Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains.

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Reduction of diagnostic and treatment delays reduces rifampicin-resistant tuberculosis mortality in Rwanda.

SETTING: In 2005, in response to the increasing prevalence of rifampicin-resistant tuberculosis (RR-TB) and poor treatment outcomes, Rwanda initiated the programmatic management of RR-TB, including expanded access to systematic rifampicin drug susceptibility testing (DST) and standardised treatment.OBJECTIVE: To describe trends in diagnostic and treatment delays and estimate their effect on RR-TB mortality.DESIGN: Retrospective analysis of individual-level data including 748 (85.4%) of 876 patients diagnosed with RR-TB notified to the World Health Organization between 1 July 2005 and 31 December 2016 in Rwanda. Logistic regression was used to estimate the effect of diagnostic and therapeutic delays on RR-TB mortality.RESULTS: Between 2006 and 2016, the median diagnostic delay significantly decreased from 88 days to 1 day, and the therapeutic delay from 76 days to 3 days. Simultaneously, RR-TB mortality significantly decreased from 30.8% in 2006 to 6.9% in 2016. Total delay in starting multidrug-resistant TB (MDR-TB) treatment of more than 100 days was associated with more than two-fold higher odds for dying. When delays were long, empirical RR-TB treatment initiation was associated with a lower mortality.CONCLUSION: The reduction of diagnostic and treatment delays reduced RR-TB mortality. We anticipate that universal testing for RR-TB, short diagnostic and therapeutic delays and effective standardised MDR-TB treatment will further decrease RR-TB mortality in Rwanda.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

Tuberculosis resistance-conferring mutations with fitness cost among HIV-positive individuals in Uganda.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is considered to be less transmissible due to the fitness cost associated with drug resistance-conferring mutations in essential genes. OBJECTIVE: To test the hypothesis that TB drug resistance-conferring mutations with fitness cost are more frequent among human immunodeficiency virus (HIV) positive than among HIV-negative patients. DESIGN: We analysed all strains from the two TB drug resistance surveys conducted in Uganda between 2008 and 2011. Strains phenotypically susceptible to rifampicin and/or isoniazid were assumed to be wild-type; in all other cases, we performed whole-genome sequencing. Mutations at the rpoB531 and katG315 codons were considered without fitness loss, whereas other rpoB codons and non-katG were considered with fitness loss. RESULTS: Of the 897 TB patients, 286 (32.1%) were HIV-positive. Mutations with fitness loss in HIV-positive and HIV-negative patients were respectively as follows: non-531 rpoB: 1.03% (n = 3), 0.71% (n = 4) (OR 1.46, 95%CI 0.58-3.68); non-katG: 0.40% (n = 1), 1.0% (n = 6) (OR 0.40, 95%CI 0.07-2.20); rpoB531: 1.49% (n = 4), 0.69% (n = 4) (OR 2.29, 95%CI 0.83-5.77); katG315: 3.86% (n = 11), 2.55% (n = 15) (OR 1.54, 95%CI 0.81-2.90). The odds of mutations with and without fitness cost were higher for patients with a history of previous anti-tuberculosis treatment. CONCLUSIONS: Our data do not support the hypothesis that resistance-conferring mutations with fitness cost are likely to be often present in HIV-positive individuals.

Discovery of an expanded set of avian leukosis subgroup E proviruses in chickens using Vermillion, a novel sequence capture and analysis pipeline [corrected].

Transposable elements (TEs), such as endogenous retroviruses (ERVs), are common in the genomes of vertebrates. ERVs result from retroviral infections of germ-line cells, and once integrated into host DNA they become part of the host's heritable genetic material. ERVs have been ascribed positive effects on host physiology such as the generation of novel, adaptive genetic variation and resistance to infection, as well as negative effects as agents of tumorigenesis and disease. The avian leukosis virus subgroup E family (ALVE) of endogenous viruses of chickens has been used as a model system for studying the effects of ERVs on host physiology, and approximately 30 distinct ALVE proviruses have been described in the Gallus gallus genome. In this report we describe the development of a software tool, which we call Vermillion, and the use of this tool in combination with targeted next-generation sequencing (NGS) to increase the number of known proviruses belonging to the ALVE family of ERVs in the chicken genome by 4-fold, including expanding the number of known ALVE elements on chromosome 1 (Gga1) from the current 9 to a total of 40. Although we focused on the discovery of ALVE elements in chickens, with appropriate selection of target sequences Vermillion can be used to develop profiles of other families of ERVs and TEs in chickens as well as in species other than the chicken.

