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1.  High Prevalence of Clustered Tuberculosis Cases in Peruvian Migrants in Florence, Italy 
Infectious Disease Reports  2014;6(4):5646.
Tuberculosis is a leading cause of morbidity for Peruvian migrants in Florence, Italy, where they account for about 20% of yearly diagnosed cases. A retrospective study on cases notified in Peruvian residents in Florence in the period 2001-2010 was carried out and available Mycobacterium tuberculosis strains were genotyped (MIRU-VNTR-24 and Spoligotyping). One hundred thirty eight cases were retrieved. Genotyping performed in 87 strains revealed that 39 (44.8%) belonged to 12 clusters. Assuming that in each cluster the transmission of tuberculosis from the index case took place in Florence, a large proportion of cases could be preventable by improving early diagnosis of contagious cases and contact tracing.
doi:10.4081/idr.2014.5646
PMCID: PMC4274404  PMID: 25568758
tuberculosis; immigrants; migrants; cluster; epidemiology; Peru; Italy
2.  Use of Third Line Antiretroviral Therapy in Latin America 
PLoS ONE  2014;9(9):e106887.
Background
Access to highly active antiretroviral therapy (HAART) is expanding in Latin America. Many patients require second and third line therapy due to toxicity, tolerability, failure, or a combination of factors. The need for third line HAART, essential for program planning, is not known.
Methods
Antiretroviral-naïve patients ≥18 years who started first HAART after January 1, 2000 in Caribbean, Central and South America Network (CCASAnet) sites in Argentina, Brazil, Honduras, Mexico, and Peru were included. Clinical trials participants were excluded. Third line HAART was defined as use of darunavir, tipranavir, etravirine, enfuvirtide, maraviroc or raltegravir. Need for third line HAART was defined as virologic failure while on second line HAART.
Results
Of 5853 HAART initiators followed for a median of 3.5 years, 310 (5.3%) failed a second line regimen and 44 (0.8%) received a third line regimen. Cumulative incidence of failing a 2nd or starting a 3rd line regimen was 2.7% and 6.0% three and five years after HAART initiation, respectively. Predictors at HAART initiation for failing a second or starting a third line included female sex (hazard ratio [HR] = 1.54, 95% confidence interval [CI] 1.18–2.00, p = 0.001), younger age (HR = 2.76 for 20 vs. 40 years, 95% CI 1.86–4.10, p<0.001), and prior AIDS (HR = 2.17, 95% CI 1.62–2.90, p<0.001).
Conclusions
Third line regimens may be needed for at least 6% of patients in Latin America within 5 years of starting HAART, a substantial proportion given the large numbers of patients on HAART in the region. Improved accessibility to third line regimens is warranted.
doi:10.1371/journal.pone.0106887
PMCID: PMC4164470  PMID: 25221931
3.  Efficacy and Duration of Immunity after Yellow Fever Vaccination: Systematic Review on the Need for a Booster Every 10 Years 
Current regulations stipulate a yellow fever (YF) booster every 10 years. We conducted a systematic review of the protective efficacy and duration of immunity of YF vaccine in residents of disease-endemic areas and in travelers to assess the need for a booster in these two settings and in selected populations (human immunodeficiency virus–infected persons, infants, children, pregnant women, and severely malnourished persons). Thirty-six studies and 22 reports were included. We identified 12 studies of immunogenicity, 8 of duration of immunity, 8 of vaccine response in infants and children, 7 of human-immunodeficiency virus–infected persons, 2 of pregnant women, and 1 of severely malnourished children. Based on currently available data, a single dose of YF vaccine is highly immunogenic and confers sustained life-long protective immunity against YF. Therefore, a booster dose of YF vaccine is not needed. Special considerations for selected populations are detailed.
doi:10.4269/ajtmh.13-0264
PMCID: PMC3771278  PMID: 24006295
4.  Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru 
Infection and Immunity  2014;82(1):124-131.
Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.
doi:10.1128/IAI.00796-13
PMCID: PMC3911835  PMID: 24126518
5.  Histopathological Features of Tungiasis in Peru 
Tungiasis is an ectoparasitic skin disease caused by Tunga penetrans and Tunga trimamillata. There is a lack of histopathological studies that evaluate the recognition of this flea in tissues. We describe the ex vivo dermoscopic and the histopathological patterns of six cases and relate the findings to the developmental stage of the parasite as defined by the Fortaleza classification: two were classified as Fortaleza 3b, 3 as 4a, and 1 as 4b. Two dermoscopic patterns were observed: a brown pigmented ring and a radial crown with a central pore. The most common histopathological findings were an eosinophilic cuticle, eggs in different stages of development, tracheal rings (parasite), and basal hyperplasia (host). The eosinophilic cuticle, eggs in different stages of evolution, and tracheal rings can help to establish the diagnosis when other parts of the parasite are lacking. The Fortaleza staging may represent a tool for pathology reporting purposes.
doi:10.4269/ajtmh.12-0645
PMCID: PMC3752826  PMID: 23478579
6.  A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis 
Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.
Author Summary
Brucellosis is a OneHealth disease reflecting the risk for human infection by interaction with and relation to affected animal populations. The disease is often difficult to diagnose because of lack of precise or accessible diagnostic reagents, and because culture is complex, hazardous and relatively insensitive. Brucellosis disproportionately affects the poor and dispossessed with human and animal burdens of disease in the Middle East, North Africa, Mongolia and other regions that are simply unknown. The diagnosis of brucellosis most often rests on serological tests—antibody detection—based on agglutination of fixed Brucella abortus. We have developed the basis for developing a new test based on the detection of the B. melitensis lipopolysaccharide, which provides rapid and definitive identification of the presence of the organism in clinically obtainable body fluids. A new approach—protein conjugation to the lipopolysaccharide antigen—was taken to enhance the affinity of the monoclonal antibodies that were generated for the test. These reagents were tested in a mouse model of B. melitensis and in humans from the brucellosis-endemic region of Peru, and provided the data for the basis of further clinical development and clinical trials for the rapid, point-of-care diagnosis of brucellosis that will also provide new tools for assessing the global burden of disease.
doi:10.1371/journal.pntd.0002926
PMCID: PMC4046965  PMID: 24901521
7.  CD4+ T cell subsets and Tax expression in HTLV-1 associated diseases 
Pathogens and Global Health  2013;107(4):202-206.
Human T lymphotropic virus type 1 (HTLV-1) infection displays variable clinical manifestations. These include inflammatory diseases such as HTLV-1 associated myelopathy (HAM) or immunosuppressive conditions such as Strongyloides stercoralis hyperinfection. The viral protein, Tax causes activation and proliferation of T cells. We hypothesize that the expression of Tax in T cell subsets characterizes the clinical manifestations of HTLV-1. To test this hypothesis, we measured T helper 1 effector cells and regulatory T cells (Tregs) among Tax expressing lymphocytes from peripheral blood mononuclear cells (PBMCs) of 32 HTLV-1 infected patients with HAM, with S. stercoralis co-infection or with asymptomatic infection. We observed increased ratios of Th1/Treg among Tax expressing lymphocytes in HAM patients. These data suggest that the expression of Tax among the different target cells may explain the variable presentation of HTLV-1.
doi:10.1179/2047773213Y.0000000091
PMCID: PMC4001472  PMID: 23816512
HTLV-1; Tax; Immunology; Regulatory T cells; Th1; Foxp3
8.  A Foodborne Outbreak of Brucellosis at a Police Station Cafeteria, Lima, Peru 
Brucella melitensis is highly infectious for humans and can be transmitted to humans in a number of epidemiological contexts. Within the context of an ongoing brucellosis surveillance project, an outbreak at a Peruvian police officer cafeteria was discovered, which led to active surveillance (serology, blood culture) for additional cases among 49 police officers who had also eaten there. The cohort was followed up to 18 months regardless of treatment or symptoms. Active surveillance estimated the attack rate at 26.5% (13 of 49). Blood cultures from four cases were positive; these isolates were indistinguishable using multiple locus variable number tandem repeat analysis. This investigation indicates the importance of case tracking and active surveillance for brucellosis in the context of potential common source exposure. These results provide rationale for public health investigations of brucellosis index cases including the bioterrorism-related dissemination of Brucella.
doi:10.4269/ajtmh.12-0606
PMCID: PMC3592540  PMID: 23382164
9.  CD80+ and CD86+ B cells as biomarkers and possible therapeutic targets in HTLV-1 associated myelopathy/tropical spastic paraparesis and multiple sclerosis 
Background
Human T-cell lymphotropic virus (HTLV-1) is the causative agent of the incapacitating, neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, there are no disease-modifying therapies with long-term clinical benefits or validated biomarkers for clinical follow-up in HAM/TSP. Although CD80 and CD86 costimulatory molecules play prominent roles in immune regulation and reflect disease status in multiple sclerosis (MS), data in HAM/TSP are lacking.
