abnormalities included pre-transplant condition, sequelae from GVHD, deconditioning, infections and medications. Our results support the 2014 NIH consensus recommendation to consider attribution when scoring organ abnormalities.

Marrow grafts from HLA-identical siblings for severe aplastic anemia: does limiting the number of transplanted marrow cells reduce the risk of chronic GvHD?

A total of 21 patients with severe aplastic anemia (SAA) underwent marrow transplantation from HLA-identical siblings following a standard conditioning regimen with cyclophosphamide (50 mg/kg/day × 4 days) and horse antithymocyte globulin (30 mg/kg/day × 3 days). Post-grafting immunosuppression consisted of a short course of methotrexate (MTX) combined with cyclosporine (CSP). The transplant protocol tested the hypothesis that the incidence of chronic GvHD could be reduced by limiting the marrow grafts to ⩽2.5 × 108 nucleated marrow cells/kg. None of the patients rejected the graft, all had sustained engraftment and all are surviving at a median of 4 (range 1-8) years after transplantation. Chronic GvHD developed in 16% of patients given ⩽2.5 × 108 nucleated marrow cells/kg. Post-grafting immunosuppression has been discontinued in 20 of the 21 patients. In conclusion, limiting the number of transplanted marrow cells may have resulted in minimal improvement in the incidence and severity of chronic GvHD.

Safety and potential efficacy of low-dose methotrexate for treatment of chronic graft-versus-host disease.

Low-dose methotrexate (MTX) is widely used in autoimmune diseases because of its anti-inflammatory activity. We report here the results of a retrospective study to review the outcomes of low-dose MTX used for treatment of refractory chronic graft-versus-host disease GVHD, with the goal of reducing the amount of prednisone needed to control the disease. In all, 14 patients with refractory chronic GVHD received MTX at a dose of 7.5 mg/m(2)/weekly for 3--0 weeks. Also, 11 patients had skin involvement, often with scleroderma or fasciitis. The median duration of chronic GVHD at the start of MTX was 38 (range 1--35) months. In this retrospective review, we found no grade 3-- toxicities, and none of the patients needed blood transfusion or growth factors. In 10 patients (71%), GVHD could be adequately controlled with prednisone at doses below 1 mg/kg every other day without the addition of other agents. Four patients decreased the amount of concomitant immunosuppressive treatment, five continued with the same regimen, four required an increase in immunosuppressive treatment, and one decided to discontinue all treatment. From this preliminary analysis, MTX appears to be a well-tolerated, inexpensive and possibly steroid-sparing agent that is worthy of further evaluation in prospective trials for treatment of chronic GVHD.

Serious graft-versus-host disease after hematopoietic cell transplantation following nonmyeloablative conditioning.

The efficacy of allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning depends on the balance between the desirable antineoplastic effects of donor cells weighed against the undesirable morbidity of graft-versus-host disease (GVHD). Development of serious acute or chronic GVHD was analyzed retrospectively in 171 consecutive patients, who had related or unrelated nonmyeloablative HCT for hematologic malignancies. GVHD was defined as serious when it resulted in (1) death, (2) disability, (3) three or more major infections in 1 year, (4) prolonged hospitalization or (5) suicide or hospitalization for suicidal ideation. According to this definition, 43 of 171 (25%) patients developed serious GVHD with a median follow-up of 30 (range, 12-65) months. The incidence of serious GVHD was similar after related and unrelated HCT. Among the 43 patients with serious GVHD, 20 had grade III-IV acute GVHD, and 30 had extensive chronic GVHD. Among the 171 patients, seven had grade III acute GVHD and 84 had extensive chronic GVHD that did not meet criteria for serious GVHD. Assessment of serious GVHD provides additional useful information to acute GVHD grades and classification of limited and extensive chronic GVHD in describing the overall risk and impact complications caused by donor cells.

Morbidity associated with anterior iliac crest bone graft harvesting in children undergoing orthopaedic surgery: a prospective review.

PURPOSE: Autologous iliac crest bone grafting is an integral part of many orthopaedic surgical procedures. Several studies have documented morbidity and prolonged pain following iliac crest bone graft harvesting in adults; however, in children there is a paucity of information. The purpose of the present study was to quantify the degree of pain and morbidity associated with anterior iliac crest graft harvesting in children undergoing non-spinal orthopaedic surgery. METHODS: Patients were prospectively enrolled prior to orthopaedic surgery. A patient self-reported visual analogue score was used to record pain at specified time points following surgery. In addition, the patients were reviewed at 2 and 6 weeks, 3 months and 1 year after surgery to record any complications. RESULTS: Data was collected on 33 patients (34 graft sites). Only one patient (2.94 %) had a complication, namely an injury to the lateral femoral cutaneous nerve. This resolved 3 months after surgery. 89 % of patients had no pain at the iliac crest graft harvest site 3 months after surgery. The three patients who had pain at 3 months had visual analogue scores of 1.0, 1.1 and 1.3, respectively. CONCLUSION: This series reveals a very low complication rate and minimal iliac crest graft harvest site pain in children undergoing non-spinal orthopaedic surgery. In addition, the pain experienced is short-lived.

Delirium affects length of hospital stay after lung transplantation.

BACKGROUND: Delirium is relatively common after lung transplantation, although its prevalence and prognostic significance have not been systematically studied. The purpose of the present study was to examine pretransplant predictors of delirium and the short-term impact of delirium on clinical outcomes among lung transplant recipients. METHODS: Participants underwent pretransplant cognitive testing using the Repeatable Battery for the Assessment of Neuropsychological Status and the Trail Making Test. After transplant, delirium was assessed using the Confusion Assessment Method until discharge. RESULTS: Sixty-three patients were transplanted between March and November 2013, of which 23 (37%) developed delirium. Among transplanted patients, 48 patients completed pretransplant cognitive testing. Better pretransplant cognitive function was associated with lower risk of delirium (odds ratio, 0.69 [95% confidence interval 0.48, 0.99], P = .043); and demographic and clinical features including native disease (P = .236), the Charlson comorbidity index (P = .581), and the lung allocation score (P = .871) were unrelated to risk of delirium, although there was a trend for women to experience delirium less frequently (P = .071). The presence (P = .006) and duration (P = .027) of delirium were both associated with longer hospital stays. CONCLUSION: Delirium occurs in more than one-third of patients after lung transplantation. Delirium was associated with poorer pretransplant cognitive functioning and longer hospital stays, after accounting for other medical and demographic factors.