Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

Candida parapsilosis, currently divided into three distinct species, proliferates in glucose-rich solutions and has been associated with infections resulting from the use of medical devices made of plastic, an environment common in dialysis centres. The aims of this study were (i) to screen for Candida orthopsilosis and Candida metapsilosis (100 environmental isolates previously identified as C. parapsilosis), (ii) to test the ability of these isolates to form biofilm and (iii) to investigate the in vitro susceptibility of Candida spp biofilms to the antifungal agents, fluconazole (FLC) and amphotericin B (AMB). Isolates were obtained from a hydraulic circuit collected from a haemodialysis unit. Based on molecular criteria, 47 strains were re-identified as C. orthopsilosis and 53 as C. parapsilosis. Analyses using a formazan salt reduction assay and total viable count, together with microscopy studies, revealed that 72 strains were able to form biofilm that was structurally similar, but with minor differences in morphology. A microtitre-based colorimetric assay used to test the susceptibility of fungal biofilms to AMB and FLC demonstrated that the C. parapsilosis complex displayed an increased resistance to these antifungal agents. The results from these analyses may provide a basis for implementing quality controls and monitoring to ensure the microbiological purity of dialysis water, including the presence of yeast.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirão Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm³ [standard deviation (SD) = 99] and a mean viral load of 5.09 log10 copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno[clinical20%] algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naÏve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.

Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.

Transmitted human immunodeficiency virus-1 drug resistance in a cohort of men who have sex with men in Belo Horizonte, Brazil - 1996-2012

The presence of transmitted human immunodeficiency virus (HIV)-1 drug-resistance (TDR) at the time of antiretroviral therapy initiation is associated with failure to achieve viral load (VL) suppression. Here, we report TDR surveillance in a specific population of men who have sex with men (MSM) in Belo Horizonte, Brazil. In this study, the rate of TDR was evaluated in 64 HIV-infected individuals from a cohort of MSM between 1996-June 2012. Fifty-four percent had a documented recent HIV infection, with a seroconversion time of less than 12 months. The median CD4+T lymphocyte count and VL were 531 cells/mm3and 17,746 copies/mL, respectively. Considering the surveillance drug resistance mutation criteria, nine (14.1%) patients presented TDR, of which three (4.7%), five (7.8%) and four (6.2%) had protease inhibitors, resistant against nucleos(t)ide transcriptase inhibitors and against non-nucleoside reverse-transcriptase inhibitors mutations, respectively. Two of the patients had multi-drug-resistant HIV-1. The most prevalent viral subtype was B (44, 68.8%), followed by subtype F (11, 17.2%). This study shows that TDR may vary according to the population studied and it may be higher in clusters of MSM.

Variation in haematological parameters in children less than five years of age with asymptomatic Plasmodium infection: implication for malaria field studies

During the season of high malaria transmission, most children are infected by Plasmodium, which targets red blood cells (RBCs), affecting haematological parameters. To describe these variations, we examined the haematological profiles of two groups of children living in a malaria-endemic area. A cross-sectional survey was conducted at the peak of the malaria transmission season in a rural area of Burkina Faso. After informed consent and clinical examination, blood samples were obtained from the participants for malaria diagnosis and a full blood count. Of the 414 children included in the analysis, 192 were not infected with Plasmodium, whereas 222 were asymptomatic carriers of Plasmodium infection. The mean age of the infected children was 41.8 months (range of 26.4-57.2) compared to 38.8 months (range of 22.4-55.2) for the control group (p = 0.06). The asymptomatic infected children tended to have a significantly lower mean haemoglobin level (10.8 g/dL vs. 10.4 g/dL; p < 0.001), mean lymphocyte count (4592/µL vs. 5141/µL; p = 0.004), mean platelet count (266 x 103/µL vs. 385 x 103/µL; p < 0.001) and mean RBC count (4.388 x 106/µL vs. 4.158 x 106/µL; p < 0.001) and a higher mean monocyte count (1403/µL vs. 1192/µL; p < 0.001) compared to the control group. Special attention should be applied when interpreting haematological parameters and evaluating immune responses in asymptomatic infected children living in malaria-endemic areas and enrolled in vaccine trials.

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis.