Methods
Using flow cytometry, we quantified ex vivo and in vitro expression of CD80 and CD86 in PBMCs of healthy controls, HTLV-1-infected individuals with and without HAM/TSP, and MS patients. We hypothesized ex vivo CD80 and CD86 expressions and their in vitro regulation by interferon (IFN)-α/β mirror similarities between HAM/TSP and MS and hence might reveal clinically useful biomarkers in HAM/TSP.
Results
Ex vivo expression of CD80 and CD86 in T and B cells increased in all HTLV-1 infected individuals, but with a selective defect for B cell CD86 upregulation in HAM/TSP. Despite decreased total B cells with increasing disease duration (p = 0.0003, r = −0.72), CD80+ B cells positively correlated with disease severity (p = 0.0017, r = 0.69) in HAM/TSP. B cell CD80 expression was higher in women with HAM/TSP, underscoring that immune markers can reflect the female predominance observed in most autoimmune diseases. In contrast to MS patients, CD80+ (p = 0.0001) and CD86+ (p = 0.0054) lymphocytes expanded upon in vitro culture in HAM/TSP patients. The expansion of CD80+ and CD86+ T cells but not B cells was associated with increased proliferation in HTLV-1 infection. In vitro treatment with IFN-β but not IFN-α resulted in a pronounced increase of B cell CD86 expression in healthy controls, as well as in patients with neuroinflammatory disease (HAM/TSP and MS), similar to in vivo treatment in MS.
Conclusions
We propose two novel biomarkers, ex vivo CD80+ B cells positively correlating to disease severity and CD86+ B cells preferentially induced by IFN-β, which restores defective upregulation in HAM/TSP. This study suggests a role for B cells in HAM/TSP pathogenesis and opens avenues to B cell targeting (with proven clinical benefit in MS) in HAM/TSP but also CD80-directed immunotherapy, unprecedented in both HAM/TSP and MS.
doi:10.1186/1742-2094-11-18
PMCID: PMC3922160  PMID: 24472094
Neuroinflammatory disease; HTLV-1; Multiple sclerosis; Interferon-alpha/beta; B cell; Costimulatory CD80; CD86; Human; Ex vivo; Disease severity; Gender
14.  Medication Possession Ratio Predicts Antiretroviral Regimens Persistence in Peru 
PLoS ONE  2013;8(10):e76323.
Objectives
In developing nations, the use of operational parameters (OPs) in the prediction of clinical care represents a missed opportunity to enhance the care process. We modeled the impact of multiple measurements of antiretroviral treatment (ART) adherence on antiretroviral treatment outcomes in Peru.
Design And Methods
Retrospective cohort study including ART naïve, non-pregnant, adults initiating therapy at Hospital Nacional Cayetano Heredia, Lima-Peru (2006-2010). Three OPs were defined: 1) Medication possession ratio (MPR): days with antiretrovirals dispensed/days on first-line therapy; 2) Laboratory monitory constancy (LMC): proportion of 6 months intervals with ≥1 viral load or CD4 reported; 3) Clinic visit constancy (CVC): proportion of 6 months intervals with ≥1 clinic visit.
Three multi-variable Cox proportional hazard (PH) models (one per OP) were fit for (1) time of first-line ART persistence and (2) time to second-line virologic failure. All models were adjusted for socio-demographic, clinical and laboratory variables.
Results
856 patients were included in first-line persistence analyses, median age was 35.6 years [29.4-42.9] and most were male (624; 73%). In multivariable PH models, MPR (per 10% increase HR=0.66; 95%CI=0.61-0.71) and LMC (per 10% increase 0.83; 0.71-0.96) were associated with prolonged time on first-line therapies.
Among 79 individuals included in time to second-line virologic failure analyses, MPR was the only OP independently associated with prolonged time to second-line virologic failure (per 10% increase 0.88; 0.77-0.99).
Conclusions
The capture and utilization of program level parameters such as MPR can provide valuable insight into patient-level treatment outcomes.
doi:10.1371/journal.pone.0076323
PMCID: PMC3788135  PMID: 24098475
15.  Ex Vivo Innate Immune Cytokine Signature of Enhanced Risk of Relapsing Brucellosis 
Background
Brucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5–40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis.
