The isolation of Balamuthia mandrillaris from environmental sources from Peru.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

The role of intrapulmonary de novo lymphoid tissue in obliterative bronchiolitis after lung transplantation.

Chronic rejection after lung transplantation is manifested as obliterative bronchiolitis (OB). The development of de novo lymphoid tissue (lymphoid neogenesis) may contribute to local immune responses in small airways. Compared with normal lungs, the lung tissue of 13 lung transplant recipients who developed OB demonstrated a significantly larger number of small, airway-associated, peripheral node addressin-positive (PNAd(+)) high endothelial venules (HEVs) unique to lymphoid tissue (p < 0.001). HEVs were most abundant in lesions of lymphocytic bronchiolitis and "active" OB infiltrated by lymphocytes compared with those of "inactive" OB. T cells in lymphocytic bronchiolitis and active OB were predominantly of the CD45RO(+)CCR7(-) effector memory phenotype. Similar lymphoid tissue was also observed in the rat lung after intrapulmonary transplantation of allograft trachea (Brown Norway (BN) to Lewis), but not after isograft transplantation. Subsequent orthotopic transplantation of the recipient Lewis lung containing a BN trachea into an F(1) (Lewis x BN) rat demonstrated stable homing of Lewis-derived T cells in the lung and their Ag-specific effector function against the secondary intrapulmonary BN trachea. In conclusion, we found de novo lymphoid tissue in the lung composed of effector memory T cells and HEVs but lacking delineated T cell and B cell zones. This de novo lymphoid tissue may play a critical role in chronic local immune responses after lung transplantation.

Zika Virus Seroprevalence in Two Districts of Chincha, Ica, Peru: A Cross-Sectional Study.

In 2017, a major outbreak of Zika virus (ZIKV) infection took place in Chincha Province, Peru, where arboviral circulation had never been reported before. We conducted a cross-sectional survey (March-May 2019) in two districts of Chincha Province: Pueblo Nuevo and Chincha Baja. We included residents who were 20 to 40 years old and who had lived in these districts for at least 1 year. Serological testing combined screening with a commercial NS1 protein-based Zika IgG ELISA, and confirmation by a cytopathic effect-based virus neutralization test (VNT). Prevalence ratios (PRs) were calculated using Poisson regression with robust error variance. Four hundred participants, divided equally among districts, were enrolled. Anti-ZIKV IgG ELISA was positive for 42 participants (10.5%) and borderline for 12 (3%). Fifty-two of these 54 samples were confirmed positive by ZIKV VNT (13% of the total population). The Pueblo Nuevo district exhibited a greater ZIKV seroprevalence based on VNT results than the Chincha Baja district (23.5% versus 2.5%), with participants from the Pueblo Nuevo district being 9.4 times more likely to have a positive ZIKV VNT result. Average monthly income greater than the minimum wage and adequate water storage were found to be protective factors (PR, 0.29 and 0.24, respectively). In multivariate analysis, living in the Pueblo Nuevo district and a personal history of fever and rash were strong predictors of ZIKV positivity by VNT. The low ZIKV seroprevalence should prompt health authorities to stimulate interventions to prevent potential future outbreaks. In the Pueblo Nuevo district, the seroprevalence was greater but presumably not sufficient to ensure protective herd immunity.

High frequency of the exoU+/exoS+ genotype associated with multidrug-resistant "high-risk clones" of Pseudomonas aeruginosa clinical isolates from Peruvian hospitals.

The type III secretion system of Pseudomonas aeruginosa is an important virulence factor contributing to the cytotoxicity and the invasion process of this microorganism. The current study aimed to determine the presence of the exoU+/exoS+ genotype in P. aeruginosa clinical isolates. The presence of exoS, exoT, exoU and exoY was determined in 189 P. aeruginosa by PCR, and the presence/absence of exoU was analysed according to source infection, clonal relationships, biofilm formation, motility and antimicrobial susceptibility. The gyrA, parC, oprD, efflux pump regulators and ß-lactamases genes were also analysed by PCR/sequencing. The exoS, exoT and exoY genes were found in 100% of the isolates. Meanwhile, exoU was present in 43/189 (22.8%) of the isolates, being significantly associated with multidrug resistance, extensively drug resistance as well as with higher level quinolone resistance. However, the presence of ß-lactamases, mutations in gyrA and parC, and relevant modifications in efflux pumps and OprD were not significantly associated with exoU+ isolates. MLST analysis of a subset of 25 isolates showed 8 different STs displaying the exoU+/exoS+ genotype. The MDR basis of the exoU+ isolates remain to be elucidated. Furthermore, the clinical implications and spread of exoU+/exoS+ P. aeruginosa isolates need to be established.