Prevalence of human papillomavirus infection, distribution of viral types and risk factors in cervical samples from human immunodeficiency virus-positive women attending three human immunodeficiency virus-acquired immune deficiency syndrome reference centres in northeastern Brazil

Human immunodeficiency virus (HIV)-positive patients have a greater prevalence of coinfection with human papillomavirus (HPV) is of high oncogenic risk. Indeed, the presence of the virus favours intraepithelial squamous cell lesion progression and may induce cancer. The aim of this study was to evaluate the prevalence of HPV infection, distribution of HPV types and risk factors among HIV-positive patients. Cervical samples from 450 HIV-positive patients were analysed with regard to oncotic cytology, colposcopy and HPV presence and type by means of polymerase chain reaction and sequencing. The results were analysed by comparing demographic data and data relating to HPV and HIV infection. The prevalence of HPV was 47.5%. Among the HPV-positive samples, 59% included viral types of high oncogenic risk. Multivariate analysis showed an association between HPV infection and the presence of cytological alterations (p = 0.003), age greater than or equal to 35 years (p = 0.002), number of partners greater than three (p = 0.002), CD4+ lymphocyte count < 200/mm3 (p = 0.041) and alcohol abuse (p = 0.004). Although high-risk HPV was present in the majority of the lesions studied, the low frequency of HPV 16 (3.3%), low occurrence of cervical lesions and preserved immunological state in most of the HIV-positive patients were factors that may explain the low occurrence of precancerous cervical lesions in this population.

Bonafide, type-specific human papillomavirus persistence among HIV-positive pregnant women: predictive value for cytological abnormalities, a longitudinal cohort study

This study investigated the rate of human papillomavirus (HPV) persistence, associated risk factors, and predictors of cytological alteration outcomes in a cohort of human immunodeficiency virus-infected pregnant women over an 18-month period. HPV was typed through L1 gene sequencing in cervical smears collected during gestation and at 12 months after delivery. Outcomes were defined as nonpersistence (clearance of the HPV in the 2nd sample), re-infection (detection of different types of HPV in the 2 samples), and type-specific HPV persistence (the same HPV type found in both samples). An unfavourable cytological outcome was considered when the second exam showed progression to squamous intraepithelial lesion or high squamous intraepithelial lesion. Ninety patients were studied. HPV DNA persistence occurred in 50% of the cases composed of type-specific persistence (30%) or re-infection (20%). A low CD4+T-cell count at entry was a risk factor for type-specific, re-infection, or HPV DNA persistence. The odds ratio (OR) was almost three times higher in the type-specific group when compared with the re-infection group (OR = 2.8; 95% confidence interval: 0.43-22.79). Our findings show that bonafide (type-specific) HPV persistence is a stronger predictor for the development of cytological abnormalities, highlighting the need for HPV typing as opposed to HPV DNA testing in the clinical setting.

Predictors of red blood cell transfusion after cardiac surgery: a prospective cohort study

Abstract OBJECTIVE To identify predictors of red blood cell transfusion (RBCT) after cardiac surgery. METHOD A prospective cohort study performed with 323 adults after cardiac surgery, from April to December of 2013. A data collection instrument was constructed by the researchers containing factors associated with excessive bleeding after cardiac surgery, as found in the literature, for investigation in the immediate postoperative period. The relationship between risk factors and the outcome was assessed by univariate analysis and logistic regression. RESULTS The factors associated with RBCT in the immediate postoperative period included lower height and weight, decreased platelet count, lower hemoglobin level, higher prevalence of platelet count <150x10 3/mm3, lower volume of protamine, longer duration of anesthesia, higher prevalence of intraoperative RBCT, lower body temperature, higher heart rate and higher positive end-expiratory pressure. The independent predictor was weight <66.5Kg. CONCLUSION Factors associated with RBCT in the immediate postoperative period of cardiac surgery were found. The independent predictor was weight.

Notas sobre a socialidade e a biologia de nidificação de Trypoxylon (Trypoxylon) asuncicola Strand, 1910 (Hymenoptera, Sphecidae)

Twenty-two nests of Trypoxylon asuncicola were sampled in Viçosa, State of Minas Gerais, Brazil, in January 2000 and the occupants' behavior of three nests was registered in 2h of direct observation. 528 brood cells were excavated (24±13.84 SD cells per nest), 129 were reused cells, some of them for seven times (meconium deposit count). The mean number of total cells, mean number of open and closed cells, parasitism rate and mean number of reused cells per nest were similar between old and new nests. Parasitism rate and cell reuse were associated with the number of building cells in the nest, but nest aggregate in the sampled area may play some role in the parasitism rate. Brachymeria sp. (Chalcididae) was the most important agent of brood mortality (80%). Other parasites were Melittobia sp. (Eulophidae) (17%) and a species of Icheumonidae (3%). The number of closed cells with immature individuals per nest was 4±4.2SD (N=17) and the mean reproductivity per female was 3±2.4SD (N=5). New nests produced more offspring (0 a 35%) than old nests (0 to 11%). Females and males can be found resting in the nest but copula or guarding behavior by the male was not observed. There is some evidence that in the sampled area the switch of nests by females is great and agonistic behavior between a nest owner and a visitor was not evident. Females were larger (3.9±0.4SD mm) than males (3.1±0.3SD mm) (measured as forewing length). The secondary sex ratio was 1.26 (±0.07 SE) in favor of females, which was not different from 1:1 ratio. The majority (97%) of the sampled larvae of T. asuncicola showed diapause. Some (5.1%) 'anomalous cells' were found.