Uso racional de antibióticos y tecnología FilmArray para identificación rápida de bacteriemias en unidad de cuidados intensivos pediátrica / Rational use of antibiotics and FilmArray technology for rapid identification of bacteremias in a pediatric intensive care unit

INTRODUCCIÓN: Las infecciones graves son la principal causa de ingreso a cuidados intensivos pediátricos. El panel FilmArray BCID permite identificar rápidamente a microorganismos causantes de bacteriemias. OBJETIVO: evaluar la eficacia de la identificación rápida de microorganismos asociado a un Programa de Uso Racional de Antibióticos (URA) en reducir los tiempos de terapias antibióticas, en un hospital pediátrico. PACIENTES Y MÉTODO: Estudio retrospectivo, que incluyó 100 pacientes, en su primer episo dio de bacteriemia, divididos en 2 grupos de 50 cada uno: Intervención (FilmArray BCID y programa URA) y Controles históricos pareados para la misma especie del microrganismo identificado (microbiología convencional). Las variables evaluadas fueron los tiempos de identificación microbiana, latencia de la terapia dirigida y de desescalar antibióticos. RESULTADOS: Los grupos fueron comparables en características demográficas, foco de infección y etiología de bacteriemia. El tiempo promedio de identificación de microorganismos fue de 23 h (IC 95% 12,4-26,7) en el grupo intervención, y 70,5 h (IC 95% 65,2-78,6) en el control (p < 0,05), mientras que la latencia de inicio de terapia dirigida fue de 27,9 h (IC 95% 22,3-32,8) y 71,9 h (IC 95% 63,2-77,8) respectivamente (p < 0,05). El tiempo de desescalar o suspender antibióticos fue de 6,4 h (IC 95% 2,76-9,49) y 22 h (IC 95% 6,74-35,6) en los grupos mencionados (p > 0,05). CONCLUSIÓN: El panel FilmArray BCID articulado a un programa URA, contribuye a la identificación de los microorganismos causantes de bacteriemias en menor tiempo que los métodos convencionales, siendo una herramienta que optimiza las terapias antibióti cas en niños críticamente enfermos.

INTRODUCTION: Severe infections are the leading cause of admission to pediatric intensive care. The FilmArray BCID panel quickly identifies microorganisms that cause bacteremia. OBJECTIVE: To evaluate if the rapid identification of the microorganisms that cause bacteremia, along with a Rational Use of Antibio tics (RUA) Program, allows optimizing the time of antibiotic therapy in a pediatric hospital. PATIENTS AND METHOD: Retrospective study which included 100 patients presenting their first episode of bacteremia, divided into 2 groups of 50 each. The first one was Intervention (FilmArray BCID and RUA program) and the second one was Historical Controls (conventional automated ID/AST). The variables evaluated were the time required for microbial identification, duration of appropriate therapy, and antibiotic de-escalation. RESULTS: The groups were comparable in terms of demographic characteristics, focus of infection, and etiology of bacteremia. The average time of microorganisms' identification of the control group was 70.5 hours (IC 95% 65.2-78.6) and 23.0 hours (IC 95% 12.4 -26.7) in the intervention one (p < 0.05). The average time of targeted therapy onset was shorter in the intervention group (27.9 h [IC 95% 22.3-32.8]) than that of the control one (71.9 h [IC 95% 63.2-77.8]) (p < 0.05). Finally, the time to de-escalate or discontinue antibiotics in the intervention group and the control one was 6.4 hours (IC 95% 2.76-9.49) hours and 22.0 hours (IC 95% 6.74-35.6 h) respectively (p > 0.05). CONCLUSION: The FilmArray panel along with the RUA Program allows the identification of the microorganisms causing bacteremia faster than conventional methods, which positions it as a tool that optimizes antibiotic therapy of critical patients.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

First molecular typing of Mycobacterium avium subspecies paratuberculosis identified in animal and human drinking water from dairy goat farms in Brazil

Abstract Mycobacterium avium subspecies paratuberculosis, the etiologic agent of Johne's disease or paratuberculosis, was identified by culture and/or polymerase chain reaction (PCR) in 50% and 30% of water samples for animal and human consumption, respectively, from ten dairy goat farms in Brazil. IS1311 restriction fragment length polymorphism analysis identified the isolates as cattle type C.

Molecular characterization of Mycobacterium tuberculosis and Mycobacterium bovis isolates by Enterobacterial Repetitive Intergenic Consensus-PCR

Tuberculosis (TB) is an infectious disease in which the molecular typing methods allow to have important information about the dynamics of transmission and to assist properly in disease control. Although the ERIC-PCR (Enterobacterial repetitive intergenic consensus-PCR) assay is fast and easy to perform, scarce studies have reported its use in epidemiological studies in TB outbreaks. In this study, we aimed to genotype Mycobacterium tuberculosis and M. bovis isolates by ERIC-PCR and compare its discriminatory power with two other classically used methods: 12 loci-MIRU (Mycobacterial Interspersed Repetitive Units) and Spoligotyping. The M. tuberculosis isolates studied were from northwestern and southwestern and M. bovis from northwestern Parana, Brazil. ERIC-PCR rendered banding patterns with great diversity (1 to 12 bands) of molecular sizes, ranging from 100 to 1600 bp. ERIC-PCR showed to be fast, simple and affordable to differentiate isolates. ERIC-PCR would be an important tool in the epidemiology of TB as screening in case of outbreak, which demands rapid intervention. However if any doubt persist, as it may occur with the application of only one genotypic method, other genotyping methods should be applied and carefully interpreted, always with additional epidemiological information.