Specific type IV pili groups in clinical isolates of Pseudomonas aeruginosa.

The relationships between specific type IV pili (TFP) groups and antibiotic resistance, biofilm formation, and bacterial motility were determined in 190 Pseudomonas aeruginosa clinical isolates. While motility and biofilm formation were determined by phenotypic assays, the presence of TFP was determined by PCR assay and antibiotic susceptibility by disk diffusion. The results showed a high ability to form biofilm (97.4%), multidrug resistance (44.7%), and the presence of a high number of motile isolates. We also found an association between strong biofilm production and multidrug resistance. Furthermore, TFP group III was associated with strong biofilm production. In contrast, the isolates with TFP group II and those without any TFP were associated with non-strong biofilm production. Regarding motility, TFP group II was associated with higher percentages of swarming, swimming, and twitching, while TFP group I showed lower percentages of swarming and twitching, and TFP group III showed lower levels of swarming and swimming. In conclusion, these findings highlight the differences in P. aeruginosa phenotypes related to the presence of specific TFP groups and their potential implications in clinical settings.

Carrion's disease: more than a neglected disease.

Infections with Bartonella bacilliformis result in Carrion's disease in humans. In the first phase of infection, the pathogen causes a hemolytic fever ("Oroya fever") with case-fatality rates as high as ~90% in untreated patients, followed by a chronical phase resulting in angiogenic skin lesions ("verruga peruana"). Bartonella bacilliformis is endemic to South American Andean valleys and is transmitted via sand flies (Lutzomyia spp.). Humans are the only known reservoir for this old disease and therefore no animal infection model is available. In the present review, we provide the current knowledge on B. bacilliformis and its pathogenicity factors, vectors, possible unknown reservoirs, established and potential infection models and immunological aspects of the disease.

Cost-effectiveness analysis of introduction of rapid, alternative methods to identify multidrug-resistant tuberculosis in middle-income countries.

BACKGROUND: Resistance to commonly used antituberculosis drugs is emerging worldwide. Conventional drug-susceptibility testing (DST) methods are slow and demanding. Alternative, rapid DST methods would permit the early detection of drug resistance and, in turn, arrest tuberculosis transmission. METHODS: A cost-effectiveness analysis of 5 DST methods was performed in the context of a clinical trial that compared rapid with conventional DST methods. The methods under investigation were direct phage-replication assay (FASTPlaque-Response; Biotech), direct amplification and reverse hybridization of the rpoB gene (INNO-LiPA; Innogenetics), indirect colorimetric minimum inhibitory concentration assay (MTT; ICN Biomedicals), and direct proportion method on Löwenstein-Jensen medium. These were compared with the widely used indirect proportion method on Löwenstein-Jensen medium. RESULTS: All alternative DST methods were found to be cost-effective, compared with other health care interventions. DST methods also generate substantial cost savings in settings of high prevalence of multidrug-resistant tuberculosis. Excluding the effects of transmission, the direct proportion method on Löwenstein-Jensen medium was the most cost-effective alternative DST method for patient groups with prevalences of multidrug-resistant tuberculosis of 2%, 5%, 20%, and 50% (cost in US$2004, $94, $36, $8, and $2 per disability-adjusted life year, respectively). CONCLUSION: Alternative, rapid methods for DST are cost-effective and should be considered for use by national tuberculosis programs in middle-income countries.

Mycobacterium ulcerans disease, Peru.

Eight adult patients (ages 18-58, 5 women) with Buruli ulcer (BU) confirmed by at least 2 diagnostic methods were seen in a 10-year period. Attempts to culture Mycobacterium ulcerans failed. Five patients came from jungle areas, and 3 from the swampy northern coast of Peru. The patients had 1-5 lesions, most of which were on the lower extremities. One patient had 5 clustered gluteal lesions; another patient had 2 lesions on a finger. Three patients were lost to follow-up. All 5 remaining patients had moderate disease. Diverse treatments (antituberculous drugs, World Health Organization [WHO] recommended antimicrobial drug treatment for BU, and for 3 patients, excision surgery) were successful. Only 1 patient (patient 7) received the specific drug treatment recommended by WHO. BU is endemic in Peru, although apparently infrequent. Education of populations and training of health workers are first needed to evaluate and understand the full extent of BU in Peru.

Novel hypertonic saline-sodium hydroxide (HS-SH) method for decontamination and concentration of sputum samples for Mycobacterium tuberculosis microscopy and culture.