Methodology/Principal Findings
Blood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM) isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively.
Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1β, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1β, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM.
Conclusions/Significance
This work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may allow for better follow-up care of brucellosis patients through improved identification of patients at risk for relapse.
Author Summary
Brucellosis is a disease caused by transmission of bacteria of the Brucella genus from infected animals to humans. The main route of infection occurs through consumption of contaminated dairy products or contact with infected animals. While most patients treated with antibiotics will be cured of the infection, between 5–40% of patients experience a relapse of brucellosis. The mechanisms underlying these recurring infections remain poorly understood. In this study, we examined blood cells from control donors, patients who previously had acute infections, and patients who previously had relapsing infections. We identified an inflammatory cytokine signature from measurements of unstimulated and stimulated cells that showed statistically significant differences between relapsing and non-relapsing brucellosis patients. Future applications of this assay system may allow for better follow-up care of brucellosis through the diagnosis of this cytokine signature and predictive or improved identification of patients at risk for relapse.
doi:10.1371/journal.pntd.0002424
PMCID: PMC3764229  PMID: 24040434
17.  Induced Sputum MMP-1, -3 & -8 Concentrations during Treatment of Tuberculosis 
PLoS ONE  2013;8(4):e61333.
Introduction
Tuberculosis (TB) destroys lung tissues and this immunopathology is mediated in part by Matrix Metalloproteinases (MMPs). There are no data on the relationship between local tissue MMPs concentrations, anti-tuberculosis therapy and sputum conversion.
Materials and Methods
Induced sputum was collected from 68 TB patients and 69 controls in a cross-sectional study. MMPs concentrations were measured by Luminex array, TIMP concentrations by ELISA and were correlated with a disease severity score (TBscore). 46 TB patients were then studied longitudinally at the 2nd, 8th week and end of treatment.
Results
Sputum MMP-1,-2,-3,-8,-9 and TIMP-1 and -2 concentrations are increased in TB. Elevated MMP-1 and -3 concentrations are independently associated with higher TB severity scores (p<0.05). MMP-1, -3 and -8 concentrations decreased rapidly during treatment (p<0.05) whilst there was a transient increase in TIMP-1/2 concentrations at week 2. MMP-2, -8 and -9 and TIMP-2 concentrations were higher at TB diagnosis in patients who remain sputum culture positive at 2 weeks and MMP-3, -8 and TIMP-1 concentrations were higher in these patients at 2nd week of TB treatment.
Conclusions
MMPs are elevated in TB patients and associate with disease severity. This matrix-degrading phenotype resolves rapidly with treatment. The MMP profile at presentation correlates with a delayed treatment response.
doi:10.1371/journal.pone.0061333
PMCID: PMC3632571  PMID: 23613834
18.  Strongyloidiasis and Infective Dermatitis Alter Human T Lymphotropic Virus-1 Clonality in vivo 
PLoS Pathogens  2013;9(4):e1003263.
Human T-lymphotropic Virus-1 (HTLV-1) is a retrovirus that persists lifelong by driving clonal proliferation of infected T-cells. HTLV-1 causes a neuroinflammatory disease and adult T-cell leukemia/lymphoma. Strongyloidiasis, a gastrointestinal infection by the helminth Strongyloides stercoralis, and Infective Dermatitis associated with HTLV-1 (IDH), appear to be risk factors for the development of HTLV-1 related diseases. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the HTLV-1-infected T-cell population (i.e. the number of distinct clones and abundance of each clone). A newly developed biodiversity estimator called “DivE” was used to estimate the total number of clones in the blood. We found that the major determinant of proviral load in all subjects without leukemia/lymphoma was the total number of HTLV-1-infected clones. Nevertheless, the significantly higher proviral load in patients with strongyloidiasis or IDH was due to an increase in the mean clone abundance, not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone. In patients co-infected with Strongyloides there was an increased degree of oligoclonal expansion and a higher rate of turnover (i.e. appearance and disappearance) of HTLV-1-infected clones. In Strongyloides co-infected patients and those with IDH, proliferation of the most abundant HTLV-1+ T-cell clones is independent of the genomic environment of the provirus, in sharp contrast to patients with HTLV-1 infection alone. This implies that new selection forces are driving oligoclonal proliferation in Strongyloides co-infection and IDH. We conclude that strongyloidiasis and IDH increase the risk of development of HTLV-1-associated diseases by increasing the rate of infection of new clones and the abundance of existing HTLV-1+ clones.