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.

Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Comparison of MALDI-TOF MS, Housekeeping Gene Sequencing, and 16S rRNA Gene Sequencing for Identification of Aeromonas Clinical Isolates

PURPOSE: The genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB). MATERIALS AND METHODS: We analyzed 65 Aeromonas isolates recovered from patients at a university hospital in Korea between 1996 and 2012. The isolates were recovered from frozen states and tested using the following four methods: a conventional biochemical method, 16S rRNA sequencing, housekeeping gene sequencing with phylogenetic analysis, and MALDI-TOF MS. RESULTS: The conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level. CONCLUSION: The most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.

Genetic diversity of Mycobacterium tuberculosis strains isolated in Algeria: results of spoligotyping

Objective/background: Molecular typing tools, including spoligotyping, are currently widely used in the monitoring and study of the dynamics of tuberculosis epidemics

Methods: A study of the molecular profile of a sample of 129 Myobacterium tuberculosis strains isolated during 2011 was carried out in the National Reference Laboratory for Tuberculosis and Mycobacteria at the Pasteur Institute of Algeria. This sample was selected at random from a set of 350 strains isolated from tuberculosis patients from central and eastern areas of the country

Results: Genotypic analysis helped to clarify the frequencies of the different genotypes in the current study population: H family, 29%; LAM family, 26%; T family, 25%; S family, 5%, and other genomic families, including orphan strains, 15%

Conclusion: The study of strains isolated between January and December 2011 has allowed insight into the frequency of different genomic families and the importance of existing clusters in the population of central and eastern Algeria

Development of RFLP-PCR method for the identification of medically important Aspergillus species using single restriction enzyme MwoI

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Molecular typing of Mycobacterium bovis isolates: a review

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA

In this study, the discriminatory power of pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods for subtyping of 54 clinical isolates of Klebsiella pneumoniae were compared. All isolates were typeable by RAPD, while 3.6% of them were not typeable by PFGE. The repeatability of both typing methods were 100% with satisfying reproducibility (≥ 95%). Although the discriminatory power of PFGE was greater than RAPD, both methods showed sufficient discriminatory power (DI > 0.95) which reflects the heterogeneity among the K. pneumoniae isolates. An optimized RAPD protocol is less technically demanding and time consuming that makes it a reliable typing method and competitive with PFGE.

Molecular epidemiology of Vibrio cholerae causing outbreaks & sporadic cholera in northern India.

Background & objectives: Several outbreaks of cholera have been reported in Chandigarh region during a span of seven years from 2002-2008. The genetic characteristics of Vibrio cholerae isolates obtained during these outbreaks have not been adequately studied. The aim of this study was to do molecular typing of V. cholerae isolated from the sporadic and outbreak cases by pulsed-field gel electrophoresis (PFGE), Rep-PCR and ribotyping. Methods: Fifty representative isolates of V. cholerae from outbreak as well as sporadic cases were subjected to molecular typing by PFGE, 173 isolates (163 clinical and 10 environmental) were typed by rep-PCR and ribotyping. Ribotyping was done by determination of rRNA restriction pattern of BglI restriction digestion and hybridization with 7.2 kb rRNA probe of pKK3535 plasmid using DIG DNA labelling and detection kit. Universal VC1 primer was used for rep-PCR. Results: PFGE generated 15 pulsotypes, of which four matched the published pulsotypes and there were 11 new pulsotypes. PFGE was the most discriminatory method that could differentiate between isolates belonging to single ribotype. Pulsotype P1 corresponding to known pulsotype H1 was the major pulsotype till 2003. Pulsotype P3 corresponding to known pulsotype L emerged in 2004. The 2007 outbreaks in Punjab and Haryana were caused by P5 though P1 and P3 were isolated from the sporadic cases from the same region. The 2008 outbreak was caused by pulsotypes P6 and P7. Ribotype IV was the most predominant followed by RIII. This ribotype was not isolated after 2003 and ribotype IV became the most predominant 2004 onwards. Of the two unknown ribotypes (UNI and UN2), UNI was more common (27 isolates). Rep-PCR was the least discriminatory and divided all clinical isolates into four major profiles. The dendrogram analysis of PFGE revealed similarity of some clinical isolates with environmental isolates indicating the genetic relatedness. Interpretation & conclusion: Our findings showed that Rep-PCR was least discriminatory method. Ribotyping was a reliable and reproducible method. Ribotype IV was predominant ribotype followed by RIII. A total of 15 pulsotypes were generated and 11 of these were not reported earlier. Genetic relatedness was shown by clinical and environmental isolates which needs to be confirmed in future studies.