This study evaluated a new decontamination and concentration (DC) method for sputum microscopy and culture. Sputum samples from patients with suspected pulmonary tuberculosis (TB) (n=106) were tested using the proposed hypertonic saline-sodium hydroxide (HS-SH) DC method, the recommended N-acetyl-L-cysteine-sodium citrate-sodium hydroxide (NALC-NaOH) DC method and unconcentrated direct smear (Ziehl-Neelsen) techniques for the presence of mycobacteria using Löwenstein-Jensen culture and light microscopy. Of 94 valid specimens, 21 (22.3%) were positive in culture and were further characterized as Mycobacterium tuberculosis. The sensitivity for acid-fast bacilli (AFB) smears was increased from 28.6% using the direct method to 71.4% (HS-SH) and 66.7% (NALC-NaOH) using DC methods. Both concentration techniques were highly comparable for culture (kappa=0.794) and smear (kappa=0.631) for AFB. Thus the proposed HS-SH DC method improved the sensitivity of AFB microscopy compared with a routine unconcentrated direct smear; its performance was comparable to that of the NALC-NaOH DC method for AFB smears and culture, but it was methodologically simpler and less expensive, making it a promising candidate for evaluation by national TB control programmes in developing countries.

A simple and improved method for diagnosis of tuberculosis using hypertonic saline and sodium hydroxide to concentrate and decontaminate sputum.

We report the use of hypertonic saline and sodium hydroxide (HS-SH) for concentration-decontamination of sputum samples for the diagnosis of tuberculosis. This method increases detection of mycobacteria in smears compared to examination of direct smears, is comparable and reduces contamination in cultures compared to NALC-NaOH.

Rapid detection of salmonellosis due to Salmonella enterica serovar Typhimurium in Peruvian commercially bred cavies, using indigenous wild bacteriophages.

INTRODUCTION: The salmonelloses are among the commonest, most widespread human zoonotic infections. They have generated international networks to attempt their control, since they cause a spectrum of ailments, ranging from inapparent carrier states to full-blown, severe, sometimes deadly diarrheal and systemic disease. Rapid diagnosis is needed for a number of reasons. The aim of this study was to standardize and validate a phage amplification test for the identification of salmonellosis to be applied to infections of Cavia porcellus. METHODS: Native bacteriophages were isolated from infected cavies and environmental residues from commercial cavy-breeding facilities. Salmonella enterica serovar Typhimurium ATCC 14028 was used to detect, isolate and propagate the bacteriophages, and to standardize a phage amplification assay to detect S. Typhimurium from rectal swabs of cavies. The phage amplification assay was tested using 2 antiviral agents, MgSO4·7H2O (MAS) and pomegranate rind extract (PRE) plus ferrous sulfate (PRE-FeSO4). RESULTS: The final assay format chosen used PRE-FeSO4 and allowed detection of S. Typhimurium in 90 min from culture, 5 h from clinical samples, with a limit of detection at 103 pfu; sensitivity was 98.2%, specificity 98%, negative predictive value (NPV) 96.1%, and positive predictive value (PPV) 99.1%. CONCLUSION: Bacteriophage amplification is therefore an appropriate, fast procedure for detection of this pathogen in clinical samples.

Interplay between MexAB-OprM and MexEF-OprN in clinical isolates of Pseudomonas aeruginosa.

MexAB-OprM and MexEF-OprN are Pseudomonas aeruginosa efflux pumps involved in the development of antibiotic resistance. Several studies developed with laboratory strains or using a few clinical isolates have reported that the regulation system of MexEF-OprN is involved in the final levels of MexAB-OprM expression. Therefore, this study was aimed to determine the interplay between MexAB-OprM and MexEF-OprN in 90 out of 190 P. aeruginosa clinical isolates with an efflux pump overexpression phenotype. Regarding oprD, 33% (30/90) of isolates displayed relevant modifications (RM) defined as frameshift or premature stop, both related to carbapenem resistance. On the other hand, 33% of the isolates displayed RM in nalC, nalD or mexR, which were significantly associated with multidrug resistance (MDR), non-susceptibility to carbapenems, OprD alterations and strong biofilm production. Meanwhile, the RM in MexS were associated with presence of pigment (p = 0.004). Otherwise, when all the regulators were analysed together, the association between RM in MexAB-OprM regulators and MDR was only significant (p = 0.039) when mexS was the wild type. These data show the modulatory effect of MexEF-OprN on MexAB-OprM in a clinical population of P. aeruginosa. Further studies may contribute to design of novel molecules acting on this interplay to fight against antimicrobial resistance.