Author Summary
HTLV-1 is a human retrovirus estimated to infect 20 million people world-wide and is causing in a small proportion of the infected individuals an inflammatory disease or a leukemia/lymphoma. HTLV-1 persists lifelong by driving clonal proliferation of infected T-cells. Strongyloidiasis, a gastrointestinal infection by an helminth (Strongyloides stercoralis) and Infective Dermatitis associated with HTLV-1 (IDH), a skin inflammation with bacterial infection, appear to increase the risk of developing HTLV-1-related diseases. It is well known that the chance of developing HTLV-1-related diseases increases with the number of cells infected by the virus (also called proviral load). It is also known that HTLV-1-infected individuals co-infected by Strongyloides or affected by IDH have a higher proviral load, but the mechanism is still unclear. Consequently, the aim of this study was to test if co-infection increases the total number and/or the abundance (or size) of HTLV-1-infected T-cell clones. We have shown that the significantly increased proviral load in HTLV-1-infected individuals with IDH or strongyloidiasis is due to an increase in the mean clone abundance (bigger clones), not to an increase in the number of infected clones. These patients appear to be less capable of restricting clone abundance than those with HTLV-1 alone.
doi:10.1371/journal.ppat.1003263
PMCID: PMC3617147  PMID: 23592987
19.  The Laboratory Diagnosis and Follow Up of Strongyloidiasis: A Systematic Review 
Background
Strongyloidiasis is frequently under diagnosed since many infections remain asymptomatic and conventional diagnostic tests based on parasitological examination are not sufficiently sensitive. Serology is useful but is still only available in reference laboratories. The need for improved diagnostic tests in terms of sensitivity and specificity is clear, particularly in immunocompromised patients or candidates to immunosuppressive treatments. This review aims to evaluate both conventional and novel techniques for the diagnosis of strongyloidiasis as well as available cure markers for this parasitic infection.
Methodology/Principal Findings
The search strategy was based on the data-base sources MEDLINE, Cochrane Library Register for systematic review, EmBase, Global Health and LILACS and was limited in the search string to articles published from 1960 to August 2012 and to English, Spanish, French, Portuguese and German languages. Case reports, case series and animal studies were excluded. 2003 potentially relevant citations were selected for retrieval, of which 1649 were selected for review of the abstract. 143 were eligible for final inclusion.
Conclusions
Sensitivity of microscopic-based techniques is not good enough, particularly in chronic infections. Furthermore, techniques such as Baermann or agar plate culture are cumbersome and time-consuming and several specimens should be collected on different days to improve the detection rate. Serology is a useful tool but it might overestimate the prevalence of disease due to cross-reactivity with other nematode infections and its difficulty distinguishing recent from past (and cured) infections. To evaluate treatment efficacy is still a major concern because direct parasitological methods might overestimate it and the serology has not yet been well evaluated; even if there is a decline in antibody titres after treatment, it is slow and it needs to be done at 6 to 12 months after treatment which can cause a substantial loss to follow-up in a clinical trial.
Author Summary
Strongyloidiasis is a parasitic infection that can occur in any place of the world. It is not easy to diagnose because the conventional tests are not good enough, especially in individuals that do not present any symptoms of the disease. This is of particular importance in immunocompromised patients, because the disease can spread causing a disseminated disease which can be fatal. In this study, authors review both conventional and novel techniques for the diagnosis of strongyloidiasis. Parasitological examinations based on the detection of the parasite in faeces are the most common techniques used until now in the majority of laboratories. However, they have some disadvantages because most of the best techniques are cumbersome and time consuming and several stool samples have to be collected to improve the diagnosis. New techniques such as the serology which is performed through a blood test are becoming available, but they have still some problems; the test sometimes does not accurately differentiate strongyloidiasis from other helminthic diseases. Another major problem in this disease is to evaluate if patient is cured after the treatment. Parasitological methods can fail to detect treatment failure, and serology has not yet been well evaluated in this context.
doi:10.1371/journal.pntd.0002002
PMCID: PMC3547839  PMID: 23350004
20.  Staphylococcus aureus Causing Tropical Pyomyositis, Amazon Basin, Peru  
Emerging Infectious Diseases  2013;19(1):123-125.