Specific gyrA gene mutations predict poor treatment outcome in MDR-TB.

OBJECTIVES: Mutations in the gyrase genes cause fluoroquinolone resistance in Mycobacterium tuberculosis. However, the predictive value of these markers for clinical outcomes in patients with MDR-TB is unknown to date. The objective of this study was to determine molecular markers and breakpoints predicting second-line treatment outcomes in M. tuberculosis patients treated with fourth-generation fluoroquinolones. METHODS: We analysed treatment outcome data in relation to the gyrA and gyrB sequences and MICs of ofloxacin, gatifloxacin and moxifloxacin for pretreatment M. tuberculosis isolates from 181 MDR-TB patients in Bangladesh whose isolates were susceptible to injectable drugs. RESULTS: The gyrA 90Val, 94Gly and 94Ala mutations were most frequent, with the highest resistance levels for 94Gly mutants. Increased pretreatment resistance levels (>2 mg/L), related to specific mutations, were associated with lower cure percentages, with no cure in patients whose isolates were resistant to gatifloxacin at 4 mg/L. Any gyrA 94 mutation, except 94Ala, predicted a significantly lower proportion of cure compared with all other gyrA mutations taken together (all non-94 mutants + 94Ala) [OR = 4.3 (95% CI 1.4-13.0)]. The difference in treatment outcome was not explained by resistance to the other drugs. CONCLUSIONS: Our study suggests that gyrA mutations at position 94, other than Ala, predict high-level resistance to gatifloxacin and moxifloxacin, as well as poor treatment outcome, in MDR-TB patients in whom an injectable agent is still effective.

The majority of cutaneous squamous cell carcinomas arise in actinic keratoses.

BACKGROUND: Retrospective studies have given conflicting results with respect to how many cutaneous squamous cell carcinomas (SCCs) arise in actinic keratoses (AK). OBJECTIVE: This study was conducted to determine what percentage of SCCs arise in AKs and to obtain more information about two histological features of SCCs, namely, thickness and ulceration. METHODS: A prospective study was done of all SCCs treated by the authors during one calendar year. RESULTS: Two hundred eight patients with SCC were entered into the study. An AK was contiguous with an SCC in 72% of the cases. This was taken as evidence that the SCC arose in the AK. Men presented with thicker and more ulcerated SCCs than women, but these were not statistically significant: p = 0.06 for thickness and p = 0.07 for ulceration. Ulcerated SCCs were more likely to arise on the head and neck (p = 0.02), on patients who had multiple skin cancers (p = 0.005), and on patients who had a family history of skin cancer (p = 0.03). CONCLUSION: Actinic keratoses need to be removed before they turn into SCCs. The prognostic significance of ulceration of cutaneous SCCs needs to be determined.

Is the incidence of malignant melanoma decreasing in young Australians?

Recent reports have detected a decrease in the incidence of malignant melanoma (MM) in young Australian women, but an increase in older Australians. The incidence rates were calculated for the entire population and no adjustments were made to take into account the change in the racial composition of the Australian population. In the past 25 years there has been an influx of immigrants from Asia, the Pacific Islands, and the Middle East who are at low risk for MM. The migrants are relatively young and there are more Asian women than men. The large increase in the percentage of the population that has a low risk for MM may be an important reason why the incidence has fallen in some population groups.

Determination of tromethamine in an eye-care pharmaceutical by capillary electrophoresis.

Tromethamine [tris(hydroxymethyl)aminomethane] is used as an emulsifying agent, alkaliser and buffering agent in pharmaceutical preparations such as eye-care solutions. A new method for the determination of this compound in an eye-care preparation was developed using capillary zone electrophoresis with indirect UV detection. The method displayed linearity between 2.5 and 25 mg l-1 with a correlation coefficient of 0.9992. The limit of detection was 2.5 mg l-1. Precision and accuracy were determined as relative standard deviation and % deviation, and were found to be 0.42% and 1.62%, respectively. Recovery studies of tromethamine in the pharmaceutical preparation gave a 102.98% recovery.

Subcutaneous granuloma annulare in a patient with rheumatoid arthritis and diabetes mellitus.

A case is reported of multiple subcutaneous nodules over the forehead and scalp of a 67-year-old male with moderately severe seropositive rheumatoid arthritis and corticosteroid-induced diabetes mellitus. The histology was initially reported as consistent with a rheumatoid nodule, but on review, was regarded as typical of subcutaneous granuloma annulare (SCGA). The nodules resolved spontaneously within 4 months, which is also in keeping with SCGA. A brief review of the relevant literature is presented.