Evaluation of rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis by a mycobacteriophage D29-based assay.

Conventional methods for determining drug susceptibility of Mycobacterium tuberculosis require several weeks to obtain results, limiting their usefulness; automated methods and those based on molecular biology techniques have been able to reduce the turnaround time, but their high cost and need for sophisticated equipment restrict their use in developing countries. The goal of the present study was to evaluate the diagnostic accuracy of a rapid (3-4 days) low-cost test based on the use of mycobacteriophage D29 to determine the susceptibility of strains of M. tuberculosis to rifampicin (RIF) and isoniazid (INH). Results obtained show that susceptibility testing for RIF has a high diagnostic accuracy as compared to the standard indirect proportion method on Löwenstein-Jensen medium (sensitivity 100% and specificity 98%). Given the association between the resistance to RIF and INH, which define multidrug resistance (MDR), this test seems suitable for rapid detection of MDR tuberculosis strains (kappa=0.978). Susceptibility testing for INH using mycobacteriophage D29 had a good but lower diagnostic accuracy as compared to the standard method (sensitivity 80.4% and specificity 80.8%); the test would then be of limited usefulness in the management of tuberculosis patients. Further studies to determine the relationship of mycobacteriophage D29 tests to in vivo correlates of sensitivity to specific antituberculosis drugs are warranted.

Evaluation of a rapid screening method for detection of antimicrobial resistance in the commensal microbiota of the gut.

The assessment of antimicrobial resistance among commensal bacteria is an indicator of the spread of antimicrobial resistance. Rapid screening methods for detection of antimicrobial-resistant faecal Escherichia coli directly on MacConkey plates have been successfully adopted but suffer from lack of standardisation. The purpose of this study was to evaluate a direct plating method (DPM) for detection of antimicrobial-resistant faecal E. coli and to compare it with a conventional method. Faecal samples were collected from 71 healthy children from Peru and Bolivia. In the DPM, a faecal swab was directly plated onto a MacConkey agar plate and antimicrobial disks were applied onto the seeded plate. Raw data were obtained by direct reading of the plate and were subjected to confirmatory analysis. Good concordance between the DPM and a conventional method was observed in detecting carriage of resistant E. coli, with a higher sensitivity for the DPM. Analysis of the results allowed interpretive criteria to be defined for DPM raw data. The DPM showed good sensitivity and specificity at very low cost (ten times cheaper than the conventional method) to investigate the faecal carriage of drug-resistant E. coli. It may represent a useful tool to conduct large-scale resistance surveillance studies and to monitor resistance control programmes cost effectively, particularly in low-resource countries.

Tomographic comparison of ventilation techniques for CT-guided thoracoscopic staple excision of subcentimeter lung nodules.

This study was planned to compare the computed tomographic detectability of lung nodules in three ventilatory conditions: total lung capacity, high-frequency ventilation, and total lung deflation. In an ex vivo lung model, 44 nodules were simulated. Using computed tomography (CT) scans, nodules were detected and compared to the actual number and excised under CT guidance. Simulated nodules measured 6.2 +/- 2.1 mm and demonstrated an attenuation of 175 +/- 14 HU. Observer confidence was highest at total lung capacity (5.00 +/- 0.00), in comparison to high-frequency ventilation and total lung deflation (4.69 +/- 0.78, 4.94 +/- 0.27, p = .24). The kappa score for total lung capacity, high-frequency ventilation, and total lung deflation was 1.00, 0.96, and 0.98, respectively, indicating a very high interrater reliability. Although surgical devices generated a substantial artifact, 90% of nodules were excised. Thus, although total lung capacity produces the highest confidence level, all three of the ventilatory techniques examined have similar detection of subcentimeter pulmonary nodules using computed tomography scans.

Rhinoscleroma: eight Peruvian cases.

Rhinoscleroma is a rare infection in developed countries; although, it is reported with some frequency in poorer regions such as Central Africa, Central and South America, Eastern and Central Europe, Middle East, India and Indonesia. Nowadays, rhinoscleroma may be erroneously diagnosed as mucocutaneos leishmaniasis, leprosy, paracoccidioidomycosis, rhinosporidiasis, late syphilis, neoplasic diseases or other upper airway diseases. From 1996 to 2003, we diagnosed rhinoscleroma in eight patients attended in the Dermatologic and Transmitted Diseases service of "Cayetano Heredia" National Hospital, in Lima, Peru. The patients presented airway structural alterations producing nasopharyngeal, oropharyngeal and, in one patient, laryngeal stenosis. Biopsy samples revealed large vacuolated macrophages (Mikulicz cells) in all patients. Ciprofloxacin 500 mg bid for four to 12 weeks was used in seven patients and oxytetracycline 500 mg qid for six weeks in one patient. After follow-up for six to 12 months the patients did not show active infection or relapse, however, all of them presented some degree of upper airway stenosis. These cases are reported because of the difficulty diagnosing the disease and the success of antibiotic treatment.

