Leishmania spp. identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana

Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism–polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.     

A simple and rapid nested polymerase chain reaction–restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp.

A rapid and specific nested polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) has been developed to detect and differentiate pathogenic and nonpathogenic Leptospira spp. Leptospiral genomic DNA was extracted from suspected human sera using an improved method of standard phenol–chloroform, and specific primers have been used to amplify 16S ribosomal RNA from all pathogenic and nonpathogenic Leptospira spp. The PCR products of all nonpathogenic species were digested with ApoI enzyme, but not pathogenic. To evaluate this assay, we analyzed 283 serum samples collected from suspected patients with leptospirosis. Nested PCR assay confirmed that 42 (14.8%) of 283 samples harbored Leptospira infection, and RFLP assay confirmed 38 (90.5%) of 42 and 4 (9.5%) of 42 positive cases had pathogenic and nonpathogenic Leptospira spp., respectively. Based on sequencing results, Leptospira interrogans, Leptospira kirschneri, and Leptospira wolffii and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected among analyzed samples.     

Report of 2 indigenous cases of leprosy from a European country: use of polymerase chain reaction–restriction fragment length polymorphism analysis of hsp65 gene for identification of Mycobacterium leprae directly from a clinical sample

In this article, we report on 2 indigenous cases of leprosy detected in a European country. We also report on the use of polymerase chain reaction–restriction fragment length polymorphism analysis of hsp65 gene for rapid identification of Mycobacterium leprae directly from the clinical sample.     

Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification of Trichosporon non–T. asahii

The VITEK 2 system was evaluated for the identification of 74 Trichosporon invasive and non-invasive clinical isolates, comparing its results with the IGS1 sequencing. The system correctly identified Trichosporon asahii but not non–T. asahii isolates, which represented nearly 50% of the invasive infections in our nosocomial setting.     

Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time–kill methodology

The in vitro activities of amphotericin B (AmB) were evaluated against 40 isolates of Cryptococcus neoformans using time–kill curves. The isolates were obtained from 20 AIDS patients with cryptococcal meningitis submitted to AmB therapy. Isolates were exposed in vitro to 1 μg/mL of AmB that represents a serum concentration of AmB, and the viable colony counts were determined over time. AmB exhibited fungicidal activity at 6 and 12 h for 70.6% of isolates, at 24 h for 7.3%, and at 48 h for 22% of isolates, respectively. This effect was not maximized when the test drug concentration was up to 4 times the AmB MIC for the isolates. Regrowth was observed in 17.5% of the isolates after fungicidal endpoint. With standard in vitro susceptibility testing, this tolerance phenomenon could not be assessed, and thus, these tests may underestimate the resistance of C. neoformans to AmB in vivo. AmB is the first-choice drug for the treatment of cryptococcosis in Brazil, and future studies using time–kill methodology are needed to estimate the predictive value of this test in the clinical failure.     

Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae–Mycobacterium abscessus group to the species level

The Mycobacterium chelonae–Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.     

Occurrence and genotyping using automated repetitive-sequence–based PCR of methicillin-resistant Staphylococcus aureus ST398 in Southeast Austria

In this retrospective study, the occurrence and genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in Austrian MRSA patients was investigated. From 2002 to 2008, 14 MRSA ST398 were detected. First occurrence of MRSA ST398 was already found in 2004. Spa ribotyping assigned 12 isolates to spa type t011 and 1 each to spa type t034 and spa type t1451. Isolated MRSA ST398 was nontypeable by pulsed-field gel electrophoresis (NT-MRSA) using restriction enzyme SmaI; therefore, genotyping was performed using automated repetitive-sequence–based PCR (rep-PCR) on the DiversiLab system. Rep-PCR results assigned 10 (71%) of the 14 MRSA ST398 into 1 cluster with a similarity >95%; there was 1 cluster consisting of 2 isolates with a similarity >99% and 2 unique MRSA ST398 isolates. In conclusion, MRSA ST398 was continuously detected in Southeast Austria; first in 2004 with up to 5 MRSA ST398 isolates in 2008. Automated rep-PCR proved as a reliable technique in determining genetic relatedness of NT-MRSA ST398 and demonstrates clonal spread of MRSA ST398 in the investigated region.     

In vitro–in vivo correlation of voriconazole resistance due to G448S mutation (cyp51A gene) in Aspergillus fumigatus

Invasive pulmonary aspergillosis continues to be associated with a high mortality despite timely and appropriate therapy. Although host immunity plays a major role in poor clinical response, antifungal drug resistance cannot be ignored. Our studies were aimed 1) to study the mechanism of drug resistance in voriconazole strains of Aspergillus fumigatus, 2) to establish a causal relationship between cyp51A mutation and voriconazole resistance (VRC-R), and 3) to determine whether VRC-R due to cyp51A mutation correlated with in vivo resistance. A point mutation (G448S) involving cyp51A gene in VRC-R isolate was associated with resistance to VRC but not to posaconazole (POS); POS had superior activity to VRC in reducing lung fungal burden and mortality in mice infected with a VRC-R mutant of A. fumigatus. Our study demonstrated that azole resistance is based on specific site of cyp51A mutation and that in vitro VRC-R correlates with in vivo resistance.     