We studied 12 Staphylococcus aureus isolates causing tropical pyomyositis in the Amazon Basin of Peru. All isolates were methicillin-susceptible; 11 carried Panton-Valentine leukocidin–encoding genes, and 5 belonged to multilocus sequence type 25 and possessed an extensive set of enterotoxins. Our findings suggest sequence type 25 is circulating in tropical areas of South America.
doi:10.3201/eid1901.120819
PMCID: PMC3557995  PMID: 23260279
tropical pyomyositis; Staphylococcus aureus; S. aureus; Panton-Valentine leukocidin; Peru; Amazon Basin; methicillin-susceptible Staphylococcus aureus; MSSA; community-associated methicillin-resistant S. aureus clones; MRSA; bacteria; staphylococci
21.  A Diagnostic Accuracy Study of Xpert®MTB/RIF in HIV-Positive Patients with High Clinical Suspicion of Pulmonary Tuberculosis in Lima, Peru 
PLoS ONE  2012;7(9):e44626.
Background
Diagnosis of pulmonary tuberculosis (TB) among human immunodeficiency virus (HIV) patients remains complex and demands easy to perform and accurate tests. Xpert®MTB/RIF (MTB/RIF) is a molecular TB diagnostic test which is rapid and convenient; the test requires minimal human resources and reports results within two hours. The majority of performance studies of MTB/RIF have been performed in high HIV burden settings, thus TB diagnostic studies among HIV patients in low HIV prevalence settings such as Peru are still needed.
Methodology/Principal Findings
From April 2010 to May 2011, HIV-positive patients with high clinical suspicion of TB were enrolled from two tertiary hospitals in Lima, Peru. Detection of TB by MTB/RIF was compared to a composite reference standard Löwenstein-Jensen (LJ) and liquid culture. Detection of rifampicin resistance was compared to the LJ proportion method. We included 131 patients, the median CD4 cell count was 154.5 cells/mm3 and 45 (34.4%) had TB. For TB detection among HIV patients, sensitivity of MTB/RIF was 97.8% (95% CI 88.4–99.6) (44/45); specificity was 97.7% (95% CI 91.9–99.4) (84/86); the positive predictive value was 95.7% (95% CI 85.5–98.8) (44/46); and the negative predictive value, 98.8% (95% CI 93.6–99.8) (84/85). MTB/RIF detected 13/14 smear-negative TB cases, outperforming smear microscopy [97.8% (44/45) vs. 68.9% (31/45); p = 0.0002]. For rifampicin resistance detection, sensitivity of MTB/RIF was 100% (95% CI 61.0–100.0) (6/6); specificity was 91.0% (95% CI 76.4–96.9) (30/33); the positive predictive value was 66.7% (95% CI 35.4–87.9) (6/9); and the negative predictive value was 100% (95% CI 88.7 –100.0) (30/30).
Conclusions/Significance
In HIV patients in our population with a high clinical suspicion of TB, MTB/RIF performed well for TB diagnosis and outperformed smear microscopy.
doi:10.1371/journal.pone.0044626
PMCID: PMC3436871  PMID: 22970271
22.  Serial Kinetics of the Antibody Response against the Complete Brucella melitensis ORFeome in Focal Vertebral Brucellosis 
Journal of Clinical Microbiology  2012;50(3):922-926.
Human brucellosis is a common zoonosis worldwide. Here we present a case of focal vertebral brucellosis in a 71-year-old Mexican-American woman who contracted infection from unpasteurized goat milk. Standard agglutination serology was negative; the diagnosis was established by the isolation of Brucella melitensis from abscess fluid. A B. melitensis protein microarray comprised of nearly all proteins encoded by the bacterial genome was used to determine the kinetics of this patient's antibody responses to the complete collection of open reading frames existing in the genome (ORFeome). Three patterns of antibody responses against B. melitensis antigens were seen for serum samples obtained on days 0 (pretreatment), 14, 49, 100, and 180: (i) stable titers over time, (ii) a steady fall in titers, and (iii) an initial rise in titers followed by declining titers. Sera from this patient with chronic brucellosis recognized some of the same B. melitensis proteins as those recognized by sera from acute/subacute, blood culture-positive brucellosis patients but also recognized a distinct set of proteins. This study is the first to determine the kinetics of the human antibody responses to the complete repertoire of proteins encoded by a bacterial genome and demonstrates fundamentally different immunopathogenetic mechanisms between acute human brucellosis and chronic human brucellosis. While an extension of these findings to a larger patient population is necessary, these findings have important clinical and diagnostic implications and lead toward new insights into the fundamental immunopathogenesis of brucellosis.
doi:10.1128/JCM.05298-11
PMCID: PMC3295165  PMID: 22219303
23.  Quinolone Resistance in Absence of Selective Pressure: The Experience of a Very Remote Community in the Amazon Forest 
Background
Quinolones are potent broad-spectrum bactericidal agents increasingly employed also in resource-limited countries. Resistance to quinolones is an increasing problem, known to be strongly associated with quinolone exposure. We report on the emergence of quinolone resistance in a very remote community in the Amazon forest, where quinolones have never been used and quinolone resistance was absent in 2002.