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.

A novel multiplex PCR for molecular characterization of methicillin resistant Staphylococcus aureus recovered from Jeddah, Kingdom of Saudi Arabia.

Background: Molecular characterization of staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) is very essential for studying the epidemiology of MRSA. Objectives: This study reports two multiplex PCR for molecular typing of MRSA collected from Jeddah, Kingdom of Saudi Arabia. Materials and Methods: A total of 101 clinical isolates of strains were collected from major hospital laboratories and public health centres, Jeddah, Kingdom of Saudi Arabia during the period from August 2009 to May 2011. All the strains were tested phenotypically by conventional methods and genotypically by a novel multiplex PCR targeting at the same time S. aureus 16S rRNA, Panton - valentine leucocidin (PVL) and mecA resistance genes. All the strains were tested also by multiplex PCR for typing of SCC mec types. Results: All the 101 strains previously identified phenotypically as S. aureus with bacteriological examination were positive for amplification of 756 base pair fragments specific for 16S rRNA of S. aureus. Moreover, all the strains were positive for amplification of 1339 base pair fragments specific for mecA gene, while only 38 strains (37.6%) showed positive amplification of 433 base pair fragments specific for PVL gene. The most predominant SCC mec type among the examined isolates is type V 43 (42.5) followed by SCCmec type III 39 (38.6%). Conclusion: The newly modified multiplex PCR is rapid and sensitive method for detection of MRSA. Moreover, the most predominant SCC mec type among the examined isolates from Jeddah, King Saudi Arabia is type V (42.5%), followed by Type III (38.6%).

Genetic and morphological characterisation of a new species of the genus Hysterothylacium (Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of the Neotropical Region, state of Rio de Janeiro, Brazil

Taking into account the difficulties of taxonomic identification of larval anisakid nematodes based on morphological characters, genetic analyses were performed, together with those usually applied, in order to identify anisakid larvae found in the flounder Paralichthys isosceles from the littoral of the state of Rio de Janeiro, Brazil. The analysis of 1,820 larvae revealed a new species, similar to Hysterothylacium MD, Hysterothylacium 2, Hysterothylacium KB and Hysterothylacium sp regarding the absence of the larval tooth, an excretory pore situated below the nerve ring level, and slender lateral alae. Moreover, the new species differs from Hysterothylacium fortalezae and Hysterothylacium reliquens with regard to the number and size of spines present on the tail end and from Hysterothylacium patagonicus by the absence of interlabia. The maximum parsimony and neighbour joining tree topologies based on the 18S ribosomal DNA gene, complete internal transcribed spacer region and cytochrome oxidase 2 (COII) gene demonstrated that the Brazilian larvae belong to Raphidascarididae and represent a unique genetic entity, confirmed as a new Hysterothylacium species. Furthermore, the new species presents COII genetic signatures and shares polymorphisms with Raphidascarididae members. This is the first description of a new anisakid species from Brazil through the integration of morphological and molecular taxonomy data.

Evaluation of Peptide Nucleic Acid Probe-based Real-time PCR for Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Respiratory Specimens

BACKGROUND: A peptide nucleic acid (PNA) probe-based real-time PCR (PNAqPCR(TM) TB/NTM detection kit; PANAGENE, Korea) assay has been recently developed for the simultaneous detection of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in clinical specimens. The study was aimed at evaluation of the performance of PNA probe-based real-time PCR in respiratory specimens. METHODS: To evaluate potential cross-reactivity, the extracted DNA specimens from Mycobacterium species and non-mycobacterial species were tested using PNA probe-based real-time PCR assay. A total of 531 respiratory specimens (482 sputum specimens and 49 bronchoalveolar washing fluid specimens) were collected from 230 patients in July and August, 2011. All specimens were analyzed for the detection of mycobacteria by direct smear examination, mycobacterial culture, and PNA probe-based real-time PCR assay. RESULTS: In cross-reactivity tests, no false-positive or false-negative results were evident. When the culture method was used as the gold standard test for comparison, PNA probe-based real-time PCR assay for detection of MTBC had a sensitivity and specificity of 96.7% (58/60) and 99.6% (469/471), respectively. Assuming the combination of culture and clinical diagnosis as the standard, the sensitivity and specificity of the new real-time PCR assay for detection of MTBC were 90.6% (58/64) and 99.6% (465/467), respectively. The new real-time PCR for the detection of NTM had a sensitivity and specificity of 69.0% (29/42) and 100% (489/489), respectively. CONCLUSIONS: The new real-time PCR assay may be useful for the detection of MTBC in respiratory specimens and for discrimination of NTM from MTBC.

Molecular Typing in Public Health Laboratories: From an Academic Indulgence to an Infection Control Imperative / 예방의학회지

Using three Austrian case studies, the variegated applications of molecular typing in today's public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.