Gene Therapy for Lung Cancer.

Gene therapy was originally conceived to treat monogenic diseases. The replacement of a defective gene with a functional gene can theoretically cure the disease. In cancer, multiple genetic defects are present and the molecular profile changes during the course of the disease, making the replacement of all defective genes impossible. To overcome these difficulties, various gene therapy strategies have been adopted, including immune stimulation, transfer of suicide genes, inhibition of driver oncogenes, replacement of tumor-suppressor genes that could mediate apoptosis or anti-angiogenesis, and transfer of genes that enhance conventional treatments such as radiotherapy and chemotherapy. Some of these strategies have been tested successfully in non-small-cell lung cancer patients and the results of laboratory studies and clinical trials are reviewed herein.

A window of opportunity study of potential tumor and soluble biomarkers of response to preoperative erlotinib in early stage non-small cell lung cancer.

BACKGROUND: Erlotinib is highly active in EGFR mutant NSCLC, but may benefit some with wild-type tumors. We examined pre-operative erlotinib in early stage NSCLC to assess response and correlation with potential biomarkers. RESULTS: Twenty-five patients were enrolled; 22 received erlotinib treatment and were evaluable (median follow-up 4.4 years). Histology was predominantly adenocarcinoma although 31% had squamous carcinoma. PET response was observed in 2 patients (9%), both with squamous carcinoma. Most (20/22) had stable disease (RECIST), with frequent minor radiographic regression and histologic findings of fibrosis/necrosis including in squamous histology. Only two had EGFR mutations identified, one with minor radiographic response and the other stable disease after 4 weeks of EGFR TKI. High pre-treatment serum levels of TGF-α correlated with primary resistance to erlotinib (p = 0.02), whereas high post-treatment soluble EGFR levels correlated with response (p = 0.03). EGFR, PTEN, cMET and AXL expression did not correlate with tumor response. METHODS: Clinical stage IA-IIB NSCLC patients received erlotinib 150 mg daily for 4 weeks followed by resection. Tumor response was assessed using CT, PET and pathological response. Tumor genotype was established using Sequenom Mass ARRAY; EGFR, PTEN, cMET and AXL expression was assessed by immunohistochemistry, circulating markers of EGFR activation (TGF-α, amphiregulin, epiregulin, EGFR ECD) by ELISA and EGFR, MET copy number by FISH. CONCLUSIONS: Erlotinib appears to demonstrate activity in EGFR wild-type tumors including squamous carcinoma. Further research is needed to characterize those wild-type patients that may benefit from EGFR TKI and predictive biomarkers including TGF-α, EGFR copy and others.

Clinical spectrum of pulmonary involvement in leptospirosis in a region of endemicity, with quantification of leptospiral burden.

BACKGROUND: Pulmonary involvement in leptospirosis remains poorly recognized in regions where it is endemic, despite reports of recent outbreaks and epidemic disease. METHODS: A prospective, population-based study was carried out to identify febrile patients exposed to Leptospira in urban and rural contexts in Iquitos, Peru. Evidence of exposure to Leptospira was obtained by serologic testing, and diagnosis of leptospirosis was confirmed in pulmonary cases by culture or quantitative real-time PCR assay. RESULTS: Of 633 consecutively enrolled febrile patients, 321 (50.7%) had antileptospiral IgM antibodies or high titers of antileptospiral antibodies. Seven patients with histories of only urban exposure to leptospires had severe pulmonary manifestations; of these, 5 patients died; 4 of the deaths were caused by pulmonary hemorrhage, and 1 was caused by acute respiratory distress syndrome and multiorgan failure. Real-time, quantitative PCR assay showed high levels of leptospiremia (>or=10(4) leptospires/mL) in most fatal cases; 1 patient, from whom tissue specimens were obtained at autopsy, had >or=10(5) leptospires/g of lung, kidney, and muscle tissue. DISCUSSION. This study demonstrates the underdiagnosis of leptospirosis in a region of high endemicity and the underrecognition of grave pulmonary complications. Pulmonary involvement in leptospirosis was present in urban but not rural areas. Presumptive treatment for leptospirosis should be initiated immediately in the appropriate epidemiological and clinical context.