Methods
The community exhibited a considerable level of geographical isolation, limited contact with the exterior and minimal antibiotic use (not including quinolones). In December 2009, fecal carriage of antibiotic resistant Escherichia coli was investigated in 120 of the 140 inhabitants, and in 48 animals reared in the community. All fluoroquinolone-resistant isolates were genotyped and characterized for the mechanisms of plasmid- and chromosomal-mediated quinolone resistance.
Principal Findings
Despite the characteristics of the community remained substantially unchanged during the period 2002–2009, carriage of quinolone-resistant E. coli was found to be common in 2009 both in humans (45% nalidixic acid, 14% ciprofloxacin) and animals (54% nalidixic acid, 23% ciprofloxacin). Ciprofloxacin-resistant isolates of human and animal origin showed multidrug resistance phenotypes, a high level of genetic heterogeneity, and a combination of GyrA (Ser83Leu and Asp87Asn) and ParC (Ser80Ile) substitutions commonly observed in fluoroquinolone-resistant clinical isolates of E. coli.
Conclusions
Remoteness and absence of antibiotic selective pressure did not protect the community from the remarkable emergence of quinolone resistance in E. coli. Introduction of the resistant strains from antibiotic-exposed settings is the most likely source, while persistence and dissemination in the absence of quinolone exposure is likely mostly related with poor sanitation. Interventions aimed at reducing the spreading of resistant isolates (by improving sanitation and water/food safety) are urgently needed to preserve the efficacy of quinolones in resource-limited countries, as control strategies based only on antibiotic restriction policies are unlikely to succeed in those settings.
Author Summary
Quinolones are broad-spectrum antibiotics which bind to type II topoisomerases (DNA gyrase and topoisomerase IV) and inhibit DNA re-ligation after enzyme cut, exerting a rapid bactericidal activity. They are widely used for the treatment of several community- and hospital-acquired infections and have become increasingly important also in resource-limited countries, following the availability of generics (which have drastically reduced drug costs) and the remarkable increase of resistance to the oldest and cheapest antibiotic classes. Resistance to quinolones is an increasing worldwide problem that challenges the efficacy of these drugs against several bacterial pathogens and is known to be strongly associated with quinolone exposure. Restriction of quinolone consumption has been advocated as an important tool for the containment of quinolone resistance and has recently been proved to succeed in reducing resistance rates in clinical isolates of Escherichia coli in a community setting from an industrialized country. This study describes the dissemination of quinolone resistant E. coli in a very remote community in the Amazon forest, with a high level isolation and minimal antibiotic use, not including quinolones. These findings demonstrate that intervention strategies based only on quinolone restriction are unlikely to succeed in resource-limited countries, unless accompanied by measures for reducing dissemination of resistant isolates by improving sanitation.
doi:10.1371/journal.pntd.0001790
PMCID: PMC3429404  PMID: 22953012
24.  Simultaneous RNA quantification of human and retroviral genomes reveals intact interferon signaling in HTLV-1-infected CD4+ T cell lines 
Virology Journal  2012;9:171.
Background
IFN-α contributes extensively to host immune response upon viral infection through antiviral, pro-apoptotic, antiproliferative and immunomodulatory activities. Although extensively documented in various types of human cancers and viral infections, controversy exists in the exact mechanism of action of IFN-α in human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1) retroviral infections.