Molecular epidemiology characterization of OXA-23 carbapenemase-producing Acinetobacter baumannii isolated from 8 Brazilian hospitals using repetitive sequence–based PCR

The typing of multidrug-resistant Acinetobacter baumannii isolates is important for the control and prevention of hospital outbreaks. This study aimed to analyze the molecular epidemiology of 46 OXA-23 carbapenemase-producing A. baumannii strains and compare them to previously described local and international clones (ICs). Isolates were recovered during May 2009–August 2011, from 8 different hospitals in the state of Parana (Brazil). The molecular profiles were determined by repetitive extragenic palindromic PCR. Seven different clusters were identified (A to G). Thirty-two isolates were clustered in the same pattern (clone A), which belong to IC 4.     

High percentage of resistance to ciprofloxacin and qnrB19 gene identified in urinary isolates of extended-spectrum β-lactamase–producing Escherichia coli in Madrid,Spain

The presence of qnr genes in 191 extended-spectrum β-lactamase–producing Escherichia coli from 2005, with 75% of resistance to ciprofloxacin, was evaluated. An SHV-12–producing E. coli carried qnrB19; both genes were transferred by conjugation. No qnrA- or qnrS-positive strains were detected. In addition, we identified 3 new parC mutations (S80W, E84R, and E84A).     

Screening Pneumocystis carinii pneumonia in non–HIV-infected immunocompromised patients using polymerase chain reaction

We evaluated the clinical value of polymerase chain reaction (PCR) for screening Pneumocystis carinii pneumonia (PCP) in non–HIV-infected immunocompromised patients. We retrospectively analyzed PCR and Grocott's methenamine silver (GMS) staining on 1044 clinical specimens obtained from 756 patients. Positive rates of PCR and GMS staining in sputum specimens were 21.1% and 9.5%, respectively (P < 0.01), and in bronchoalveolar lavage (BAL), specimens were 31.9% and 25.5%, respectively. Among 5 patient groups, the highest GMS staining and PCR positive rates were observed in immunosuppressed patients. In 28 GMS staining-positive patients, positive rates of PCR and GMS staining after a short anti-PCP treatment were 39.3% and 17.9%, respectively. In 5 patients with positive PCR but negative GMS staining results in initial sputum examination, GMS staining result turned positive in subsequent BAL specimens. In conclusion, PCR is sensitive for detection of Pneumocystis in sputum specimens and is useful for screening PCP in non–HIV-infected high-risk patients.     

Analysis of relationship between cerebral infarction and behavioral factors and gene polymorphism of Bβ-FBG-455G/A by Logistic analysis

EpidemiologicalsurveyindicatedFBGincreaseisanimportantriskfactorofembolismdiseasesofcerebralvessels．RelationshipbetweenFBGpolymorphismandserumFBGlevelandembolismdiseasereceivedmoreattentioninrecentyears．ItwasreportedpolymorphismofBβ－FBG－４４５G／AcorrelatedwithFBGlevelratherthancerebralinfarction．Aimofpresentstudywastoinvestigatere－lationshipcerebralinfarctionbetweenabovefactorsbyusingLogisticanalysis．１Subjectandmethod１．１Subject９９patientswereadoptedincluding５９menand４０…     

Evaluation of IgG ELISA using N-lauroyl-sarcosine–soluble proteins of Bartonella henselae for highly specific serodiagnosis of cat scratch disease

Conventional IgG-ELISA methods for diagnosing cat scratch disease (CSD) caused by Bartonella hensela are still poor in sensitivity and specificity, which generally employ bacterial whole-cell proteins or N-lauroyl-sarcosine–insoluble proteins as the antigen. By Western blot analysis, we found that sarcosine-soluble fraction of proteins (SSP) showed highly specific reaction to immunofluorescence assay (IFA)–positive sera obtained from CSD patients compared with the above antigens. Clinical utility of the new ELISA employing SSP was evaluated using sera from 118 patients with clinically suspected CSD (sera positive by IFA: titers ≥ 1:256, n = 46; negative: titers < 128, n = 72) and 88 sera from healthy individuals. Sensitivity and specificity of distinguishing IFA-positive patients from healthy individuals were 95.7% and 97.7%, respectively. Fifteen discordant results were observed (13 ELISA(+)/IFA(−); 2 ELISA(−)/IFA(+)). However, all 15 sera reacted with SSP by Western blot analysis, indicating superiority of the new ELISA over IFA. The ELISA employing SSP greatly improved the accuracy of diagnosing CSD.     