Lymphocyte counts of patients who have had skin cancer.

BACKGROUND: Investigations of lymphocyte counts in patients with skin cancer have given conflicting results, possibly because homogeneous groups of patients were not studied. OBJECTIVE: Our purpose was to measure lymphocyte counts in patients with skin cancer to determine whether any abnormalities were associated with the number of cancers removed and to determine whether a lymphocyte count could identify patients at risk of the development of large numbers of cancers. METHODS: Apparently otherwise normal patients who had histologically confirmed skin cancers removed were studied. One group consisted of patients who had one skin cancer removed but had not had another within a minimum of 5 years. The other group consisted of patients who had had three or more skin cancers. Standard flow cytometry was used to determine the total lymphocyte count, CD4 (helper cell) count, and CD8 (cytotoxic cell) count. RESULTS: Ninety-six patients with multiple skin cancers, and 24 with one skin cancer were studied. Only basal cell carcinomas (BCCs) were removed from 84 patients and the results from this homogeneous group were as follows: women had a higher CD4 cell count than men (p < 0.05); patients with 20 or more BCCs had a lower lymphocyte count (p < 0.01); and patients with one BCC had a higher CD4/CD8 ratio than those who had multiple BCCs (p < 0.05). CONCLUSION: Differences were found between men and women, as well as between subgroups of patients with skin cancer. However, the range of lymphocyte counts was large and it was not possible to determine a threshold below which patients had a worse prognosis. A lymphocyte count is not a reliable way of predicting which patients will have a large number of skin cancers.

Nonmelanoma skin cancer: number of cancers and their distribution in outpatients.

BACKGROUND: Nonmelanoma skin cancer (NMSC) is increasing in incidence and more are developing on the trunk and limbs. The objectives were to determine how many outpatients have more than one NMSC at the time they present for treatment and to determine the anatomical distribution of the cancers. METHODS: All outpatients with histologically confirmed NMSC visited by the authors during 1992 received a total body examination and the number and sites of NMSC were recorded. RESULTS: A total of 952 outpatients were seen. A single NMSC was present in 69.4%, two in 16%, three in 6.4%, four in 3.5%, five to nine in 4.2%, and 0.5% had ten or more. Of the 291 patients with more than one NMSC, one anatomical region was involved in 53.4%, two were involved in 38.4%, and 8.2% had three or more regions involved. CONCLUSION: Patients with NMSC required a total body examination to detect unsuspected skin cancers.

Idiopathic megacolon in the BB rat.

It has been assumed that idiopathic megacolon occurring in the spontaneously diabetic BB rat results from metabolic decompensation associated with diabetes. In a large prospective necropsy study we have documented the incidence of idiopathic megacolon and its distribution between diabetic and non-diabetic animals in the BB rat colony in Edinburgh. A significantly increased incidence of megacolon was found in non-diabetic rats in the diabetes-prone subline compared with diabetic rats in the diabetes-prone subline and with non-diabetic rats in the diabetes-resistant subline. Affected animals were found to have an inflammatory infiltrate in the wall of the large bowel with destruction of both autonomic ganglia and muscularis propria. These necropsy observations indicate that megacolon cannot be a result of the metabolic disturbance due to the hyperglycaemia associated with diabetes.

Lymphoma in the BB/E rat: c-myc translocation identified.

In a prospective necropsy study involving 257 animals in the BB rat colony in Edinburgh, there was an increased incidence of lymphoma (average 10.9 per cent in all three subgroups: 8 per cent in diabetic, 20 per cent in non-diabetic diabetes-prone, and 3.2 per cent in diabetes-resistant rats). The incidence was significantly increased (P < 0.05) in the non-diabetic diabetes-prone subgroup. These results differ markedly from previous results and indicate that the relationship between lymphoma and diabetes is more complex than previously suggested. All the lymphomas bar one involved the ileocaecal nodes and were classified as immunoblastic lymphomas of B-cell origin. There was a striking resemblance both in tissue distribution and in histological classification to the lymphoma seen in the established B-cell lymphoma model, the LOUVAIN rat. Southern blot analysis carried out on the BB rat lymphomas revealed a translocation of variable length involving the c-myc oncogene. Such a translocation has not been demonstrated in the BB rat before.