Results
IFN-α displayed strong anti-HIV-1 effects in HIV-1/HTLV-1 co-infected MT-4 cells in vitro, demonstrated by the dose-dependent inhibition of the HIV-1-induced cytopathic effect (IC50 = 83.5 IU/ml, p < 0.0001) and p24 levels in cell-free supernatant (IC50 = 1.2 IU/ml, p < 0.0001). In contrast, IFN-α treatment did not affect cell viability or HTLV-1 viral mRNA levels in HTLV-1 mono-infected cell lines, based on flow cytometry and nCounter analysis, respectively. However, we were able to confirm the previously described post-transcriptional inhibition of HTLV-1 p19 secretion by IFN-α in cell lines (p = 0.0045), and extend this finding to primary Adult T cell Leukemia patient samples (p = 0.031). In addition, through microarray and nCounter analysis, we performed the first genome-wide simultaneous quantification of complete human and retroviral transciptomes, demonstrating significant transcriptional activation of interferon-stimulated genes without concomitant decrease of HTLV-1 mRNA levels.
Conclusions
Taken together, our results indicate that both the absence of in vitro antiproliferative and pro-apoptotic activity as well as the modest post-transcriptional antiviral activity of IFN-α against HTLV-1, were not due to a cell-intrinsic defect in IFN-α signalisation, but rather represents a retrovirus-specific phenomenon, considering the strong HIV-1 inhibition in co-infected cells.
doi:10.1186/1743-422X-9-171
PMCID: PMC3492208  PMID: 22917064
Retrovirus; IFN-α; HIV-1; HTLV-1; IFN-α signaling; Antiviral activity
25.  Ascorbic Acid Has Superior Ex Vivo Antiproliferative, Cell Death-Inducing and Immunomodulatory Effects over IFN-α in HTLV-1-Associated Myelopathy 
Background
Clear therapeutic guidelines for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are missing due to the lack of randomized double-blind controlled clinical trials. Moderate yet similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (AA) monotherapy in a large open clinical trial. However, there is a lack of in vivo and in vitro studies exploring and comparing the effects of high-dose AA and IFN-α treatment in the context of HAM/TSP. Therefore, we performed the first comparative analysis of the ex vivo and in vitro molecular and cellular mechanisms of action of IFN-α and high-dose AA in HAM/TSP.
Principal Findings
Through thymidine incorporation and quantification of Th1/Th2/Th17 cytokines, we demonstrate that high-dose AA displays differential and superior antiproliferative and immunomodulatory effects over IFN-α in HAM/TSP PBMCs ex vivo. In addition, high-dose AA, but not IFN-α, induced cell death in both HAM/TSP PBMCs and HTLV-1-infected T-cell lines MT-2 and MT-4. Microarray data combined with pathway analysis of MT-2 cells revealed AA-induced regulation of genes associated with cell death, including miR-155. Since miR-155 has recently been demonstrated to up-regulate IFN-γ, this microRNA might represent a novel therapeutic target in HAM/TSP, as recently demonstrated in multiple sclerosis, another neuroinflammatory disease. On the other hand, IFN-α selectively up-regulated antiviral and immune-related genes.
Conclusions
In comparison to IFN-α, high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti-HTLV-1 effects. Differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets.
Author Summary
HAM/TSP is a chronic and disabling neuroinflammatory disease, for which clinical management is mostly empirical and symptomatic rather than evidence-based, due to the lack of biomarkers and controlled clinical trials. Although similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (vitamin C) in one major open clinical trial with 200 patients, their cellular and molecular mechanisms of action remain unexplored in HAM/TSP. We demonstrate that high-dose ascorbic acid strongly inhibits lymphoproliferation of HAM/TSP mononuclear cells in ex vivo cultures, in contrast to IFN-α. Furthermore, high-dose ascorbic acid, but not IFN-α, significantly decreased ex vivo TNF-α and IFN-γ pro-inflammatory cytokine levels in supernatant of mononuclear cells from HAM/TSP patients. In addition, ascorbic acid, but not IFN-α, induced cell death in HTLV-1-infected T-cell lines, which was confirmed by gene expression profiling, revealing cell death-associated pathways activated by high-dose ascorbic acid, including miR-155. This microRNA has previously been shown up-regulated in HTLV-1-infected cells, as well as in blood and brain samples of multiple sclerosis patients, another neuroinflammatory disease. In addition, miR-155 has also been reported to up-regulate IFN-γ production in human natural killer cells, thus linking both cell death and cytokine signaling pathways, rendering it a potential therapeutic target in neuroinflammatory disorders. Thus, our findings reveal molecular mechanisms of action as well as candidate biomarkers for high-dose ascorbic acid therapy and provide a rational basis, rather than an empirical basis, for its use in HAM/TSP treatment.
doi:10.1371/journal.pntd.0001729
PMCID: PMC3404116  PMID: 22848768

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