Progression to bacteremia in critical care patients colonized with methicillin-resistant Staphylococcus aureus expressing Panton–Valentine leukocidin

The role of Panton–Valentine leukocidin (PVL) in methicillin-resistant Staphylococcus aureus (MRSA) infections is unclear. PVL has been long associated with soft tissue infections and necrotizing pneumonia, but inconsistently with other site infections or mortality. The retrospective cohort study explores the association between PVL and bacteremia in colonized medical intensive care unit (ICU) patients with surveillance isolates and blood cultures. A total of 840 patients were screened by nasal swab, with 266 patients found to be colonized and 46 with bacteremia. Colonization by PVL⁺ MRSA increased the odds of bacteremia (odds ratio, 2.40; confidence interval, 1.23–4.57), and invasive infection developed earlier in these patients (relative risk, 0.44; confidence interval 0.25–0.85) compared to those colonized with PVL⁰ MRSA. PVL was not associated with infections at other sites, length of ICU stay, or mortality. PVL decreases the time to bacteremia in colonized patients but does not otherwise contribute to disease course or clinical outcome.     

Cutaneous manifestations of Nocardia brasiliensis infection in Taiwan during 2002–2012—clinical studies and molecular typing of pathogen by gyrB and 16S gene sequencing

To observe the clinicopathologic and resistance profiles of the Nocardia brasiliensis causing cutaneous nocardiosis in Taiwan, 12 N. brasiliensis isolates were prospectively collected from patients with cutaneous nocardiosis in a hospital during 2002–2012. Clinicopathologic data were obtained, and isolates were identified by biochemical methods and 16S rRNA sequencing. Susceptibilities to 14 antimicrobial compounds were tested. Isolates were further genotyped by sequencing of 16S rRNA, secA1, hsp65, and gyrB genes. The nodulopustular pyoderma associated with sporotrichoid spreading was the most common skin presentations caused by N. brasiliensis. All of the isolates were susceptible to amikacin, gentamicin, tobramycin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole and resistant to kanamycin, erythromycin, and oxacillin, while susceptibilities to imipenem, vancomycin, penicillin-G, tetracycline, clindamycin, and ciprofloxacin varied among the 12 isolates. GyrB genotyping delineated the 12 isolates into 2 major groups, which was coincident with different single nucleotide substitutions at position 160 (G versus T) of 16S rRNA, different levels of imipenem minimum inhibition concentration (4–32 versus 0.25–0.75 mg/L), and prevalence of lymphadenitis (66.7 versus 16.7%). We have noted that tiny pustular lesions can be the first sign of cutaneous nocardiosis, which we believe has not been previously emphasized. No resistance to trimethoprim and sulfamethoxazole was found; therefore, sulphonamide drugs remain effective for treatment of cutaneous nocardiosis in Taiwan.     

Role of transforming growth factor-β2 in, and apossible transforming growth factor-β2 gene polymorphism as a marker of, renal dysfunction in essential hypertension: A study in Turkish patients

Background:

Many studies have shown that transforming growth factor(TGF)-β has a major role in renal scarring in many renal diseases and hypertension.

Objectives:

The primary aim of this study was to investigate both the relationship between hypertension and serum and urinary levels of TGF-β₂ (a more sensitive isoform for glomeruli than TGF-β₁), and the effects of combination therapy with perindopril + indapamide on microalbuminuria, which becomes an early indicator of hypertensive benign nephropathy, and serum and urinary TGF-β₂ levels in patients with mild to moderate essential hypertension. In addition, we examined the possible relationship between TGF-β₂ gene polymorphism and essential hypertension.

Methods:

This study was conducted at the Department of Nephrology, Medical Faculty, Gazi University, Ankara, Turkey. Patients aged ≥18 years with newly diagnosed mild to moderate essential hypertension (systolic/diastolic blood pressure [SBP/DBP] >120/>80 mm Hg) who had not previously received antihypertensive treatment were included in the study. Patients with stage I hypertension received perindopril 2 mg + indapamide 0.625 mg (tablet), and patients with stage lI hypertension received perindopril 4 mg + indapamide 1.125 mg (tablet). All study drugs were given OD (morning) PO with food for 6 months. Serum and urinary TGF-β₂ and creatinine levels and serum and urinary albumin levels were measured before and after perindopril + indapamide administration. Amplified DNA fragments of the TGF-β₂ primer region were screened using amplification refractory mutation system polymerase chain reaction analysis, and the number of ACA repeats was confirmed by DNA sequencing. Genetic studies were performed using a commercial TGF-β₂ kit.

Results:

Forty patients were enrolled in the study, and 38 patients (27 women, 11 men; mean [SD] age, 46.3 [6.5] years) completed it. SBP and DBP were significantly decreased from baseline with perindopril/indapamide (both, P < 0.001). Microalbuminuria and urinary TGF-β₂ levels also decreased significantly from baseline (P = 0.04 and P < 0.001, respectively), whereas the serum TGF-β₂ level did not change significantly. Three patients, all of whom were found to have TGF-β₂ gene mutations, had increased urinary TGF-β₂ levels despite good blood pressure control.

Conclusions:

The results of this study in patients with mild to moderate hypertension suggest that, despite good clinical control of blood pressure, the persistence of microalbuminuria and high urinary TGF-β₂ levels might predict renal impairment. When treating these patients, genetic tendencies and possible polymorphisms on the TGF-β₂ locus should be kept in mind.